Functional Food: Safety Aspects: Symposium (Forschungsberichte (DFG))

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Deutsche Forschungsgemeinschaft Functional Food: Safety Aspects Symposium edited by the Senate Commission on Food Safety SKLM Gerhard Eisenbrand (Chairman) Scientific Secretariat Sabine Guth, Monika Kemény and Doris Wolf

Deutsche Forschungsgemeinschaft Functional Food: Safety Aspects Symposium

Deutsche Forschungsgemeinschaft Functional Food: Safety Aspects Symposium edited by the Senate Commission on Food Safety SKLM Gerhard Eisenbrand (Chairman) Scientific Secretariat Sabine Guth, Monika Kemény and Doris Wolf

Deutsche Forschungsgemeinschaft Geschäftsstelle: Kennedyallee 40, D-53175 Bonn Postanschrift: D-53170 Bonn Telefon: 49/228/885-1 Telefax: 49/228/885-2777 E-Mail: (Internet RFC 822): [email protected] Internet: http://www.dfg.de

This book was carefully produced. Nevertheless, editors, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Library of Congress Card No.: applied for

A catalogue record for this book is available from the British Library.

Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographical data is available in the Internet at http://dnb.ddb.de.

ISBN 3-527-27765-X

c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Printed on acid-free and chlorine-free paper. All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Cover Design and Typography: Dieter Hüsken Composition: Hagedorn Kommunikation, Viernheim Printing: betz-druck gmbh, Darmstadt Bookbinding: J. Schäffer GmbH & Co. KG, Grünstadt Printed in the Federal Republic of Germany

Inhaltsverzeichnis

Vorwort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I

XI XIII

Hauptschlussfolgerungen und Empfehlungen . . . . . . . . . . . 1 Einleitung . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Allgemeine Aspekte der Sicherheitsbewertung . . . . . . . . . 3 Spezielle Aspekte der Sicherheitsbewertung . . . . . . . . . . 4 Erkenntnislücken und Empfehlungen zum Forschungsbedarf für die Sicherheitsbewertung . . . . . . . . . . . . . . . . . . . 5 Literatur . . . . . . . . . . . . . . . . . . . . . . . . . . .

II Kriterien zur Beurteilung Funktioneller Lebensmittel . . . . 1 Vorbemerkung . . . . . . . . . . . . . . . . . . . . . 2 Abgrenzung von Funktionellen Lebensmitteln gegenüber anderen Lebensmitteln und Produkten . . . . . . . . . 3 Bewertung der gesundheitlichen Unbedenklichkeit . . . 4 Funktionalität und Auslobung . . . . . . . . . . . . . . 5 Beobachtung nach Markteinführung . . . . . . . . . . . 6 Schlussbemerkung . . . . . . . . . . . . . . . . . . . 7 Literatur . . . . . . . . . . . . . . . . . . . . . . . . III Main Conclusions and Recommendations . . . . 1 Introduction . . . . . . . . . . . . . . . . 2 General Aspects of the Safety Evaluation . . 3 Special Aspects of the Safety Evaluation . . . 4 Gaps in Knowledge and Recommendations for Safety Evaluation . . . . . . . . . . . . . . 5 References . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . Research . . . . . . . . . .

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10 11 15 17 17 17

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V

Inhaltsverzeichnis IV Criteria for the Evaluation of Functional Foods . . . . . . . . . 1 Preamble . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Differentiation of Functional Foods From Other Foodstuffs and Products . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Evaluation of the Safety to Health . . . . . . . . . . . . . . 4 Functionality and Claims . . . . . . . . . . . . . . . . . . 5 Observation after the Market Introduction . . . . . . . . . . 6 Concluding Remarks . . . . . . . . . . . . . . . . . . . . 7 References . . . . . . . . . . . . . . . . . . . . . . . . . V

VI

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27 27

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28 29 32 34 34 35

Contributions . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Diet, Genes, and Cancer Risk . . . . . . . . . . . . . . . . . Cornelia M. Ulrich 2 Regulatory Requirements for Functionality and Safety: A European View . . . . . . . . . . . . . . . . . . . . . . Burckhard Viell 3 Functional Foods Research and Regulation in Japan . . . . . . Shaw Watanabe 4 Aspect on the Chinese Functional Food and their Safety . . . . Yi-Min Wei 5 Regulatory Framework for Functionality and Safety: A North American Perspective . . . . . . . . . . . . . . . . Barbara O. Schneeman 6 Dose-Response Relationships with Special Reference to Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . Aalt Bast and Guido R. M. M. Haenen 7 Low Dose Competition of Flavonoids with Endogenous Thyroid Transport Proteins: Potential Relevance to the Thyroid Hormone Axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Josef Köhrle 8 Host Factors Relevant for Bioavailability: Transporters, Metabolizing Enzymes and Genetic Polymorphisms . . . . . . Dieter Schrenk 9 Food Matrix and Related Factors Affecting Bioavailability . . . Karin H. van het Hof and Johan M. M. van Amelsvoort 10 Toxicokinetics/Toxicodynamics . . . . . . . . . . . . . . . . Ron Walker 11 Biomarkers to Assess Safety Aspects and Functional Effects of Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Beatrice L. Pool-Zobel 12 Biomarkers Relevant to Cardiovascular Disease . . . . . . . . Margreet R. Olthof 13 Biomarkers of Effect on the Immune System . . . . . . . . . . Raymond Pieters

37 37

50 67 85

90

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137 144 152

164 178 179

Inhaltsverzeichnis 14 Mechanistic Understanding of Potential Adverse Effects of b-Carotene Supplementation . . . . . . . . . . . . . . . . Xiang-Dong Wang 15 Potential Adverse Mechanisms of Antioxidants During Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . Hans Konrad Biesalski 16 Cholesterol Lowering Vegetable Oil Spreads: Results of a Post Launch Monitoring Programme . . . . . . . . . . . . . . . Linda J. Lea 17 Effect of Flavonoids on Human Topoisomerases . . . . . . . Doris Marko 18 Risk/Benefit Aspects of Phytoestrogen Consumption . . . . . Catherine Boyle 19 Influence of Phytoestrogens on the Biotransformation of Endogenous Estrogens . . . . . . . . . . . . . . . . . . . Manfred Metzler 20 How Selective are Prebiotics? . . . . . . . . . . . . . . . . Walter P. Hammes 21 Pathogen or Probiotic – Where is the Boundary? . . . . . . . Maria Saarela

. 189

. 216

. 217 . 231 . 237

. 238 . 247 . 264

VI Posters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 In vitro Study of Prebiotic Properties of Levan-Type Exopolysaccharides from Lactobacilli and non-Digestible Carbohydrates using Denaturing Gradient Gel Electrophoresis . . . . . . . . F. Dal Bello, J. Walter, C. Hertel, and W. P. Hammes 2 Effects of Butyrate on Glutathione S-Transferase P1 Expression in Human Colonic Adenoma Cells . . . . . . . . . . . . . . G. Festag, N. Haag, G. Beyer-Sehlmeyer, M. N. Ebert, B. Marian and B. L. Pool-Zobel 3 In vitro Fermentation Supernatants of Inulin-associated Prebiotics Modulate Proliferation and Glutathione S-Transferase Activity in Human Colon HT29 Cells . . . . . . . . . . . . . Eva Gietl, Annett Klinder, Stella Pistoli, Beatrice Pool-Zobel 4 b-Carotene Effects in a Tobacco Smoke Carcinogen-induced Lung Cancer Model in vivo . . . . . . . . . . . . . . . . . . R. Goralczyk, H. Bachmann, G. Riss, C.-P. Aebischer, B. Lenz, and K. Wertz 5 Influence of Daidzein and its Metabolites on the Expression of Catalase in Rat Hepatoma Cells . . . . . . . . . . . . . . . . A. Kampkötter, E. Röhrdanz, K. Iwami, S. Ohler, W. Wätjen, Y. Chovolou, S. E. Kulling, and R. Kahl1 6 Flavonoids and the Central Nervous System: The Anxiolytic Flavone Hispidulin . . . . . . . . . . . . . . . . . . . . . . D. Kavvadias, P. Sand, P. Riederer, E. Richling, P. Schreier

279

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281

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VII

Inhaltsverzeichnis 7 Comparative Bioavailability of Synthetic and Tomato-Based Lycopene in Humans? . . . . . . . . . . . . . . . . . . . . Peter P. Hoppe, Klaus Kraemer, Henk van den Berg, Gery Steenge, Trinette van Vliet 8 The Mycotoxin Ochratoxin A Induced DNA Damage in MDCK Cells and Primary Cultured Porcine Urinary Bladder Epithelial Celles (PUBEC) in vitro . . . . . . . . . . . . . . . . . . . . S. Lebrun, H. Schulze, and W. Föllmann 9 Genotoxic Potential of the Phytoestrogen Resveratrol in Cultured V79 Chinese Hamster Fibroblasts . . . . . . . . . . . . . . . Leane Lehmann, Erika Pfeiffer, and Manfred Metzler 10 Investigation of Oxidative Stress in H4IIE Cells: Modulation by the Flavonoid Kaempferol . . . . . . . . . . . . . . . . . . . P. Niering, W. Wätjen, S. Ohler, I. Köhler, Y. Chovolou, A. Kampkötter, Q.-H. Tran-Thi, and R. Kahl 11 In Vitro Studies on the Estrogenic Activity and the Metabolism of Curcumin . . . . . . . . . . . . . . . . . . . . . . . . . Erika Pfeiffer, Harald L. Esch, Simone Höhle, Aniko M. Solyom, Barbara N. Timmermann and Manfred Metzler 12 Structural Studies of Sphingolipids, a Class of Chemopreventive Compounds in Food . . . . . . . . . . . . . . . . . . . . . W. Seefelder, N. Bartke, T. Gronauer, S. Fischer, H.-U. Humpf 13 Modulation of (Oxidative) DNA Damage by Constituents of Carob Fibre . . . . . . . . . . . . . . . . . . . . . . . . . S. Schäfer, H. G. Kamp, C. Müller, B. Haber, G. Eisenbrand, C. Janzowski 14 Pro- and Antiapoptotic Effects of Flavonoids in H4IIE-Cells: Implication of Oxidative Stress . . . . . . . . . . . . . . . . W. Wätjen, Y. Chovolou, P. Niering, A. Kampkötter, Q.-H. Tran-Thi, and R. Kahl 15 Analysis of the Hypotheses on b-Carotene/Tobacco Smoke Interactions in the A/J Mouse Lung Cancer Model . . . . . . . Goralczyk, R., Wertz, K., Riss, G., Bachmann, H., Buchwald, P., Hansen, T., Niehof, M., Dangers, M., and Borlak J. 16 Carob Fibre – Functional Effects on Human Colon Cell Line HT29 . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stefanie Klenow, Michael Glei, Gabriele Beyer-Sehlmeyer, Bernd Haber, Beatrice L. Pool-Zobel 17 Influence of Food Constituents on Cytochrome P450 1A Activity Annette Baumgart, Melanie Schmidt, Hans-Joachim Schmitz, and Dieter Schrenk 18 Red Grape Products and Ethanol Modulate Coagulation and Fibrinolysis in Healthy Male Volunteers . . . . . . . . . . . . Achim Bub, Bernhard Watzl, Gerhard Rechkemmer

VIII

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Inhaltsverzeichnis 19 Long-Term Treatment with a Prebiotic Modulates the Gut-Associated Immune System of Azoxymethane-Treated F344 Rats . . . . . . . . . . . . . . . . . . . . . . . . . . M. Roller, G. Caderni, G. Rechkemmer, B. Watzl 20 b-Carotene Inhibits Growth of Human Colon Carcinoma Cells (HT 29) in vitro by Induction of Apoptosis . . . . . . . . . . . Karlis Briviba, Kerstin Schnäbele, Elke Schwertle, Gerhard Rechkemmer 21 The Influence of Short-Chain Fatty Acids on Intracellular pH and Calcium-Concentration in the HT-29 Colon Carcinoma Cell Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kerstin Rebscher, Stephan W. Barth, Gerhard Rechkemmer 22 Effect of Short Chain Fatty Acids on Cytotoxicity, Proliferation, and Apoptosis in Human Colon Carcinoma Cell Lines . . . . . Silvia Roser, Heike Lang, Gerhard Rechkemmer 23 Investigation of the Antihormonal Potential of Xanthohumol, the Major Prenylated Flavonoid of Humulus Lupulus . . . . . . . . Anette Höll, Sabine Guth, Gerhard Eisenbrand 24 Anthocyanidins Potently Interfere with Signalling Cascades Regulating Cell Proliferation . . . . . . . . . . . . . . . . . Doris Marko, Monika Keme´ny, Michael Habermeyer, Edda Bernardy, Susanne Meiers, Ulrike Weyand

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VII Participants and Contributors of the Symposium . . . . . . . . . 377

IX

Vorwort

Der Begriff „Funktionelle Lebensmittel“ wird für solche Lebensmittel verwendet, für die über den reinen Ernährungszweck hinaus, gesundheitlich vorteilhafte Wirkungen beansprucht werden. Solche „Funktionellen Lebensmittel“ (FLM) dürfen keine unerwünschten, gesundheitlich nachteiligen Effekte auslösen. Daher ist eine wissenschaftlich fundierte Sicherheitsbewertung als integraler Bestandteil einer Risiko/Nutzen-Analyse unerlässlich. Die gesundheitliche Unbedenklichkeit muss hinreichend belegt sein, bevor Untersuchungen zur Funktionalität am Menschen durchgeführt werden können. Rechtliche Regelungen für das Inverkehrbringen von FLM existieren in Deutschland und anderen europäischen Ländern bisher nicht. Allerdings bestehen in einigen europäischen Staaten, z. B. in Schweden, dem Vereinigten Königreich oder den Niederlanden, bereits rechtsähnliche Vereinbarungen („Codes of Practice“), bzw. befinden sich solche in Vorbereitung, in denen insbesondere die Verwendung gesundheitsbezogener Auslobungen und deren wissenschaftlicher Beleg überprüft werden. Die Senatskommission der Deutschen Forschungsgemeinschaft (DFG) zur Beurteilung der gesundheitlichen Unbedenklichkeit von Lebensmitteln (SKLM) hat vom 5. bis 7. Mai 2002 unter Beteiligung von Experten aus dem In- und Ausland ein Symposium zu „Funktionellen Lebensmitteln“ abgehalten, bei dem der thematische Schwerpunkt auf den Sicherheitsaspekten lag. Ziel war eine kritische Bestandsaufnahme und Bewertung des gegenwärtigen Erkenntnisstands. In Wahrnehmung ihres Beratungsauftrags hat die SKLM hierzu Schlussfolgerungen und Empfehlungen erarbeitet. Die SKLM hat darüber hinaus als Resultat gründlicher wissenschaftlicher Beratungen und unter Auswertung der Ergebnisse des Symposiums „Kriterien zur Beurteilung Funktioneller Lebensmittel“ erarbeitet (Teil II), die in Verbindung mit den Symposiumsbeiträgen (Teil V) sowie den Hauptschlussfolgerungen und Empfehlungen (Teil I) in diesem Symposiumsband veröffentlicht werden. Mein Dank gilt den Teilnehmern des Symposiums für ihre wissenschaftlichen Beiträge sowie den Mitgliedern und Gästen der Senatskommission für ihre Mithilfe bei der Abfassung der vorliegenden Veröffentlichung. BesonXI

Vorwort ders zu danken ist der Arbeitsgruppe „Funktionelle Lebensmittel“ für die Erarbeitung der „Kriterien zur Beurteilung Funktioneller Lebensmittel“. Die SKLM vertraut darauf, dass die hier vorgelegten Schlussfolgerungen und Empfehlungen in Verbindung mit den „Kriterien zur Beurteilung funktioneller Lebensmittel“ auf breite Beachtung stoßen werden. Das wissenschaftliche Sekretariat der SKLM mit Dr. S. Guth, Dr. M. Keme´ny und Dr. D. Wolf hat wesentlich zum Zustandekommen dieses Bandes beigetragen. Ihnen gilt mein herzlicher Dank, ebenso der Leiterin des Fachreferats Frau Dr. H. Velke und dem Lektorat der Deutschen Forschungsgemeinschaft. Die SKLM dankt der Deutschen Forschungsgemeinschaft dafür, dass sie dieses Symposium und die Veröffentlichung ermöglicht hat. Prof. Dr. Gerhard Eisenbrand Vorsitzender der DFG-Senatskommission zur Beurteilung der gesundheitlichen Unbedenklichkeit von Lebensmitteln

XII

Preface

The term “Functional Food” has been coined for foods claimed to exert advantageous human health effects that go beyond solely nutritional effects. Such functional foods (FFs) must not cause any effects that may be undesirable and disadvantageous for health. A scientifically based safety evaluation is essential as an integral component of a risk-benefit analysis. The safety to health of FFs must be demonstrated adequately before investigations on their functionality can be carried out in humans. Legal regulations for controlling the placing of FFs on the market do not yet exist, neither in Germany nor other European countries. However, in some European states, e. g. Sweden, the United Kingdom and The Netherlands, quasi-legal arrangements (Codes of Practice) exist or are in preparation; in these the use of, and scientific evidence for, health-related claims in particular is examined. The Senate Commission on Food Safety (SKLM) of the Deutsche Forschungsgemeinschaft (DFG) organised with the participation of experts from Germany and abroad a symposium on functional foods held on 5th to 7th May 2002. This had safety aspects as its main topic and the aim was to provide a critical survey and evaluation of the existing state of knowledge. In pursuance of its advisory task the SKLM has formulated conclusions and recommendations. In addition, following extensive scientific consultations and using the evaluation of the results of the symposium the SKLM formulated an opinion paper entitled “Criteria for the Evaluation of Functional Foods” (Part IV) which, in combination with the contributions to the symposium (Part V) and the “Main Conclusions and Recommendations” (Part III), is published in this symposium volume. My thanks go to the participants of the symposium for their scientific contributions as well as to the members and guests of the Senate Commission for their help in preparing the present publication. Special thanks go to the working group “Functional Foods” for preparing the paper on “Criteria for the Evaluation of Functional Foods”. The SKLM is confident that the conclusions and recommendations published here in combination with the “Criteria for the Evaluation of Functional Foods” will find broad acceptance.

XIII

Preface The SKLM is especially grateful to Dr. Peter Elias and Dr. John Greig for the great effort they took in preparing the English translation. The scientific secretariat of the SKLM with Dr. S. Guth, Dr. M. Keme´ny, and Dr. D. Wolf has contributed substantially to the production of this volume. My sincere thanks go to them as well as to the head of the DFG referate Dr. H. Velke and the reviewers of the DFG. The SKLM thanks the DFG for making possible the holding of this symposium and of the publication of this volume. Prof. Dr. Gerhard Eisenbrand Chairman of the DFG-Senate Commission on Food Safety

XIV

I Hauptschlussfolgerungen und Empfehlungen

1 Einleitung Das Interesse an Funktionellen Lebensmitteln und das entsprechende Marktangebot wachsen weltweit. Auch in Europa spielen Funktionelle Lebensmittel eine zunehmende Rolle, wie das Beispiel der Einführung Phytosterolesterangereicherter Brotaufstriche zur Reduktion des Plasmacholesterolspiegels bei entsprechend disponierten Personen zeigt. Funktionelle Lebensmittel sollen gegenüber herkömmlichen Lebensmitteln über den Ernährungszweck hinausgehende, gesundheitsförderliche bzw. das Erkrankungsrisiko vermindernde Wirkungen aufweisen. Das Ziel einer günstigen Beeinflussung bestimmter Körper- und Organfunktionen, aber auch spezifischer Erkrankungsrisiken stellt hohe Anforderungen, sowohl an den wissenschaftlichen Nachweis solcher in Form von so genannten „Health Claims“ ausgelobten Effekte, als auch an eine wissenschaftlich fundierte Bewertung. Darüber hinaus gilt für Funktionelle Lebensmittel als Grundvoraussetzung, dass sie ebenso wie alle anderen Lebensmittel im Rahmen des empfohlenen bzw. abzusehenden Verzehrs gesundheitlich unbedenklich sein müssen. Wie dieses generell akzeptierte Grunderfordernis im Einzelnen aber zu sichern bzw. plausibel zu verifizieren ist, ist bisher nicht angemessen diskutiert und einheitlich geregelt. Für diese Diskussion bedarf es klarer Definitionen, z. B. auch hinsichtlich einer Unterscheidung zwischen Funktionellen Lebensmitteln und Nahrungsergänzungsmitteln. Im asiatischen Raum ist die Nutzung von Nahrungsmitteln bzw. ihrer Bestandteile zur Beeinflussung des gesundheitlichen Wohlbefindens von Alters her in der so genannten traditionellen Medizin verankert. Erste systematische und marktorientierte Ansätze zur Entwicklung von Lebensmitteln mit gesundheitsförderndem Effekt stammen aus Japan. Standardisierung und Sicherheit solcher Lebensmittel mit festgelegtem Nutzen für die Gesundheit, den so genannten FOSHU (Food for Specified Health Uses), werden seit 1991 per Gesetz geregelt. Jedes Produkt durchläuft ein individuelles Genehmigungsverfahren, bei dem wissenschaftliche Daten zu Funktionalität, Sicherheit und der abgeschätzten täglichen Aufnahme vorzulegen sind. Die 1

I

Hauptschlussfolgerungen und Empfehlungen

FOSHU Regelung erfasst nur Lebensmittel, die im Rahmen der üblichen Ernährung verzehrt werden, jedoch keine Nahrungsergänzungsmittel. Weitergehende Informationen finden sich im Beitrag „Functional Foods Research and Regulation in Japan“ von Dr. Watanabe (Part V, Kapitel 3). In den USA stellen Funktionelle Lebensmittel im Gegensatz zu Nahrungsergänzungsmitteln keine eigenständig geregelte Lebensmittel-Kategorie dar. Sie werden dort den allgemeinen Lebensmitteln zugerechnet, die bei vorgesehener Verwendung den üblichen Sicherheitsanforderungen (reasonable certainty of no harm) genügen müssen. Health Claim und entsprechende Kennzeichnung werden im Rahmen gesetzlicher Regelungen bewertet. Allerdings gelten diese – anders als in Japan – nicht nur für spezielle Produkte (Funktionelle Lebensmittel), sondern beziehen sich auf allgemein anerkannte Eigenschaften von Inhaltsstoffen, die Lebensmitteln zugesetzt werden (generic health claims). Nahrungsergänzungsmittel sind als „dietary supplements“ mit der Verabschiedung des „Dietary Supplement Health and Education Act“ von 1994 (DSHEA) gesetzlich geregelt. Im Jahre 2002 hat das Institute of Medicine (IOM) der National Academy of Sciences (NAS) im Auftrag der Food and Drug Administration (FDA) Verfahren und Vorgehensweisen zur systematischen Sicherheitsbewertung von Nahrungsergänzungsmitteln erarbeitet. Ausführlichere Informationen sind im Beitrag „Regulatory Framework for Functionality and Safety: A North American Perspective“ von Dr. Schneeman (Part V, Kapitel 5) sowie im Internet [1] zu finden. In Europa beschäftigen sich Expertengremien bisher vor allem mit Anforderungen an den wissenschaftlichen Nachweis für gesundheitsfördernde bzw. das Erkrankungsrisiko vermindernde Wirkungen. Ein erstes Konsenspapier eines Expertengremiums, FUFOSE (Functional Food Science in Europe), das sich mit der wissenschaftlichen Basis für die Entwicklung von Funktionellen Lebensmitteln und mit den verschiedenen darauf bezogenen Health Claims beschäftigt, wurde 1999 veröffentlicht [2]. Auf diesem Konzept aufbauend hat die Diskussion zu den wissenschaftlichen Anforderungen an solche Health Claims in verschiedenen Gremien, u. a. des Europarats und der EU-Kommission (PASSCLAIM, „A Process for the Assessment of Scientific Support for Claims on Foods“), begonnen und entsprechende Kriterien werden derzeit entwickelt. Seit Mitte 2002 besteht eine EU-Richtlinie zur einheitlichen Regelung des Zusatzes von Vitaminen und Mineralstoffen zu Nahrungsergänzungsmitteln [3]. Als erste Schritte wurden in dieser Richtlinie für Vitamine und Mineralstoffe Positiv-Listen erstellt, Kennzeichnungsregeln erlassen sowie die Vorgehensweise für die Festsetzung von Höchstund gegebenenfalls Mindestmengen definiert. Die SKLM hielt eine gründliche wissenschaftliche Diskussion auch von potenziellen Risiken Funktioneller Lebensmittel für notwendig und hat aus diesem Grund den Schwerpunkt des Symposiums auf die Sicherheitsaspekte gelegt. Als Resultat ihrer eigenen gründlichen wissenschaftlichen Beratungen und unter Berücksichtigung der Ergebnisse des Symposiums hat die SKLM darüber hinaus eine Stellungnahme „Kriterien zur Beurteilung Funktioneller Lebensmittel“ erarbeitet. Diese Stellungnahme wird im Verbund mit 2

2

Allgemeine Aspekte der Sicherheitsbewertung

den Beiträgen des Symposiums und den hieraus abgeleiteten Schlussfolgerungen und Empfehlungen im vorliegenden Band veröffentlicht.

2 Allgemeine Aspekte der Sicherheitsbewertung Lebensmittel sind meist komplexe Mischungen von Makro- und Mikrobestandteilen, deren Unbedenklichkeit und Nährwert gemeinhin außer Frage stehen. Die Vorgehensweise zur Sicherstellung der gesundheitlichen Unbedenklichkeit von Lebensmitteln ist von dieser Grundvoraussetzung geleitet und konzentriert sich deshalb klassischerweise auf die Bewertung von Zusatzstoffen, Hilfsmitteln für die Verarbeitung, Kontaminanten und anderen Begleitstoffen sowie auf Herstellungs- bzw. Verarbeitungsverfahren [4]. Neuere Regelungen betreffen neuartige Lebensmittel, unabhängig davon, ob sie gesundheitliche Wirkungen beanspruchen oder nicht. Marktentwicklungen mit dem Ziel, Lebensmittel neuartig zusammenzusetzen bzw. Lebensmitteln Stoffe mit ernährungsphysiologischer bzw. gesundheitsförderlicher Zweckbestimmung zuzusetzen, werfen neue Fragen, insbesondere nach der Sicherheitsbewertung dieser Stoffe bzw. Lebensmittel auf. Rein ernährungswissenschaftliche Beurteilungen solcher Lebensmittel oder Kostformen können in der Regel nicht als Grundlage für die Bewertung der gesundheitlichen Unbedenklichkeit dienen. Für Funktionelle Lebensmittel ist daher eine Sicherheitsbewertung nach generell akzeptierter Vorgehensweise erforderlich. Die einzelnen Schritte der Sicherheitsbewertung sind allerdings den speziellen Anforderungen an die Bewertung Funktioneller Lebensmittel anzupassen. Basis ist zunächst die Problemdefinition und die Sammlung von Vorwissen unter Einbezug der Erfahrungen aus dem Verzehr in anderen Kulturkreisen. Darauf aufbauend erfolgt die Ermittlung sicherheitsrelevanter Daten, die Expositionsabschätzung und die Risikocharakterisierung als Grundlage für die Sicherheitsbewertung. Auch bei Funktionellen Lebensmitteln, die nur für spezielle Zielgruppen bestimmt sind, ist die gesundheitliche Unbedenklichkeit für alle Gruppen zu sichern, die als potenzielle Konsumenten in Frage kommen, insbesondere für Risikogruppen wie z. B. Kleinkinder, Schwangere und stillende Mütter sowie ältere und chronisch kranke Menschen. Prospektive Abschätzungen Verbrauchergruppen-spezifischer Expositionen können dabei beispielsweise anhand zuverlässiger Markt- und Verzehrsanalysen bereits im Handel befindlicher Vergleichsprodukte vorgenommen werden, wobei besonderes Augenmerk auf den Gesamtverzehr funktioneller Bestandteile aus verschiedenen wirkungsähnlichen Produkten zu richten ist. Nach Inverkehrbringen eines Funktionellen Lebensmittels ist dessen Verzehr auf geeignete Weise zu überprüfen (Post-Launch Monitoring). Dieses sollte ermöglichen, vorhergesagte Exposition und Zielgruppenspezi3

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fität zu verifizieren sowie u. U. eintretende Veränderungen von Ernährungsgewohnheiten frühzeitig zu erkennen. Das Post-Launch Monitoring soll nicht dem Wirksamkeitsnachweis dienen. Es ist jedoch wünschenswert, das PostLaunch Monitoring dazu zu nutzen, funktionelle Eigenschaften zu überprüfen bzw. zu verifizieren und gegebenenfalls sogar eventuell zu beobachtende unerwünschte Begleitwirkungen frühzeitig aufzudecken. Epidemiologische Studien zur Erfassung von langfristigen Auswirkungen Funktioneller Lebensmittel sollten die vorliegenden Informationen ergänzen. Besonders erwünscht ist die Entwicklung und Validierung geeigneter Biomarker für Wirkung und Exposition, die eine frühzeitige Erfassung auch minimaler Effekte und eine genauere Darstellung der Exposition ermöglichen.

3 Spezielle Aspekte der Sicherheitsbewertung Funktionelle Inhaltsstoffe können eine Vielzahl biologischer Wirkungen auslösen, die über verschiedene zelluläre Wege vermittelt werden. Zu nennen sind beispielsweise die Beeinflussung von zellulären Signalketten, von Enzymen des Fremdstoffmetabolismus oder von Proteinen des Transmembrantransports, darüber hinaus die Beeinflussung der Integrität des Erbmaterials bzw. der Aktivität DANN prozessierender Enzyme, des Immunsystems sowie der Homöostase zwischen pro- und antioxidativen Wirkungen. Ebenso können die Beeinflussung der hormonellen Homöostase, z. B. von Biosynthese, Stoffwechsel und Ausscheidung von Hormonen, sowie die Interaktion mit Transport- bzw. Rezeptorproteinen von Bedeutung sein. Potenzielle Interaktionen mit Resorption, Verteilung, Stoffwechsel und Ausscheidung von Nährstoffen sowie Arzneimitteln sind zu berücksichtigen. Werden einem Produkt mehrere funktionelle Inhaltsstoffe zugesetzt, sind potenzielle Wechselwirkungen zwischen den einzelnen Komponenten zu untersuchen. In der Regel sind solche biologischen Wirkungen dosis- bzw. konzentrationsabhängig und werden somit in erster Linie von der jeweiligen Aufnahmemenge und der Bioverfügbarkeit der Stoffe bestimmt. Ob sich eine Beeinflussung der genannten zellulären Angriffspunkte negativ oder positiv auf die Gesundheit auswirkt, ist nicht immer klar zu beantworten, denn die Balance zwischen den einzelnen Wirkungen unterliegt in vivo komplexen Regelsystemen. Auch können sich bei manchen Stoffen nichtlineare DosisWirkungs-Beziehungen ergeben oder sogar Wirkqualitäten umkehren, z. B. von antioxidativen zu prooxidativen Wirkungen. Die Vielfalt an biologischen Angriffspunkten mit gesundheitlich relevanten Auswirkungen erfordert eine dosisbezogene wirkmechanistische Analyse, um eine zuverlässige Datenbasis für die Sicherheitsbewertung zu erarbeiten. 4

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Spezielle Aspekte der Sicherheitsbewertung

Neben der Aufnahmemenge bestimmen im Wesentlichen Absorption, Verteilung, Metabolismus und Ausscheidung die Bioverfügbarkeit eines Stoffs und damit die Konzentration am Wirkort bzw. die resultierende Wirkqualität. Zusätzlich zu berücksichtigende individuelle Einflussgrößen sind genetische bzw. funktionelle Polymorphismen, aber auch Alter, Geschlecht und Ernährungsstatus. Darüber hinaus sind auch weitere, vom Individuum unabhängige Einflussgrößen wie beispielsweise die Matrix des Lebensmittels, die Interaktion mit anderen Lebensmittelinhaltsstoffen sowie mit bestimmten Arzneimitteln zu untersuchen. Detaillierte Ausführungen zur Vielfalt der biologischen Wirkungen sowie zu Kinetik und Bioverfügbarkeit funktioneller Lebensmittelinhaltsstoffe sind in einer exemplarischen Stellungnahme der SKLM „Aspekte potenziell nachteiliger Wirkungen von Polyphenolen/Flavonoiden zur Verwendung in isolierter oder angereicherter Form“ beschrieben [7]. Bei Funktionellen Lebensmitteln deren funktionelle Bestandteile Einzelsubstanzen, Substanzgemische und Extrakte darstellen, sind anerkannte toxikologische Untersuchungs- und Bewertungsmethoden, wie sie beispielsweise für Zusatzstoffe in Lebensmitteln beschrieben sind, anzuwenden [4]. Die Vorgehensweise im Einzelnen ist im Teil II, „Kriterien zur Beurteilung Funktioneller Lebensmittel“, genauer erläutert. Bei Funktionellen Lebensmitteln mit präbiotischen, d. h. mit mehr oder weniger unverdaulichen Stoffen, die das Wachstum bestimmter Bakteriengruppen in der Mikroflora des Darms fördern sollen, ist die Vorhersage von selektiven Effekten auf eine einzelne, definierte Gruppe von Mikroorganismen in der Regel nicht möglich. Funktionelle Lebensmittel mit probiotischen Mikroorganismen, die die Balance der Darmflora erhalten und verbessern sollen, befinden sich bereits seit einigen Jahren auf dem Markt. Da es beim Verzehr solcher Lebensmittel zur Aufnahme von lebenden Bakterien kommt, ist die Absicherung der Unbedenklichkeit dieser Mikroorganismen von essentieller Bedeutung. Die Auswirkungen einer Aufnahme von probiotischen Bakterien hängen sowohl vom Wirtsorganismus als auch vom Bakterium ab, insofern kann es grundsätzlich kein Null-Risiko für den jeweiligen Wirtsorganismus geben. Insgesamt aber ist das Gesundheitsrisiko durch probiotische Mikroorganismen aufgrund von Langzeiterfahrungen am Menschen als vergleichsweise gering einzustufen. Richtlinien zur Bewertung von Mikroorganismenkulturen zur Verwendung als oder in so genannten Probiotischen Lebensmitteln sind von einer Joint FAO/WHO Working Group herausgegeben worden. In diesen wird darauf hingewiesen, dass historisch Lactobazillen und Bifidobakterien in Lebensmitteln immer als sicher angesehen worden sind [5].

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4 Erkenntnislücken und Empfehlungen zum Forschungsbedarf für die Sicherheitsbewertung

Die wissenschaftliche Diskussion im Rahmen des Symposiums hat eine Reihe von Erkenntnislücken und den sich hieraus ergebenden Forschungsbedarf aufgezeigt. Beides soll im Folgenden näher erläutert werden.

4.1 Expositionsermittlung Forschungsbedarf wird hinsichtlich der Entwicklung neuer, zuverlässiger und geeigneter Marktbeobachtungsverfahren gesehen, die es ermöglichen sollen, Verzehrsmengen von Produkten sowie von Produktgruppen mit ähnlicher Wirkung durch bestimmte Zielgruppen und Risikogruppen zuverlässig zu erfassen. Entwicklung und Evaluierung von „Biomarkern der Exposition“, die eine indirekte Erfassung von Verzehrsmengen sowie ein personenbezogenes Monitoring ermöglichen, sind für die Expositionsermittlung von besonderer Bedeutung.

4.2 Wirkungsanalyse Die Kenntnis der dosisabhängigen Wirkungen funktioneller Lebensmittelbestandteile und deren Mechanismen ist neben der zuverlässigen Ermittlung der Exposition die Grundlage der Sicherheitsbewertung. Der Schwerpunkt weiterer Forschungsarbeit sollte auf der Erfassung und dem zuverlässigen Ausschluss potenziell gesundheitlich nachteiliger Wirkungen für den Menschen liegen. Zu berücksichtigen sind dabei auch potenzielle Wechselwirkungen zwischen mehreren funktionellen Inhaltsstoffen oder zwischen funktionellen Inhaltsstoffen und anderen Nahrungsbestandteilen. Wirkmechanistische Untersuchungen können anhand von in vitro und von tierexperimentellen Modellen wesentliche Erkenntnisse liefern, die Plausibilität für den Menschen ist dabei aber zu sichern, z. B. durch Vergleich mit geeigneten Humansystemen wie Biopsiematerial [6]. Innovative Ansätze, z. B. solche, die den Einfluss von Stoffen auf spezifische Gruppen von Genen (Nährstoff-Gen-Interaktionen) untersuchen, sowie die Weiterentwicklung von Biomarkern können neue mechanistische Einblicke verschaffen und u. a. das Verständnis für individuelle Empfindlichkeiten vertiefen. Diese neuen Techniken bieten nicht nur die Möglichkeit, die Plausibilität experimenteller Ergebnisse für den Menschen zu zeigen, 6

4

Erkenntnislücken und Empfehlungen

sondern darüber hinaus auch ein großes Potenzial für die Entwicklung von „Biomarkern der Wirkung“ für molekular-epidemiologische Studien.

4.3 Stoffkinetik Stoffspezifische Parameter, wie z. B. Absorptions- und Eliminationsrate, Bioverfügbarkeit, Metabolismus, Gewebespiegel, Bindungsverhalten sowie eine möglicherweise gewebespezifische Akkumulation von Stoffen sind bisher häufig noch unzureichend geklärt. Weitere Forschung ist erforderlich zur Abklärung der Bedeutung individuell bestimmter Einflussgrößen für Stoffkinetik und Bioverfügbarkeit, beispielsweise genetischer bzw. funktioneller Polymorphismen. Zu berücksichtigen sind aber auch Alter, Geschlecht oder Ernährungs- und hormoneller Status. Ebenso ist die Untersuchung anderer Einflussgrößen, die nicht in erster Linie durch das exponierte Individuum bestimmt werden, erforderlich. Zu nennen sind beispielsweise Einflüsse auf die Bioverfügbarkeit durch die Lebensmittelmatrix oder als Folge einer Therapie mit bestimmten Arzneimitteln, aber auch Interaktionen mit anderen Lebensmittelinhaltsstoffen. Nicht zuletzt ist die Beeinflussung der Stoffkinetik durch die Darmflora und deren Beitrag zum Metabolismus von Belang.

4.4 Epidemiologie Forschungsbedarf besteht hinsichtlich der Entwicklung aussagekräftiger Verfahren zur Analyse der Wirkungsweise eines Funktionellen Lebensmittels beim Menschen. Die Entwicklung, Standardisierung und Validierung von „Biomarkern der Wirkung“, die durch funktionelle Inhaltsstoffe induziert bzw. beeinflusst werden und die möglichst auch eine zuverlässige Aussage über sicherheitsrelevante Wirkungsmuster zulassen sollten, ist besonders vordringlich. Ein weiterer Schwerpunkt der Forschung sollte auf die Identifizierung genetischer oder Lebensstil bedingter Prädispositionen und entsprechender molekularer Marker (Biomarker) gelegt werden, um das frühzeitige Erkennen von Risikogruppen zu ermöglichen.

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5 Literatur 1. Proposed Framework for Evaluating the Safety of Dietary Supplements – For Comment (2002); http://www.nap.edu/books/NI000760/html/ 2. Diplock AT, Aggett PJ, Ashwell M, Bornet F, Fern EB, Roberfroid MB: Scientific Concepts of Functional Foods in Europe: Consensus Document. British Journal of Nutrition 81 (1999) Suppl. 1. 3. Richtlinie 2002/46/EG des Europäischen Parlaments und des Rates vom 10. Juni 2002 zur Angleichung der Rechtsvorschriften der Mitgliedstaaten über Nahrungsergänzungsmittel, Amtsblatt Nr. L 183 vom 12/07/2002, S. 0051–0057. 4. Guidance on submissions for food additive evaluations by the Scientific Committee on Food, SCF 12. July 2001; http://europa.eu.int/comm/food/fs/sc/scf/out98_en.pdf 5. Guidelines for the Evaluation of Probiotics in Food. Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food, London, Ontario, Canada, April 30 and May 1, 2002. 6. Eisenbrand G, Pool-Zobel B, Baker V, Balls M, Blaauboer BJ, Boobis A, Carer A, Kevekordes S, Lhuguenot JC, Pieters R, Kleiner J: Methods of in vitro toxicology. Food Chemistry and Toxicology 40 (2/3) (2002), 193–236. 7. Beschluss der SKLM vom 08. 07. 2003: Aspekte potenziell nachteiliger Wirkungen von Polyphenolen/Flavonoiden zur Verwendung in isolierter oder angereicherter Form. Im Originalwortlaut über das Sekretariat der SKLM, Kaiserslautern erhältlich. Publikation vorgesehen in Mitteilung 7 „Lebensmittel und Gesundheit II“, WileyVCH Verlag, Weinheim, 2004.

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II Kriterien zur Beurteilung Funktioneller Lebensmittel

1 Vorbemerkung Die Senatskommission zur Beurteilung der gesundheitlichen Unbedenklichkeit von Lebensmitteln (SKLM) der Deutschen Forschungsgemeinschaft (DFG) hat diese Empfehlung „Kriterien zur Beurteilung Funktioneller Lebensmittel“ mit dem Ziel erarbeitet, Mindestanforderungen für die Bewertung der gesundheitlichen Unbedenklichkeit von Funktionellen Lebensmitteln (FLM) und für den wissenschaftlichen Nachweis ihrer Funktionalität zu definieren. Die SKLM bezieht in der hier vorgelegten Empfehlung weder zu den unter 2 zusammengefassten rechtlichen Aspekten von Funktionellen Lebensmitteln Stellung, noch sieht sie sich veranlasst, zu einer rechtlichen oder rechtsähnlichen Bewertung von gesundheitsbezogenen Auslobungen (so genannten Health Claims) Stellung zu nehmen. Dazu wird auf die einschlägigen Bemühungen anderer Gremien (Codex Alimentarius Kommission, Europarat) sowie auf die Initiative der EU-Kommission zur Erstellung einer Rechtsverordnung auf europäischer Ebene verwiesen. Fällt das FLM in den Geltungsbereich der Novel Food Verordnung der EU (Nr. 258/97), unterliegt das Inverkehrbringen des Produkts dem spezifischen Antragsverfahren [1]. Die SKLM geht davon aus, dass die Kriterien zur Beurteilung „Funktioneller Lebensmittel“ nach dem jeweiligen Stand der Wissenschaft zu modifizieren bzw. zu ergänzen sind.

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2 Abgrenzung von Funktionellen Lebensmitteln gegenüber anderen Lebensmitteln und Produkten

2.1 Funktionelle Lebensmittel Bisher gibt es keine rechtlich verbindliche Definition für FLM. Die SKLM lehnt sich daher an die Definition eines im Rahmen einer EU-Initiative erarbeiteten Consensus Documents der so genannten FUFOSE-Arbeitsgruppe an [2]. Danach kann ein Lebensmittel als „funktionell“ angesehen werden, wenn es über adäquate ernährungsphysiologische Effekte hinaus einen nachweisbaren positiven Effekt auf eine oder mehrere Zielfunktionen im Körper ausübt, so dass ein verbesserter Gesundheitsstatus oder gesteigertes Wohlbefinden und/oder eine Reduktion von Krankheitsrisiken erzielt werden. Funktionelle Lebensmittel werden ausschließlich in Form von Lebensmitteln angeboten und nicht wie Nahrungsergänzungsmittel in arzneimittelähnlichen Darreichungsformen. Sie sollten integraler Bestandteil der normalen Ernährung sein und ihre Wirkungen bei üblichen Verzehrsmengen entfalten. Ein funktionelles Lebensmittel kann ein natürliches Lebensmittel sein oder ein Lebensmittel, bei dem ein Bestandteil angereichert bzw. hinzugefügt oder abgereichert bzw. entfernt worden ist. Es kann außerdem ein Lebensmittel sein, in dem die natürliche Struktur einer oder mehrerer Komponenten modifiziert oder deren Bioverfügbarkeit verändert wurde. Ein funktionelles Lebensmittel kann für alle oder für definierte Bevölkerungsgruppen funktionell sein (z. B. definiert nach Alter oder genetischer Konstitution).

2.2 Nährstoffangereicherte Lebensmittel Nährstoffangereicherte Lebensmittel sind laut einer Definition der Codex Alimentarius Kommission Lebensmittel, denen essentielle Nährstoffe – d. h. Stoffe, für die allgemein akzeptierte Zufuhrempfehlungen vorliegen – in Form einer Anreicherung oder Ergänzung zugesetzt wurden mit dem Ziel, einen Mangel an einem oder mehreren Nährstoffen in der Bevölkerung oder bestimmten Bevölkerungsgruppen vorzubeugen [3]. Eine derartige Modifikation eines Lebensmittels im Rahmen anerkannter, von Fachgesellschaften ausgesprochener Ernährungsempfehlungen bietet jedoch keine funktionelle Wirkung über die „übliche“ Ernährung hinaus, die sich entsprechend den nachfolgend beschriebenen Kriterien nachweisen ließe. Aus diesem Grunde kann die einfache Anreicherung mit essentiellen Nährstoffen 10

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Bewertung der gesundheitlichen Unbedenklichkeit

nicht als funktionelles Prinzip im Sinne der Definition für Funktionelle Lebensmittel (siehe oben) gelten. Ähnliche Überlegungen treffen auch für die Abreicherung zu.

2.3 Nahrungsergänzungsmittel Nahrungsergänzungsmittel sind nach einer Richtlinie des Europäischen Parlaments und des Rates Lebensmittel, die aus Einfach- oder Mehrfachnährstoff-Konzentraten bestehen, in dosierter Form in den Verkehr gebracht werden und dazu bestimmt sind, die Zufuhr dieser Nährstoffe im Rahmen der normalen Ernährung zu ergänzen [4]. Der Begriff Nährstoffe umfasst in diesem Entwurf lediglich Vitamine und Mineralstoffe. „In dosierter Form“ bedeutet in arzneimittelähnlichen Darreichungsformen wie z. B. Kapseln, Tabletten, Pillen oder Ampullen.

3 Bewertung der gesundheitlichen Unbedenklichkeit

3.1 Allgemeine Anforderungen FLM müssen für den Verbraucher gesundheitlich unbedenklich sein und sind in dieser Hinsicht einer eingehenden Prüfung und Bewertung zu unterziehen. Ist nach aktuellem Kenntnisstand kein Anhaltspunkt für ein gesundheitliches Risiko erkennbar, können gezielte Untersuchungen zur funktionellen Wirkung am Menschen beginnen. Die Sicherheitsbewertung sollte den Empfehlungen über neuartige Lebensmittel des Wissenschaftlichen Lebensmittelausschusses (Scientific Committee on Food, SCF) der EU Kommission folgen [5], unabhängig davon, ob das FLM in den Definitionsbereich der Verordnung (EG) Nr. 258/97 bzw. ihrer Nachfolgeverordnungen über neuartige Lebensmittel und Lebensmittelzutaten fällt oder nicht [1]. Nach diesen Empfehlungen wird das neuartige Lebensmittel unter dem Gesichtspunkt der wesentlichen Gleichwertigkeit bzw. Unterschiedlichkeit beurteilt, d. h. auf der Basis eines Vergleichs mit einem entsprechenden traditionellen Produkt. In der Regel wird sich ein FLM durch die An- oder Abwesenheit bzw. die erhöhte oder verminderte Konzentration bzw. Bioverfügbarkeit eines oder mehrerer funktioneller Bestandteile von einem vergleichbaren Produkt unterscheiden. In diesen Fällen kann sich die Bewer11

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tung der gesundheitlichen Unbedenklichkeit auf die funktionell wirksamen Bestandteile konzentrieren. Der Einfluss der Lebensmittelmatrix ist gegebenenfalls zusätzlich zu berücksichtigen. Aufgrund der zu erwartenden Verschiedenartigkeit von FLM bzw. der Produktzusätze ist eine Einzelfallbetrachtung erforderlich. Art und Umfang der erforderlichen Untersuchungen hängen von den Eigenschaften der funktionellen Bestandteile bzw. des Wirkprinzips sowie der zu erwartenden Exposition der Zielgruppe bzw. potenzieller Risikogruppen ab. Eine systematische Zusammenstellung der gesamten relevanten Vorinformationen zu den Eigenschaften der funktionellen Bestandteile bzw. zu den möglichen nachteiligen Wirkungen ist unerlässlich. Dabei sollten auch Daten aus nicht publizierten bzw. nicht nach anerkannten Kriterien durchgeführten Studien mit berücksichtigt werden, wie es beispielsweise auch für bestimmte diätetische Lebensmittel gefordert wird [6]. Erfahrungen am Menschen, beispielsweise aus langjährigem Verzehr in anderen Kulturkreisen, aus epidemiologischen Studien oder aus anderen Studien am Menschen sind hierbei besonders zu berücksichtigen. Auf der Grundlage von Verzehrsdaten sollte die erwartete Exposition der Bevölkerung sowie der Zielgruppe des FLM unter Einschluss potenzieller Risikogruppen abgeschätzt werden. In diese Abschätzung ist auch der Gesamtverzehr gleichartiger oder auf ähnlichem Wirkprinzip basierender funktioneller Bestandteile mit einzubeziehen.

3.2 Einzelsubstanzen, Substanzgemische und Extrakte Die SKLM empfiehlt, die Prüfanforderungen zur Beurteilung der gesundheitlichen Unbedenklichkeit von funktionellen Bestandteilen an international anerkannten Prüfkriterien für Zusatzstoffe auszurichten, wie sie in einer Stellungnahme des SCF veröffentlicht worden sind: x

Guidance on submissions for food additive evaluations by the Scientific Committee on Food [7]

Im Wesentlichen wird eine hinreichende Charakterisierung der funktionellen Bestandteile gefordert, d. h. die Beschreibung ihrer chemischen Zusammensetzung, der physikalisch-chemischen und der mikrobiologischen Eigenschaften sowie eine Beschreibung ihrer Herkunft, ihrer Isolierung bzw. ihres Herstellungsprozesses. Darüber hinaus sind Spezifikationen, Reinheitskriterien und praktikable Analysenmethoden vorzulegen. Erforderlich sind auch Informationen zur Stabilität im Lebensmittel, zu möglichen Abbau- und Reaktionsprodukten sowie zu möglichen Interaktionen mit Nährstoffen und zur Beeinflussung ihrer Bioverfügbarkeit.

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Für eine Sicherheitsbewertung sind die in den SCF-Leitlinien [7] aufgeführten toxikologischen Eckdaten vorzulegen. Gegebenenfalls können im Einzelfall ergänzende Studien, wie sie ebenfalls in den Leitlinien beschrieben sind, erforderlich werden.

3.3 Enzyme Werden Enzyme bzw. Enzympräparate über eine rein technologische Anwendung hinaus als funktionelle Bestandteile zugesetzt, empfiehlt die SKLM, die Prüfkriterien zur Bewertung der Unbedenklichkeit im Rahmen einer Einzelfallbetrachtung an den nachfolgend aufgeführten Leitlinien auszurichten: x x x

Leitlinien des SCF zur Vorlage von Daten über Enzyme für Lebensmittel [8] Empfehlungen der SKLM zur Bewertung von Starterkulturen und Enzymen für die Lebensmitteltechnik [9] Empfehlungen der SKLM zur Beurteilung von neuen Proteinen, die durch gentechnisch modifizierte Pflanzen in Lebensmittel gelangen können [10]

Nach der SCF-Leitlinie [8] sind Informationen zu Herkunft und Herstellungsverfahren, zur katalytischen Aktivität und zur Stabilität im Produkt sowie zum Verwendungszweck des Produkts erforderlich. Für eine Sicherheitsbewertung sind die in den SCF-Leitlinien für Enzyme unterschiedlicher Herkunft jeweils genannten toxikologischen Eckdaten vorzulegen. Da die katalytische Funktion des Enzyms sowohl Veränderungen im Lebensmittel herbeiführen als auch nach der Aufnahme in den Verdauungstrakt auf Verdauungsprozesse und die Bioverfügbarkeit der Nährstoffe wirken kann, ist auch dies zu prüfen. Nach den Empfehlungen der SKLM [10] ist die gesundheitliche Unbedenklichkeit in Form einer Einzelfallbetrachtung durch eine Kombination verschiedener Untersuchungen zu belegen. Dazu können auch Homologievergleiche zu bekannten toxischen Proteinen und Allergenen dienen. Notwendig sind weiterhin Informationen zur Abbaubarkeit des Proteins im Gastrointestinaltrakt.

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3.4 Mikroorganismenkulturen Sofern die Wirkung eines FLM auf der Anwesenheit von Mikroorganismenkulturen beruht, empfiehlt die SKLM die Prüfkriterien zur Bewertung der gesundheitlichen Unbedenklichkeit an den folgenden Empfehlungen und Leitlinien auszurichten: x x x

Empfehlung der SKLM zu Starterkulturen und Enzymen für die Lebensmitteltechnik [9] BgVV-Empfehlung zu Probiotischen Mikroorganismenkulturen in Lebensmitteln [11] FAO/WHO-Leitlinie zur Beurteilung von Probiotika in Lebensmitteln [12]

Bevorzugt sind Stämme solcher Spezies zu verwenden, die sich während ihres langfristigen Einsatzes in der Lebensmittelproduktion, beim Verzehr durch den Menschen oder als Kommensale im menschlichen Intestinaltrakt als sicher erwiesen haben. Erforderlich sind die Charakterisierung der taxonomischen Position sowie Informationen zur möglichen Infektiosität, zur Virulenz sowie zur Persistenz. In-vitro-Tests zur Sicherheitsprüfung sind in der FAO/WHO-Leitlinie genannt [12]. Zum Ausmaß der Anforderungen an den Beleg der Sicherheit von probiotischen Mikroorganismenstämmen wird in der FAO/WHO-Richtlinie darauf hingewiesen, dass historisch Lactobazillen und Bifidobakterien in Lebensmitteln immer als sicher angesehen worden sind und dass ihr Vorkommen als normale Kommensale im menschlichen Intestinaltrakt sowie der nachgewiesenermaßen sichere Einsatz in Lebensmitteln und Nahrunsergänzungsmitteln diese Annahme stützt. Jedoch können theoretisch Nebenwirkungen wie eine systemische Infektion, nachteilige metabolische Aktivitäten, exzessive Immunstimulation bei empflindlichen Individuen und ein möglicher Gentransfer ausgelöst werden, auf die gegebenenfalls zu prüfen ist. Darüber hinaus können Prüfungen hinsichtlich spezifischer, potenziell nachteiliger Stoffwechselleistungen oder Eigenschaften erforderlich werden. Beispiele sind die Bildung von biogenen Aminen oder Toxinen, die Aktivierung von Prokanzerogenen, die Beeinflussung der Blutgerinnung bzw. eine mögliche hämolytische Aktivität, die Verursachung von allergischen Reaktionen sowie Wirkungen auf das Immunsystem.

14

4

Funktionalität und Auslobung

4 Funktionalität und Auslobung

Ein FLM muss – entsprechend der angestrebten Auslobung – eine oder mehrere Wirkungen aufweisen, die über diejenigen hinausgehen, die durch vergleichbare Produkte und mit vergleichbaren Verzehrsmengen im Rahmen einer ausgewogenen Ernährung erreicht werden. Der Nachweis der besonderen Wirkung ist Voraussetzung für die angestrebte Auslobung. Die Auslobung stellt die sprachliche Fixierung der produktspezifischen Eigenschaften dar, die über die Eigenschaften eines vergleichbaren Lebensmittels hinausgehen. Sie dient damit als Grundlage für die Festlegung von Art und Umfang der notwendigen Studien. Für den wissenschaftlichen Nachweis der Funktionalität ist nach Sicherstellung der gesundheitlichen Unbedenklichkeit die Durchführung von prospektiven Studien am Menschen erforderlich. Der Beleg der ausgelobten Wirkung muss am Produkt geführt werden. Für den wissenschaftlichen Nachweis einer Funktionalität muss a priori eine Studienhypothese formuliert sein. Vorläufige Pilotstudien sind oftmals nützlich zur Festlegung des endgültigen Studiendesigns und der Zielparameter, analog zu Forderungen aus [6]. Art und Umfang der notwendigen Studien am Menschen sind dabei jeweils in Abhängigkeit vom konkreten FLM, seinem funktionellen Prinzip und der angestrebten Auslobung festzulegen. Erwünscht sind mindestens zwei unabhängige Studien, unabdingbar ist mindestens eine Studie am Menschen, möglichst nach Art einer kontrollierten, randomisierten Doppelblindstudie gegen ein nichtfunktionelles vergleichbares Produkt. Die Wahl des Studienkollektivs richtet sich nach der angestrebten Zielgruppe. Darüber hinaus sind übliche Verzehrsmengen zugrunde zu legen und Bedingungen zu wählen, die für die entsprechende Zielgruppe eine charakteristische Ernährungsweise darstellen. Die Planung der Studie muss so ausgelegt sein, dass das Studienziel mit ausreichender Genauigkeit erreicht werden kann. Insgesamt zeigen solche Studien prinzipielle Parallelen zu Studien, wie sie für die Zulassung von Arzneimitteln erforderlich sind. Zwar können Art und Umfang der Studien bei FLM von denen bei Medikamenten abweichen, doch darf ihre Qualität hinsichtlich Konzeption, Durchführung und Auswertung nicht hinter jener bei der Arzneimittelprüfung zurückstehen. Sie müssen auf der Basis allgemein akzeptierter wissenschaftlicher Kriterien und unter Einhaltung der aktuellen wissenschaftlichen Qualitätsstandards erfolgen. Bei solchen Humanstudien sind GLP- und GCP-Bedingungen [13] zu beachten (good laboratory practice, good clinical practice). Die Studien sollten so angelegt sein, dass auch unerwünschte Wirkungen erfasst werden können. Um Art und Ausmaß unerwünschter Wirkungen zuverlässig abschätzen zu können, müssen hinreichend viele Beobachtungen an genügend Probanden vorliegen. Die SKLM empfiehlt, den Nachweis der funktionellen Wirkung probiotischer Lebens15

II

Kriterien zur Beurteilung Funktioneller Lebensmittel

mitteln an den Kriterien der BgVV-Arbeitsgruppe „Probiotische Mikroorganismenkulturen in Lebensmitteln“ [11] auszurichten. Wesentliche Qualitätskriterien von Humanstudien zum Nachweis der funktionellen Wirkung eines Lebensmittels sind, in Stichworten: x x x x x x x x x x x

x x x x x x x

hypothesengeleitetes Vorgehen prospektiver Charakter vor Beginn der Studien festgelegte Prüfparameter der Wirkung Kontrollgruppen Studienplan Biometrie ausreichende „Power“ (Macht der Studie) Probandeneinwilligung (informed consent), Ethikvotum Randomisation Doppelblindstudie Stratifikation nach Einflussfaktoren der Wirkung, z. B. Alter, Geschlecht, Ernährungs- und Gesundheitsstatus oder sonstiger, die gewählten Endpunkte definierender Einflussgrößen Abbruchkriterien „Compliance“, d. h. Einhaltung von Verzehrsmenge und -häufigkeit sowie Dokumentation der Parameter (Konkordanz) begrenzte Ausfallrate in Studienkollektiven adäquate biometrische Auswertung Monitoring zur Sicherung der Diätenqualität Berücksichtigung nachteiliger Wirkungen Bericht der Studienergebnisse nach anerkannten Kriterien, CONSORTstatement [14]

Fragen, die zur Bewertung von Relevanz und Validität der Ergebnisse im Vordergrund stehen: x

x x x

x x x

16

Sind alle relevanten Befunde und Erkenntnisse aus der verfügbaren Literatur und anderen Quellen hinreichend berücksichtigt und nach welchen Kriterien wurden sie zusammengestellt? Stehen die Studienergebnisse in direkter Beziehung zur Hypothese? Gibt es Belege für die beobachtete Funktionalität – auch aus tierexperimentellen Studien? Wenn der Befund die Beeinflussung eines so genannten Surrogat Markers betrifft, ist der Bezug des Surrogat Markers zur Hypothese gesichert und validiert? Ist die untersuchte Personengruppe repräsentativ für die Zielgruppe des Produktes? Liegt eine Bestätigung von Art und Stärke des Effektes durch eine oder mehrere nach anerkannten Kriterien durchgeführte Studie(n) vor? Gibt es vergleichbare Studien mit negativen Befunden?

7 x

Literatur

Wurde das Langzeitverhalten der Prüfparameter unter besonderer Berücksichtigung von Adaptation des Organismus bzw. Reversibilität von Effekten erfasst?

5 Beobachtung nach Markteinführung Das Marktbeobachtungsverfahren muss geeignet sein, Konsumentengruppen und deren tatsächliche Verzehrsmengen zu erfassen. Auf der Grundlage dieser Daten ist ein Vergleich von tatsächlicher und erwarteter Verzehrsmenge sowie der Zielgruppenspezifität des Produkts durchzuführen. Nach der Markteinführung eines FLM kann die Erfassung von funktionellen Wirkungen und gegebenenfalls auftretenden unerwünschten Wirkungen sinnvoll sein (Post-Launch Monitoring).

6 Schlussbemerkung Die SKLM hat diese Kriterien zur Bewertung der gesundheitlichen Unbedenklichkeit Funktioneller Lebensmittel sowie zum wissenschaftlichen Nachweis ihrer funktionellen Wirkung auf dem Erkenntnisstand des Jahres 2002 zusammengestellt. Die SKLM ist sich bewusst, dass diese Stellungnahme einer ständigen Aktualisierung nach dem jeweiligen Stand der Wissenschaft bedarf.

7 Literatur 1. Verordnung (EG) Nr. 258/97 des europäischen Parlaments und des Rates vom 27. Januar 1997 über neuartige Lebensmittel und neuartige Lebensmittelzutaten; Amtsblatt Nr. L 043 vom 14/02/1997, S. 1–6. 2. Diplock AT, Aggett PJ, Ashwell M, Bornet F, Fern EB, Roberfroid MB: Scientific Concepts of Functional Foods in Europe: Consensus Document. British Journal of Nutrition 81 (1999) Suppl. 1. 3. General Principles for the Addition of Essential Nutrients to Foods. 1987 (amended 1989, 1991). Codex Alimentarius Commission CAC/GL 09-1987. 4. Richtlinie 2002/46/EG des Europäischen Parlaments und des Rates vom 10. Juni 2002 zur Angleichung der Rechtsvorschriften der Mitgliedstaaten über Nahrungsergänzungsmittel. Amtsblatt der Europäischen Gemeinschaften 183/51 vom 12.7.2002.

17

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Kriterien zur Beurteilung Funktioneller Lebensmittel

5. 97/618/EG: Empfehlung der Kommission vom 29. Juli 1997 zu den wissenschaftlichen Aspekten und zur Darbietung der für Anträge auf Genehmigung des Inverkehrbringens neuartiger Lebensmittel und Lebensmittelzutaten erforderlichen Informationen sowie zur Erstellung der Berichte über die Erstprüfung gemäß der Verordnung (EG) Nr. 258/97 des Europäischen Parlaments und des Rates; Amtsblatt Nr. L 253 vom 16/09/1997, S. 1–36. 6. Aggett PJ, Agostini C, Goulet O, Hernell O, Koletzko B, Lafeber HL, Michaelsen KF, Rigo J, Weaver LR: The Nutritional and Safety Assessment of Breast Milk Substitutes and Other Dietary Products for Infants: A Commentary by the ESPGHAN Committee on Nutrition. Journal of Pediatric Gastroenterology and Nutrition 32 (2001) 256–258. 7. Guidance on Submissions for Food Additive Evaluations by the Scientific Committee on Food, SCF 12. Juli 2001. 8. Report of the Scientific Committee for Food 27th Series, 1992: Guidelines for the Presentation of Data on Food Enzymes (Opinion Expressed on 11 April 1991). 9. Starterkulturen und Enzyme für die Lebensmitteltechnik. DFG, Deutsche Forschungsgemeinschaft (Hrsg.), Wiley-VCH Verlag, Weinheim 1987; ISBN 3-52727362-X. 10. Beschluss der SKLM vom 2./3. Juni 1997: Beurteilungskriterien neuer Proteine, die durch gentechnisch modifizierte Pflanzen in Lebensmittel gelangen können. Im Originalwortlaut über das Sekretariat der SKLM, Kaiserslautern erhältlich, Publikation vorgesehen innerhalb der Beschlüssesammlung 1997–2004. 11. Probiotische Mikroorganismenkulturen in Lebensmitteln, „Arbeitsgruppe „Probiotische Mikroorganismenkulturen in Lebensmitteln“ am Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin (BgVV), Berlin. Ernährungs-Umschau 47 (2000) 191–195. 12. Guidelines for the Evaluation of Probiotics in Food. Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food. London, Ontario, Canada, April 30 and May 1, 2002. 13. ICH Topic E6; Guideline for Good Clinical Practice. http://www.emea.eu.int/pdfs/ human/ich/013595en.pdf 14. Moher D, Schulz KF, Altmann DG; CONSORT GROUP (Consolidated Standards of Reporting Trials). The CONSORT statement: revised recommendations for improving the quality of reports of parallel-group randomized trials. Ann Intern Med 134 (2001) 657–62; http://www.consort-statement.org

18

III Main Conclusions and Recommendations

1 Introduction The interest in functional foods and the corresponding range of products marketed are increasing worldwide. Functional foods now play an increasing role also in Europe as shown by the example of the introduction of spreads enriched with phytosterol esters to reduce plasma cholesterol levels in individuals with a disposition to hypercholesterolaemia. When compared to traditional foods, functional foods should exert effects on the improvement of health and the reduction of the risk of developing disease, which go beyond their nutritional effect. The aim of favourably influencing certain body and organ functions as well as the risks for developing specific diseases requires high standards both of the scientific evidence for any effects, which are the subject of so-called health claims, and of their scientifically based evaluation. In addition, for functional foods, as for all other foodstuffs, the basic premise is that they must be safe within the limits of the recommended or foreseeable amounts consumed. How this generally accepted basic requirement can be assured or plausibly verified in every case, has so far not been discussed in depth nor uniformly regulated. Clear definitions are needed for this discussion in order to differentiate between functional foods and food supplements. In Asia, the use of foodstuffs or food ingredients to influence well-being has been practiced for ages in traditional medicine. The first systematic and market-orientated approaches to the development of foods with healthimproving effects originated in Japan. Standardisation and the safety of such foods with defined benefits for health, the so-called FOSHU (Food for Specified Uses), have been regulated by law since 1991. Each product is subjected to an individual approval procedure, for which scientific data have to be submitted on functionality, safety and on the estimated daily amounts taken in. The FOSHU regulations cover only foods which are consumed within the context of normal nutrition but not food supplements. More detailed information can be found in the contribution by Watanabe (Chapter 3 in Part V) entitled “Functional Foods Research and Regulation in Japan”. 19

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Main Conclusions and Recommendations

In the USA, functional foods, in contrast to food supplements, are not a distinct and separately regulated food category. They are included among the general foodstuffs, which in the course of their intended use must satisfy the usual safety provisions to ensure reasonable certainty of their causing no harm. Health claims and corresponding labelling are evaluated in the context of legal regulations. However, these regulations apply – in contrast to Japan – not only to special products (functional foods) but also to the generally recognised properties of ingredients, which are added to foods (generic health claims). Food supplements have been legally regulated as “dietary supplements” by the coming into force of the “Dietary Supplement Health and Education Act” of 1994 (DSHEA). In the year 2002, the Institute of Medicine (IOM) of the National Academy of Sciences (NAS) prepared at the request of the Food and Drug Administration (FDA) processes and procedures for the systematic safety evaluation of food supplements. More extensive information may be found in the contribution by Schneeman entitled “Regulatory Framework for Functionality and Safety: A North American Perspective” (Chapter 5 in Part V) and on the Internet [1]. In Europe, expert committees are occupied at present mainly with the requirements for the scientific proof of health-promoting or disease risk-reducing effects. An initial consensus document of an expert committee, FUFOSE (Functional Food Science in Europe) was published in 1999 [2], which was concerned with the scientific basis for the development of functional foods and with the different health claims related thereto. Building on this concept, a discussion has been initiated by various authorities, e. g. the Council of Europe and the EU-Commission (PASSCLAIM, a Process for the Assessment of Scientific Support for Claims on Foods), on the scientific requirements for such health claims and the corresponding criteria are being developed. Since mid-2002, a Directive exists for the uniform regulation of the addition of vitamins and minerals to food supplements [3]. As first steps this Directive established a positive list for vitamins and minerals, set forth labelling rules and defined the procedure for establishing maximum or eventually minimum levels. The SKLM felt the need for a thorough scientific discussion, including potential risks of functional foods, and for this reason has directed the emphasis of this symposium to the safety aspects. As a result of its own extensive scientific consultations and taking into account the results of the symposium, the SKLM has, in addition, prepared an opinion entitled “Criteria for the Evaluation of Functional Foods”. It will be published as a separate article, but is also included in this symposium volume (Part IV), which contains the contributions to the symposium and the Conclusions and Recommendations derived from it as set out in this summary.

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General Aspects of the Safety Evaluation

2 General Aspects of the Safety Evaluation

Foods are mostly complex mixtures of macro- and micro-components, their safety and nutritional value are generally established. The procedure to establish the safety to health of foods usually follows this basic principle and therefore focuses classically on the evaluation of food additives, processing aids, contaminants, other ingredients, and of manufacturing and processing procedures [4]. More recent regulatory activities cover novel foods with and without associated health claims. Market developments with the aim of combining foods in a novel way or the addition of substances with nutritional, physiological or health-promoting effects to foods raise new questions particularly concerning the safety evaluation of such novel substances or foods. A purely nutritional scientific evaluation of such foods or diets cannot normally serve as a basis for the evaluation of the safety to health. For functional foods therefore a safety evaluation following generally accepted procedures is needed. However, the individual steps of the safety evaluation need to be adjusted to the special requirements for the evaluation of functional foods. Initially, the basis is the definition of the problem and the collection of existing knowledge including the experiences from consumption in other cultures. Building on this, follows the determination of data relevant to safety, of an estimate of the exposure and the preparation of a risk characterisation as a basis for the final safety evaluation. For functional foods, destined solely for special target population groups it is necessary to ensure safety to health for all groups likely to be potential consumers, particularly sensitive groups, e. g. infants, pregnant women, breast-feeding mothers, and elderly and chronically sick people. Prospective estimates of the exposure of specific consumer groups can be made, for example, by using reliable market and consumption analyses of comparable products already marketed with special attention being paid to the total consumption of functional ingredients from all different products claiming similar effects. After a functional food is put on the market its consumption needs to be examined by appropriate means (post-launch monitoring). This should enable the verification of previously predicted exposure and target group specificity as well as, for example, the early recognition of any changes in nutritional habits. Although post-launch monitoring cannot provide any evidence for efficacy, it is desirable to utilise it as an early check to verify the claimed functional properties or to detect eventually any undesirable accompanying effects. Epidemiological studies to discover long-term effects of functional foods should complete the existing information. Particularly desirable is the development and validation of suitable biomarkers for effect and for exposure which would enable an early detection of even minimal effects and a more accurate determination of the exposure. 21

III

Main Conclusions and Recommendations

3 Special Aspects of the Safety Evaluation

Functional ingredients are able to induce a multitude of biological effects, which are mediated through different cellular pathways. Examples are the influence on cellular signalling pathways, on enzymes of the xenobiotic metabolism or on the proteins of the transmembrane transport systems, and, in addition, the influence on the integrity of the hereditary material or the activity of DNA-processing enzymes, on the immune system and the homoeostasis between pro-oxidant and anti-oxidant effects. Similarly, the influence on the hormonal homoeostasis may be of importance, e. g. on biosynthesis, metabolism and secretion of hormones and the interaction with transport or receptor proteins. Potential interactions with absorption, distribution, metabolism and excretion of nutrients as well as drugs must be considered. If several functional ingredients are added to a product, the potential interaction between the individual components needs to be examined. Such biological effects are normally dose- and concentration-dependent and are determined primarily by the actual intake and the bioavailability of the substance. Whether an influence on the cellular target sites produces a positive or negative effect on health cannot always be clearly determined, because the balance between the individual effects in vivo is subject to complex regulatory systems. Some substances may exert non-linear dose-effect relationships or the nature of the effect may be reversed, e. g. an anti-oxidative effect becoming a pro-oxidative effect. The multitude of biological points of attack that may show health-relevant effects requires an analysis of dose dependency and of the mechanism of action, in order to provide a reliable database for a safety evaluation. The ultimate nature of the effect depends not only on the amount of the functional food taken in but also on its absorption, distribution, metabolism and excretion and thus on the concentration at the target site. Other parameters that can influence the response of individuals and of which account must be taken are genetic and functional polymorphisms, age, sex and nutritional status. Additional further influence parameters – independent of the subject involved, e. g. the matrix of the food, the interaction with other food components as well as with certain drugs – also require investigation. Detailed explanations of the multiplicity of biological effects and of the kinetics and the bioavailability of functional-food ingredients are exemplified in an opinion of the SKLM entitled “Aspects of potentially adverse effects of flavonoids/polyphenols to be used in isolated or enriched form in so-called functional foods or food supplements”. For functional foods, in which the functional components are represented by single substances or mixtures of substances and extracts, recognised toxicological test and evaluation methods are mandatory, such as

22

4

Gaps in Knowledge and Recommendations

those described for food additives [4]. The actual procedure is described in greater detail in Part IV “Criteria for the Evaluation of Functional Foods”. In functional foods with prebiotic ingredients, i. e. containing more or less indigestible materials, which are intended to support the growth of certain bacterial strains in the microflora of the gut, it is generally impossible to predict the selective effects on individual, defined group of microorganisms. Functional foods with probiotic microorganisms, which are designed to maintain or improve the microbial balance in the gut flora, have been marketed for several years. Because the consumption of such foods involves the intake of living bacteria, it is most important to ensure the safety of these microorganisms. The effects of the intake of probiotic bacteria depend on the host organism and also on the bacterium and therefore, in principle, a null risk does not exist for the host organism involved. However, in view of the long-term experience in man, the health risk from probiotic microorganisms altogether is considered to be comparatively small. Guidelines on the evaluation of cultures of microorganisms for use as or in so-called probiotic foodstuffs have been issued by a Joint FAO/WHO Working Group. It is pointed out there [5] that historically Lactobacilli and Bifidobacilli in foods have always been regarded as safe.

4 Gaps in Knowledge and Recommendations for Research for the Safety Evaluation The scientific discussion within the scope of the symposium has revealed a series of gaps in the existing knowledge and, consequently, the resulting research needs are explained in more detail below.

4.1 Exposure Assessment A need for research has been noted regarding the development of newer, reliable and suitable procedures for monitoring the marketing, which should enable a reliable determination of the amounts of products and of product groups with similar effects consumed by defined target population groups and sensitive groups. The development and evaluation of “biomarkers of exposure”, which permit an indirect ascertainment of the amounts consumed and the monitoring of individual subjects, are of special importance for the determination of the exposure.

23

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Main Conclusions and Recommendations

4.2 Analysis of Effects Knowledge of the dose-related effects of functional food ingredients and their mechanisms of action together with a reliable determination of the exposure provide the basis for a safety evaluation. The emphasis of further research work should be directed to the identification and the reliable exclusion of potentially adverse health effects for man. It is necessary to take account of the potential interactions between several functional ingredients or between functional ingredients and other nutritional components. Investigations of the mechanisms of action by means of in vitro and animal experimental models might furnish important knowledge but the plausibility of the mechanisms for man must be always ensured, e. g., by comparison with appropriate human data such as that obtained from biopsy samples [6]. Innovatory models, e. g. examining the effect of substances on specific groups of genes (nutrient/gene interaction) or the further development of biomarkers, might yield new mechanistic insights and also deepen the understanding of individual sensitivities. These new techniques open not just the possibility for demonstrating the plausibility of the experimental results for man but also offer a great potential for the development of “biomarkers for effects” for use in studies in molecular epidemiology.

4.3 Kinetics of the Substance Substance-specific parameters such as absorption and elimination rates, bioavailability, metabolism, tissue levels, adduct formation as well as a possible tissue-specific accumulation of substances have not yet been examined adequately. Further research is needed to clarify the importance of individually determined parameters for substance kinetics and bioavailability, e. g. genetic or functional polymorphisms. Account must also be taken of age, sex, nutritional and hormonal status . Furthermore, it is necessary to examine other parameters, which are not directly determined through the exposed subject. Examples are the influences of the food matrix on bioavailability or the consequences of therapy with certain medicaments as well as interactions with other food ingredients. Finally, the importance of the influence of the gut flora on the substance kinetics and the contribution to metabolism should be considered.

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5

References

4.4 Epidemiology Research needs exist for the development of well-substantiated procedures for the analysis of the mechanisms of the effects of a functional food in man. Of primary importance is the development, standardisation and validation of “biomarkers of the effect” induced or affected by functional ingredients, which would permit a reliable statement to be made about effect patterns relevant to safety. A further point of emphasis for research is the identification of genetic or lifestyle-associated predisposition and of corresponding molecular markers (biomarker) in order to enable an early recognition of population groups at risk.

5 References 1. Proposed Framework for Evaluating the Safety of Dietary Supplements – For Comment (2002); http://www.nap.edu/books/NI000760/html/ 2. Diplock AT, Aggett PJ, Ashwell M, Bornet F, Fern EB, Roberfroid MB: Scientific Concepts of Functional Foods in Europe: Consensus Document. British Journal of Nutrition 81, Suppl. 1, (1999). 3. Directive 2002/46/EU of the European Parliament and the Council of 10. June 2002 on the harmonization of the laws of Member States on food supplements. O. J. No. L 183 of 12/07/2002, pp 0051–0057. 4. Guidance on submissions for food additive evaluations by the Scientific Committee on Food, SCF 12. July 2001, http://europa.eu.int/comm/food/fs/sc/scf/out98_en.pdf 5. Guidelines for the Evaluation of Probiotics in Food. Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food, London, Ontario, Canada, April 30 and May 1, 2002. 6. Eisenbrand G, Pool-Zobel B, Baker V, Balls M, Blaauboer BJ, Boobis A, Carer A, Kevekordes S, Lhuguenot JC, Pieters R, Kleiner J: Methods of in vitro toxicology. Food Chemistry and Toxicology 40, (2/3), (2002), 193–236. 7. Opinion of the SKLM: “Aspects of potentially adverse effects of flavonoids/polyphenols to be used in isolated or enriched form in so called functional foods or food supplements”. Published in Report No. 7 “Lebensmittel und Gesundheit II”, Wiley-VCH Verlag, Weinheim, 2004, in preparation.

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IV Criteria for the Evaluation of Functional Foods

1 Preamble The Senate Commission on Food Safety (SKLM) of the DFG has elaborated the following recommendations with the objective of defining the minimum requirements for the evaluation of the safety to health of functional foods (FFs) and for the scientific proof of their functionality. With these recommendations the SKLM does not take a position regarding the legal aspects of functional foods set out below, nor does the SKLM discern a need for taking any action on the legal or quasi-legal assessment of health-related claims (so-called health claims). For this purpose attention is drawn to the efforts of other authoritative bodies (Codex Alimentarius Commission, Council of Europe) and to the initiative of the EU Commission in preparing a Directive at the European level. If the FF falls within the field of application of the EU Novel Food Directive (No. 258/97), then the market placement of any such product is subject to a specified approval procedure [1]. The SKLM assumes that the criteria for the evaluation of “functional foods” will be modified and updated in conformity with the state of science existing at the relevant time.

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Criteria for the Evaluation of Functional Foods

2 Differentiation of Functional Foods From Other Foodstuffs and Products

2.1 Functional Foods No legally binding definition exists as yet for FFs. The SKLM therefore relies on the definition described in a consensus document elaborated in the context of an EU initiative, the so-called FUFOSE working group [2]. According to this definition, a foodstuff may be considered as being “functional”, if it exerts a demonstrable positive effect beyond its normal nutritional physiological effects on one or several target functions in the human body, thereby achieving an improved state of health or an increased feeling of wellness and/or a reduction in the risk of developing a disease. FFs are offered for sale exclusively in the form of foodstuffs and not, as are food supplements, in the form of medicinal preparations resembling medicines. They must be an integral component of normal nutrition and should already exert their effects when consumed in normal amounts. A FF may be a natural foodstuff or one containing an ingredient that has been added, enriched, reduced or removed. Additionally, it may be a foodstuff in which the natural chemical structure of one or several components or their bioavailability has been modified. A FF may be functional for the whole population or only for a defined population group (e. g. defined by age or genetic constitution).

2.2 Foodstuffs Enriched With Certain Nutrients According to the definition of the Codex Alimentarius Commission, foodstuffs enriched with certain nutrients are foodstuffs to which essential nutrients, i. e. substances for which generally accepted intake recommendations exist, have been added as enrichment or supplementation with the aim of preventing a deficiency of one or several nutrients in the general population or only in certain population groups [3]. Such a modification of a foodstuff, covered by accepted nutritional recommendations as issued by societies recognized as peer advisory bodies in this field, does not provide any functional effects over and above those of normal nutrition that could be identified by the criteria described below. Therefore, simple enrichment with essential nutrients cannot be regarded as a functional principle within the meaning of definition for functional foods mentioned above. Similar considerations apply to any reductions in the content of any food component.

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Evaluation of the Safety to Health

2.3 Food Supplements Food supplements are defined in a Draft Directive of the European parliament and Council as foodstuffs consisting of concentrates of single or multiple nutrients, marketed in the form of dosed preparations and destined to supplement the intake of these nutrients within the scope of normal nutrition [4]. In this draft the concept of nutrients encompasses merely vitamins and minerals. “In the form of a dosed preparation” signifies a form of preparation similar to medicines, e. g. capsules, tablets, pills or ampoules.

3 Evaluation of the Safety to Health

3.1 General Requirements FFs must not be hazardous to the health of the consumer and it is required that they are thoroughly investigated and evaluated in this respect. Targeted investigations of their functional effects in humans may only be initiated, when no indication of a risk to health is apparent in the light of current knowledge. Any safety evaluation should comply with the guideline recommendations for novel foods of the EU Scientific Committee on Food (SCF) [5], independent of whether the FF does or does not fall within the scope of the definition in the EC Directive No. 258/97 and any subsequent amendments concerning novel foodstuffs and novel-food ingredients [1]. According to these recommendations, any novel food is evaluated following the principle of substantial equivalence or difference, i. e. on the basis of a comparison with an equivalent traditional product. A FF usually differs from its comparable product either by the presence or absence or an increased or reduced concentration or bioavailability of one or several functional components. It is possible in such circumstances to restrict the safety evaluation to the functionally effective ingredients. If necessary, the additional influence of the matrix of the foodstuff has to be considered. Because of the expected diversity of FFs or of the added ingredients, an individual case-by-case evaluation is essential. The nature and extent of the required investigations depend on the properties of the functional components or active principle and also on the expected future exposure of the target population or the potential population group at risk. It is essential to prepare a systematic summary of the entire available information on the properties of the functional components and 29

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their possible adverse effects. In carrying out this task information from unpublished studies and from studies not performed according to accepted criteria should also be considered, as already required for certain dietary products [6]. Human experience, e. g. from lifespan consumption in other cultures, from epidemiological studies or from other human studies, has to be specially considered in this context. The exposure of the general population including the targeted population as well as potential population groups at risk should be estimated. This estimate should also include the total consumption of all other functional components having similar functionality or exerting comparable effects.

3.2 Single Substances, Mixtures of Substances and Extracts The SKLM recommends that the testing requirements for the evaluation of the safety to health of functional ingredients should follow internationally recognised testing criteria for food additives as published in an opinion paper of the SCF: x

Guidance on submissions for food additive evaluations by the Scientific Committee on Food [7].

Essentially, the requested information includes an adequate characterisation of the functional ingredients, i. e. a description of their chemical composition, their physico-chemical and microbiological properties, their sources, and the processes employed for their isolation or production. Additionally, specifications, purity criteria and practical methods of analysis must be provided. Information must be supplied on their stability in the foodstuff, their possible degradation and reaction products and on possible interactions with nutrients and any influences on the bioavailability of these nutrients. For the safety evaluation, the basic data listed in the SCF guidelines [7] must be submitted. In certain cases it may be necessary to perform supplementary studies as described in the guidelines.

3.3 Enzymes If pure enzymes or enzyme preparations are added as functional components over and above their use for purely technological purposes, the SKLM recommends that the testing criteria chosen for the safety evaluation should be defined within the context of a case-by-case consideration according to the guidelines mentioned below: 30

3 x x x

Evaluation of the Safety to Health

Guidelines of the SCF on the submission of data for enzymes for foodstuffs [8], Recommendations of the SKLM on the evaluation of starter cultures and enzymes used in food technology [9], Recommendations of the SKLM on the evaluation of novel proteins, which may enter foodstuffs through the use of genetically modified plants [10].

According to the SCF guidelines [8], information is required on the source of the enzyme, the method of production, the catalytic activity, the stability in the food product, and the intended use of the product. For the safety evaluation of enzymes of different origin the basic toxicological data listed in the SCF guidelines must be submitted for each individual enzyme. Also, as the catalytic function of the enzyme may cause not only changes in the foodstuffs but also in the digestive processes and in the bioavailability of nutrients after uptake from the intestinal tract, this aspect has to be examined. According to the recommendations of the SKLM [10], evidence for the safety to health must be provided in the form of a case-by-case consideration using a combination of various investigations. These may include comparisons for homology with toxic proteins and allergens. Furthermore, information is needed on the degradability of the enzyme protein in the gastrointestinal tract.

3.4 Cultures of Microorganisms Whenever the functionality of a functional food depends on the presence of cultures of microorganisms, the SKLM recommends that the testing criteria for the evaluation of the safety to health should be in accordance with the following recommendations and guidelines: x x x

Recommendations of the SKLM on starter cultures and enzymes for food technology [9], Recommendations of the BgVV on cultures of probiotic microorganisms in foods [11], FAO/WHO guidelines for the evaluation of probiotics in foodstuffs [12].

Preferentially such strains of species should be used which during their traditional long-term employment in food production have proven to be safe for consumption by man or to be commensals in the human intestinal tract. It is necessary to characterize the taxonomic position and to provide information on the possible infectivity, virulence and persistence. The FAO/WHO guidelines mention in-vitro tests for the safety evaluation [12]. The FAO/WHO recommendation also points out in connection 31

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with the extent of the requirements for demonstrating the safety of probiotic strains of microorganisms, that historically Lactobacilli and Bifidobacteria in foodstuffs have always been regarded as safe and that this assumption is supported by their presence as natural commensals in the human intestinal tract as well as their proven safe use in foodstuffs and food supplements. However, there is the theoretical possibility of their causing side-effects, such as systemic infections, adverse metabolic activities, excessive stimulation of the immune system and a possible gene transfer in sensitive individuals. These possibilities must be investigated. In addition, tests may become necessary for specific, potentially adverse metabolic activities or properties. Examples would be the formation of biogenic amines or of toxins, the activation of pro-carcinogens, an influence on blood coagulation or a possible haemolytic activity, the induction of allergic reactions as well as effects on the immune system.

4 Functionality and Claims A FF must produce – according to the intended claim – one or several effects, which exceed those that may be achieved by a comparable product consumed in comparable amounts as part of a balanced diet. Evidence for a special effect is the precondition for any desired claim. A claim represents the linguistic description of product-specific properties, which extend beyond the properties of a comparable foodstuff. This claim serves as the basis for defining the type and extent of the necessary studies. For the scientific proof of any functionality it is necessary to carry out prospective studies in humans after assurance of the safety to health. Evidence of the claimed effect should be produced for the product under examination. For the scientific proof of functionality a study hypothesis must be formulated a priori. Preliminary pilot studies are frequently useful for deciding about the final study design and the targeted parameters, analogous to requirements in [6]. In this connection the type and extent of the necessary studies in humans are to be determined depending on the actual FF, its functional principle, and the intended claim. A minimum of two independent studies is desirable, of which at least one human study is essential, preferably following the design of a controlled, randomised double-blind study against a nonfunctional comparable product. The choice of the study population depends on the intended target population group. Apart from this, the study has to be based on normal amounts consumed and conditions have to be chosen which represent a characteristic nutritional habit for the selected target population group. 32

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The study plan must be designed in the way that the study goal can be reached with an adequate precision. Generally such studies will have parallels to studies required for the registration of medicinal preparations. Although the type and extent of studies with FFs may deviate from those performed for medicines, yet their quality – as concerns concept, execution and evaluation – must not be lower than that required for the testing of medicines. They must be carried out on the basis of generally accepted scientific criteria and in compliance with acceptable scientific quality standards. In these human studies GLP and GCP conditions (good laboratory practice, good clinical practice) must be observed [13]. The studies should be so designed that they also record undesirable effects. In order to estimate reliably the type and extent of adverse effects, a sufficient number of observations on sufficient subjects are needed. The SKLM recommends that the demonstration of a functional effect with probiotic foodstuffs should follow the criteria of the BgVV-working group “Probiotic microorganism cultures in foodstuffs” [11]. Important quality criteria for human studies to demonstrate the functional effect of a food are listed as key phrases: x x x x x x x x x x x

x x x x x x x

procedure to follow a hypothesis prospective character test parameters for the effect to be fixed in advance of study control groups study plan biometry adequate power of the study informed consent of participants, agreement of ethics commission randomisation double-blind study stratification according to factors influencing the functional effect, e. g. age, sex, nutritional status, health status, other parameters defining the chosen endpoints criteria for discontinuing the study compliance, i. e. maintaining the amounts consumed and the consumption frequency as well as documentation of the parameters (concordance) limited default rate for participants in the study group adequate biometric evaluation monitoring to confirm the quality of the diet accounting for adverse reactions report of results of the study to follow recognised criteria; CONSORT statement [14].

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Questions, which are of paramount importance in the evaluation of the relevance and validity of the results: x

x x x

x x x x

Have all relevant findings and knowledge from the available literature and other sources been appropriately considered and according to which criteria were they collected? Are the results of the studies directly correlated with the hypothesis? Is there evidence for the observed functionality, also from experimental studies in animals? If the finding concerns an effect on a so-called surrogate biomarker, has the relationship of the surrogate biomarker to the hypothesis been ascertained and validated? Is the group of individuals examined representative of the target population group for the product? Does confirmation exist of the nature and strength of the effect through one or more studies carried out according to recognised criteria? Do comparable studies exist with negative findings? Are long-term changes of the test parameters included among the observations with special attention being paid to adaptive responses of the organism to or reversibility of the effects?

5 Observation after the Market Introduction The procedure for the post-marketing observation must be suitable for sampling the actual consumer groups and measuring the amounts actually consumed. On the basis of these data, a comparison should be made between the actual and the expected amounts consumed and of the specificity of the product for the target population. After market placement of a FF, it is sensible to determine the functional effects and any potential undesirable effects appearing as a consequence (post-launch monitoring).

6 Concluding Remarks The SKLM has assembled these criteria for the evaluation of the safety to health of FFs as well as for the scientific proof of their functional effects in conformity with the state of knowledge in 2002. The SKLM is aware that this opinion will require constant updating according to the state of science existing at the relevant time. 34

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References

7 References 1. Directive 258/97/EU of the European Parliament and the Council of 27. January 1997 on Novel Foods and Novel Food Ingredients. O. J. No.: L 043 of 14/02/ 1997, pp. 1–6. 2. Diplock AT, Aggett PJ, Ashwell M, Bornet F, Fern EB, Roberfroid MB: Scientific Concepts of Functional Foods in Europe: Consensus Document. British Journal of Nutrition 81 (1999) Suppl. 1. 3. General Principles for the addition of essential nutrients to foods (1987) (amended 1989, 1991). Codex Alimentarius Commission CAC/GL 09-1987. 4. Directive 2002/46/EU of the European Parliament and the Council of 10. June 2002 for the Harmonisation of the Laws of Member States on Food Supplements. O. J. 183/51 of 12.7.2002. 5. 97/618/EU: Recommendations of the Commission of 29. July 1997 on the scientific aspects and the submission of applications with the required information for the permission to market novel foods and food ingredients as well as the provision of reports on the initial testing according to the Directive No. 258/97/EU of the European Parliament and the Council. O. J. No.: L 253 of 16/09/1997, pp. 1–36. 6. Aggett PJ, Agostini C, Goulet O, Hernell O, Koletzko B, Lafeber HL, Michaelsen KF, Rigo J, Weaver LR: The Nutritional and Safety Assessment of Breast Milk Substitutes and Other Dietary Products for Infants. A Commentary by the ESPGHAN Committee on Nutrition. Journal of Pediatric Gastroenteritis and Nutrition 32 (2001) 256–258. 7. Guidance on submissions for food additive evaluations by the Scientific Committee on Food, SCF 12. July 2001. 8. Report of the Scientific Committee on Food 27th series, 1992: Guidelines for the presentation of data on food enzymes (Opinion expressed on 11 April 1991). 9. Starterkulturen und Enzyme für die Lebensmitteltechnik. DFG, Deutsche Forschungsgemeinschaft, Wiley-VCH-Verlag, Weinheim 1987; ISBN 3-527-27362-X. 10. Beschluss der SKLM vom 2./3. Juni 1997: Beurteilungskriterien neuer Proteine, die durch gentechnisch modifizierte Pflanzen in Lebensmittel gelangen können. Im Originalwortlaut über das Sekretariat der SKLM, Kaiserslautern erhältlich, Publikation vorgesehen innerhalb der Beschlüssesammlung 1997–2004. 11. Probiotische Mikroorganismenkulturen in Lebensmitteln, Arbeitsgruppe “Probiotische Mikroorganismenkulturen in Lebensmitteln” am Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin (BgVV), Berlin. Ernährungs-Umschau 47 (2000) 191–195. 12. Guidelines for the Evaluation of Probiotics in Food. Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food. London, Ontario, Canada, April 30 and May 1, 2002. 13. ICH Topic E6, Guideline for Good Clinical Practice. http://www.emea.eu.int/pdfs/ human/ich/013595en.pdf. 14. Moher D, Schulz KF, Altmann DG; CONSORT GROUP (Consolidated Standards of Reporting Trials). The CONSORT statement: revised recommendations for improving the quality of reports of parallel-group randomized trials. Annual International Medicine 134 (2001) 657–62, http://www.consort-statement.org

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1 Diet, Genes, and Cancer Risk Cornelia M. Ulrich* and John D. Potter

Abstract Dietary factors are important contributors to cancer development. The World Cancer Research Fund expert panel has published a detailed report, “Food, Nutrition and the Prevention of Cancer: A Global Perspective”, that summarizes the epidemiologic literature on diet-cancer relationships. Some consistent findings are that high intakes of vegetables and fruit are associated with a decreased risk of cancers of the stomach, lung, and colorectum. The biologic mechanisms for these associations are beginning to be understood, as plant foods can induce biotransformation enzymes, and contain many potent anticarcinogens. Folate may also be a key nutrient responsible for the inverse associations observed with high intakes of fruit and vegetables. There are inherited differences in the ability to absorb and metabolize nutrients or excrete harmful components of foods. An emerging body of research is addressing the relationship between this inherited genetic variability and cancer risk. In many studies it has become apparent that common genetic variants (polymorphisms) rarely display a strong “main effect”, but become important only in the context of a relevant exposure. For example, polymorphisms in folate-metabolizing enzymes have been shown to modify risk of colorectal carcinogenesis but only in the presence of variation in folate intake. Genetic variability in folate metabolism has also been shown to affect risk of cancers of the pancreas, breast, and lung. Polymorphisms in epoxide

* Fred Hutchinson Cancer Research Center and University of Washington, Seattle, Washington, 98109-1024, USA

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hydrolase – an enzyme that detoxifies polycyclic aromatic hydrocarbons – appear to modify the risk of colorectal adenomas seen with high intakes of well-cooked meat. Investigating these associations furthers our understanding of the underlying biological mechanisms linking dietary factors to carcinogenesis. Observational epidemiologic studies have limitations, and causality cannot be inferred from these alone. Several randomized controlled trials of nutrient supplementation or other dietary interventions have been conducted. Only a few of these trials have yielded promising results, and, in some instances, harm has resulted. These findings question the approach to cancer prevention using single components, especially at high doses. Again, in this setting, understanding the genetic make-up of study participants may help elucidate whether there are individuals who may benefit versus groups that will not.

1.1 Introduction The relationship between diet and cancer has been investigated in a large body of research, ranging from epidemiologic studies and human intervention studies to in vitro experiments and animal studies. Recently, the World Cancer Research Fund published a comprehensive report summarizing knowledge in this area, mostly from the epidemiologic perspective [1]. Some associations with cancer risk are more consistent across the literature than others: for example, an inverse association between vegetable and fruit consumption and risk of several types of cancer has been quite consistently observed, whereas the relationship between meat intake and cancer risk is less well established. Although some of these differences may represent organ-specific effects, some of the inconsistencies may be explained by inherited genetic susceptibility in some individuals and not others. Utilizing information on genetic variability may provide further information on the underlying biological mechanisms. It has been hypothesized that a cell needs to undergo at least six alterations to acquire the necessary capabilities for becoming cancerous [2]. Dietary factors may be involved in increasing the likelihood of such alterations, e. g., by increased genotoxic damage due to a high consumption of mutagens, or by effects on an individual’s ability to repair DNA damage. A model integrating several dietary factors and risk of colorectal cancer is shown in Fig. 1.1. Genetic variability extends beyond the well-known “cancer genes”, or high-penetrance alleles, that carry a high individual risk of disease, but are relatively rare. Examples of these include BRCA1 and BRCA2, and the genetic defects in mismatch-repair enzymes responsible for hereditary nonpolyposis colorectal cancer (HNPCC). More interesting from a public health 38

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Figure 1.1. Relationship between dietary factors, folate, DNA damage and repair, and risk of colorectal neoplasia.

perspective are genetic mutations with low penetrance that may be very common in the population. These polymorphisms often occur in metabolizing enzymes and, although the alteration in risk for the individual is low, based on their high frequency in the population their public health impact may be substantial [3]. Although this paper will predominantly focus on the interaction of diet with these common polymorphisms, the role of environmental factors in determining the phenotype of families that carry high-risk alleles should not be disregarded. An excellent example stems from a multigenerational HNPCC family with a mismatch repair deficiency. This family was described first in 1913, and the first three generations were characterized mostly by cancers of the stomach and the uterus [4]. However, within the 4th generation, 23 of the 26 cancers described in this family were of colorectal origin, thus showing a typical HNPCC phenotype [5]. Since the genetic predisposition in this family has not changed over time, changes in environmental factors, perhaps including diet, must have caused this shift in phenotype. It is conceivable that with a diet higher in mutagens and lower in foods containing protective factors, the colon of these individuals was subject to greater DNA damage, and a reduced repair capacity became crucial. Meat intake increased markedly from the early to mid 20th century, which may explain part of these findings.

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1.2 Meat Intake 1.2.1

Epidemiologic Evidence

As summarized in the WCRF report, the evidence with respect to the amount of meat intake and cancer risk is inconsistent [6]. Rather than total meat intake, the cooking method may be relevant. High temperature cooking results in the production of heterocyclic amines (HCAs, e. g., PhiP and MeIQx) and polycyclic aromatic hydrocarbons (PAHs, e. g., benzo[a]pyrene) [7]. Both of these classes of compounds have been shown to be highly mutagenic. Based on a nutritional database estimating the content of HCAs in the diet, Sinha and colleagues were able to show a significantly increased risk of colorectal adenomas [8] and breast cancer [9] with high intakes of PhiP. Several groups of biotransformation enzymes are responsible for the excretion (and in some instances activation) of these compounds, including the Nacetyltransferases (NATs), glutathione transferases (GSTs), cytochrome p450 enzymes, and epoxide hydrolase. Thus, the rate of adduct formation to DNA may differ depending on the activity of these enzymes.

1.2.2

Genetic Variability in Epoxide Hydrolase

Microsomal epoxide hydrolase (mEH) detoxifies epoxides that can be generated during the oxidative metabolism of PAHs [10]. However, in some instances, trans-dihydrodiols generated from PAHs are highly toxic and mutagenic [11]. An example is ( )-anti-7,8,diol-9,10-epoxide derived from benzo[a]pyrene [12]. The mEH protein and nucleic acid sequences are highly conserved across species [13]. Several-fold variation in epoxide hydrolase activity in humans has been reported [13, 14] and some of this variation is attributable to known genetic polymorphisms. Hassett et al. [10] have described two common mEH coding-region variants that result in amino acid substitutions at two positions (Tyr113His and His139Arg). In vitro expression analyses of the corresponding proteins showed a 40 % decrease (slow phenotype), and 25 % increase (rapid phenotype), respectively, with the variant alleles, presumably the result of altered protein stability rather than changes in enzyme activity [10, 14]. Our group investigated the association between these polymerphisms and colorectal polyps within a case-control study of colorectal polyps conducted in Minnesota [15]. Cases were diagnosed with colonoscopically confirmed adenomas (n = 530); controls (n = 649) were seen at the same gastroenterology practice and found polyp-free at colonoscopy. Information on smoking status and meat consumption were obtained from self-administered questionnaires prior to colonoscopy. The increase in risk associated with smoking was more pronounced among individuals with the exon 3 His/His genotype (slow phenotype), and a similar pattern was seen with respect to fried, baked, or broiled meat intake (j 2 ser40

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vings/wk (high) compared to J 1 serving/wk): A greater increase in risk was seen among those with the mEH exon 3 homozygous variant compared to those with the wildtype genotype. For the exon 4 polymorphism, no such increase in risk was observed, which is consistent with the opposite in vitro effect of this mutation. Although epoxide hydrolase polymorphisms are not associated with an increased risk of colorectal polyps overall, genotypes conferring a slow phenotype appear to be associated with an increased risk when combined with smoking and high intakes of fried, baked, or broiled meat [15].

1.3 Vegetable Intake 1.3.1

Epidemiologic Evidence

High intakes of vegetables and fruit have been found consistently to be associated with a decreased risk of cancer, especially those of the gastrointestinal tract [6]. There seems to be a clear dose-response relationship for some associations, e. g., vegetable consumption and reduced risk of lung cancer, or fruit consumption and reduced stomach cancer risk. One exception may be the lack of benefit with respect to breast cancer, based on a pooled analyses of prospective studies [16]. There are many possible explanations for these inverse associations, including a confounding effect by other health behaviors. However, plants contain numerous potent phytochemicals that may be responsible for these anticarcinogenic effects (for a review see [17]). In the following we will discuss two potential mechanisms linking fruits and vegetables to a reduced risk to cancer, including their interplay with genetic factors.

1.3.2

Potential Mechanisms – Induction of Biotransformation Enzymes

As noted previously, biotransformation enzymes are important for the metabolism and excretion of mutagenic compounds. Lampe et al. conducted a feeding study examining the effects of three botanically defined diets on CYP1A2 and NAT2 activity, and GSTm activity in lymphocytes [18, 19]. 43 individuals were randomly assigned to a diet high in brassica vegetables (e. g., broccoli and cabbage), allium vegetables (e. g., leeks and onions); apiaceous vegetables (e. g., carrots and dill weed), and a diet free of vegetables and their phytochemicals. It was observed that brassica vegetables significantly increased Cyp1A2 activity, whereas apiaceous vegetables significantly decreased Cyp1A2 activity [19]. Brassica vegetables induced GST-a, whereas both brassica and allium vegetables increased GSTm activity in peripheral blood lymphocytes. Further the GSTM1 genotype significantly modi41

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fied the response to diet [18]. These studies illustrate that active compounds in vegetables can indeed affect the expression and activity of biotransformation enzymes. However, it also becomes clear that the underlying mechanisms are complex, and far from being fully understood.

1.3.3

Potential Mechanisms – Folate Intake

Folate is a B-vitamin that commonly occurs in green vegetables, legumes, and some fruit. Folate acts as a one-carbon donor in the synthesis of methionine and nucleotides (see Fig. 1.2). Thus, it is important for DNA synthesis and repair, as well as methylation reactions. The methylation of DNA at CpG islands is a common mechanism for gene regulation. Fig. 1.2 demonstrates that other dietary factors also play a role in folate metabolism, specifically vitamins B6 and B12, B2 (as a cofactor of MTHFR), methionine, and also

Figure 1.2. Folate metabolism, and links to DNA synthesis and methylation (adapted from [30]). Key enzymes are denoted as ovals, substrates as rectangles. THF = tetrahydrofolate; DHF = dihydrofolate; RFC = reduced folate carrier; hFR = human folate receptor; MTHFR = 5,10-methylenetetrahydrofolate reductase; DHFR = dihydrofolate reductase; GART = glycinamide ribonucleotide transformylase; AICARFT = 5-aminoimidazole-4-carboxamide ribonucleotide transformylase; AICAR = 5-aminoimidazole-4-carboxamine ribonucleotide; GAR = glycinamide ribonucleotide; SAM = S-adenosylmethionine; SAH = S-adenosylhomocysteine; dUMP = deoxyuridine monophosphate; dTMP = deoxythymidine monophosphate; X = a variety of substrates for methylation.

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alcohol (which appears to affect folate metabolism in several ways under very high intakes). In a number of studies, high dietary folate intakes have been shown to be associated with a decreased risk of colon cancer [20–22], possibly cervical cancer [23, 24], esophageal/gastric cancer [25, 26], breast cancer (especially in combination with low alcohol intakes ) [27–30] and pancreatic cancer [31, 32]. For example, in the study population of the a-tocopherol b-carotene intervention trial of male smokers, folate intakes in the highest quintile (i 373 mg) compared to those in the lowest quintile (I 280 mg) were associated with a halving in risk of pancreatic cancer (RR = 0.52 95 % CI 0.31– 0.87) [32] and similar results were seen for serum folate levels [31].

1.3.4

Genetic Variability in Folate Metabolism

Several common polymorphisms in enzymes in folate metabolism have been described that appear to affect protein function (for a review see [30]). Most commonly investigated is 5,10-methylenetetrahydrofolate reductase (MTHFR), with two common polymorphisms C677T and A1298C [33, 34]. The 677 TT genotype is associated with 30 % in vitro enzyme activity, lower serum folate levels, higher homocysteine levels and lower DNA methylation in white blood cells [35–37]. The variant is very common, with approximately 40 % of Caucasian populations carrying the CT genotype (intermediate enzyme activity), and about 10–15 % having the TT genotype. In the Minnesota polyp case-control study described previously, individuals with the TT genotype were at increased risk for adenomas only if they consumed a diet low in folate, or in vitamins B6 and B12 [38]. This pattern was more pronounced among the elderly. Several other studies on colorectal adenomas or cancer have shown similar results [39–41], and this is probably one of the most consistent patterns of gene-diet interaction known to date: lower MTHFR activity (TT genotype) is associated with no effect (or possibly a decreased risk) in the presence of a high folate status, whereas under conditions of a low folate status, risk is increased. More recently, polymorphisms in thymidylate synthase have been investigated. Thymidylate synthase is important for the provision of the nucleotide thymidine for DNA synthesis and repair. Its substrate, 5,10-methylenetetrahydrofolate, is a central metabolite in folate metabolism, as it can be diverted into three different branches (see Fig. 1.2). TS is also a primary target for chemotherapeutic agents, including 5-fluorouracil. Two common polymorphisms in TS have been described, the first one consisting of a variable number of repeats (usually 2 or 3 repeats) in the promoter enhancer region (TSER polymorphism) [42], and the second one being a 6bp deletion in the 3’ untranslated region of TS [43]. The TSER 2rpt/2rpt genotype is associated with 2.6-fold decreased gene expression compared to the 3rpt/3rpt genotype [44]. In the Minnesota polyp case-control study, a significant gene-diet interaction with respect to the TSER polymorphism was observed: the variant 43

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allele was associated with a decreased risk in the presence of low folate or vitamin B12 intakes, whereas risk was increased under conditions of a high folate or vitamin B12 intake [45]. These studies on the MTHFR and TS polymorphism illustrate our increasing understanding of how genetic factors may affect interindividual differences in metabolism, and eventually cancer risk.

1.3.5

Can There Be Too Much Folate?

In 1992, the US Public Health Service recommended that all reproductiveaged women should consume at least 400 mg of folate per day for the prevention of neural-tube defects. Since compliance was low, it was decided to fortify enriched grain products with folic acid. The addition of folic acid to the US food supply was mandatory by January 1, 1998, and results in about 100 mg supplemental folic acid for the average person. The fortification program seems to be effective, as the birth prevalence of neural tube defects has since decreased by about 19 % [46]. However, in a preliminary cross-sectional study of folate and immune function among postmenopausal women, an inverted U-shaped relationship was observed (Ulrich et al. unpublished results). When stratified by source and amount of folate, it was observed that women who consumed a diet low in folate (I 233 mg) and used supplements up to 400 mg of folate per day had higher natural killer cell activity than those with a low-folate diet and no supplementation. However, women, who consumed a diet already relatively high in folate (j 233 mg) and also ingested more than 400 mg folic acid from supplements had significantly lower NK activity than those with a low intake from diet and no supplement intake. These results should be considered preliminary due to some study limitations, and require, minimally, replication. In animal experiments of colorectal carcinogenesis, excess folate intakes (40x the requirement) were not beneficial, and findings suggested that folate supplementation administered later in carcinogenic progression may enhance carcinogenic progression rather than prevent it [47, 48].

1.4 Supplements and Cancer Prevention Dietary instruments measure foods, not nutrients. Thus, it is questionable whether we can draw conclusions about nutrients, especially since there are a multitude of constituents in foods. A 1997 review of the epidemiologic evidence regarding vitamin supplements and cancer risk indicates only modest evidence for protective effects of nutrients from supplements [49]. The b-carotene intervention trials (CARET and ATBC) showed that supplemental b-carotene is not effective in cancer prevention, and appears to increase 44

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cancer risk in high-risk populations [50, 51]. More recently, Neuhouser et al. have reported that the inverse association with fruits and vegetables and lung cancer risk was only seen among CARET participants in the placebo arm, but not in those receiving b-carotene [52]. Overall, randomized controlled trials have been only occasionally successful in either the chemoprevention of cancer, or the reduction of precursors of cancer (e. g., adenoma recurrence). Some of the successes (e. g., retinoids in head and neck cancer [53] and tamoxifen) are, of course, of considerable importance. Unfortunately, apart from a true lack of effect, null results from chemoprevention trials can have a multitude of explanations. For example, the treatment duration may not have been long enough, or not at the correct time; the wrong dose/formulation may not have been used; adherence may have been poor; or the wrong outcome may have been measured. Last but not least, in the multifactorial etiology of cancer, other risk factors may be important, possibly in a synergistic fashion with the nutrient/food under question. Chemoprevention agents that increase risk also raise many other issues – as well as the possibility that some agents may indeed be deleterious at inappropriate doses – or at the wrong time in the cancer process. Thus, the relationship between nutritional supplements and cancer risk should be considered unresolved, and the outcomes from major trials investigating a calcium/vitamin D, or a low-fat diet high in fruits and vegetables (both Women’s Health Initiative) or selenium (SELECT trial) will hopefully provide more insight.

1.5 Summary and Recommendations In summary, there is strong evidence that diet influences cancer risk, and epidemiologic studies report some consistent, as well as some inconsistent associations. Taking inherited genetic variability into account can further our understanding of the underlying biologic mechanisms, and may help resolve some of the inconsistencies in the epidemiologic literature. However, we need to consider that, with respect to common polymorphisms themselves, usually no strong associations are observed; the genetic variation often becomes relevant in the presence of specific exposures (e. g., meat intake and mEH or folate and MTHFR). It is also important to keep in mind that humans usually consume foods, not nutrients, and any interpretation about nutrient-disease associations needs to be made carefully. Investigating genetic polymorphisms in specific nutritional pathways may help to solidify our understanding of the involvement of a particular nutrient. Randomized controlled trials of chemoprevention have sometimes been problematic, and safety aspects need to be considered. Again, genetics may help identify subgroups that may benefit from chemoprevention. Over the next decade, our understanding of genetic variability and gene-diet interactions will increase dramatically. Our knowledge regarding 45

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genetically definable population groups, who may either be at risk of toxicity with high intakes, or gain benefit from them, will grow. This has obvious implications for the introduction of functional foods and their safety aspects. Recommendations for individuals are currently premature, although this has not prevented a growing industry in advice on nutritional intake based on genotype, and nutritional supplements. Nonetheless, more individualized counseling on nutrient intakes, based on genetic make-up may be possible in the future. Practicing nutritionists will increasingly need to be able to evaluate the literature on molecular epidemiology [54]. Thus, nutrition students as well as clinical nutritionists should receive adequate training in genetics, as well as epidemiologic study design and research methods.

References 1. World Cancer Research Fund (WCRF) (1997) Diet, nutrition, and the prevention of cancer: a global perspective. WCRF/American Institute for Cancer Research. 2. Hanahan, D. and Weinberg, R. A. (2000) The hallmarks of cancer. Cell 100, 57–70. 3. Potter, J. D. (2001) At the interfaces of epidemiology, genetics and genomics. Nature Reviews Genetics 2, 142–7. 4. Warthin, A. S. (1913) Heredity with reference to carcinoma. Arch. Int. Med. 12, 546–555. 5. Lynch, H. T., Lynch, P. M., Albano, W. A. and Lynch, J. F. (1981) The cancer family syndrome: a status report. Diseases of the Colon & Rectum 24, 311–22. 6. World Health Organization (WHO) (1997) The world health report. In: (Ed.) WHO. 7. Sinha, R., Knize, M. G., Salmon, C. P., Brown, E. D., Rhodes, D., Felton, J. S., Levander, O. A. and Rothman, N. (1998) Heterocyclic amine content of pork products cooked by different methods and to varying degrees of doneness. Food & Chemical Toxicology 36, 289–97. 8. Sinha, R., Kulldorff, M., Chow, W. H., Denobile, J. and Rothman, N. (2001) Dietary intake of heterocyclic amines, meat-derived mutagenic activity, and risk of colorectal adenomas. Cancer Epidemiology, Biomarkers & Prevention 10, 559–62. 9. Sinha, R., Gustafson, D. R., Kulldorff, M., Wen, W. Q., Cerhan, J. R. and Zheng, W. (2000) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, a carcinogen in hightemperature-cooked meat, and breast cancer risk. Journal of the National Cancer Institute 92, 1352–4. 10. Hassett, C., Aicher, L., Sidhu, J. S. and Omiecinski, C. J. (1994) Human microsomal epoxide hydrolase: genetic polymorphism and functional expression in vitro of amino acid variants [published erratum appears in Hum. Mol. Genet. 1994 Jul;3(7):1214]. Human Molecular Genetics 3, 421–8. 11. Oesch, F. (1973) Mammalian epoxide hydrolases: inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica 3, 305–40. 12. Sims, P., Grover, P. L., Swaisland, A., Pal, K. and Hewer, A. (1974) Metabolic activation of benzo(a)pyrene proceeds by a diol-epoxide. Nature 252, 326–8. 13. Kitteringham, N. R., Davis, C., Howard, N., Pirmohamed, M. and Park, B. K. (1996) Interindividual and interspecies variation in hepatic microsomal epoxide hydrolase

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activity: studies with cis-stilbene oxide, carbamazepine 10, 11-epoxide and naphthalene. Journal of Pharmacology & Experimental Therapeutics 278, 1018–27. Hassett, C., Lin, J., Carty, C. L., Laurenzana, E. M. and Omiecinski, C. J. (1997) Human hepatic microsomal epoxide hydrolase: comparative analysis of polymorphic expression. Archives of Biochemistry & Biophysics 337, 275–83. Ulrich, C. M., Bigler, J., Whitton, J. A., Bostick, R., Fosdick, L. and Potter, J. D. (2001) Epoxide hydrolase Tyr113His polymorphism is associated with elevated risk of colorectal polyps in the presence of smoking and high meat intake. Cancer Epidemiology, Biomarkers & Prevention 10, 875–82. Smith-Warner, S. A., Spiegelman, D., Yaun, S. S., Adami, H. O., Beeson, W. L., van den Brandt, P. A., Folsom, A. R., Fraser, G. E., Freudenheim, J. L., Goldbohm, R. A., Graham, S., Miller, A. B., Potter, J. D., Rohan, T. E., Speizer, F. E., Toniolo, P., Willett, W. C., Wolk, A., Zeleniuch-Jacquotte, A. and Hunter, D. J. (2001) Intake of fruits and vegetables and risk of breast cancer: a pooled analysis of cohort studies. JAMA 285, 769–76. Steinmetz, K. A. and Potter, J. D. (1991) Vegetables, fruit, and cancer. II. Mechanisms. Cancer Causes & Control 2, 427–42. Lampe, J. W., Chen, C., Li, S., Prunty, J., Grate, M. T., Meehan, D. E., Barale, K. V., Dightman, D. A., Feng, Z. and Potter, J. D. (2000) Modulation of human glutathione S-transferases by botanically defined vegetable diets. Cancer Epidemiology, Biomarkers & Prevention 9, 787–93. Lampe, J. W., King, I. B., Li, S., Grate, M. T., Barale, K. V., Chen, C., Feng, Z. and Potter, J. D. (2000) Brassica vegetables increase and apiaceous vegetables decrease cytochrome P450 1A2 activity in humans: changes in caffeine metabolite ratios in response to controlled vegetable diets. Carcinogenesis 21, 1157–62. Freudenheim, J. L., Graham, S., Marshall, J. R., Haughey, B. P., Cholewinski, S. and Wilkinson, G. (1991) Folate intake and carcinogenesis of the colon and rectum. International Journal of Epidemiology 20, 368–74. Benito, E., Obrador, A., Stiggelbout, A., Bosch, F. X., Mulet, M., Munoz, N. and Kaldor, J. (1990) A population-based case-control study of colorectal cancer in Majorca. I. Dietary factors. International Journal of Cancer 45, 69–76. Giovannucci, E., Rimm, E. B., Ascherio, A., Stampfer, M. J., Colditz, G. A. and Willett, W. C. (1995) Alcohol, low-methionine – low-folate diets, and risk of colon cancer in men. Journal of the National Cancer Institute 87, 265–73. Weinstein, S. J., Ziegler, R. G., Frongillo, E. A., Jr., Colman, N., Sauberlich, H. E., Brinton, L. A., Hamman, R. F., Levine, R. S., Mallin, K., Stolley, P. D. and Bisogni, C. A. (2001) Low serum and red blood cell folate are moderately, but nonsignificantly associated with increased risk of invasive cervical cancer in U. S. women. Journal of Nutrition 131, 2040–8. Alberg, A. J., Selhub, J., Shah, K. V., Viscidi, R. P., Comstock, G. W. and Helzlsouer, K. J. (2000) The risk of cervical cancer in relation to serum concentrations of folate, vitamin B12, and homocysteine. Cancer Epidemiology, Biomarkers & Prevention 9, 761–4. Zhang, Z. F., Kurtz, R. C., Yu, G. P., Sun, M., Gargon, N., Karpeh, M., Jr., Fein, J. S. and Harlap, S. (1997) Adenocarcinomas of the esophagus and gastric cardia: the role of diet. Nutrition & Cancer 27, 298–309. Mayne, S. T., Risch, H. A., Dubrow, R., Chow, W. H., Gammon, M. D., Vaughan, T. L., Farrow, D. C., Schoenberg, J. B., Stanford, J. L., Ahsan, H., West, A. B., Rotterdam, H., Blot, W. J. and Fraumeni, J. F., Jr. (2001) Nutrient intake and risk of subtypes of esophageal and gastric cancer. Cancer Epidemiology, Biomarkers & Prevention 10, 1055–62.

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27. Shrubsole, M. J., Jin, F., Dai, Q., Shu, X. O., Potter, J. D., Hebert, J. R., Gao, Y. T. and Zheng, W. (2001) Dietary folate intake and breast cancer risk: results from the Shanghai Breast Cancer Study. Cancer Research 61, 7136–41. 28. Sellers, T. A., Kushi, L. H., Cerhan, J. R., Vierkant, R. A., Gapstur, S. M., Vachon, C. M., Olson, J. E., Therneau, T. M. and Folsom, A. R. (2001) Dietary folate intake, alcohol, and risk of breast cancer in a prospective study of postmenopausal women. Epidemiology 12, 420–8. 29. Rohan, T. E., Jain, M. G., Howe, G. R. and Miller, A. B. (2000) Dietary folate consumption and breast cancer risk. Journal of the National Cancer Institute. 92, 266–9. 30. Ulrich, C. M., Robien, K. and Sparks, R. (2002) Pharmacogenetics and folate metabolism – a promising direction. Pharmacogenomics 3, 299–313. 31. Stolzenberg-Solomon, R. Z., Albanes, D., Nieto, F. J., Hartman, T. J., Tangrea, J. A., Rautalahti, M., Sehlub, J., Virtamo, J. and Taylor, P. R. (1999) Pancreatic cancer risk and nutrition-related methyl-group availability indicators in male smokers. Journal of the National Cancer Institute 91, 535–41. 32. Stolzenberg-Solomon, R. Z., Pietinen, P., Barrett, M. J., Taylor, P. R., Virtamo, J. and Albanes, D. (2001) Dietary and other methyl-group availability factors and pancreatic cancer risk in a cohort of male smokers. American Journal of Epidemiology 153, 680–7. 33. Frosst, P., Blom, H. J., Milos, R., Goyette, P., Sheppard, C. A., Matthews, R. G., Boers, G. J., den Heijer, M., Kluijtmans, L. A., van den Heuvel, L. P. and Rozen, R. (1995) A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nature Genetics 10, 111–3. 34. Weisberg, I., Tran, P., Christensen, B., Sibani, S. and Rozen, R. (1998) A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Molecular Genetics & Metabolism 64, 169–72. 35. Harmon, D. L., Woodside, J. V., Yarnell, J. W., McMaster, D., Young, I. S., McCrum, E. E., Gey, K. F., Whitehead, A. S. and Evans, A. E. (1996) The common lthermolabilel variant of methylene tetrahydrofolate reductase is a major determinant of mild hyperhomocysteinaemia. Qjm 89, 571–7. 36. Bagley, P. J. and Selhub, J. (1998) A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells. Proceedings of the National Academy of Sciences of the United States of America 95, 13217–20. 37. Stern, L. L., Mason, J. B., Selhub, J. and Choi, S. W. (2000) Genomic DNA hypomethylation, a characteristic of most cancers, is present in peripheral leukocytes of individuals who are homozygous for the C677T polymorphism in the methylenetetrahydrofolate reductase gene. Cancer Epidemiology, Biomarkers & Prevention 9, 849–53. 38. Ulrich, C. M., Kampman, E., Bigler, J., Schwartz, S. M., Chen, C., Bostick, R., Fosdick, L., Beresford, S. A., Yasui, Y. and Potter, J. D. (1999) Colorectal adenomas and the C677T MTHFR polymorphism: evidence for gene-environment interaction? Cancer Epidemiology, Biomarkers & Prevention 8, 659–68. 39. Levine, A. J., Siegmund, K. D., Ervin, C. M., Diep, A., Lee, E. R., Frankl, H. D. and Haile, R. W. (2000) The methylenetetrahydrofolate reductase 677CpT polymorphism and distal colorectal adenoma risk. Cancer Epidemiology, Biomarkers & Prevention 9, 657–63. 40. Ma, J., Stampfer, M. J., Giovannucci, E., Artigas, C., Hunter, D. J., Fuchs, C., Willett, W. C., Selhub, J., Hennekens, C. H. and Rozen, R. (1997) Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer. Cancer Research 57, 1098–102.

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References 41. Chen, J., Giovannucci, E., Hankinson, S. E., Ma, J., Willett, W. C., Spiegelman, D., Kelsey, K. T. and Hunter, D. J. (1998) A prospective study of methylenetetrahydrofolate reductase and methionine synthase gene polymorphisms, and risk of colorectal adenoma. Carcinogenesis 19, 2129–32. 42. Horie, N., Aiba, H., Oguro, K., Hojo, H. and Takeishi, K. (1995) Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5l-terminal regulatory region of the human gene for thymidylate synthase. Cell Structure and Function 20, 191–7. 43. Ulrich, C., Bigler, J., Velicer, C., Greene, E., Farin, F. and Potter, J. (2000) Searching expressed sequence tag databases: discovery and confirmation of a common polymorphism in the thymidylate synthase gene. Cancer Epidemiology, Biomarkers & Prevention 9, 1381–5. 44. Kawakami, K., Omura, K., Kanehira, E. and Watanabe, Y. (1999) Polymorphic tandem repeats in the thymidylate synthase gene is associated with its protein expression in human gastrointestinal cancers. Anticancer Research 19, 3249–52. 45. Ulrich, C. M., Bigler, J., Bostick, R., Fosdick, L. and Potter, J. D. (2002) Thymidylate synthase promoter polymorphism, interaction with folate intake, and risk of colorectal adenomas. Cancer Research 62, 3361–4. 46. Honein, M. A., Paulozzi, L. J., Mathews, T. J., Erickson, J. D. and Wong, L. Y. (2001) Impact of folic acid fortification of the US food supply on the occurrence of neural tube defects. Jama 285, 2981–6. 47. Song, J., Medline, A., Mason, J. B., Gallinger, S. and Kim, Y. I. (2000) Effects of dietary folate on intestinal tumorigenesis in the apcMin mouse. Cancer Research 60, 5434–40. 48. Song, J., Sohn, K. J., Medline, A., Ash, C., Gallinger, S. and Kim, Y. I. (2000) Chemopreventive effects of dietary folate on intestinal polyps in Apc /-Msh2-/-mice. Cancer Research 60, 3191–9. 49. Patterson, R. E., White, E., Kristal, A. R., Neuhouser, M. L. and Potter, J. D. (1997) Vitamin supplements and cancer risk: the epidemiologic evidence. Cancer Causes & Control 8, 786–802. 50. Omenn, G. S., Goodman, G. E., Thornquist, M. D., Balmes, J., Cullen, M. R., Glass, A., Keogh, J. P., Meyskens, F. L., Jr., Valanis, B., Williams, J. H., Jr., Barnhart, S., Cherniack, M. G., Brodkin, C. A. and Hammar, S. (1996) Risk factors for lung cancer and for intervention effects in CARET, the b-Carotene and Retinol Efficacy Trial. Journal of the National Cancer Institute 88, 1550–9. 51. The a-Tocopherol, b-Carotene Cancer Prevention Study Group (1994) The effect of vitamin E and b-carotene on the incidence of lung cancer and other cancers in male smokers. New England Journal of Medicine 330, 1029–35. 52. Neuhouser, M. L., Patterson, R. E., Thornquist, M. D., Omenn, G. S., King, I. B., Goodman, G. E. Fruits and vegetables are associated with lower lung cancer risk only in the placebo arm of the beta-carotene and retinol efficacy trial (CARET). [Clinical Trial. Journal Article. Multicenter Study. Randomized Controlled Trial] Cancer Epidemiology, Biomarkers & Prevention. 12 (4): 350–8, 2003 Apr. See http://www.fhcrc.org/library/journals.html for holdings. UI: 12692110. 53. Khuri, F. R., Lippman, S. M., Spitz, M. R., Lotan, R. and Hong, W. K. (1997) Molecular epidemiology and retinoid chemoprevention of head and neck cancer. Journal of the National Cancer Institute 89, 199–211. 54. Patterson, R. E., Eaton, D. L. and Potter, J. D. (1999) The genetic revolution: change and challenge for the dietetics profession. Journal of the American Dietetic Association 99, 1412–20.

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2 Regulatory Requirements for Functionality and Safety: A European View Burckhard Viell*

2.1 Introduction In Europe, an increasing number of new foods appeared on the market, like energy drinks, probiotic products, soft drinks with special vitamin combinations (e. g. ACE) or bread with omega-3-fatty acids – just to give a few examples. The striking feature of these products is recognizable in two aspects. They contain unusual ingredients like carnitine, taurine or probiotic bacteria, and/or they are claimed for effects, which are not yet ascribed to foods, like improvement of health, support of the immune system or balance of the gut flora. Are these foods messengers of a new trend in food development [1]? The protagonists of the so-called functional foods suggest, that we stand today at the threshold of a new frontier in nutritional sciences. The concepts of food are changing from a past emphasis on survival, hunger satisfaction, absence of adverse effect on health, and health maintenance to an emphasis on the promising use of foods to promote better health and well-being, thus helping to reduce the risk of chronic illnesses such as cardiovascular disease, some cancers and obesity. This statement can be read in the foreword of a supplement to the British Journal of Nutrition in 1999, which is completely dedicated to Functional Food Science in Europe [2]. The basic idea about functional foods is, that it should be able to modify foods in such a way that they become healthier, and the main strategy to modify foods is to add health-promising substances [3]. There is much evidence that many foods like cereals, fruits and vegetables contain such health promising substances. They can be extracted or isolated. Foods enriched with these substances, could – theoretically – gain a special physiological function, a function beyond normal nutrition. However, such an idea is not new. It is customary for a long time to add substances like vitamins and minerals to foods to improve their nutritional value. It is difficult to understand how additional amounts of vitamins and minerals or of other new substances like fibres, fatty acids or for example soy isoflavones could bring additional health effects. The crucial point is to recognize the strict scientific approach, which is followed by the functionalfood concept for the devolopment of health-promoting products.

* Federal Institute for Health Protection of Consumers, Berlin

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The idea of proven additional functionality of functional foods was strongly promoted during the late 80ths of the last century in Japan and resulted in the development of several hundred products. Meanwhile such products are regulated in Japan as “foods of special health use” (FOSHU). They are defined as usual foods to which natural substances or ingredients were added and which must be licensed (the ingredient and the claim) by a special approval process. On the market FOSHU’s can be distinguished from ordinary foods by a special logo. Many products like the FOSHU’s in Japan can also be found in Europe, and functional foods are discussed for several years, see e. g. [4]. It is allowed in Europe to add calcium, fibre or other substances to a food and to make a claim related to that fortification. Likewise, it is not forbidden to announce a product for a health-promoting effect, if a disease-related effect is not suggested. Therefore, the question about all of these new products is still aimed at the underlying concept and at the exceptional features, by which they are distinguished from ordinary foods. Are these products functional foods and what does this exactly mean? Did we already cross the threshold of a new frontier to new foods or are consumers only carried into a glittering area of food marketing?

2.2 A Problem in the Discussion about Functional Foods is the Definition There are many different definitions of functional foods, depending on the different interests of the stakeholders [5]. Industry typically defines functional foods as fortified processed foods which are presented as healthy and discloses its interest as primarily aligned with marketing chances. Functional foods are interpreted simply as a new marketing trend, in which it depends only on the subjective decision of the producer, how to present a product. If it is offered under the headline “health” in the supermarket or, if it is announced as a “lifestyle product”, then such foods are believed to be functional foods. Following more scientific orientated protagonists, functional foods offer more than that. They are not only claimed for health promotion or effects beyond nutrition; they offer scientifically based effects [3]. According to that a functional food is a food (not a supplement) modified by the addition of a functional ingredient for which a health claim is proven – to bring it in a very short form. Such a science-oriented definition is the basis of the ongoing discussion in Europe [2, 6, 7], but it should be emphasized that functional foods are not yet regarded in Europe as a special group of foods. Unlike the situation in Japan, no legal definition exists. Instead, the discussion is focused on the problem, how to verify the concept of functional foods and how the scientific 51

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requirements can be fulfilled for such products [8]. A convincing definition could be found eventually, but it is more probable that the discussion in Europe remains restricted to the problem of health claims. In fact, the health-claim problem is the only real new aspect. From a regulatory point of view it is less important to define functional foods and more important to identify those areas which touch issues of consumer protection. Based on a very general and tentative definition of functional foods (see Fig. 2.1), three areas can be identified. The first is related to safety. Therefore, it must be considered, how the safety of an added functional ingredient can be assured. The second question is related to the claim and consists in fact of two separate questions. Since functionality of one or several ingredient(s) is suggested in functional foods and since functionality is by definition more than simply delivering nutrient substances, a claim on a functional food exceeds naturally that what is normally offered by foods. Existing law determines that all claims on foods are forbidden, which are related to the prevention treatment or cure of a disease. Therefore, in most cases it appears questionable whether a claim on a functional food is compatible with existing rules. It is also important to make sure that a claim on a food label is true or in scientific wording, that the claimed effect is proven. In essence, the scientific proof is the issue with the key problem of the functional food concept.

Figure 2.1. Functional foods – definition and regulatory questions.

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2.3 Safety Considerations about Functional Foods 2.3.1

The Principle of Positive Lists

In Germany, there are many products on the market, to which different vitamins and minerals are added in variable amounts. Similarly, many other substances can be found in foods, like the examples given in Tab. 2.1 – a list which is by far not complete. All these substances are added for nutritional or physiological purposes, despite the fact that such a purpose is seldom based on a proof. It is obvious that many producers wish to upgrade their products by the addition of promising substances, probably in order to get a better chance for marketing. The addition of substances to foods raises questions about safety. Accordingly, risk assessment of chemicals in food and diet was thoroughly reviewed in the FOSIE project [9]. Many substances are added in varying numbers and dosages, which bears the possibility of overdosing, at least when different products are enriched by the same substance and the substance is ingested in a “cumulative way”. Therefore, there is an urgent need to clarify, which substances can be permitted, which sources of nutrients or substances can be used, and to which total amount such substances can be added to foods. Regulatory bodies also have to look on food categories. It is meaningful for the safety evaluation, whether a substance is added to ordinary foods (like for example spread or yoghurt) or whether it is added to products of new food categories (like bars or sports drinks). It has also to be considered, whether a

Table 2.1. “Functional ingredients”, which can be found in products marketed in Germany. The figures are based on 241 fortified foods and 217 dietary supplements, which were found on the market (9 supermarkets in Berlin and Cologne (during 1997 to 2002)) in a non-representative explorative survey.

Vitamins Minerals Trace elements Lecithine Carnitine Taurine Tyrosine Choline Coenzyme Q10 Omega-3-FA Inuline Flavonoids

“usual” foods (n = 241) [ %]

food supplements (n = 217) [ %]

46 17.8 3.3 2.9 4.1 7.3 0.4 1.7 0.8 2.5 4.1 0.0

46.1 17.5 21.6 7.0 1.7 1.3 0.5 0.9 4.6 2.3 2.3 1.8

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nutrient or a substance with physiological function is presented in a pharmaceutical form or whether such substances are contained in ordinary foods. Publications about the safety of functional foods are available [10, 11]. Several regulatory projects can be mentioned with regard to safety of functional substances. Since 1997, the Novel Food Regulation exists. It prescribes an evaluation procedure for any new substance, which must be regarded as novel under this regulation [12]. There are specific evaluation procedures established under this regulation, which guarantee a high standard of safety for approved novel substances. Several substances are already permitted by this procedure, for example phytosterole in margarine [13]. With respect to “conventional” substances (vitamins, minerals etc.) it must be stated that most of them are tolerated or even explicitly permitted (sometimes as an additive, for example b-carotene), but it is questionable, whether they can be presumed as safe in higher doses. Its intended use has changed completely and therefore it would be commendable to have them evaluated again. In its White Paper from 1999 the EU-Commission has inter alia announced proposals for two directives: one on dietary supplements and the other on fortified foods [14]. The primary task of these initiatives is to harmonize the European market, which is characterized by many conflicts, especially in the area of dietary supplements and fortified foods, because such products are differently regulated in several EU-countries. The common denominator for harmonisation is safety, because nobody would accept a loss of safety, just in order to remove trade barriers between the EU-countries. Therefore, safety can be regarded as the cornerstone for an EU-based regulation of these foods. It will now challenge the intellectual resources in Europe to clarify how safety of “conventional substances” should be defined – and assured. As a first step the EU-commission has proposed a directive on dietary supplements. Meanwhile, a Common Position is adopted by the Council – and the European Parliament. The directive will be set in force probably in the middle of 2002 and the directive on fortified foods will follow in the same year. Both directives can be regarded as a starting point for the regulation of isolated nutrients. The supplement directive is for the time being only aimed at vitamins and minerals, which are covered by a positive list in annex 1 and 2 of the directive, but the scope of the directive is broader. Any substance with a nutritional or physiological effect is going to be covered. This is the result of a very laborious agreement process in Europe during the last two years. It is planned to include e. g. fatty acids and amino acids on the positive list in the near future, and thereafter all other substances which are used in dietary supplements. The supplement directive is a kind of pacemaker for the safety evaluation of nutrients and other physiological substances in foods, because in the long run any substance in Europe must be included in the positive list, if the substance shall be marketed in dietary supplements. The substance 54

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Table 2.2. Concept of a positive list of nutrients and other substances with physiological functions verified in three regulatory projects. 1.

Directive about PARNUT’s substances*) Categories: 1. Vitamins 2. Minerals 3. Amino acids 4. Carnitine/Taurine 5. Nucleotides 6 Choline/Inositole e.g for vitamin B1 only thiamin hydrochloride and thiamin mononitrate are allowed as the only sources, or for choline the chloride, citrate and bitartrate salt can be used as sources. *) 2001/15/EG of the EU-Commission of 15. February 2001

2.

Directive on dietary supplements**) Categories: 1.Vitamins 2. Mineral 3., 4. etc. to be extended in the future **) common position accepted, to be set in force in the middle of 2002

3.

Directive on fortificated foods***) Categories: 1.Vitamins 2. Minerals ***) expected in 2002

3., 4. etc. to be extended in the future

will only be included in the positive list after an adequate evaluation. A directive constructed in a similar way will be set in force also for fortified foods (see Tab. 2.2).

2.3.2

The SCF Evaluation

Another safety problem is the question, how much of a substance can be permitted in a dietary supplement or in a fortified food. Clear figures are needed to accept a given dose in various foods for a life-long ingestion. Risk managers are obliged to know, what dose is without harm, a question similar to the situation with additives. The basis for future evaluations of all substances of concern added to a food is laid down by a project in which the SCF was charged to evaluate the risk of 27 vitamins and minerals. According to the working plan [15] the European Commission has requested the SCF to review the upper levels of daily intakes of individual vitamins and minerals that are unlikely to pose a risk of adverse health effects. The aim is to provide the basis for the establishment of safety factors, where necessary, for individual vitamins and minerals, which would ensure the safety of fortified foods and food supplements containing these nutrients. In this project, the existing data in the literature are reviewed and brought into a consistent picture, which can be considered under safety aspects. A tolerable upper intake level (UL) for each of the vitamins and minerals is derived, see the example for manganese [16]. In fact, the proce55

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dure, which is followed by the SCF, is a retrospective literature review with all well-known disadvantages. However, such a review is necessary in order to make up the balance of the existing evidence, to compare it with other scientific reviews and – most importantly – to gain a consistent safety evaluation procedure, which can be applied for all nutrients and other substances with physiological effects in the future. The SCF has already evaluated several vitamins and minerals and published its opinion in the internet – for several substances with a relatively restrictive statement e. g. for manganese [16] or for b-carotene [17]. A similar project was begun by the Food and Nutrition Board of the Institute of Medicine in the United States, see for example [18]. This expert group has already finished its task (see Tab. 2.3), and the opportunity is given to compare these two independent scientific performances. Basically, the work of these two expert groups is an indication of a world-wide consensus about the need for safety evaluations about all nutrients and other substances, which are added to foods, even for the widely applicated vitamins and minerals.

Table 2.3. Projects to derive the upper levels of safe intake (UL) for vitamins and minerals. Nutrient

FNB

Vitamin A Vitamin D Vitamin K Vitamin E Thiamine Riboflavin Niacin Vitamin B6 Vitamin B12 Folate Biotin Panthotenic Acid Vitamin C Calcium Magnesium Phosphorus Iron Zinc Selenium Fluoride Choline Manganese

SCF

FNB: Food and Nutrition Board of the Institute of Medicine (USA), SCF: Scientific Committee on Food of the EU Commission. Indicated are several of the micronutrients for which an UL is established.

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Regulatory requirements exist also with respect to the clarification of the question, which sources of nutrients can be permitted. Many substances are available as different compounds, and these may pose special problems. Here again a project of the Scientific Committee on Food (SCF) is a starting point for a consistent safety evaluation procedure in Europe. The SCF has evaluated all nutrients and nutrient sources, which could be regarded suitable for the addition to dietetic foodstuffs in order to set harmonized rules for the existing dietetic directives. Based on that opinion [19] the EU-Commission has set in force a directive about all substances, which are permitted for the addition to dietetic foods on the first of April 2002 [20]. Six groups of substances are compiled, in which all sources of the proper nutrients are listed. Only the listed sources are permitted. For example, choline is permitted in the form of three salts, thiamine as thiamine hydrochloride or monocitrate. For both of these substances other sources are not allowed. The question by which procedure this list could be enlarged is decisive for the future development of an adequate evaluation procedure in Europe. In the political agreement about the directive on supplements, the European Parliament proposed to include more nutrients like boron, nickel, silicon or vanadium. This proposal was accepted under the condition, that a dossier is provided to the SCF for each of these substances and that the SCF is in favour of the addition of these nutrients to foods. The SCF has published a detailed list of information in the internet, which is required when a dossier is submitted [21]. According to the SCF, the aim of these guidelines is to outline a framework of general principles for evaluation of the adverse effects of micronutrients in humans and for establishing upper levels of intake of micronutrients, which are unlikely to result in adverse effects in the general

Figure 2.2. Scheme for safety evaluations of nutrients or other substances with physiological functions by the SCF as it is outlined by different regulatory projects.

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population. The requirements are very similar to those of the novel food procedure. From a regulatory point of view it can be seen that the initiatives for the supplement directive and that for fortified foods make the SCF evaluation to a kind of safety-bottleneck in Europe for all substances, which are intended to be added to foods, see the scheme in Fig. 2.2.

2.4 Functionality and Health Claims 2.4.1

The Definition of Claims

Under a regulatory point of view functionality bears not one, but even two problems, which should be carefully distinguished. One is the question of truthfulness of the claimed functionality on a food label and the other the question of claim acceptability. According to article 2 of the directive 2000/13/EG ... the labelling of a food must not attribute the property of preventing, treating or curing a human disease, or refer to such properties. Therefore, a functionality related claim should and will be checked, whether it is in conformity to existing rules. Considering the acceptability of a functionality related claim, it must be cleared, whether health or disease is meant. The problem is even more complex, when it is noticed that claims on food labels are mainly discussed under the catchword “health claims”, but in fact, enhanced function claims and disease risk reduction claims are meant. In earlier days it was very clear and rather simple to define the borderline between allowed and forbidden claims and to separate the scope and limits of food and drug regulations. According to an old WHO definition, health was regarded as an absence of disease. A healthy person didn’t need any special care for his health, and no special health foods at all. A claim related to health was self-evident a hidden drug claim, indicating to a disease. Today, our understanding of health has changed. Health – in agreement with a change in the WHO definition – is understood more as a continuum from “very bad health” to “optimal health” [22]. Now we are convinced that health can be influenced by many measures, especially by nutrition, and therefore foods are also believed to influence health and well-being. The regulatory borderline defined by the food and drug laws and supported by the old definition of “health” doesn’t convince any more. This has led in practice to the tendency to accept more and more health related claims, for example so-called nutrient-function claims and in consequence the old interpretation of the claim-borderline between foods and drugs was steadily shifted from position 1 to 2 during the last years (see Fig. 2.3).

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In Europe, it is considered today to further shift the borderline to the right side, perhaps to position 3 in Fig. 2.3. In principle, it is discussed, whether disease-related claims could be allowed and if so, what kind of claims. It must be emphasized that this is primarily a question to the society, which has to decide, if such claims could be accepted on food labels. Completely different from that is the question, whether such claims are justified from a scientific point of view and how rigid the proof has to be brought in order to support such claims (see row B in Fig. 2.3). Similar to the definition of functional foods, it has to be defined what is meant by claims, if the discussion about the requirements for the claims shall be pushed a little step further. The most accepted definition is the classification of the Codex Alimentarius, in which different groups of claims are distinguished. They are shown in Fig. 2.3. On the left side of a “claim scale” (row A in Fig. 2.3) nutrient claims are indicated (for example, ...rich in protein or ...contains calcium), followed by function claims, which equals to nutrient function claims (for example, ... calcium is an important factor in bone formation), enhanced function claims, which indicate specific functional (health) benefits, (for example ...promotes a healthy digestion or ...stimulates immune function), and disease risk reduction claims (for example ...may help to lower blood cholesterol as a risk factor).

Figure 2.3. Different types of claims on food labels (row A) and the corresponding underlying scientific evidence (row B).

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The classification enables a closer look at the scientific requirements, which have to be fulfilled in order to support the claims. It makes a difference in the type and amount of evidence, whether it is necessary to proof a nutrient claim or a disease risk reduction claim. For the first type a well-performed, validated laboratory analysis of representative quantities of a food may be sufficient. For the latter several clinical studies may be required. At the EU level, the EU Commission has provided a discussion paper about nutrient and function claims based on the Codex Alimentarius and has invited comments to gain an impression about the different positions in Europe [23]. The discussion in Europe is probably going to shift the regulatory borderline from position 2 to 3 (in Fig. 2.3), possibly by distinguishing the “reduction of a disease risk” from “prevention”. Again, it must be emphasized that the discussion about claims, or more precisely about the acceptability of health claims, must be clearly separated from the discussion about the evidence (row B in Fig. 2.3), which can be regarded as sufficient for a functionality-related claim or claim group in row A [24].

2.4.2

Discussion Platforms for Functionality

In 1996, a project about Functional Food Science in Europe (FUFOSE) was started with a first plenary meeting in Nice. Based on the results of this meeting, six areas in human physiology were identified to be reviewed by individual theme groups which critically reviewed the science base of the concept. The resulting documents were published as a State of the Art on functional foods in Europe [2]. Thereafter, a consensus document was approved by 73 experts from all over Europe. It was agreed that there is evidence to support the hypothesis, that, by modulating specific target functions in the body, diet can have beneficial physiological and psychological effects beyond the widely accepted nutritional effects. It was stated that we are progressing from a concept of “adequate nutrition” to one of “optimal nutrition”. Diet may not only help to achieve optimal health and development, but it might also play an important role in reducing the risk of disease [7]. An important point in this consensus document is the agreement that all of the statements made are hypothetical and especially functional foods are to be regarded more as a concept than a well-defined group of foods. Within the Council of Europe, a group of experts worked out a paper on the scientific requirements to justify a health claim on functional foods [6]. The basic message of this paper is dealing with a more precisely tailored definition of functional foods. It is suggested that a benefit beyond normal nutrition can only be achieved by a modified food, which is not part of the usual diet (otherwise “normal nutrition” would illogically be improved by a “usual diet”). Functional foods, which are by definition products for which a claim beyond nutrition is made, are best defined by the unequivocal 60

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demonstrability of an effect beyond nutrition, that means beyond the usual diet. A functional food is defined as “a food for which a health claim is proven”. In that definition “proven” is the key word, i. e. the scientific proof of functionality. The so-called PASSCLAIM (Process for the Assessment of Scientific Support for Claims on Foods) action is also worth to mention [25]. In this programme several groups are looking after specific requirements for claims, which have to be fulfilled in the different themes, worked out in the FUFOSE project. The project has started with four theme groups: diet-related cardiovascular disease (A), bone health and osteoporosis (B) and physical performance and fitness (C), and review of existing processes (D). The project objectives are to set up a European network involving many experts in different fields of physiological functions relating to health claims [25]. In Germany, one of the first activities to stimulate the discussion about functional foods and to support their development was started by the Senate Commission on Food Safety (SKLM) of the Deutsche Forschungsgemeinschaft (DFG) in 1999. A working group on functional foods was founded and a catalogue of criteria, which are regarded as essential for the evaluation of functional foods with regards to safety and functionality, was collected in a working paper. In the same year, a report about the situation of functional foods in Germany was published by the Technikfolgen-Abschätzungsbüro of the German Parliament [26], which was followed by a similar report about the situation in Switzerland one year later. Furthermore, there were two requests of the German Parliament to the German Government – a third request was recently dispatched to the Ministry of Consumer Protection, Nutrition and Agriculture. All three requests raised questions about functional foods and health claims. In 2000, there was a workshop arranged by the Society of German Chemists (GDCh) and the German Nutrition Society (DGE), from which recommendations were published, how to support the scientific development of functional foods [27]. Functional foods were in this status seminar expressively called the “foods of the future”.

2.4.3

Acceptance of Different Claims in the EU Countries

Six countries in Europe are developing voluntary Codes of Practice on health claims in order to follow the demand of transparency in the handling of food claims – and of course in order to support producers. The Code applied in Sweden should be mentioned here, because it was probably the first regulatory initiative for health claims on food labels – one year earlier than Japan has set its FOSHU regulation in force. In Sweden, a self-regulating programme on health claims was introduced in 1990 and revised in 1996 [28]. The programme permits claims (in Sweden they are called function-based claims), which consist out of two parts: information about eight approved diet-health relationships, listed in Tab. 2.4, followed by a second information 61

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Table 2.4. Self-regulating programme on health claims in Sweden: accepted diet-health relationships for health claims in the labelling and marketing of food products. “Health” status

Diet constituents

Obesity Blood cholesterol Blood pressure Atherosclerosis Constipation Osteoporosis Dental caries Iron deficiency

energy (fat) fat quality (less sat. FA) sodium n-3-PUFA in fish dietary fibre calcium easily fermentable carbohydrates iron

*) Revised self-regulating programme 28.08.1996/The food industry‘s rules

about the composition of the product or the content of the special nutrient – so called two-step claims. In the United Kingdom, the Joint Health Claim Initiative (JHCI) was recently created, a non-government institution. This foundation has published its Code of Practice in the internet. The scientific requirements for a claim are described in a very detailed and exemplary manner. According to that the totality of evidence and the hierarchy of evidence has to be observed, modern systematic methods have to be applied to reach all existing evidence from the literature, and good clinical studies are preferred [29]. The JHCI distinguishes generic from innovative claims. Generic claims may be applied to all foods for which special compositional conditions are outlined. Innovative claims are foreseen only for special foods. This approach is very similar to that followed in the USA, and, besides, the catalogue of evaluation criteria in the JHCI is very similar to what the SKLM Group of the German DFG has worked out for the evaluation of functional foods (published at the end of this book).

2.5 Conclusions To sum up the activities and to illustrate the European view it can be stated that Europe is struggling to find a common way to assure safety of all substances added to foods by an SCF evaluation. Furthermore, in different platforms and discussion groups it is searched for the highest scientific standard by which appropriate claims could be permitted without opening the door to misleading or fraudulent claims. Witht respect to the acceptability of health claims a rather moderate and somehow multicentric way is recognizable in Europe represented by several voluntary initiatives. It is remarkable that the accepted claims (e. g. by the JHCI) are very cautiously formulated, which demonstrates on the one side 62

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that it is intended to follow a strict scientific way. For example, there are no objections against a claim like ...people with a healthy heart tend to eat more wholegrain foods as part of a healthy lifestyle. On the other hand and in due caution and high diligence with the formulation of health claims, moderate claims like that cited above show the limits. Clearly, a scientific message on a food label must be formulated as precisely as possible. However, claims should also be “honestly correct”. Therefore, there is a need to ask consumers how such a message is understood. Presumably the message of a claim like that cited above is very simply understood as nothing else than the product is good for my heart. We should avoid a discussion about health claims, which is more a linguistic exercise with the critique of authoritative bodies in mind. Another point is credibility of the authorities and science. Set the possibility that an accepted claim must be withdrawn, because new study results speak against the accepted link between a food constituent and health. Who will decide to withdraw the claim and how could the credibility of the institutions be preserved, which did accept the claim. The situation with b-carotene [30] is a good example for a dramatic change in scientific knowledge. Possibly we will learn in the near future, that the link between fat and adipositas and/or cancer is by far not so well established as suggested now. See the accepted health claims in Sweden (Tab. 2.4) or the health claims evaluated by the FDA [31]. We have to check very carefully, whether functionality is definitely established. New situations, e. g. with b-carotene or with manganese challenge the authorities to look for restrictive measures. Science is not definitive. The truth of today is the error of tomorrow. Therefore, it is a critical question how authoritative bodies should be positioned in the upcoming framework of functional foods and health claims. In Japan a semi-authoritative approval process is prescribed by law. In the USA the FDA is committed to the truthfulness of a claim and also to the evaluation process [31]. From a strategic point of view, authoritative bodies should keep a little bit apart from the evaluation process for functionality (not for safety) until there is no doubt about the validity and effectiveness. It seems to be prudent for authoritative bodies to leave the scientific evaluations to the scientific community like it was begun in the described voluntary initiatives in Europe. The acting bodies could work in a triangle where science and industry create a main axis and authoritative bodies are collaborating and supervising from the edge of the triangle, see Fig. 2.4. In Europe, the voluntary initiatives of different countries, e. g. of Sweden and the UK (Fig. 2.4), could merge into one European Authority (perhaps the European Food Safety Authority). National institutions could take linguistic peculiarities into account and make sure that the rules are kept. Authorities should not play outside the game, rather they should act as a third player in order to lead science and industry onto a prudent way, and to make convincingly sure that functionality offers an unambiguous benefit to 63

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Figure 2.4. Possible position of authoritative bodies in the upcoming regulatory framework for functional foods (and health claims).

consumers. It is not justified to involve consumers into a scientific dispute or to strike a scientific battle on food labels. It must be avoided that today we are in favour of a link, and that tomorrow we tell consumers, that we found another link.

Summary The European view about functional foods with respect to the regulatory requirements for functionality and safety can be characterized as two strategic lines. Safety evaluations of nutrients and other substances with physiological function will increasingly be carried out by the Scientific Committee on Food. An approach, which is mainly driven by two EU-directives, one about food supplements and the other about fortified foods. Both are based on positive lists, in which all permitted substances are listed, which have to be evaluated before inclusion. Regarding functionality (and health claims), discussion platforms and voluntary initiatives in different countries of the EU can be mentioned. It is extensively explored, which scientific requirements have to be fulfilled before a health claim could be accepted.

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References 1. Viell, B. (1997) Neue Lebensmittel und gesundheitlicher Verbraucherschutz – Wünsche, Möglichkeiten und Grenzen? Verbraucher Rundschau 1–2, 51–62. 2. Bellisle, F.; Diplock, A. T.; Hornstra, G.; Koletzko, B.; Roberfroid, M.; Salminen, S.; Saris, W. H. M. (1998) Functional Food Science in Europe. British Journal of Nutrition, Supplement 1, 3. 3. Goldberg, I. (1994) Functional Foods. Chapman & Hall, New York, London. 4. Pascal, G. (1996) Functional Foods in the European Union. Nutrition Rev. 54, 29–32. 5. Viell, B. (2001) Funktionelle Lebensmittel und Nahrungsergänzungsmittel – wissenschaftliche Gesichtspunkte. Bundesgesundheitsbl.-Gesundheitsforsch.-Gesundheitsschutz 44,193–204. 6. Council of Europe’s Policy Statements Concerning Nutrition, Food Safety and Consumer Health (2001) Technical Document. Guidelines Concerning Scientific Substantiation of Health Related Claims for Functional Foods. www.coe.fr/soc-sp. 7. Diplock, A. T.; Aggett, P. J.; Ashwell, M.; Bornet, F.; Fern, E. B.; Roberfroid, M. B. (1999) Scientific Concepts of Functional Foods in Europe: Consensus Document. British Journal of Nutrition 81, Supplement 1, 1–27. 8. Roberfroid, M. B. (1999) Concepts in functional foods: a European perspective. Nutrition Today 34 (4), 162–165. 9. Barlow, S.; Dybing, E.; Edler, L.; Eisenbrand, G.; Kroes, R.; Van den Brandt, P. Guest Editors. (2002) Food Safety in Europe (FOSIE): Risk Assesssment of Chemicals in Food and Diet. Food and Chemical Toxicology 40, 137–427. 10. Curtis, G. L.; Cichoracki, J. R. (1994) Food Safety and Health Claims: The Need for Clinical Research. Food Technology May, 92–95. 11. Huggett, A. C.; Verschuren, P. M. (1996) The safety assurance of functional foods. Nutrition Reviews 54, (11/96), 132–140. 12. Verordnung (EG) Nr. 258/97 des Europäischen Parlaments und des Rates vom 27. Januar 1997 über neuartige Lebensmittel und neuartige Lebensmittelzutaten. Amtsblatt der Europäischen Gemeinschaften Nr. L 043 vom 14.02.1997, p. 0001–0007, http://europa.eu.int/eur-lex/de/lif/dat/2000/de_300D0500.html (acc. on 16.11.2000). 13. EU Kommission der Europäischen Gemeinschaften (2000) 2000/500/EG: Entscheidung der Kommission vom 24. Juli 2000 über die Genehmigung des Inverkehrbringens von “gelben Streichfetten mit Phytosterinesterzusatz“ als neuartige Lebensmittel oder neuartige Lebensmittelzutaten gemäß der Verordnung (EG) Nr. 258/ 97 des Europäischen Parlaments und des Rates (Bekanntgegeben unter Aktenzeichen K(2000) 2121) (only the English text is binding). Amtsblatt Nr. L 200 vom 08.08.2000, p. 0059–0060. 14. EU Kommission der Europäischen Gemeinschaften (2000) Weissbuch zur Lebensmittelsicherheit, Brüssel, 12. Januar KOM (1999) 719. 15. Scientific Committee on Food of the EU Commission (2001) Working plan ‘Tolerable upper intake levels for vitamins and minerals’ (updated on September 2001) http://europa.eu.int/comm/food/fs/sc/scf/out80_en.html (acc. on 16.10. 2001). 16. Scientific Committee on Food of the EU Commission (2000) Opinion of the Scientific Committee on Food on the Tolerable Upper Intake Level of Manganese (expressed on 19 October 2000) SCF/CS/NUT/UPPLEV/21; Final 28. November 2000. http://europa.eu.int/comm/food/fs/sc/scf/index_en.html. 17. Scientific Committee on Food of the EU Commission (2000) Opinion of the Scientific Committee on Foodon the safety of use of beta carotene from all dietary

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24. 25.

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29. 30. 31.

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sources (Opinion adopted by the SCF on 7 September 2000) SCF/CS/ADD/COL/ 159 Final. http://europa.eu.int/comm/food/fs/sc/scf/index_en.html. Food and Nutrition Board (FNB), Committee on Diet and Health; National Research Council, Commission on Life Sciences (1998) Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Institute of Medicine, National Acadamy Press, Washington, D. C. Scientific Committee on Food of the EU Commission (1999) Opinion on substances for nutritional purposes which have been proposed for use in the manufacture of foods for particular nutritional purposes (‘PARNUTS’) (expressed on 12.5.1999) SCF/CS/ADD/NUT/20/Final 12/05/99. http://www.europa.eu.int/comm/dg24/ health/sc/scf/index_en.htmlEU KOM Parnuts RL (acc. on 27.07.2000). EU Kommission: Richtlinie 2001/15/EG der Kommission vom 15. Februar 2001 über Stoffe, die Lebensmitteln, die für eine besondere Ernährung bestimmt sind, zu besonderen Ernährungszwecken zugefügt werden dürfen. Amtsblatt der Europäischen Gemeinschaften Nr. L 52/19 vom 22.2.2001. Scientific Committee on Food of the EU Commission (2000) Guidelines of the Scientific Committee on Food for the development of tolerable upper intake levels for vitamins and minerals (adopted on 19 October 2000). SCF/CS/NUT/UPPLEV/ 11. Final from 28 November 2000. Bengel, J.; Strittmaier, R.; Willmann, H. (1999) Was erhält Menschen gesund? Antonovsky’s Modell der Salutogenese – Diskussionsstand und Stellenwert. Bundeszentrale für gesundheitliche Aufklärung (BzgA) Forschung und Praxis der Gesundheitsförderung, Bd. 6. Köln 4. Aufl. EU Commission: SANCO/1341/2001 Discussion Paper on Nutrition Claims and Functional Claims. Prepared by Directorate General Health and Consumer Protection (SANCO D4). http://europa.eu.int/comm/dgs/health_consumer/index_en.htm (acc. on 29.05.2001). Viell, B. (1999) Lebensmittelanreicherung und “health claims” – besondere Produkte oder nur Werbung im Gesundheitstrend? Consumer Voice 3(4), 17–18. International Life Sciences Institute (2002) Process for the Assessment of Scientific Support for Claims on Foods (PASSCLAIM). A European Commission (EC) Concerted Action Organised by International Life Sciences Institute – ILSI Europe. http://europe.ilsi.org/passclaim/structure/index.html (acc. on 27.04.2002). Hüsing, B.; Menrad, K.; Menrad, M.; Scheef, G. (1999) Functional Food – Funktionelle Lebensmittel. Gutachten im Auftrg des Büros für Technikfolgen-Abschätzung beim Deutschen Bundestag. Lebensmittelchemische Gesellschaft – Fachgruppe in der GDCh – in Zusammenarbeit mit der Deutschen Gesellschaft für Ernährung (2001) Funktionelle Lebensmittel – Lebensmittel der Zukunft. Erwartungen, Wirkungen, Risiken. Band 25 der Schriftenreihe Lebensmittelchemie, Lebensmittelqualität, Behr’s Verlag, Hamburg. Sjölin, K. (2001) Zehn Jahre schwedisches Selbstregulierungsprogramm für „health claims” bei Lebensmitteln. Bundesgesundheitsbl.-Gesundheitsforsch.-Gesundheitsschutz 44, 219–226. Joint Health Claims Initiative (JHCI). http://www.jhci.co.uk (acc. on 06.04.2002). Rowe, P. M. (1996): Beta-carotene takes a collective beating. The Lancet 347, 249. Food and Drug Administration (FDA, U. S.) Guidance for Industry. Significant Scientific Agreement in the Review of Health Claims for Conventional Foods and Dietary Supplements. Center for Food Safety and Applied Nutrition. Office of Special Nutritionals, Dec. 22, 1999 http://www.cfsan.fda.gov/zdms/guidance.html lab (acc. 10.01.2000).

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Functional Foods Research and Regulation in Japan

3 Functional Foods Research and Regulation in Japan Shaw Watanabe*, Xing Gang Zhuo, and Mitsuru Kimira

Abstract In most countries in Asia and other parts of the world, cancer and other chronic diseases, such as cardiovascular disease and diabetes mellitus, are increasing. These diet-related diseases could be controlled by appropriate dietary habits and/or use of supplements. Recent studies on functional aspects of non-nutrient phytochemicals suggest important roles in cancer and other chronic disease prevention. Functional food system has started by the Japanese Government in 1990. In Japan, foods with health claim have two categories: food with nutrition claims and food for specified health uses. Other dietary supplements with health claims have also appeared. More than 600 functional non-nutrient food factors (FFFs) in vegetables and fruits are considered to be effective for health promotion and disease prevention. Our isoflavone rich tablets improved climacteric symptoms, bone metabolism and hypertension among climacteric women. We started to make a database of non-nutrient FFFs to estimate the kinds and total amount of FFF intake for providing scientific evidence for health research. So far, flavonoids, terpenoids and carotenoids, sulfur compounds and functional peptides are included in our FFF database. We can estimate the intake of various phytochemicals per capita from dietary records, and evaluate the most effective combination of FFF for human health by using population-based cohorts. The recent occurrence of bovine spongiform encephalopathy and scandals in food industries in Japan have revealed a serious lack of coordination between the agriculture and health ministries, although Food Sanitation Law and Health Improvement Law control the standardization, safety and food labeling. The ad hoc committee required changes from product-first policy to give top priority to consumer protection. The proposed Food Safety Agency would take over food safety-related operations from the Ministry of Agriculture, Forestry and Fisheries and the Ministry of Health, Labor and Welfare in the future.

* Department of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan

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3.1 Brief History of Functional Food Research in Japan The relationship between dietary habits and diseases has a long history in nutritional study [1]. Major nutrients were found in the late 19th century, vitamins and minerals were discovered in early 20th century, and effects of nonnutrient phytochemicals were found in late 20th century. These three periods could be called the 1st, 2nd and 3rd generation of nutritional science, respectively. The Japanese scientists distinguished three functional aspects as different elements in foods: nutrition, sensory satisfaction, and fortification and/or modulation of physiological systems [2]. The nutrients are nutritionally functional for human body (primary function). Flavor and aroma have function by giving sensory satisfaction (secondary function). Fortification and modulation of physiological function are called tertiary function. Foods that put emphasis on the tertiary functional aspect are accepted as “functional foods” in Japan. The term “functional food” was proposed in 1984 and the idea of functional foods attracted many food industries by adding new values to their products. The study group, “Systematic analysis and development of food function”, was organized by Masao Fujimaki, under the support by the Ministry of Education, Science and Culture in 1984. Hideo Chiba succeeded the study group under the title, “Analysis of food function on the modulation of physiological systems”, from 1988 to 1990. Soichi Arai expanded the study group under the title, “Analysis of functional foods and their derivatives”, from 1992 to 1994. The Japanese government standardized the functional food system in 1990. Five categories of foods were designated special dietary uses (FOSDU); (i) foods for the ill, (ii) milk powder for pregnant and lactating

Figure 3.1. Food category by the Ministry of Health, Labour and Welfare.

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women, (iii) formulated milk powder for infants, (iv) foods for the aged, and (v) foods for specified health uses (FOSHU). Foods for the ill consisted of two categories: single foods and packed meals. Low-calorie foods, low-protein foods, no/low-protein and high-calorie foods, high-protein foods, allergenremoved foods, and lactose-free foods belonged to the single foods. Packed meals consisted of four types; sodium-reduced meals, meals for diabetes mellitus, meals for liver diseases, and meals for adult obesity. Foods for the aged had two types; foods for people with difficulty in masticating and foods for people with difficulty in both masticating and swallowing. In the 2000’s revision of the Health Improvement Law, the foods were divided in to two categories: food with nutrition claims and food for specified health uses (FOSHU) (Fig. 3.1). Foods with nutrient-function claims should contain one or a combination of vitamins and minerals defined by the Government.

3.2 Functional Food Development in Japan 3.2.1

Birth of FOSHU

Since the 1980’s, the health food market has expanded along with the healthoriented movement of the society in Japan. Some products, however, carried exaggerated health claims without scientific evidence, and were sold with unfair or illegal methods. The Pharmaceutical Bureau of the Ministry of Health and Welfare needed to strengthen the enforcement of the Pharmaceutical Affairs Law to control these products for consumer protection. The Environmental Health Bureau of the Ministry of Health and Welfare set up the Office of Health Policy on Newly Developed Foods in 1988, in view of the rising number of health and food issues in the field of public health. In 1989, Report on Functional Foods was summarized by the Functional Foods Round Table, which grew into the Functional Foods Study Committee in the bureau to systematize functional foods. Functional foods were defined as a group of foods for special dietary uses (FOSDU). The word “functional” was restricted by the Pharmaceutical Affairs Law, which defined anything to affect function of humans or animals as a “drug”. So, the word functional foods was substituted by the word FOSHU. FOSHU referred to all foods having positive effects on human physiological functions. In 1991, FOSHU became active by the Nutrition Improvement Law [3]. Individual FOSHU products required permission from the Ministry of Health and Welfare to label health claims on their products. Functional food is a concept that appeared from food science, while FOSHU focuses more on the health and nutritional aspects of the human body. This was a paradigm change in nutrition in Japan. 69

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Figure 3.2. Mark of food for special health use (FOSHU).

3.2.2

Labeling of FOSHU

FOSHU has been clinically proven to be suitable for promotion of health and permitted to label on the products. The label should describe product name, name and address of manufacturer, source of nutrients and contained permitted/approved health claim, amount of nutrients, calories, and food ingredients, use-bye date or date of minimum durability, a statement to the effect that it is a food for specified health use, contents, recommended daily intake, notices on consumption, and proportion of the target nutrient to the corresponding dietary allowance, notice about how to consume, prepare and preserve, and specified permission/approval mark (Fig. 3.2). Health claims, however, are only limited to health promotion and reduction of disease risk. In the United States, health claims were approved with the enforcement of the Nutrition Labeling and Education Act in 1990. The scientifically proven relationship between ingredients in the foods and chronic non-communicable diseases can be stated. In Japan, FOSHU cannot print claims stating effects in prevention of diseases or for treatment. So, most claims appear to be indirect and give a very vague impression. New pharmacological effects of phytochemicals have opened a wide business chance in many countries. An increasing number of dietary supplements requires more comprehensive labeling on foods and education for consumers in Japan.

3.2.3

Standards for Foods with Nutrient-Function Claims

Vitamins, minerals, herbs, proteins, fatty acids and dietary fibers are regarded as components of foods with nutrient-function claims. Twelve kinds of vitamins (vitamin A, B1, B2, B6, B12, niacin, vitamin C, D, E, K, folic acid, panthothenic acid and biotin) and two kinds of minerals (Ca, Fe) are nutrients that could be used for dietary supplements. The upper and lower levels in daily consumption were determined [4]. Products should indi70

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cate energy, protein, fat, carbohydrates (or sugars and dietary fibers), sodium, and others. The amount of each nutrient may be indicated per 100 g or 100 ml, or per serving, per package, or per other appropriate unit. When the amount of a nutrient is indicated as being a certain level, the actual level should remain within the following range: vitamin A, D, E, Ca and Fe, –20 % to 50 %; other water soluble vitamins, –20 % to 80 %; other nutrients, –20 % to 20 %. A food bearing the claim, “high” or “rich”, must include the given level or more of the nutrient. Claim using the term “non”, “free” or “zero” could be applied when the energy is under 5 kcal/ 100 g, fat under 0.5 g/100 g, cholesterol 5 mg/100 g, sugar 0.5 g/100 g or sodium 5 mg/100 g. Claims, “low” or “reduced” for major nutrients are allowed when energy is less than 40 kcal/100 g, fat less than 3 g/100 g, cholesterol 20 mg/100 g, sugars 5 g/100 g or sodium 120 mg/100 g. This requirement is not applied to a food for which single serving size is 15 g or less.

3.3 Dietary Supplements 3.3.1

Dietary Supplements in FOSHU

As described in the previous chapter, dietary supplements can make nutrient claims, if the nominated vitamins and minerals were included. When there are more health-oriented effects, they can apply to FOSHU. Herbs could be used in FOSHU, because of their various effects on physiological systems. After the revision of the Pharmaceutical Affairs Law in 1999, manufacture of tablet and small bottle-type supplements were allowed [5]. The number of such supplements is steadily increasing and it may reach 300 in 2002. Most of them are prebiotics and probiotics, and absorbable iron and calcium at beginning, and supplements to lower cholesterol and/or triacylglycerol,

a

b

Figure 3.3. (a) Target of dietary supplements in FOSHU; (b) Type of FOSHU for sales.

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lower blood glucose level, lower blood pressure, and prevent dental caries has increased after setting “Healthy Japan 21” in 2000 (Fig. 3.3a). Food type of FOSHU was mostly liquid (Fig. 3.3b).

3.3.2

Isoflavone-Rich Supplement for Intervention Study

Recently, more functional effects of phytochemicals have been disussed, so new supplements have been developed. We made an isoflavone (IF)-rich tablet from soy hypocotyl, which contains 5 times more isoflavones than that made from soy seed (Tab. 3.1). There are many epidemiological reports describing the human health effects of soy-rich food on the prevention of estrogen-related cancers and cardiovascular diseases, decrease of climacteric symptoms, and prevention of osteoporosis [6, 7]. Japanese consume a lot of soybean products that contain phytoestrogen isoflavones (IFs), such as daidzein and genistein, and IF levels in plasma and urine are much higher compared to those among Caucasians [8, 9]. Numerous laboratory studies indicate that isoflavones exhibit a large number of diverse biologic effects, both in vivo and in vitro [7, 10]. We studied the effect of soy-hypocotyl by PHIP-induced mammary carcinogenesis, and succeeded to inhibit 60 % of breast cancer in cumulative cancer incidence rate. A combination diet containing soy-hypocotyl and matured garlic powder prevented nearly 70 % of PHIP induced cancer (Fig. 3.4). Antioxidant activity and decreased amount of DNA damage were confirmed after hypocotyl containing diet intervention [12]. Various effects of IFs should work in different stages of carcinogenesis (Fig. 3.5).

Table 3.1 Contents of soy(Kogane)-isoflavone by different parts of soy and growth temperature. Temperature

Chemicals

High temperature

Low temperature

Cotyledon

daidzein genistein malonyldaidzein malonylgenistein total

0.9e0.6 1.1e0.6 3.5e0.5 5.2e1.4 10.7e3.1

10.2e1.8 15.0e1.5 69.0e10.3 100.7e5.1 194.9e15.1

Hypocotyl

daidzein glycitein genistein malonyldaidzein malonylglycitein malonylgenistein total

14.5e4.0 4.3e1.8 9.1e0.9 84.3e19.1 13.2e5.1 10.2e3.9 135.6e33.1

48.3e13.0 39.8e14.6 45.9e10.2 428.6e69.5 155.5e31.2 119.9e24.1 838.0e161.4

Contents of isoflavones are remarkably influenced by the growth condition. (Modified from Tsukamoto et al. J Agric Food Chem 43 (1995), 1184–1192)

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In human intervention studies, various types of soy proteins, soymilk and soy-rich foods have been used, so the results are often controversial [11]. Newly developed isoflavonoid rich soya-hypocotyl tea showed antioxidant effects in humans as demonstrated by lowering level of phosphatidylcholine hydroperoxides in red blood cells [13]. Urinary excretion of 8ohdG, which is a

Figure 3.4. Cumulative rate of PHIP-induced breast cancer. Hypocotyl powder feed in both initiation and post-initiation phase suppresses the cumulative incidence of PhiPinduced breast cancer in SD rats. Addition of matured garlic powder inhibit more than 70 % breast cancer.

Figure 3.5. Possible mechanism to inhibit cancer by isoflavone at initiation, promotion and progression during carcinogenesis.

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Figure 3.6. (a) Changes in biomarker of bone metabolism by IF tablets; (b) Changes in climacteric symptoms by IF tablets; (c) Changes in blood pressure by IF tablets.

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c

Figure 3.6. Continued.

biomarker of DNA damage, also decreased among tea drinkers. It was more effective among equol (a metabolite of daidzein) producers. Complaints of climacteric syndrome and osteoporosis among Japanese are not so severe compared to Western Caucasians. We carried out an intervention using IF-rich tablets in 56 perimenopausal women to study the longterm effects of IFs. Bone density at baseline showed significant correlation between the age and bone density (CC = –0.57, p I 0.01). Women with high deoxypyridinoline excretion showed significant reduction by IF tablets within a month, but plasma osteocalcin increased at 4 months (Fig. 3.6a, b, c). Longer intervention study was necessary to find low-dose effects in humans. As for the menopausal symptoms, we use the simplified menopausal index (SMI). The median value of SMI decreased from 19 to 13 by IF tablet (p I 0.05). Twenty women complained of hot flashes before the study, but the incidence decreased to only seven after one-month intervention (Wilcoxon’s p I 0.05). Cold feeling of extremities was a complaint of 26 women, but it also decreased to 15 women (x2 p I 0.05). We also found the significant improvement of hypertension, systolic BP dropped from 157e16 mmHg to 141e16 mmHg and diastolic PB from 91e5 mmHg to 85e6 mmHg, respectively. The number of hypertensive women decreased from 29 to 15 after one month IF tablet intake (Uesugi and Watanabe, in preparation). Generally, administration of IF showed decreased plasma estradiol level and a prolongation of menstruation cycle [14–16]. Does it cause any adverse 75

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Figure 3.7. In vitro effects of isoflavones by different concentration.

effects? Regulation of the menstrual cycle occurs as a result of a number of interactions between the hormones of the hypothalamo-pituitary-gonadal axis [17–24]. We found that a depressed FSH level in preovulatory phase was prolonged by IF tablet intake, resulting in a prolongation of an LH surge [11]. This suggested that isoflavones affect the hypothalamo-pituitary level through a feedback-like mechanism. Changes in androstendione, testosterone, and thyroxin, caused by IF tablet intake, would also be caused by an altered function of the hypothalamus and hypophysis. Further study is necessary along the hypothalamo-pituitary-gonadal axis. Metabolites of isoflavones, such as equol, could doubly influence the endocrine functions [25]. Upper intake levels should be determined to avoid adverse effects (Fig. 3.7). Recently, supplement of IF has become very popular in the US and Japan. Setchell et al. [26] found that IF contents in the commercial supplements were varied, and insisted on a necessity to standardize IFs contents. Further multipotent effects of phytoestrogens should be clarified before recommendation of extensive use for prevention of the diseases.

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3.4 Need for New Database of non-Nutrient Functional Phytochemicals for Epidemiology 3.4.1

Consumption of Flavonoids and Isoflavonoids by Japanese

We have studied the health effects of IFs among Japanese, but contributions of other phytochemicals can not be ruled out. Intake amount of flavonoids and isoflavonoids was reevaluated based upon the preliminary FFF-database covering 40 food items, which covered at least 80 % food consumption by Japanese [27]. Average intake of flavonoids was 4.9 mg kaempferol, 8.3 mg quercetin, 1.5 mg rutin, 0.6 mg myricetin, 0.3 mg luteolin, 0.01 mg myricitrin, 0.4 mg fisetin, and 0.3 mg eriodictyol per day per capita. Daidzein and genistein were taken 13 mg (3.2–35.6 mg) and 40 mg (4.6–52.1 mg) per day, respectively. The total amount of flavonoids was less than 20 mg from vegetables and fruits. The total isoflavone intake was more than 50 mg, 3-fold higher than of flavonoids and 10-fold higher than carotenoids. This suggests various physiological functions are enhanced by various competitive and/or collaborative interactions of FFFs in the body. Recent studies clarify that many non-nutrient food factors (FFF) could prevent various diseases [28–32]. Carotenoids, flavonoids, isoflavonoids, and other phytochemicals are representative examples. The antioxidant property of flavonoids, such as quercetin and kaempferol, is considered to decrease the risk of coronary heart disease [28]. The plasma quercetin level was significantly correlated to LDL cholesterol level [32] Interactions among these FFFs should be present inside the body, so further study of metabolites and biomarkers in relationship to the end results is necessary (Fig. 3.8).

Figure 3.8. Necessity of amount of intake, proper biomarkers and end-results for nutritional epidemiology.

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FFF-DB to Estimate Intake Amount of Phytochemicals

More than 600 food factors in vegetables, fruits and seaweeds are considered to influence the various metabolic stages in the body [35–37] (Tab. 3.2). Recently introduced tropical vegetables and fruits in Japan may have unknown good effects for health. Interaction and effects of many FFFs among each other or with macromolecules in the body could be found by the appropriate biomarkers and endpoints. End results could be obtained by a cross-sectional study (ecological study), case-control study, prospective cohort study, and intervention study in humans. The goal of nutrition recommendation has to be directed to diets that provide all the essential nutrients and non-nutrient FFF to meet individual requirements. We started to make FFF-database (FFF-DB) for consumers, researchers, health professionnals and food industries. It is a relational DB, and includes a code number of foods, name of foods, nutrient content, FFF content, basic characteristics of each FFF, effects of FFF, evaluation for health evidence and references (Fig. 3.9). Data are obtained by the measurement of our group, supported by the Ministry of Education, Science, Sport and Culture and contributors of the food industries, and from the literature [33, 34]. If we succeed in finding associations among various FFFs intake and health or disease, we can save time and cost to select an agent for the human intervention studies. This approach would be a breakthrough in the nutritional epidemiology to find health effects of chronic low dose exposure of FFFs. The intake dose of safety and toxicity could also be obtained from the study. Table 3.2. Cancer preventive food factors in vegetables and fruits. Sulfur compounds (diallyl sulfide, allyl methyl trisulfide) Sulphorophane Isothiocyanate Indol-3-carbinol Carotenoids, terpenoids (a-carotene, b-carotene, cryptoxanthin, lutein, lycopene, D-lymonen) Polyphenols Coumarins Flavonoids (quercetin, kaempherol, rutin) Isoflavones (genistein, daidzein, equol, biochanin A) Lignans (enterolacton, sesaminol, etc.) Catechins (epigalocatechin, teophillin, etc.) Saponin Diketons Curcumin Phytosterol Inositol-6-phosphate Dietary fibers Short chain fatty acids Vitamins (ascorbic acid, tocopherol, folic acids) Minerals (selenium, zinc)

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Figure 3.9. Concept of FFF-database.

3.5 Legal Aspects for Controlling Food Safety

3.5.1

Food Sanitation Law

This law is aimed at prevention of the occurrence of health hazards arising from eating and drinking, so it covers of all foods hygiene. The law is roughly divided into two parts: (i) establishment of standards for labeling (Article 11), standards for management/operation (Article 18–19), and standards for facilities (Article 20): and (ii) inspections and guidance by local and municipal governments for domestic food-related businesses. The minister of Health, Labour and Welfare established each standard, after hearing the Pharmaceutical Affairs and Food Sanitation Council. The same council also approves food additives. Food sanitation inspectors (7.799 inspectors in total) are working in health centers (594 centers in Japan). They have to visit food-related facilities (about 4.22 million facilities) to conduct on-site inspection, in order to see whether the established standards for food and facilities are met.

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Nutrition Improvement Law (Revised as Health Promotion Law Since 2002)

This law is aimed at raising public awareness for nutrition improvement, clarifying nutrition standards for the public, and maintaining and promoting public health. The Health Service Bureau has conducted the national nutrition survey since 1947 to identify the nutrition intake of the public, health status and the relationship between nutrition intake and household financial burden. Nearly 7000 people are randomly selected surveyed each year through the health center. The Department of Food Safety regulates labeling of foods as described earlier. The Health Promotion Law supports the “Healthy Japan 21”. Practice and attainable goals were planned by the committee for nine categories: (i) Nutrition and Diet, (ii) Physical Activities and Exercises, (iii) Relaxation and Mental Health (“Healthy Mind”), (iv) Tobacco, (v) Alcohol Consumption, (vi) Dental Health, (vii) Diabetes, (iix) Cardiovascular Diseases, and (ix) Cancer. Nutrition and diet have a strong relationship with life style-related diseases. In the past, enriching rice with vitamin B and iron have helped to eliminate the risks of beri-beri, pellagra and anemia, use of iodizing salt was effective for decreasing goiter, fortifying margarine with vitamin A and milk with vitamins A and D prevented rickets, and adding vitamin C to fruit juices was effective for scurvy. They are also strongly associated with quality of life. The control of body weight is one of important targets, and intake of vegetables is recommended. Three steps were planned in the “Healthy Japan 21”: (i) adequate intakes of nutrients (food); (ii) behavioral changes for the adequate intakes of nutrients; and (iii) creation of a supportive environment for such behavioral change. The “Healthy Japan 21” set the attainable goal in 2010 for each item.

3.5.3

International Relationship

Japanese consumers have been skeptical of genetically modified foods and food additives. The Phamaceutical Affairs and Food Sanitation Council evaluate genetically modified foods according to species/type. The items to be evaluated include allergy-inducing properties and the productivity of harmful substances. Products for which safety evaluation was completed were permitted for distribution. So far 7 foods, such as soybean and corn, are allowed to be imported. International trade should provide high-quality healthy food. Stepwise negotiation for the international trade and standardization of food safety has been carried out. The FAO/WHO Codex Alimentarius Commission was made in 1962, the Uruguay Round of Multilateral Trade Negotiations was obtained in 1994, and the World Trade Organization (WTO) was established in 1995 to achieve these purposes. 80

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Application of Sanitary and Phytosanitary Measures (SPS Agreement) would become more important. Codex Alimentarius plays an important role to protect human and animal health, and the phytosanitary situation. Food additives, veterinary drug and pesticide residues, and other contaminants and toxins need cautious investigation [38]. Methods of analysis and sampling are proposed by Codex Alimentarius. Hazard Analysis and Critical Control Points (HACCAP) has been introduced to many companies to reduce the risk of dietary hazard.

3.6 New Movement 3.6.1

Recent Food Scandals in Japan

Many scandals in food industries that happened in the last year have undermined consumer confidence in food labeling. Snow Brand Foods a unit of Snow Brand Milk Products Co., was rocked by a scandal when about 13 000 people in the Osaka region fell ill after drinking the companyls recycled old milk in June 2000. In January 2002, Snow Brand Foods Co. caused a national uproar when it was revealed that it mislabeled foreign beef as domestic meat to obtain government subsidies for producers hit by the mad cow disease scare. In March 2002, it was revealed that Zenno Chicken Foods had been labeling imported chicken meat as higher-priced domestic chicken. The company, a subsidiary of the National Federation of Agricultural Cooperative Associations (Zenno), was also found to have sold chickens raised with feed that contained antibiotics, as organically raised chicken. Japanls laws, systems, policies and administrative organizations related to agriculture seem to be legacies from the period of food shortages. Diet members elected from rural constituencies also promoted the producer-first policy. In 1990, when the number of BSE-infected cattle sharply increased in Britain, the British government sent a report to the Ministry of Agriculture, Forestry and Fisheries on its measures to ban the use of meat-and-bone meal, but little heed was given to the document. Warnings from the World Health Organization and the European Union were also ignored, and were not disclosed to the public in Japan. The first confirmed case of mad cow disease in Japan was found September 2001, and two more infected cows have been found. The BSE problem has revealed a serious lack of coordination between the agriculture and health ministries, which followed their own separate safety policies.

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Proposal of a New Agency for Food Safety

The ad hoc committee in the government proposed to reform the system: (i) giving top priority to consumer protection, (ii) establishing an integrated safety system reaching from “the farm to the table”, (iii) introducing risk-analysis methods, and (iv) respecting advice from scientists and other specialists. The proposed Food Safety Agency would take over food safety-related operations from the Ministry of Agriculture, Forestry and Fisheries and the Ministry of Health, Labor and Welfare. A new Agency for Food Safety, like Food Safety Agency in France, Food Standardization Agency in England and States Consumer Protection Food Agriculture Agency in Germany. The Ministry of Agriculture, Forestry and Fisheries plans to tighten inspections of fresh foods for granting Japanese Agricultural Standards certification (JAS). The new seal will be different from the current JAS seal of approval, and a government-designated third party will determine, which companies are authorized to use it. Coordination with health-claim labels is necessary. Food additives, remnant of herbicides and animal drug use, chemicals in the food and safety of containers should be covered. The new law should guarantee preventive surveillance and research, inspection and supportive system. Participation of consumers and disclosure of information are essential.

3.7 Conclusion Functional foods were developed in Japan, and widely accepted in the world. The FOSDU, including FOSHU, regulates functional foods by scientific evaluation, and is approved by the government to make health claims. Some problems remain in the regulations since health claims used for FOSHU are quite vague and sometimes difficult to understand. The problems arise because the Pharmaceutical Affairs Law divides foods and drugs. More scientific evidence is necessary to evaluate health effects on humans by dietary supplements with FFFs. Non-nutrient FFF-DB could contribute to clarify the good combination of FFFs for promoting health and/or preventing diseases by well-designed epidemiological studies. A more comprehensive approach of FFFs in nutrition can be done by this database in the future.

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Acknowledgments A part of this study was supported by the research fund from the Ministry of Education, Science Technology, Culture and Sport, and the Ministry of Health, Labour and Welfare. The authors appreciate Drs. S. Arai, Professor of Tokyo University of Agriculture and Norimasa Hosoya President of Japan Health Food & Nutrition Food Association, for their valuable suggestions and comments.

References 1. Lyons AS, Petrucelli R. Medicine. An illustrated history. Harry N. Abrams. Inc. NY, 1978. 2. Arai S, Osawa T, Ohigashi H, Yoshikawa M, Kaminogawa S, Watanabe M, Ogawa T, Okubo K, Watanabe S, Nishino H, Shinohara K, Esashi T, Hirahara T. A mainstay of functional food science in Japan-History, present status, and future outlook. Biosci Biotechnol Biochem 65 (2001), 1–13. 3. Ministry of Health, Labour and Welfare. Health Promotion Law. Ministry of Health, Labour and Welfare, Japan Aug, 2002. 4. Ministry of Health, Labour and Welfare. Dietary Reference Intake, 6th edition. Ministry of Health, Labour and Welfare, Japan, 2000. 5. Ministry of Health, Labour and Welfare. Pharmacological Affair Law. Ministry of Health, Labour and Welfare, Japan Dec, 1999. 6. Watanabe S, Koessel S. Colon cancer; approaches from molecular epidemiology. J Epidemiol 3 1993, 47–61. 7. Adlercreutz H, Foldin BR, Gorbach SL, Hockerstedt KAV, Watanabe S, Hamalainen EK, Markkanen MH, Makela TH, Hase TA, Fotsis T. Soybean phytoestrogen intake and cancer risk. J Nutr 125 (1995), 757s–770s. 8. Adlercreutz H, Honjo H, Higashi A, Fotsis T, Hamalainen E, Hasegawa T, Okada H. Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet. Am J Clin Nutr 54 (1991), 1093–1100. 9. Adlercreutz H, Markkanen H, Watanabe S. Plasma concentrations of phytoestrogens in Japanese men. Lancet 342 (1993),1209–1210. 10. Watanabe S, Uesugi S, Kikuchi Y. Isoflavones for prevention of cancer, cardiovascular diseases, gynecological problems and possible immune potentiation. BioMed Pharmacother 56 (2002), 302–312. 11. Watanabe S, Terashima K, Sato Y, Arai S, Eboshida A. Effects of isoflavone supplement on healthy women. Biofactors 12 (2000), 233–241. 12. Haba R, Watanabe S, Arai Y, Chiba H, Miura T. Suppression of lipid-hydroxyperoxide and DNA-adduct formation by isoflavone-containing soy hypocotyl tea in rats. Env Health Prev Med 7 (2002), 64–73. 13. Watanabe S, Haba R, Tarashima K, Arai Y, Miura T, Chiba D, Takamatsu M. Antioxidant activity of isoflavones in humans. BioFactors 10/12 (2000), 227–232.

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14. Lu LJ, Anderson KE, Grady JJ, Nagamani M. Effects of soya consumption for one month on steroid hormones in premenopausal women: implications for breast cancer risk reduction. Cancer Epidemiol Biomarkers Prev 5 (1996), 63–70. 15. Lu LJ, Anderson KE, Grady JJ, Kohen F, Nagamani M. Decreased ovarian hormones during a soya diet: implications for breast cancer prevention. Cancer Res 60 (2000), 4112–4121. 16. Lu LJ, Anderson KE, Grady JJ, Nagamani M. Effects of soya consumption for one month on steroid hormones in premenopausal women: implications for breast cancer risk reduction. Cancer Epidemiol Biomarkers Prev 5 (1996), 63–70. 17. Cassidy A, Bingham S, Setchell K. Biological effects of isoflavones in young women: Importance of the chemical composition of soybean products. Br J Nutr 74 (1995), 587–601. 18. Cassidy A, Bingham S Setchell KD. Biological effects of a diet of soy protein rich in isoflavones on the menstrual cycle of premenopausal women. Am J Clin Nutr 60 (1994), 333–340. 19. Duncan AM, Merz BE, Xu X, Nagel TC, Phipp WR, Kurzer MS. Soy isoflavones exert modest hormonal effects in premenopausal women. J Clin Endocrinol Metab 84 (1999), 192–197. 20. Xu X, Duncan AM, Merz BE, Kurzer MS. Effects of estrogen and phytoestrogen metabolism in premenopausal women. Cancer Epidemiol Biomarkers Prev 7 (1998), 1101–1108. 21. Zhang Y, Song TT, Cunnick JE, Murphy PA, Hendrich S. Daidzein and genistein glucuronides in vitro are weakly estrogenic and activate human natural killer cells at nutritionally relevant concentration. J Nutr 129 (1999), 399–405. 22. Kao YC, Zhou C, Sherman M, Laughton CA, Chen S. Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: A site-directed mutagenesis study. Environ Health Perspect 106 (1998), 85–92. 23. Lu W L-J, Anderson KE, Nagamani M. Reproductive endocrine effects of a soya diet in premenopausal women. 3rd International Symposium on the Role of Soy in Preventing and Treating Chronic Diseases, Washington DC, 1999, p. 22. 24. Lu LJ, Anderson KE. Sex and long-term soy diets affect the metabolism and excretion of soy isoflavones in humans. Am J Clin Nutr 68 (1998), 1500S–1504S. 25. Duncan AM, Merz-Demlow BE, Xu X, Phipps WR, Kurzer MS. Premenopausal equal excretors show plasma hormone profiles associated with lowered risk of breast cancer. Cancer Epidemiol Biomarker Prev 9 (2000), 581–586. 26. Setchell KD, Brown NM, Desai P, Zimmer-Nechemias L, Wolfe BE, Brashear WT, Kirschner AS, Cassidy A, Heubi JE. Bioavailability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. J Nutr 131 (4 Suppl) (2001), 1362S–1375S. 27. Kimira M, Arai Y, Shimoi K, Watanabe S. Japanese intake of flavonoids and isoflavonoids from foods. J Epidemiol 8 (1998), 168–175. 28. Ho C-T. Antioxidant properties of plant flavonoids. In: Food Factors for Cancer Prevention, Ohigashi H, et al. (eds.) Springer-Verlag, Heidelberg, Tokyo, 1997, pp593. 29. Hertog MGL, Feskens EJM, Hollman PCH, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease: The Zutphen Elderly Study. Lancet 342 (1993), 1007–1011. 30. Frankel EN, Kanner J, German JB, Parks E, Kinsella JE. Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. Lancet 341 (1993), 454–57. 31. Hertog MGL, Kromhout D, Aravanis C, Blackburn H, Buzina R, Fidanza F, Giampaoli S, Jansen A, Menotti A, Nedeljkovic S, Pekkarinen M, Simic BS, Toshima H,

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Feskens EJM, Hollman PCH, Katan MB. Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Arch Intern Med 155 (1995), 381–386. Y Arai, S Watanabe, M Kimira, K Shimoi, R Mochizuki, N Kinae. Dietary intake of flavonols, flavone and isoflavones in Japanese women: The quercetin intake inversely correlated with the plasma LDL-cholesterol concentration. J Nutr 10 (2000), 127–135. Dr. Dukes. http:www. Dr. Dukes/USDA, Beltcity, BA. Hertog MGL, Hollman PCH, Katan MB. Content of potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in the Netherlands. J Agric Food Chem 40 (1992), 2379–2383. Cancer Research Fund. Foods for Cancer Prevention. Cancer Research Fund, London, 1996. Xu X, Wang H-J, Murphy PA, Cook L, Hendrich S. Daidzein is a more bioavailable soymilk isoflavone than is genistein in adult women. J Nutr 124 (1994), 825–832. Frankel EN. Nutritional benefits of flavonoids. In: Food Factors for Cancer Prevention, Ohigashi H, et al. (eds.) Springer Verlag, Heidelberg, Tokyo, 1997, pp. 613-616. DeVries J (ed.). Food Safety and Toxicity. CRC Press, Boca Raton, New York, London, Tokyo, 1997.

4 Aspect on the Chinese Functional Food and their Safety Yi-Min Wei*, Quo-Quan Zhang, and Jun-Ling Shi

Abstract Traditional Chinese Medicine (TCM) originated mainly from application of plants, animals, minerals and acupuncture. Chinese medicated diet has a long history. There is an ancient legend “Agricultural god Shennong tastes a hundred grasses”. A Chinese medic Simiao Sun (541–682 after Christ in West Wei to Tang Dynasty of China) wrote two books: “Prescriptions Worth a Thousand Gold for Emergencies” and “A Supplement to Essential Prescriptions Worth a Thousand Gold for Emergencies”. He cured some patients with food materials through diets. Another Chinese medic Shizhen Li (1518–1593 in Ming Dynasty of China) developed Chinese traditional medicine and wrote the book “Dietotherapy of Materia Medica”. In this book, pharmacological properties of plants, animals and minerals were reported.

* Institute of Food Science & Technology, CAAS, P. O. Box 5109, 100094, Beijing, PR China

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Chinese medics indicated that pharmacy came from similar natural resources as food. A functional food can be defined as food that exerts a beneficial health effect beyond the recognized traditional nutritional value of such a food. The functional food has a long history and is widely popular in Chinese consumers. Chinese government has recognized the problems of functional-food administration and their safety. Law on Food Hygiene of People’s Republic of China was approved on October 30, 1995. According to the Law on Food Hygiene, Regulation on the Functional Food was approved on July 1, 1996 by the Ministry of Public Health. This paper will introduce the history, traditional applications, administration, and markets of functional food in China.

4.1 History of Functional Food in China Traditional Chinese Medicine (TCM) originated mainly from application of plants, animals, minerals and acupuncture. There is an ancient legend “Agricultural god Shennong tastes a hundred grasses”. The earliest Chinese medical monograph on materia medica “Shennong’s Herbal Classic” was published approximately between Qing and Han Dynasty of China. Chinese medic Simiao Sun (after Christmas 541–682 in West Wei to Tang Dynasty of China) wrote two books: “Prescriptions Worth a Thousand Gold for Emergencies” and “A Supplement to Essential Prescriptions Worth a Thousand Gold for Emergencies”. He cured some patients with food materials through diets. Another Chinese medic Shizhen Li (1518–1593 in Ming Dynasty of China) developed Traditional Chinese Medicine and wrote a book of “Dietotherapy of Materia Medica”. In this book, he studied pharmacological properties of plants, animals and minerals, indicated and conformed some crops, fruits and vegetables that could be used as medical material, such as barley malting, ginger, garlic etc.. Chinese believed that some plants could be used as food and medicaments at the same time, which could be used to cure some illnesses. Chinese medics indicated that pharmacy and food came from similar natural resources.

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4.2 Definition of Functional Food There is a growing interest from both consumers and food producers in so-called “functional foods”. Terms such as “functional foods” and “novel foods” are often used together and are randomly interchanged. Although functional foods and novel foods can overlap, there is a clear distinction. A functional food can be defined as food that exerts a beneficial health effect beyond the recognized traditional nutritional value of such a food, but can’t be used as medicaments to cure diseases. A novel food is a food that is produced by a new concept of technology – so far, that has not been used in food production. The beneficial health effects of a functional foods are often achieved by adding, enriching or eliminating dietary components derived from plants, animals, minerals and chemical materials. Chinese consumers have understood and accepted that functional foods are being made of plants (especially of Chinese medical plant material), animals, minerals and chemical addictives, and have one or two effects on human health.

4.3 Laws on Functional Food Food Hygiene Law regulates food hygiene, hygiene of food additives, hygiene of food containers, packaging as well as utensils and equipments used for foods, creation of food hygiene standards and food hygiene management measures, management on food hygiene, and supervision over food hygiene. The law came into effect on October 30, 1995. It includes three Articles on functional food:

Chapter II Food Hygiene Article 10 Food must not contain medicinal substances, with the exception of those materials that have traditionally served as both food and medicaments and are used as raw materials, condiments or nutrition fortifiers.

Chapter VI Food Hygiene Control Article 22 With regard to food indicated to have specific health functions, the products and its description must be submitted to the administrative department of public health under the State Council for examination and approval; its hygiene standards and the measures for control of its production and marketing shall be formulated by the administrative department of public health under the State Council. 87

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Article 23 The food indicated to have specific health functions may not be harmful for human health; the content of the product description shall be true, and the functions and ingredients of the product shall be identical with the information given in the product description and there shall be no false information. It is stipulated in the law of the People’s Republic of China on Food Hygiene that ‘The product and the introduction claiming the food having special health functions must be examined and approved by the administrative department for health of the State Council’. The administrative department for health of the State Council is the Ministry of Public Health, People’s Republic of China.

4.4 Regulations on Functional Food in China The Regulations on the Functional Food have been approved on July 1, 1996 by the Ministry of Public Health. The Ministry of Public Health examines and approves the health-food importation cautiously and strictly, aiming at ensuring the safety, viability and quality of the products and protecting the consumer’s health and related rights. This provision shows a system of examination and approval in respect to health food (including nutritional supplement) being applied in China. It is illegal to sell any product or nutritional supplement that claim to have health function without approval of the Ministry of Public Health on the territory of the People’s Republic of China. Function tests on functional food include the following 22 properties. A functional food shall contain no more than two of the following properties: 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 88

regulation of the immune system regulation of blood fat regulation of blood glycogen alleviation of aging improvement of memory improvement of sight reduction of Bp content in blood improvement of voice regulation of blood pressure improvement of sleeping promotion of secreting milk anti-mutation anti-fatigue tolerance of oxygen shortage anti-radiation reduction of figure weight promotion of growth and development

4 18) 19) 20) 21) 22)

Aspect on the Chinese Functional Food and their Safety

improvement of bone density improvement of nutritional-induced anemia protection of the chemical-induced liver damage pharynx de-phlogistication and moistening larynx improvement of gastrointestinal function

4.5 Characteristics of Chinese Medicated Diet At present, medicated diet has begun to be valued in trades of both medicine and food, and drink. Some Chinese medical institutions have developed scientific researches on medicated diets; some hospitals have established departments of dietetic therapy. Personnel of academic, industrial and commercial circles abroad have paid close attention to Chinese medicated diets-functional foods. Chinese medicated diet has begun to go abroad. Chinese medicated diet should have the following characteristics: x x x x x

Laying stress on the whole, selecting medicated diet on the basis of differential diagnosis. Suitable for both prevention and treatment, outstanding in effect. Good in taste, convenient for taking. Right amount and perseverance, moderate in eating and drinking. Correct handling of the relationship between medicine therapy and dietetic therapy.

Medicated diet differs from food and drink in common sense; when it is prepared and used, attention should be paid to the nature and flavor of edible Chinese drugs, the compatibility and incompatibility of medicated diet, selection and processing of materials, cookery and so on. The applied basic principles of medicated diet must be grasped, too.

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5 Regulatory Framework for Functionality and Safety: A North American Perspective Barbara O. Schneeman*

5.1 Introduction As a North American perspective, this chapter will focus primarily on the regulatory frameworks in the United States. The term functionality can refer to either the functional properties of a food ingredient within a food product or the biological activity of the ingredient or food that affects health or prevention of disease. In this perspective only the later aspect of functionality is considered. To discuss safety, a context of intended use of the product must be considered. Three major use categories include drugs, foods and dietary supplements. While food, drugs and dietary supplements are considered under the same regulatory act, the Food, Drug and Cosmetic Act, the Act recognizes key differences among these categories based on the intended use. A substance is considered a drug, if it is “(A) recognized in the official United States Pharmacopoeia, official Homoeopathic Pharmacopoeia of the United States, or official National Formulary, or any supplement to any of them; and (B) articles intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in man or other animals; and (C) articles (other than food) intended to affect the structure or any function of the body of man or other animals; and (D) articles intended for use as a component of any article specified in clause (A), (B), or (C).” Drugs undergo premarket evaluation resulting in a decision regarding approval, including any limitations on use of the drug if approved. Risk-benefit assessment can enter into the decision about allowing certain drugs on the market. Once approved, post-market surveillance is required. The definition primarily used for food comes from a court case and includes substances “consumed primarily for taste, aroma, or nutritive value”. According to the Dietary Supplement Health and Education Act of 1994, the label dietary supplement “means a product (other than tobacco) intended to supplement the diet that bears or contains one or more of the following dietary ingredients (A) a vitamin, (B) a mineral, (C) an herb or other botanical, (D) an amino acid, (E) a dietary substance for use by man to supplement the diet by increasing the total dietary intake, or (F) a concentrate, metabolite, constituent, extract, or combination of any ingredient described in clause (A), (B), (C), (D), or (E).” In addition, a dietary supplement is a product that “is not represented for

* Department of Nutrition, University of California, Davis CA 95616, USA

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use as a conventional food or as a sole item of a meal or the diet, and is labeled as a dietary supplement”. Unlike drugs, foods and dietary supplements have not required pre-market approval or notification to the Food and Drug Administration (FDA) to be marketed; however, FDA notification is required for dietary supplements that were not on the market prior to October 15, 1994. Likewise, new food products developed with genetic engineering must comply with FDA guidelines for biotechnology. Under the law governing food, substances categorized as additives have to undergo pre-market approval or be reviewed for status as “Generally Recognized as Safe” or GRAS. The differences in intended use impact the approach for assessing the safety and functionality of products in the market place and this chapter will not focus on products that are intended for use as drugs. In the area of functional foods, whether the product is designed for consumption as a food or as a supplement to the diet, the regulatory framework that exists in the USA appears to expect that assessment for safety and functionality will be food-like [1–3].

5.2 Legislation in the USA that Creates the Regulatory Framework for Food and Dietary Supplements Regulations to insure a safe, wholesome food supply that is not adulterated nor labeled in a misleading manner were first established in 1906 as the Federal Food and Drug Act. The law provided little in the way of enforcement authority for the government and was replaced in 1938 with the Food, Drug and Cosmetic Act (FDCA). Through the internet it is possible to get copies of the act as well as information regarding current FDA policies for implementtation of the Act (www.fda.gov or www.cfsan.fda.gov for food and dietary supplement specific information). Table 5.1 highlights the impact of several federal laws on the regulatory framework for safety and functionality of food and dietary supplements in the United States. The Federal Food, Drug and Cosmetic Act, as amended, is the current law governing regulations for food, drugs and cosmetics. Additional amendments and laws that influence the regulatory framework for foods and dietary supplements are the Food Additive Amendment of 1958, which established the need for pre-market approval of food additives that are not considered GRAS, the Proxmire Amendment of 1976, which primarily affects dietary supplements, the Nutrition Labeling and Education Act of 1990 (NLEA), which authorized the use of health claims on foods in the United States, the Dietary Supplement Health and Education Act of 1994 (DSHEA), which categorized dietary supplements as similar to foods with respect to safety, and the FDA Modernization Act of 1997, which established the use of authoristative statements for approval of health claims. Several principles emerge as the regulatory framework for safety and functionality related to health pro91

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Table 5.1. Impact of federal laws in the United States on the regulatory framework for safety and functionality. Regulation

Food safety

Functionality or health effects of food

Federal Food, Drug and Cosmetic Act

– safe, wholesome, and of fair value – creation of food standards – defines adulteration, misbranding and tolerances – requires new drug applications and placed the burden of proof on manufacturers for safety of drugs – established the need for premarket approval of food additives and gave manufacturers the burden of proof for safety – allowed certain additives to be considered Generally Recognized as Safe (GRAS) prevented certain supplements from being categorized as drugs because of potency

label cannot be misleading

Food Additive Amendment Law of 1958

Proxmire Amendment of 1976 Nutrition Labeling and Education Act of 1990

Dietary Supplement Health and Education Act of 1994

FDA Modernization Act of 1997

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– defines dietary supplements – established the manufacturer’s responsibility to ensure that its products are safe and properly labeled prior to marketing, similar to foods – FDA is responsible for proving an ingredient presents a significant or unreasonable risk of illness or injury – excluded dietary supplement ingredients from food additives – requires FDA notification for supplements not marketed in the USA prior to 15 October 1994

– authorized the use of health claims on food products based on significant scientific agreement – made nutrition labeling on processed foods mandatory – defines dietary supplements as certain products designed to supplement the diet and not intended as a conventional food or sole item of a meal – provisions for use of health claims and structure-function claims on products – claims cannot be misleading

authorizes use of authoritative statements from government agencies or the National Academy of Sciences for health claims and established a 120 day notification system

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motion and disease prevention from the concepts outlined in these laws. Food and dietary supplements are viewed as inherently safe and FDA has the burden of proof to demonstrate that either a food or a dietary supplement currently on the market is unsafe. In conjunction with this concept is the recognition that the manufacturer is responsible for insuring the safety of products on the market. However, the manufacturer is not required to submit data to FDA regarding the safety of foods or dietary supplements for products already on the market. A provision in DSHEA does require that manufacturers notify FDA prior to marketing a dietary supplement that was not on the market in the United States prior to October 15, 1994. In contrast, food additives – as defined under the regulation – must be approved for use in food either by petition or an affirmation of GRAS status (Generally Recognized As Safe). FDA has developed an in-depth reference guide, Toxicological Principles for the Safety of Food Ingredients, referred to as The Redbook 2000, which provides information on the type of data needed for approval of a food additive. This guide incorporates a risk-analysis approach to determine, if there is reasonable certainty of no harm for the item in its intended use. Under the current regulations, FDA recognizes three major types of claims for nutritional or health benefit; these include nutritional content claims, health claims or structure function claims. The nutritional content claim typically refers to the amount of a nutrient relative to a standard such as the RDA or the Daily Values or makes a comparison between products in nutritive content. Data supporting the nutrient content claim for foods must be submitted to FDA. Health claims are statements about the association between a food or food component and reduced risk for a certain disease. These associations are based on significant scientific agreement and submitted to FDA prior to their use in marketing. Under the provisions of NLEA, 7 claims, based on approved monographs were initially allowed, and this has now expanded to include about 12 claims. Because health claims are making an association with disease-risk reduction, it is important that they reflect the various factors in addition to the specific food product that can impact risk of the disease and be worded in a manner that facilitates consumer understanding of the relationship. Initially, a petition containing scientific data for review by FDA was required before the claim could be used; however, FDAMA allows pre-market submission of a claim based on an approved authoritative statement. FDA has a certain time frame to raise objections before the claim can be used on food products. (Note: Since the paper was originally prepared, FDA has allowed qualified health claims, see the CFSAN web site for information.) Structure function claims describe the association between a food, food component, dietary supplement and the structure or function of the body. These claims cannot make statements about diagnosing, treating, curing or mitigating disease. Structure function claims must be truthful and not misleading and they are not pre-approved by FDA. It is expected that structure function claims will be submitted to FDA 30 days before appearing in the market. The types of statements that 93

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have been approved for nutrient content claims or approved health claims as well as details regarding the use of these claims can be found at the web site for the FDA Center for Food Safety and Nutrition (www.cfsan.fda.gov). A major focus in the development of functional foods is their use in health promotion and disease prevention, and the laws governing the use of claims regarding nutritional benefit create the regulatory framework for conveying this information to the consumer through the food or dietary supplement label. The regulations regarding safety and functionality make it clear that the intended use of these products differs from that of drugs for which additional issues regarding side effects and risk-benefit analysis also factor into the decision-making process. Thus, in the context of the current regulations, foods referred to as functional foods do not exist in a separate category but remain foods. The regulations governing safety as promulgated under FDCA, including the Food Additive amendment, and, in conditions when the intended use is a supplement, DSHEA applies to this category. Likewise, the conditions for allowing claims on functional foods are outlined in the FDCA, NLEA, DSHEA, and FDAMA [1–4]. Health Canada is currently reviewing a framework for evaluating foods with health claims and has outlined three components of product and claim acceptance that include product safety, claim validity and quality assurance. Table 5.2 summarizes the proposed key elements of each of these components [5].

Table 5.2. Standards of evidence for evaluating foods with health claims: A proposed framework, Health Canada. Framework component

Key provisions

Product safety

– assessment of potential negative nutritional and toxicological impacts – assessment of expected total exposure to the food or bioactive substances – extent of assessment is proportional to product novelty and uncertainty about product safety – good manufacturing practices – good laboratory practices – good practices concerning data collection and analysis – documentation – strength of evidence to support a causal relationship between food and bioactive substance in the food and claimed benefit – strength of evidence required is dependent on the nature of the claim – information that characterizes the relationship between the claimed benefit and the food or the bioactive substance – efficacy: can the product produce the effect as claimed? – effectiveness: does the product produce the effect as claimed?

Quality assurance

Claim validity

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In both Canada and the USA, the regulatory frameworks separate the evaluation of safety from functionality; one is based on establishing a reasonable certainty of no harm using risk analysis and the other is based on appropriate validation of the claimed benefit.

5.3 Emerging Issues Regarding Substantiation of Safety and Functionality The traditional approach for establishing safety of new food ingredients has been to establish reasonable certainty of no harm through toxicological testing and risk analysis. This approach is based on identifying toxic substances and characterizing the potential for harm based on levels of exposure [6, 7]. If the functional ingredient is added to a food as a micro component, then the traditional risk-analysis approach can be used because it is possible to test the substance and establish an acceptable daily intake level that includes a safety margin. Risk analysis is also being used to establish Tolerable Upper Levels (UL) of Intake or ULs for vitamins and minerals as a component of Dietary Reference Intakes (DRI) developed by the Food and Nutrition Board [8]. The UL is the intake level of a nutrient above which risk of adverse effects increases. However, this risk-analysis approach is not applicable in the safety assessment of whole foods or of functional ingredients that are macro-components of the food system because the foods are a complex mixture containing many components and the food or macro-components may be consumed at a high level in the diet. Under these circumstances the nutritional and metabolic impact of the product must be considered along with more traditional safety factors, and traditional safety testing is complicated by the difficulty of producing acute effects in response to consumption of the product. Examining the FDA review of Olean illustrates some of the emerging issues for safety evaluation of this type of macro-component of the diet. Olean is a sucrose molecule to which fatty acids are esterified. Because of its unique structure it cannot be digested by human lipolytic enzymes and passes through the small intestine undigested. In foods it functions as a fat but without adding the energy traditionally associated with fat in the diet since it is not digested. Thus, when added to food, it replaces fat as a bulk ingredient of the food. Traditional toxicology testing had demonstrated a reasonable certainty of no harm; however, Olean cannot be fed to animals at levels that exceed potential maximal intakes because it is a macro-replacer in the diet. During the review of Olean as a food additive several nutritional and metabolic questions were raised regarding the long-term impact of Olean consumption. Information on gastrointestinal tolerance, ability of large bowel microflora to metabolize, impact on the absorption of fatsoluble vitamins, interaction with drug bioavailability, and potential impact on overall dietary balance were also considered in the approval process. 95

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These types of factors as well as potential for allergenicity become a component of the framework for evaluation of safety when macro components are to be evaluated. Claims for functionality and nutritional benefit must be validated and based on scientific evidence. The most straightforward types of claims to validate are those based on nutrient content. Reliable, acceptable analytical methods that are standardized, adequately referenced and agreed upon are required. To avoid claims that are confusing to consumers, the regulatory framework needs to specify the level of a nutrient or the level of change in nutrient content that allows a claim based on nutrient content to be made. For example, FDA specifies that a food can claim to be a good source of an essential vitamin or mineral if it provides 10–19 % of the RDA for that nutrient or must have its energy content reduced by at least 25 % of a reference amount to qualify for a claim of reduced. The regulations can limit the types of nutrient content claims that can be made based on the total composition of the food and its potential contribution to a healthful diet. The validation of health claims or structure function claims is more challenging and dependent on significant scientific agreement about substantiation. Health claims, which are approved by the FDA, are defined statements that refer to the total diet as well as other factors that affect disease risk and generally indicate the probability of a relationship. While the health claim makes a statement about reducing risk of disease, the degree of risk reduction is not specified. In addition, these claims cannot be misleading and should be phrased for consumer understanding of the relationship and the importance of the nutrient in the context of the daily diet. Most regulatory agencies agree that scientific substantiation of a health claim is based on the total evidence available, the scientific plausibility of the association, several lines of evidence including both human and animal data and meet standards for both statistical and biological significance. In addition, the amount of food or food component needed for a biologically significant effect must be a reasonable portion of the diet. These claims are typically based on reduction or modification of a risk factor that predicts risk of disease in which case validation of the claim is dependent on validation of the biomarker or risk factor for predicting disease. The traditional approach to safety has included evaluation of the safety of the ingredients in foods, adulteration, risk analysis, misbranding and allergenicity, while health issues have more commonly focused on the scientific basis for claims, the appropriate context for the claim to be truthful and not misleading, and verification of the product composition related to the claim. As new products enter the market, additional issues, such as level of consumption, interactions, concerns for vulnerable groups and physiological tolerance, emerge as issues to consider under the safety and functionality frameworks.

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5.4 Development of a Safety Framework for Dietary Supplements in the United States With passage of DSHEA in 1994, dietary supplements were defined as a new category of products. The law indicated that, like food, dietary supplements on the market were assumed to be safe unless proven to cause harm. Thus, the manufacturers of dietary supplements are responsible for producing a product that presents no significant or unreasonable risk; however, like food, the manufacturer is not required to submit safety data to FDA for products on the market. In responding to the regulatory framework for dietary supplements created by DSHEA, FDA anticipates the need for a cost-effective, scientifically based approach for evaluating safety of dietary supplement using existing data. In other words, FDA needs to be able to effectively evaluate information to decide, which dietary supplement ingredients are most likely to present unreasonable risk to the public and further investigate these materials to determine their safety. In order to develop such a framework, FDA requested the Institute of Medicine of the National Academies of Science (IOM-NAS) to convene a group of experts to develop such a framework. The NAS is a private corporation established by federal charter in 1863. It responds to appropriate requests from government and the private sector, although its primary responsibility is to provide expert, independent, objective scientific advise to government agencies. This project to develop a safety framework for dietary supplement ingredients is managed by the Food and Nutrition Board of the IOM. FDA requested that the expert panel develop a proposed framework for categorizing and prioritizing dietary supplement ingredients sold in the United States based on safety issues, describe a process for developing a system of scientific reviews with specification for evaluating the safety of dietary supplement ingredients, and develop at least 6 scientific reviews or monographs as prototypes for the system. The committee has developed a framework in response to this charge. The proposed framework is currently under review; once published, copies can be obtained through the National Academy Press (www.nap.edu). The Food and Nutrition Board provides updates on committee activities on its web site (www4.nas.edu/cp.nsf and search for dietary supplements). Comments on the proposed framework can be submitted through the NAS. The committee will consider these comments as it finalizes the proposed framework and recommendations for its final report.

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References 1. 21 Code of Federal Regulations (CFR) § 301. 2. U. S. Food and Drug Administration. Nutrition Labeling and Education Act of 1990. Public Law, 101–535. 3. U. S. Food and Drug Administration. Dietary Supplement Health and Education Act of 1994. Public Law, 103–417. 4. U. S. Food and Drug Administration. Food and Drug Modernization Act of 1997. Public Law, 105–115. 5. Bureau of Nutrition Science, Food Directorate, Health Products and Food Branch, Health Canada. Standards of Evidence for Evaluating Foods with Health Claims: A Proposed Framework. June 2000. 6. Committee on the Institutional Means for Assessment of Risks to Public Health, National Research Council. Risk Assessment in the Federal Government: Managing the Process, National Academy Press, 1983. 7. Center for Food Safety and Nutrition, U. S. Food and Drug Administration. Toxicological Principles for the Safety of Food Ingredients: 1993 Draft Redbook II, Redbook 2000 is available at www.cfsan.fda.gov/Zredbook/red-toca.html. 8. Food and Nutrition Board, Institute of Medicine. Dietary Reference Intakes: A Risk Assessment Model for Establishing Upper Intake Levels for Nutrients. National Academy Press, Washington DC, 1998.

6 Dose-Response Relationships with Special Reference to Antioxidants Aalt Bast* and Guido R. M. M. Haenen

6.1 Introduction Not only for scientists but also for the lay public, antioxidants have a huge appeal. Antioxidants are used to combat oxidants which have been associated with many disorders (Tab. 6.1). Notably, many age related pathologies can be found among these oxidant related disorders [1]. In fact, advertisements on antioxidants promise a healthier and extended life. Some nuance is brought to this marketing-type message by the following joke. Two men are discussing the putative benefits of antioxidants. Man one: “Did you hear about this miraculous story of antioxidants? Apparently, they

* University of Maastricht, Faculty of Medicine, Department of Pharmacology and Toxicology, P. O. Box 616, 6200 MD Maastricht, The Netherlands

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Table 6.1. Disorders associated with oxidants. Lung Bronchopulmonary dysplasia, inhaled oxidants (NOx, O3), asbestosis, silicosis, chemicals (e. g. paraquat, bleomycin), cigarette smoke, idiopathic pulmonary fibrosis, cystic fibrosis, ARDS, asthma, COPD, cancer Brain Hyperbaric oxygen, Parkinson’s disease, neurotoxins (e. g. aluminium, MPTP, 6-hydroxydopamine), vitamin E deficiency, stroke, trauma (intracranial hemorrhage), ALS, neuroal ceroid lipofuscinoses, multiple sclerosis Kidney Autoimmune nephrosis (inflammation), chemicals (e. g. aminoglycosides, heavy metals, cisplatin) Cardiovascular system Myocardial infarction, transplantation, endotoxic shock, Keshan disease (selenium deficiency), chemicals (e. g. doxorubicin), atherosclerosis, (pre-)eclampsia, vasculitis Gastrointestinal system Inflammatory bowel disease, reperfusion, chemicals (nonsteroidal antiinflammatory agents, iron), emesis Liver Chemicals (e. g. halogenated hydrocarbons, iron, paracetamol, ethanol, quinones), endotoxin, reperfusion, fibrosis, nonalcohol and alcohol induced steatohepatitis Blood Thallassemia, anemia (sickle cell, favism, Fanconi’s), chemicals (phenylhydrazine, sulfonamides, lead, primaquine) malaria, protoporphyrin photooxidation Eye Cataractogenesis, ocular hemorrhage, photochemical retinopathy, retrolental fibroplasia Muscle Muscular dystrophy, exercise Skin Radiation (ionizing, solar), thermal injury, chemicals (i. e. tetracyclines), contact dermatitis, porphyria, photodynamic therapy Miscellaneous Aging, radiation injury, inflammation in general, rheumatoid arthritis, chemicals (e. g. radiosensitizers), alcoholism, nutritional deficiencies, diabetes mellitus

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are able to prolong your life”. Man two replies: “A pity that those years come at the end of your life when you are already old”. Man two could also have replied: “Which antioxidant should I use and what is the optimal dose to use?”. This reply would have formed an intriguing but rather difficult question. In this paper, we will pursue this question.

6.2 Concentration-Response Curves In pharmacology the receptor concept originated from an observed doseeffect relationship. At the end of the nineteenth century, Ehrlich performed work on selective tissue staining with various dyes and on the development of dyes with specific toxic actions. The notion that the dye molecules reacted with “receptive substances” in order to give staining and specific toxicity, led to the idea that the action of drugs should also arise through receptors, i. e. compounds to which the drug molecules could bind. A famous quote of Ehrlich, ‘corpora non agunt nisi fixata’ (substances do not act unless bound) illustrates the receptor concept. This, at that time theoretical idea, led to an elaborate mathematical framework in which binding of drugs to a receptor could be connected to a pharmacological effect of the drug. It was necessary to introduce the term intrinsic activity to explain the different efficacies of several receptor-activating compounds (agonists). The occupation theory assumes that the response is a function of the occupation of receptors (R) by an agonist (A). In equilibrium, the agonistreceptor interaction can be described by the dissociation constant (KA). It is further assumed that the number of receptors is low compared to the number of agonist molecules and the agonist-receptor complex (AR) does not appreciably alter the receptor concentration. In that case, the response (E) as a fraction of the maximal response (Emax) against the concentration of the agonist [A] gives a hyperbolic curve that approaches Emax assymptotically. The plot of E/Emax against the logarithm of [A] produces a symmetrical sigmoid curve. Interestingly, it took several decades before this receptor concept could be materialized. Now we understand how receptors look like and we are even able to make single amino acid modifications to receptor proteins in order to mechanistically envision the binding site and the signal transduction characteristics of the receptor. Dose dependency in toxic effects was already recognized by Paracelsus (1493–1541), born in Switzerland as Theophrastus Phillipus Aureolus Bombastus von Hohenheim. His adage that every compound can become toxic, as long as the dose is high enough, is still the opening sentence in many handbooks on toxicology. In fact, this forms the basis for defining safe dosages for xenobiotics (vide infra).

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In contrast to compounds that act through specific interactions with receptors or enzymes, redox active compounds might become inactive upon binding. ‘Corpora non agunt nisi fixata’ can be paraphrased into ‘antioxidants don’t act when bound’. We recently found that binding of antioxidants to protein could prevent their antioxidant action [2].

6.3 Which Antioxidant Should be Used? Antioxidants are abundantly present in nature. Their presence is presumably required in order to cope with the natural free radical generating machinery that is formed by UV light and oxygen. A nice example is formed by the coloured flower. Flavonoids, that form the colour, are antioxidants and protect the flower against UV light-induced free radical damage. Fat that is rich in unsaturated fatty acid is vulnerable to oxidation. Especially in polyunsaturated fatty acids a free radical may abstract a hydrogen atom thereby initiating the chain process of lipid peroxidation (Fig. 6.1). The lipid radical (L·), that is initially produced, reacts with oxygen. The

Figure 6.1. The chain process of lipid peroxidation.

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resulting lipid peroxyl free radical (LOO·) reacts with a second lipid molecule (LH) and abstracts a hydrogen atom. LOO· is changed into a lipidhydroperoxide (LOOH) and LH transforms into L·. Transition metals like Fe or Cu can transform LOOH into lipid oxyradicals (LO·), LOO· and hydroxyl radicals (·OH). All these formed free radicals may sustain this lipid-peroxidation process. Nature uses the antioxidants tocopherols and tocotrienols to prevent lipid peroxidation to occur. Tocopherols and tocotrienols may donate a hydrogen atom to LOO· thus breaking the chain reaction of lipid peroxidation and are therefore referred to as “chain breaking antioxidants”. An antioxidant is a compound that in a relatively low concentration inhibits, delays or prevents the oxidation of other molecules. According to this definition, many compounds can be regarded as antioxidants. The definition takes away mystics that surround antioxidants. It is easy to find new antioxidants in novel components of our diet. It is just a matter of smart comparison of the new and readily oxidisable component with a second molecule that is difficult to oxidise. The new component can be regarded as an antioxidant for the second one. Further, chelators of transition metals that prevent the radical catalytic behaviour of these transition metals can be regarded as antioxidants. Some of the flavonoids, mentioned earlier are in fact good iron and copper chelators. Moreover, certain enzymes (as catalase , glutathione peroxidase and superoxide dismutase) belong to the antioxidant armoury of nature (Fig. 6.2). Superoxide dismutase was discovered in the sixties in erythrocytes as a Cu-containing protein and was called erythrocuprein. The finding that it dismutates superoxide anion radicals formed the beginning of the free radical research in biological sciences. Both non-enzymatic and enzymatic antioxidants form an intricate network in the protection against oxidative damage (Fig. 6.3).

glutathione peroxidase H2O2 + 2GSH

2H2O + GSSG

catalase 2H2O2

2H2O + O2

superoxide dismutase 2O2.- + 2H+

O2 + H2O2

Figure 6.2. Antioxidant enzymes, superoxide dismutase, gluthathione peroxidase and catalase.

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Figure 6.3. Illustration of the network of antioxidants in the protection against free radicals.

The antioxidant network differs between different organs [3]. Interestingly, when two extracellular fluids like blood plasma and the epithelial lining fluid (ELF) of the lungs are compared, huge differences are in the level of antioxidants are discerned (Table 6.2). In the pulmonary ELF relatively high concentrations of glutathione are found. Undoubtedly, these differences will dictate the character of optimal supplementation regimes for either blood plasma or the lung ELF. Thus far, knowledge on organ or suborgan antioxidant differences is scarce. Changes that occur in the antioxidant network as a result of diseases are also not well described. This hampers adequate antioxidant supplementation with regards to both choice (which one?) and dose (how much?).

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Table 6.2. Differences in antioxidant levels in blood plasma and lung epithelial lining fluid (ELF). ascorbic acid glutathione uric acid bilirubin alpha-tocopherol beta-carotene albumin-SH

Plasma mM 40 1.5 300 10 25 0.4 500

ELF mM 100 100 90 – 2.5 – 70

6.4 Flavonoids: Apparently Uncomplicated Dose-Response Relationships Flavonoids are ubiquitous in photosynthesising plants. Flavonoids have a historical use in therapy against chronic venous insufficiency, because they decrease vascular permeability and protect vascular endothelial cells. Moreover, beneficial effects of flavonoids have been reported in various diseases like diabetes mellitus, asthma, radiation damage, cancer, viral infections, stomach- and duodenal ulceration and inflammation. This class of compounds have long been recognised as excellent scavengers of hydroxyl radicals, superoxide anion radicals, peroxyl radicals and singlet oxygen. They are potent inhibitors of lipid peroxidation and form complexes with all kinds of transition metal ions, as Fe2 , Fe3 , Cu2 , Al3 . The combination of these properties renders them excellent antioxidants [4, 5]. We have been investigating ways to prevent the doxorubicin-induced cardiotoxicity. The anthracycline doxorubicin is a widely used antitumour agent and ranks among the best agents in the treatment of a variety of haematological malignancies and solid tumours. Apart from common side-effects in anticancer therapy, such as bone marrow suppression, alopecia, nausea and vomiting, its clinical use is largely limited by the occurrence of a cumulative dose-related cardiotoxicity, which manifests itself as congestive heart failure. It is hypothesised that the generation of free radicals by doxorubicin or doxorubicin-transition metal complexes in heart tissue are responsible for the cardiotoxicity. We anticipated that flavonoids could possess the right characteristics, viz. radical scavenging and metal complexing properties, to prevent the doxorubicin-induced cardiotoxicity. We were able to come up with a semi-synthetic flavonoid, 7-monohydroxyethylrutoside that dose dependently protects against doxorubicin-induced cardiotoxicity without affecting the antitumour effect of the anthracycline [6]. In the selection process for this flavonoid we investigated the quantum chemical and physicochemical explanation for the activity of the flavonoids as well as the iron chelating and the antioxidant properties of the compounds. The chemical selec104

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tion process was followed with efficacy studies in which the flavonoids were studied with regard to their protection against the negative inotropic action of doxorubicin in the isolated electrically paced left atrium of the mouse. By means of telemetry in the mouse, the doxorubicin-induced ECG changes, i. e. the lengthening of the ST-interval were dose dependently inhibited by 7-monohydroxyethyl rutoside.

6.5 Dose Dependent pro-Oxidant Activity of Vitamin C and Superoxide Dismutase Antioxidants cannot distinguish between radicals that play a physiologic role and those that cause damage. When a general use of antioxidants is advocated, this seems to be often disregarded. Moreover, antioxidants don’t only function as such but they intrinsically can also behave as pro-oxidants. This is nicely illustrated with vitamin C. Ascorbic acid (vitamin C) has well-known antioxidant properties. It may also act as a distinct pro-oxidant as shown in Fig. 6.4. The oxidative breakdown of polyunsaturated fatty acids – the process of lipid peroxidation – in rat liver microsomes is not stimulated with Fe3 . Fe2 mildly activated this lipid peroxidation process. Vitamin C by itself does not have an effect, but the combination of vitamin C with either Fe3 or Fe2 caused intense lipid peroxidation. Addition of vitamin C up to a concentration of 0.2 mM potentiated 10 mM Fe2 -induced lipid peroxidation, since the maximal amount of thiobarbituric acid reactive material increased. Increasing the vitamin C concentration above 0.2 mM showed the antioxidant capacity of vitamin C, since a lag

Figure 6.4. Time course of the oxidative breakdown of polyunsaturated fatty acids in rat liver microsomes. This process, lipid peroxidation, was followed by determination of thiobarbituric acid-reactive material (DA at 535–600 nm). Lipid peroxidation is induced by 10 mM Fe3 (1), 10 mM Fe2 (2), 0.2 mM vitamin C (3), 10 mM Fe3 and 0.2 mM vitamin C (4), or 10 mM Fe2 and 0.2 mM vitamin C (5).

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Figure 6.5. Lipid peroxidation in rat liver microsomes in the presence of 10 mM Fe2 and vitamin C. The modulation of lipid peroxidation is determined by using various concentrations of vitamin C. Curves 1–10 are: 0, 0.025, 0.1, 0.2, 0.35, 0.6, 1, 1.5, 2 and 4 mM vitamin C, respectively.

time in the time course of lipid peroxidation appeared (Fig. 6.5). With high concentrations of vitamin C, no lipid peroxidation was observed within the incubation period of 45 minutes. The pro-oxidant activity of vitamin C probably results from the reduction of Fe3 . In this reaction the vitamin C radical is formed. The vitamin C radical might decay by disproportionation and produce vitamin C and dehydroascorbate or it might reduce another Fe3 atom. During the oxidation of vitamin C, H2O2 is also formed. These reactions provide all the ingredients for the Fenton reaction in which Fe2 reacts with H2O2 to form hydroxyl radicals. The reduction of Fe3 is a probable explanation for the pro-oxidant action of vitamin C [7]. It has been suggested that the Fe/vitamin C supplements that are used to enhance the intestinal absorption of iron in its reduced form, result in intestinal damage through lipid peroxidation [8]. A comparable pro-oxidant function has been described for the enzyme superoxide dismutase. We have recently established a good protective effect of lecithinized CuZn-superoxide dismutase (SOD1) against the doxorubicininduced cardiotoxicity (not yet published). These experiments were performed reluctantly, because it is known that SOD1 generates hydroxyl radicals when it is incubated with H2O2 [9]. With EPR experiments the formation of hydroxyl radicals was proven. The CuZn-SOD comprises a positively charged channel that ends near the active site at the Cu-ion. This channel conducts the substrate superoxide anion radical, which also explains the high rate for the dismutation reaction. The Cu-ion in SOD1 probably catalyses a Fenton-like reaction which yield hydroxyl radicals and leads to inactivation of the enzyme. Figure 6.6 shows a U-shaped curve in radical scavenging/forming activity for SOD1. At low concentrations of SOD1 the superoxide anion radical is scavenged, which is measured as a decrease in nitroblue tetrazolium reduction. At higher SOD1 concentrations the formation of hydroxyl radicals in increased, which is measured as the hydroxylation of 106

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Figure 6.6. Superoxide anion radical scavenging and hydroxyl radical formation by superoxide dismutase 1 in a system consisting of xanthine and xanthine oxidase and detector molecules for superoxide anion radicals (nitroblue tetrazolium, NBT) and hydroxyl radicals (coumarin-3-carboxylic acid, 3-CCA). The rate of NBT reduction is plotted on the left y-axis. On the right y-axis the extent of 3-CCA hydroxylation is plotted.

coumarin-3-carboxylic acid. Thus far, the pro-oxidant effects of SOD1 have only been demonstrated in cell culture systems or isolated organs.

6.6 Multiple Actions and Metabolites of Antioxidants It is hard to understand that large epidemiological intervention trials are conducted with compounds for which neither the kinetics nor the biotransformation or the precise mode of action has been reported. This is in sharp contrast to trials with pharmaceuticals. This matter can be illustrated with vitamin E [10, 11]. Vitamin E is the collective term for all biological active tocopherols and tocotrienols and their derivatives which exhibit the biological activity of R,R,R,-a-tocopherol, the natural vitamer with the highest activity. As shown in Fig. 6.7 by acting as a membrane-bound antioxidant, tocopherol is transformed into a fairly stable chromanoxyl radical. Interaction with either vitamin C or glutathione may lead to a conversion of this chromanoxyl free radical into tocopherol again. Tocopherol can also be transformed into a quinone, which may be reduced to a hydroquinone. This hydroquinone has a superior antioxidant activity compared to tocopherol. It has been hypothesised that a-tocopherol might just serve as a reservoir for the a-tocopherol quinone. 107

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Figure 6.7. Oxidation and regeneration of a-tocopherol. Tocopherol is converted in a tocopherol radical once it reacts with a radical. The tocopheryl or semi-quinone radical can be regenerated to tocopherol by vitamin C or reduced glutathione. In the oxidation of the tocopheryl radical to a tocopheryl quinone, one of the rings of the chroman moiety is opened. The tocopheryl quinone can be reduced to tocopheryl hydroquinone. The tocopheryl hydroquinone is an excellent antioxidant. In vivo the tocopheryl hydroquinone can be converted in tocopherol.

We recently found that tocopherols and several tocopherol esters inhibit glutathione S-transferase P1-1 (GST P1-1) [12]. It is known that GST P1-1 is present in the skin. Mice lacking GST P1-1 have an increased risk for skin tumorgenesis. Vitamin E has been reported to be a complete tumour promoter in mouse skin. Combining these data, it is tempting to suggest that the promoter effect of vitamin E might be caused by GST P1-1 inhibition [10]. The inhibition of the proliferation of smooth muscle cells by R,R,R,-atocopherol has been attributed to its inhibition of protein kinase C [13]. Recently, a metabolite of g-tocopherol that is formed by b-oxidation 2,7,8-trimethyl-2-(b-carboxyethyl)-6-hydroxychroman was reported to possess a strong natriuretic effect [14]. A thought that offers new possibilities is that vitamin E should in first instance not be regarded as an antioxidant but rather as a hormone-like compound that controls several cellular functions. Its oxidisability renders it suitable to act as a sensor for oxidative stress. The primary function of vitamine E would then not be to detoxify free radicals but rather to modify the cellular machinery as response to oxidative stress. The new biological effects of vitamin E request a re-evaluation of the dose-new response relationship in which also the effects of the vitamin E metabolites should be included. Similar reasoning might apply for other antioxidants.

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6.7 Hormetic Dose Responses High doses of oxidants are clearly toxic to cells. On the other hand is now recognised that low levels of oxidants play a significant role in the regulation of numerous cellular activities [15]. Low levels of oxidants may lead to modulation of protein structures and functions with subsequent activation of signal-transduction pathways and modulation of gene expression. The adaptive responses to oxidative stress that may arise in this way can lead to biphasic (hormetic) dose responses. Oxidants may activate NF-kB, AP-1 and mitogen activated kinases [16]. They can also stimulate receptor tyrosine kinases as well as various components downstream in the signal-transduction pathways [17]. The influence of antioxidants in these hormetic dose-response relations has not extensively been investigated thus far.

6.8 Safety Factors Knowledge on the dose-response curves for beneficial and adverse effects are valuable to decide on the safe use of compounds. For chemicals extensive information on in vitro studies and animal studies are used to establish the acceptable daily intake (ADI). The ADI is defined as ‘an estimate of the amount of a substance in food or drinking water, expressed on a body weight basis, which can be consumed daily over a lifetime by humans without appreciable health risk’. The highest No Observed Adverse Effect Level (NOAEL) in animal studies is usually divided by a safety factor of 100 to derive the ADI for humans. The safety factor of 100 consists of a factor of 10 to account for the extrapolation from animal to human and a factor of 10 to account for differences between individuals. The Scandanavian Alpha-Tocopherol, Beta-Carotene (ATBC) study showed that a supplementation with 20 mg b-carotene resulted in an 18 % increase in lung cancer incidence. This dose of b-carotene apparently has an effect and is not the NOAEL in humans. The NOAEL is less than 20 mg. In the aforementioned classical toxicological approach the NOAEL is divided by a safety factor of 10 to account for intra-individual differences. If we use this factor the ADI would be 2 mg. In fact the ADI should be lower because the NOAEL in human is apparently lower than 20 mg. The ADI of 2 mg b-carotene per day is below the average daily intake of 3 mg in the Netherlands. The classical toxicological paradigm is apparently not valid here [10]. We have to accept that safety factors might be much smaller than we assent for chemicals. We could accept smaller safety levels if extensive epidemiological studies have defined a safe dose. Also plasma/serum levels (or even better tissue levels) of the antioxidant could guide the decisions on safety. In supplementation would lead to excessively exceeding of 109

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the normal plasma/serum levels (in the ATBC study 17.5 times) additional research should be required. To study antioxidants in a food matrix it is sometimes not possible to increase the dose in the diet of the test animals to reach an exposure of greater than 100 times the human exposure.

6.9 Conclusions To avoid toxicity of antioxidants in the future on the one side and to circumvent unrealistic precautionary measures at the other side several recommendations could be given [10]. x

Application of the classical toxicological safety factor paradigm is not always feasible for (food derived) antioxidants.

x

Knowledge on the biokinetics and the biotransformation of antioxidants and their metabolites as well as on their profile of action and toxicity should increase.

x

Bio-kinetic or bio-efficacy modelling might be needed if the normal tissue or plasma level of (food derived) antioxidants is excessively exceeded after supplementation.

x

A more accurate risk/benefit analyses is warranted if the administered dose of an antioxidant is high and (inherently) the health claim becomes more explicit.

References 1. Bast, A.: Antioxidant pharmacotherapy. Drug News & Perspectives 7 (1994), 465–472. 2. Arts, M. J. T. J.; Haenen, G. R. M. M.; Voss, H.-P.; Bast, A.: Masking of antioxidant capacity by the interaction of flavonoids with protein. Food Chem. Tox. 39 (2001), 787–791. 3. Van der Vliet, A; O’Neil, C. A.; Cross, C. E.; Kootstra, J. M.; Volz, W. G.; Halliwell, B.; Louie, S.: Determination of low-molecular-mass antioxidant concentrations in human respiratory tract lining fluids. Am. J. Med. 276 (1999), L289–L296. 4. Van Acker, S. A. B. E.; Van den Berg, D.-J.; Tromp, M. N. J. L.; Griffioen, D. H.; Van Bennekom, W. P.; Van der Vijgh, W. J. F.; Bast, A.: Structural aspects of antioxidant activity of flavonoids. Free Rad. Biol. Med. 20 (1996), 331–342.

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References 5. Van Acker, S. A. B. E.; Van den Berg, D.-J.; Tromp, M. N. J. L.; Griffioen, D. H.; Van Bennekom, W. P.; Van der Vijgh, W. J. F.; Bast, A.: A quantum chemical explanation for the antioxidant activity of flavonoids. Chem. Res. Tox. 9 (1996), 1305–1312. 6. Van Acker, S. A. B. E.; Kramer, K.; Grimbergen, J. A.; Van den Berg, D.-J.; Van der Vijgh, W. J. F.; Bast, A.: Monohydroxyethylrutoside as protector against chronic doxorubicin-induced cardiotoxicity. Br. J. Pharmacol. 115 (1995), 1260–1264. 7. Bast, A.; Haenen, G. R. M. M.; Doelman, C. J. A.: Oxidants and antioxidants: State of the art. Am. J. Med. 91 (Suppl 3C) (1991), 2S–13S. 8. Slifka, A; Kang, J.; Cohen, G.: Hydroxyl radicals and the toxicity of oral iron. Biochem. Pharmacol. 35 (1986), 553–556. 9. Yim, M. B.; Chock, P. B.; Stadtman, E. R.: Copper zinc superoxide dismutase catalyzes hydroxyl radical production from hydrogen peroxide. Proc. Natl. Acad. Sci. USA 87 (1990), 5006–5010. 10. Bast, A.; Haenen, G. R. M. M.: The toxicity of antioxidants and their metabolites. Environ. Toxicol. Pharmacol. 11 (2002), 251–258. 11. Van Acker, S. A. B. E.; Koymans, L. H. M.; Bast A.: Molecular pharmacology of vitamin E. Free Rad. Biol. Med. 15 (1993), 311–328. 12. Van Haaften, R. I. M.; Evelo, C. T. A.; Penders, J.; Eijnwachter, M. P. F. G. R. M. M. Haenen, G. R. M. M.; Bast A.: Inhibition of human glutathione S-transferase P1-1 by tocopherols and a-tocopherol derivatives. Biochim. Biophys. Acta 1548 (2001), 23–28. 13. Azzi, A; Stocker, A.: Vitamin E: non-antioxidant roles. Prog. Lipid Res. (2000) 39, 231–255. 14. Wechter, W. J.; Kantoci, D.; Murray, E. D.; D’Amico, D. C.; Jung, M. E.; Wang, W.-H.: A new endogenous natriuretic factor: LLU-alpha. Proc. Natl. Acad. Sci. USA 93 (1996), 6002–6007. 15. Finkel, T.: Redox-dependent signal transduction. FEBS Letters 476 (2000), 52–54. 16. Powis, G.; Gadaska, J; Baker, A.: Redox signalling and the control of cell growth and death. Adv. Pharmacol. 38 (1997), 329–359. 17. Van der Vliet, A; Bast, A.: Effect of oxidative stress on receptors and signal transmission. Chem.-Biol. Interactions 85 (1992), 95–116.

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7 Low Dose Competition of Flavonoids with Endogenous Thyroid Transport Proteins: Potential Relevance to the Thyroid Hormone Axis Josef Köhrle*

7.1 Introduction Thyroid hormones are synthesized by the thyroid gland under the control of the hypothalamic-pituitary-peripheral feedback system depending on the supply of two essential trace elements, i. e. iodine and selenium [1]. The thyroid gland system, characterized by its ability to concentrate iodide from environment and circulation against a gradient into the gland evolved very early in lower vertebrates probably in the primitive gastrointestinal tract. Most of the relevant transcriptions factors controlling thyroid development and migration to its final location in front of the larynx as well as its pituitary feedback control elements have been identified during the last years (thyroid transcription factor 1 and 2 (TTF1,2) and PAX 8) [2]. Similarly, most of the required proteins and enzymes instrumental for thyroid hormone biosynthesis, serum transport, tissue uptake, intracellular metabolism and action are known. However, only limited knowledge is available on interference of drugs, nutrients or components contained in food or derived from intestinal digestion of food, its components or contaminants with the complex pathways involved in thyroid hormone synthesis, metabolism and action. Especially as thyroid hormones are highly hydrophobic, aromatic and phenolic compounds, several drugs, xenobiotics and a series of phenolic secondary metabolites of plants might interfere with thyroid hormone binding, metabolism and action. Therefore, even low levels of compounds might exert significant action by synergistic interference at several levels of control. This hypothesis had also been proposed to explain the potent action of folk remedies with antithyroid efficacy still available and in use in some treatment regimens for thyroid disease [3]. There is even evidence, that subgroups of phenolic compounds contained as secondary metabolites of plants in our daily nutrition act as hormones themselves [4, 5], such as isoflavonoids in the steroid hormone network or derivatives of unsaturated fatty acids in the prostanoid and prostacyclin network. Less is known on interference of plant-derived nutritional compounds in the thyroid hormone network, which controls

* Institut für Experimentelle Endokrinologie und Endokrinologisches Forschungszentrum der Charite´, Medizinische Fakultät Charite´ der Humboldt Universität zu Berlin, Schumannstr. 20/21, 10098 Berlin, Germany

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growth, differentiation, development, basal metabolic rate, thermogenesis, and many pathways of anabolic and catabolic reaction of intermediary as well as structural metabolism in vertebrates including man.

7.2 The Thyroid Hormone Network Thyroid hormone is exclusively produced, stored and secreted by the thyroid gland. Iodide is concentrated by the sodium-iodide symporter (NIS) in the basolateral plasma membrane of thyrocytes which form an unicellular layer engulfing the colloid space [6]. A related transporter, the apical iodide transporter (AIT) delivers iodide to the apical colloid space [7]. Thyrocytes produce and secrete thyroglobulin (Tg) via the apical membrane into the colloid. Tg provides the molecular matrix for hormone synthesis at the apical extracellular space and the storage form of thyroid hormone in the colloid [8]. Thyroxidases (Thox1,2) provide H2O2 required by extracellularly oriented Thyroperoxidase (TPO) for iodination of tyrosine residues and coupling of iodotyrosine residues to iodothyronines of the Tg backbone [9, 10]. Specific condensation processes enable storage of Tg [11]. Decondensation and redox-regulated proteolytic release reactions liberate thyroid hormones from Tg in the colloid or in thyrocytes, respectively [12]. Synthesis, storage and secretion of thyroid hormones is under the control of thyrotropin (TSH), the pituitary hormone mainly addressing thyrocytes via a G-Protein coupled serpentine receptor on the basolateral membrane, stimulating cAMP production [13]. Once secreted by the gland, the highly hydrophobic thyroid hormone (Lthyroxine, T4 and to a minor extent 3,3l,5-triiodo-L-thyronine, T3) reach their target organs and cells bound to three major transport or distributor proteins (Tab. 7.1) [14, 15]. Thyroxine-binding globulin (TBG), a high affinity low capacity binding proteins found only in higher vertebrates, transthyretin (TTR), a medium affinity higher capacity binding protein which is the most conserved binding protein in most phyla which take advantage of thyroid hormones (TTR also binds retinol binding protein and Vitamin A in one complex with T4). Albumin is a low affinity high capacity binder of T4 and T3 and a minor fraction also bind to serum lipoproteins. The free thyroxine concentration circulating in serum is below 0.1 %! Uptake of free T4 or T3, amphipathic charged amino acid-derivatives, into target cells occurs by energy- and Na-dependent transporters, which are related to the organic anion transporter family (OAT) [16]. Intracellular cytosolic binding proteins buffer thyroid hormones. Activation of the prohormone T4, which is devoid of action at nuclear or mitochondrial TR-receptors (TRa, TRb) to the thyromimetically active thyroid hormone T3, the most potent ligand of TRs occurs by two iodothyronine-5l-deiodinase enzymes, 5lDI (Type I-5l-deiodinase) and 5lDII (type II 5l-deiodinase). These two enzymes control the last step of thyr113

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oid hormone synthesis at the cellular level (5lDII, and 5lDI) and provide the majority of systemically circulating T3 (5lDI), thus acting as guardians of the gate to the thyroid hormone receptor mediated action of T3 [17]. Inactivation of T4, or the active T3 occurs either by type III 5-deiodinase producing 3,3l-5l-reverse T3 (rT3) from T4 or 3,3l-T2 from T3 or alternatively by 5lDI which is somewhat promiscuous in its substrate specificity and also acts on some sulphated iodothyronines. Conjugation of the phenolic 4l-OH group of iodothyronines occurs either by specific action of members of the sulfotransferase family (SULT) or by UDG-glucuronyl transferases and leads to inactivation, enterohepatic recycling or excretion of iodothyronines [18]. Action of thyroid hormones mainly occurs via T3 binding to ligand activated nuclear receptors (TRa, TRb), but also the rapid and direct effects on targets of the plasma membrane, the mitochondria and other cellular structures are known [19–23]. Table 7.1. Thyroid hormone transport proteins. Thyroid hormone binding protein

Occurrence

Characteris- Affinity tics Ka

Thyroxine binding globulin (TBG)

higher mammals only, old or pregnant rats

high affinity, low capacity binding protein, binding T4 (and T3)

Transthyretin (TTR)

most highly conserved T4 binding protein, produced in liver (and choroid plexus)

Albumin

low affinity, high capacity

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Pathophysiology

1010 M-1

Xq21-q22

polymorphic variants (Australian aborigines, French Canadians, Japanese, Africans), complete or partial deficiency resp. excess

complex with retinol-binding protein (vitamin A)

108 M-1

18q11.2q12.1

variants with increased or reduced affinity, familial amyloidoic neuropathy (FAP)

abundant

106 M-1

4q11-q13

familial dysalbuminemic hyperthyroxinemia, analbuminemia (very rare)

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7.3 Flavonoids and Thyroid Hormones – Competition by Structural Similarity? Studies over the last twenty years revealed a marked interference of some representatives of the huge family of secondary metabolites of plants at several levels of the thyroid hormone feedback system [24, 25]. Tab. 7.2 summarizes the main published findings. Among the most prominent and best characterized interactions is the binding and competition of flavonoids for T4 with TTR, leading to major disturbances of the thyroid feedback axis both in acute and in chronic exposure experiments. Both in vitro and in vivo experiments revealed detailed structure activity relationships, evidence for low dose effects, alterations of total and free thyroid hormone levels, which lead to (reversible) changes in pituitary TSH-secretion, disturbed thyroid hormone synthesis and secretion, alterations of tissue deiodinase and thyroid hormone levels, and last but not least, modification of thyroid hormone action at various cellular and systemic endpoints [3, 26–36]. At this moment it remains unclear, which of the observed effects are due to direct

Table 7.2. Components of the thyroid hormone system affected by naturally occurring and synthetic flavonoids. Tissue or target

Effect

CNS choroid plexus hypothalamus anterior pituitary, content and secretion Thyroid NIS AIT ThOx TPO Tg Serum binding, distribution, and transport TBG TTR albumin lipoproteins Cellular uptake and efflux Intracellular cytosolic thyroid hormone binding proteins Deiodinases 5’DI 5’DII 5’DIII Nuclear T3 Receptors (TR) Thyroid Hormone Sulfotransferases Thyroid Hormone UDPG-Glucuronyl-Transferases

yes T4-binding to TTR ? inhibition of TSH yes ? ? ? potent ? yes no potent no weak ? ? yes potent weak weak no ? ?

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Figure 7.1. Structural formula of the synthetic flavonoid F21388 (4,6,4’-(OH)3,3-CH3,3’,5’-Br2-flavonone), a derivative of the naturally occurring flavonoid luteolin. F21388 is a very useful tool for studying interference of flavonoids in the thyroid hormone network.

flavonoid action or indirect adaptations to disturbances in the fine-tuned network of thyroid hormone homeostasis in vivo. Especially in vitro and in vivo studies in rodent models with a synthetic, rather stable brominated derivative (F21388) (Fig. 7.1) of the naturally occurring luteolin, the lead compound among natural flavonoids in displacement of T4 from binding to TTR, revealed a rather detailed picture of flavonoid interference in the thyroid network: flavonoids not only displace T4 from TTR, they also alter free thyroid hormone concentrations and cellular hormone levels, inhibit deiodinases, and most importantly cross placental and blood-brain barrier and reach the foetus including the foetal brain, This indicates a prominent role for some of these naturally occurring or man-made compounds in altering setpoint or homeostasis of (thyroid) hormone networks during development and differentiation especially under conditions of iodine-deficiency, where the thyroid feedback network is already markedly disturbed and/or stimulated.

7.3.1

Flavonoid Effects at the Pituitary Level of Control of the Thyroid Axis

Several effects of plant extracts containing secondary metabolites of plants including flavonoids (Fig. 7.2) have been reported in rodent experimental animals [26, 37–39]. However, at this moment it remains unclear whether these effects are exerted directly by flavonoids or indirectly by altered levels of circulating thyroid hormone, which act on thyrotrophs via local negative feedback control. Administration of both plant extracts as well as isolated naturally occurring or synthetic flavonoids leads to inhibition of TSH secretion, either transient or persistent. The most detailed studies employing the synthetic flavonoid F21388 (Fig. 7.1) revealed that administration of this com116

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Figure 7.2. Structures of some natural flavonoids with antithyroidal activity (from Divi & Doerge (1996) [48]).

Figure 7.3. Effect of a single injection (i. p.) of F21388 on serum TSH in intact rats.

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Figure 7.4. Displacement of 125-I-T4 from binding to Transthyretin (TTR) by the flavonoid F21388 in human serum. (Alb: albumin, TBG: thyroxine-binding globulin).

pound rapidly increases total free and % free T4 and T3 levels, which subsequently suppress TSH-secretion (Fig. 7.3). Elevation of free T4 is achieved by displacement of T4 from binding to transthyretin (Fig. 7.4), the major T4binding protein in normal adult rats. F21388 titrates the circulating TTR and forms a 1:1 complex [26]. In addition, flavonoids also affect pituitary deiodinase activity. No information is yet available on possible direct effects of flavonoids such as F21388 or naturally occurring compounds on TSH synthesis, storage or release. As rodent anterior pituitaries show marked sexual dimorphism [40, 41] indirect effects of flavonoids on the steroid hormone axis might also disturb the steroid-sensitive thyrotroph function. 7.3.2

Flavonoid Effects at the Thyroid Level

Antithyroid effects of flavonoids are well known from studies in several in vitro and in vivo models. Most consistent data are available for their interference with TPO, the key enzyme of thyroid hormone biosynthesis. Here extensive structure activity relationships have been established and the potent goitrogenic action of flavonoids is relevant for production and maintenance of life stock as well as goitrogenesis in human populations exposed to high nutrient load of some flavonoids such as in millet in third world countries [42–51]. Among the most potent goitrogenic compounds are vitexin, quercetin and some other flavonoids. Divi and Doerge reported extensive data on structure-activity relationships and mechanism of inhibition of TPO [48, 52]. Similarly aqueous extracts of a Brazilian medicinal herb, Kalanchoe brasiliensis, which contains flavonoids was shown to inhibit TPO and to scavenge H2O2 [53]. In addition to inhibition of TPO a recent report also indicates a rather efficient inhibition of 5’DI in rat thyroid [46], which contributes both to local and systemic T3 production in rats. Here IC50 values for in vitro inhi118

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bition of 5’DI were obtained in the 11 to 77 mM concentration range with baicalein and quercetin being the most potent, and kaempferol and biochanin A the least effective compounds tested (Fig. 7.2). These studies indicate that some of the flavonoids are more powerful in inhibiting 5’DI than TPO at the thyroid level; others exert their action mainly by inhibiting TPO. Currently no data are available on possible effects of flavonoids on other components of thyroid hormone synthesis, storage or secretion except several reports on inhibition of TSH binding to its receptor or competition for TSHreceptor stimulating antibodies [54–57]. Detailed structure-activity and dose-response analyses revealed, however, that for this aspect of flavonoid interference in the thyroid hormone axis not monomeric but oxidized polymeric complexes of flavonoids and phenolic compounds of plant extracts are the most potent agents in contrast to monomeric, non oxidized free phenolic flavonoids which interfere with hormone binding to TTR and inhibit deiodinases (see below).

7.3.3

Interaction of Flavonoids with Serum Transport and Distribution Proteins for Thyroid Hormones

The iodinated phenolic and highly hydrophobic thyroid hormones which are released by the thyrocytes upon TSH-stimulation by yet unknown mechanisms reach the blood compartment and are found there bound to three major thyroid hormone binding proteins (see above and Tab. 7.1). This tight binding with graded affinity to the three major binding proteins in higher mammals results in very low free T3 and even lower free T4 concentrations. Remarkably, flavonoids have been shown to specifically interfere with T4 and T3 binding to TTR (Fig. 7.4) but not with TBG or albumin to a significant extent [26, 35, 36, 58, 59]. Other pharmaceuticals and aromatic compounds such as salicylates, antiphlogistic drugs and chemicals are able to displace T4 (and T3 (?)) from TBG or albumin respectively albeit with rather low affinity and potency compared to the flavonoid competition for T4 binding to transthyretin [60, 61]. IC 50 values for TTR interference by flavonoids have been described in the submicromolar to micromolar range with detailed structure activity relationships [34, 35, 62]. Here, similarity of flavonoids to the three dimensional structure of T4 or even rT3 (the latter is a weak ligand for TTR) seems to govern T4-competition. Chemical modification of natural lead compounds of the flavonoid, aurone or chalcone class of compounds, in a sense to better mimic thyroid hormone skewed or antiskewed conformation, even increased potency in TTR-competition assays in vitro. In vivo administration of these ‘designer flavonoids’ in rat and porcine models confirmed the in vitro observations of T4 (and T3) competition specifically from TTR, even shifting the thyroid hormone to albumin or, in case of human serum in vitro, to TBG binding (Fig. 7.4) [26, 30, 34, 35, 37, 63, 64]. These alterations led to marked but transient increases in serum free thyroid hormone level both % free and total free hormones. The latter changes were 119

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subsequently reflected by immediate but transient suppression of circulating TSH, enhanced tissue transfer and also enhanced elimination of thyroid hormone from the circulation probably by the renal excretion. Whether faecal excretion was also affected needs to be examined in more detail. Apart from inhibition of TSH secretion also hepatic readouts of enhanced cellular content of thyroid hormone were observed such as elevated cytosolic malic enzyme or mitochondrial aGAPDH levels (unpublished observations). In these studies no direct inhibitory effect of administered flavonoids on deiodinase inhibition could be found. Observed changes especially of 5’DII activity (inhibitory) or increased activity of 5’DI in selected tissues in the rat rather have to be interpreted as indirect effects of the compounds due to increase of cellular thyroid hormones levels caused by altered thyroid hormone distribution between the vascular, (interstitial?) and cellular compartments subsequent to potent T4 and T3 displacement from TTR in the rat [31, 34, 36, 65, 66]. It has to be taken into consideration that in the normal adult rat expression of TBG is not detectable [67, 68] and therefore no compensation and buffering of altered free thyroid hormone levels can occur by this high affinity protein. However, as similar alterations of total and free thyroid hormone levels have also been found in the porcine model after administration of similar doses of F21388, and TBG is expressed and circulating in porcine serum, these findings indicate that nevertheless flavonoids are potent competitors of thyroid hormone binding to TTR and thus even in presence of the high affinity binder TBG will alter thyroid hormone economy and homeostasis. Recent unpublished experiments on consumption of parsley which contains high amounts of the flavonoids apigenin revealed that also in human volunteers this orally administered natural dose of flavonoids results in rapid alteration of serum thyroid levels and markedly increased renal excretion of intact thyroid hormone (D. van der Heide, unpublished, personal communication) again suggesting that this mechanism of competition for thyroid hormone binding to transthyretin by flavonoids even in presence of high concentrations of TBG efficiently interferes with thyroid hormone economy and homeostasis. Another set of observations of effects of flavonoids is remarkable. The in vivo administration of the synthetic T4-analogue flavonoid compound F21388 allowed to exactly titrate the serum concentration of TTR in the rat [26]. This suggests, that after saturation of circulating (and tissue?) TTR excessive flavonoid might be available to reach the tissue or alternatively be excreted. If this mechanism also holds for other related flavonoid congeners experimental or therapeutic administration of flavonoids might need to take into consideration the available amount of circulating TTR. As TTR is not only produced and secreted by the liver but also synthesized by the choroid plexus into the CSF [63, 69], agents with similarity to flavonoids either of nutritional, environmental or pharmceutical origin might be efficiently sequestered by hepatic and or choroid plexus TTR and in addition efficiently reach the CSF by this directed secretion of TTR into the CSF. Whether this ‘trojan horse’ approach can be used as efficient tool to target drugs via TTR secretion to the CNS remains to be examined. 120

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In vitro experiments using choroid plexus cell culture models in monolayer culture and bicameral systems demonstrated the feasibility of this approach, indicating that flavonoids such as F21388 are able to compete for T4 at choroid plexus TTR and to interfere with T4 economy [29, 63]. Findings of administration of brominated or iodinated flavonoids to pregnant rats in constant infusion protocols using Alzet minipumps also support that flavonoids reach not only the foetus but also the foetal brain, indicating that not only the choroid plexus barrier but also the placental barrier might be crossed by flavonoids [27, 28, 31]. These observations are of eminent importance, as flavonoids are ubiquitous nutritional compounds consumed in high amount in normal diet and at even greater extent by vegetarians. Therefore, on the background of the still persisting iodine deficiency in middle Europe and especially in Germany, compounds as potent as flavonoids in disturbing thyroid hormone economy and balance might exert marked antithyroidal, goitrogenic and persistent effects on thyroid hormone synthesis, secretion and action. In order to understand these complex and interrelated networks of thyroid hormone control further experimental studies in appropriate animal models but also in human volunteers, both adult and in children appear to be mandatory. A further consequence of T4-displacement from TTR is enhanced elimination of thyroid hormone from circulation probably via renal excretion (D. van der Heide, Wageningen, NL; personal communication) and/or enhanced transfer of T4 (and T3?) to tissues. These alterations are rapidly reversed after single dose administration of F21388. However, after continuous treatment (e. g. by alzet minipumps) both serum and tissue hormone levels are changed in rodent models, suggesting, in addition to altered hormone distribution resulting from TTR displacement, also inhibition of tissue deiodinase activity. Both, 5’DI and 5’DII seem to be affected as indicated by altered T3/T4 ratios in relevant organs [27, 28, 31]. Whether these altered tissue hormone levels result in altered expression of T3-dependent genes remains to be studied. Up to now, no evidence has been presented that flavonoids in spite of their similarity to thyroid hormones are also effective in displacing T3 from its binding to the nuclear T3-receptor family. Initial pilot experiments in cooperation with the group of Juan Bernal, Madrid, using nuclear extracts enriched in T3-receptors did not reveal any significant competition of natural and synthetic flavonoids for T3 binding to TR in reasonable concentrations. It has to be noted however, that the flavonoids used in these experiments were representative for T4-binding and competition and not selected as possible T3-analogues, Therefore definitive conclusions are not yet possible.

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Flavonoid Effects in Target Tissues of Thyroid Hormone Action

The potent binding of flavonoids to TTR in blood raises the question whether flavonoids reach intracellular tissues at all to exert antithyroid action (Fig. 7.5). Currently, no systematic information is available on possible interference of flavonoids with thyroid hormone uptake or export from tissues. Several transporter molecules for thyroid hormones, specific for either T4 and/or T3 have been identified in the recent years, apparently with tissue specific distribution and distinct characteristics with respect to their substrate specificity, Na- and energy-dependent action [16, 70–72]. Apparently specific transporters also mediate thyroid hormone efflux from thyrocytes. It is conceivable that some or all of these molecules also are targets for specific flavonoids competing for thyroid hormone transport across membranes. However, also indirect effect of flavonoids might occur not related to direct competition for the thyroid hormone ligand-binding site of these transporters. It has been shown that flavonoids are potent inhibitors of several kinases, phosphatases and enzymes with nucleotide binding sites [24, 73–75]. These effects are exerted already at low micromolar concentrations. As thyroid hormone transporters are energy dependent, indirect blockade of cellular uptake of efflux might thus occur. The most prominent effects of flavonoids on transpor-

Figure 7.5. Components of the thyroid hormone system. The major systemic and cellular proteins and enzymes involved in transport, uptake, metabolism and action of the highly hydrophobic thyroid hormone are illustrated, indicating possible site of interference of phenolic secondary metabolites of plants such as (iso-)flavonoids with the thyroid hormone network. (TTR: Transthyretin, TBG: thyroxine-binding globulin).

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ters has been described for phlorhizin and its aglycon phloretin [59], which is a potent and specific inhibitor of glucose transport. Interestingly phloretin and its derivatives are also both potent competetors of T4 binding to transthyretin and of type I 5’-deiodinase both in vitro and in intact isolated cells [34, 59]. Therefore, complex patterns of interference with metabolic pathways have to be expected for some of the flavonoids. Currently the focus of analysis is directed towards anti-steroid action of flavonoids especially isoflavonoids such as genistein, which is a potent ligand for the estrogen receptor ERb [52,76]. In many studies the potent action of these compounds in both in vitro, cellular or intact animal models on kinases, phosphatases or enzymes and transporters with nucleotide binding sites is completely neglected and some effects assigned to the endpoint of interest in these studies merely might reflect secondary or indirect events caused by other target molecules and signalling cascades. Using primary isolated rat liver hepatocytes in suspension we could clearly show inhibitory action of naturally occurring flavonoids (aurones, chalcones and flavonoids) on hepatic type I 5’deiodinase activity in dose dependent manner. As some of these redox active flavonoids alter their colour depending on their chemical environment, the observation of coloured hepatocytes after incubation with flavonoids or staining of their intracellular compartments give clear evidence that some if not all flavonoids are taken up by the cells and reach several intracellular targets or subcellular compartments. In favour of this statement is also the observation that several of the compounds affect gluconeogenesis in intact hepatocytes in suspension and that others affect viability of the hepatocytes [32, 36, 77, 78]. As effects of some flavonoids on deiodinase inhibition or on gluconeogenesis are observed far below the concentrations were cellular integrity is affected, as indicated by trypan blue exclusion or other tests of cellular viability, these studies support the notion that some flavonoids reach intracellular deiodinases and act there as potent inhibitors, similar to observations resulting from comprehensive in vitro inhibition studies using deiodinase enriched microsomal membranes from different tissues expressing either 5’DI or the two other enzymes, 5’DII or 5DIII [32, 34, 36, 59, 62]. The in vitro and in vivo rat studies indicate however, that those flavonoids analysed so far are more potent inhibitors of 5’DI compared to 5’DII or even 5DIII. Again, this is clear evidence for distinct structure activity relationships among these huge classes of natural phenolic secondary metabolites of plants and also reminds of the distinct differences of competition for thyroid hormone transport proteins, which all bind T4, but have distinct active sites being distinguished by the flavonoid class of ligands.

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Flavonoids are Potent Inhibitors of 5’DI but Affect the Two Other Iodothyronine Deiodinases to a Minor Extent

As mentioned above, the extensive analysis of structure-activity relationships of ligands, both substrates but also inhibitors of type I 5’-deiodinase mainly of rat hepatic origin revealed a detailed picture of the active site of this key enzyme in activation of the prohormone T4 to its thyromimetically active form T3 (Fig. 7.6) [34, 35, 58, 62, 79, 80]. Remarkably, a comparative analysis of thyroid hormone binding to the serum transport and distributor protein TTR revealed such a high similarity of both ligand-binding sites not only for thyroid hormone derivatives but also for flavonoids. The well characterized ligand binding site of the homotetrameric structure of TTR, known from detailed X-ray crystal structure analysis without or with several ligands bound in the active site [81–83], could be used as active site model for 5’DI in order to develop and design new potent ligands for 5’DI and also TTR, both substrates of 5’DI but also inhibitors. The most detailed in vitro and in vivo functional studies have been performed for the two halogenated compounds F21388 and F49209 [26, 28, 30, 31, 63, 66, 84–90]. So far the 5’DI protein is not available in amounts required for structure analysis, as this enzyme is a homodimeric integral membrane protein of the

Figure 7.6. Deiodination of the thyroid prohormone L-Thyroxine T4 to the thyromimetically active T3, catalysed by either type 1 or type 2 5’-deiodinase (5’-D) and inactivation of T4 to the regulatory active reverse T3 (rT3) catalysed by the type 3 5-deiodinase (5-D).

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ER in liver and the baso-lateral membrane in the kidney [91–93]. 5’DI occurs in very low abundance as a hormone metabolising protein and its selenoprotein nature even impairs its purification or in vitro expression as recombinant protein. The same is true for the 5’DII enzymes which might have an even more complex structure and for 5DIII where almost no data exist on physicochemical properties or structural models [17, 94]. Flavonoids are then potent ligands of 5’DI if they are able to adopt a T4 skewed or rT3-like antiskewed structure, if their polarized or dissociated phenolic groups occupy the space at sites comparable to the 4’-phenolate group of T4 or the carboxylate group of the alanine side chain of T4 and if bulky substituents are positioned in the space normally required by the iodine atoms of T4 or rT3. In general methylation or conjugation of the phenolic groups of flavonoids as found in many natural compounds reduces their inhibitory potency both for 5’DI and TTR. Aglycones are in most cases more potent than their glycosides (in contrast to some transporters or other target proteins). Structures where the flavonoid conformation is fixed in a way that rotation between the A-C and B-ring is impaired, especially nonplanar structures are generally more potent in 5’DI inhibition [3, 35, 36, 77, 79, 80]. The position of phenolic groups also markedly affects their inhibitor potency: ortho-position of substituents and formation of a 5- or 6-membered intramolecular hydrogen bond between the 4-keto and the 3- or 6-OH group also favours inhibitory potency. A detailed summary of these observations has been presented before [3, 35, 36, 77, 79, 80].

7.4 Are There Other Effects of Flavonoids Known on the Thyroid Hormone Network of Metabolism and Action? Apart from reductive metabolism of thyroid hormone by the selenoenzyme family of deiodinases the highly reactive 4’-phenolic group of thyroid hormones, polarized by the electron-rich bulky iodine substituents, is a metabolic target relevant for metabolism by conjugating enzymes, both sulfotransferases and UDPG-glucuronyltransferases [79, 95]. So far no evidence has been presented that flavonoids interfere with these metabolic pathways of thyroid hormones, which result in inactivation, enterohepatic recycling or excretion. However, as thyroid hormones are conjugated in 4’-position by enzymes whose major substrates are steroid hormones or phenolic pharmaceuticals a possible interference of flavonoids in these reactions cannot be excluded, as it is well established that some of the flavonoids interfere with steroid hormone conjugation reactions [5]. Again, also no data are available on potential effects of flavonoids on side chain metabolism of thyroid hormones. The alanine side chain of thyroid hormones is substrate both to oxidative decarboxylation and related pathways known for amino acids [79]. However, these reactions are of minor importance in thyroid hormone metabolism, 125

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which is predominantly governed by reductive deiodination and 4’-O-conjugation reactions. Oxidative cleavage of the iodothyronine diphenylether ring has also been reported under special conditions of oxidative burst, leucocyte or macrophage activation during inflammation, sepsis or other infections [79, 96–98]. However, no direct relationship has to be expected for flavonoids in this metabolic pathway as flavonoids do not contain a similar diphenylether ring structure as found in iodothyronines and might solely interfere by their phenolic chemical nature in such oxidative reactions.

7.5 Possible Endpoints for Identification of Nutritional or Environmental Agents Interfering with the Thyroid Hormone System The most prominent endpoint of antithyroid action is goitrogenesis (Tab. 7.3) as initially observed on rabbits kept on goitrogenic cabbage diet irrespective of iodine deficiency induced goitre, In many instances goitrogenesis manifests only at inadequate iodine supply [45]. However, the molecular mechanisms involved in growth of the thyroid gland are not completely understood. The driving force generally is elevated TSH, the main stimulator of thyroid hormone synthesis and secretion [13, 99–101]. As TSH elevations are observed under several conditions leading to hypothyroidism or disturbance of the negative feedback regulation, serum determination of TSH is probably the most prominent parameter to be analysed, as goitrogenesis is a long-term process. However, it has to be considered that TSH elevations also result from other pathophysiological conditions not related to frank hypothyroidism, such as drug effects, non-thyroidal illness (the euthyroid sick syndrome) and other constellations. Due to the strong binding of T4 and its rather long half-life T4 serum levels are a less sensitive but rather specific parameter of disturbed thyroid function especially if a more detailed analysis of total and free hormone levels is performed which takes into consideration altered binding protein levels. The determination of serum binding proteins for thyroid hormones or analysis of occupation of their binding sites by thyroid hormones or potential competitors is not a routine analysis. Other thyroid related parameters such as expression or function of NIS, AIT, ThOx, TPO or TSH-receptor are too meticulous to be used as screening parameter or indicator of interference. In some constellations serum Tg levels might be useful to monitor both the secretory activity of the thyroid gland or as hint towards destructive processes or even as thyroid tumour marker. Also determination of autoantibodies to thyroid components (directed against TPO, Tg or the TSH-receptor), frequently used in clinical practice for diagnosis or monitoring for autoimmune stimulating or destructive processes affecting thyroid function will not be use126

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Table 7.3. Markers, endpoints and tissue-specific readouts of disturbed thyroid function or interference of nutrients, environmental agents, xenobiotics or drugs with the thyroid hormone axis. Parameter

Endpoint or Readout

Possible Interferences

Serum TSH

pituitary, hypo- and hyperthyroidism, systemic indicator

various diseases, stress, drugs,circadian rhythm

Serum T4

thyroid function, hypo- and hyperthyroidism, sytemic indicator

altered binding proteins, drugs

Serum T3

thyroid function, deiodinase action, starvation, systemic indicator

‘euthyroid sick syndrome’

Serum Tg

secretory activity and destructive processes in the thyroid gland

Tg autoantibodies, thyroid tumours

Serum binding proteins for thyroid hormones

altered hepatic function, systemic parameter

drugs, genetic abnormalities, malnutrition (TTR)

Tissue specific proteins or enzymes

malic enzyme, mitochondrial aGPDH, heart MHC, lipid parameters, brain proteins and enzymes

specific regulators of protein expression or enzyme function

ful parameters indicative of early or persistent effects of nutritive or environmental agents interfering with thyroid function. It needs to be remembered, however, that a large fraction of aromatic, phenolic or polymeric synthetic compounds, newly screened in animal models for possible industrial, chemical or pharmaceutical use, fail application due to their prominent interference with thyroid function. One possible explanation for this persistent observation in toxicology might be that thyroid is one of the most vascularized and perfused organs in the body [102, 103]. In addition it is equipped with highly efficient uptake systems not only for iodide but also for other nutrients and metabolites required to perform the synthesis of the huge molecule and Tg and its storage, which serves as a matrix for life-long oxidative H2O2-catalyzed thyroid hormone synthesis in the follicular extracellular colloid space. This compartment is exposed to H2O2 and reactive oxygen species derived therefrom already at the end of the first trimester of foetal development until death of the adult organism. This peculiar and unique prooxidative environment and organisation of the thyroid follicle might provide the basis not only for accumulation and action of the highly reactive phenolic secondary metabolites of plants but also for many other nutritive components, chemicals, xenobiotics and drugs. In addition to TSH, serum thyroid hormones and Tg tissue-specific endpoints of thyroid hormone action might be analysed as indicators of disturbed thyroid hormone status. However, there is no generally valid or integrative 127

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marker or endpoint of thyroid hormone action suitable for simple screening analysis. Several thyroid hormone dependent genes, proteins or enzyme activities will provide evidence for interference with thyroid status [19–21, 104], but many of these parameters are also under influence and control of other nutrients, hormones, agents or (patho-)physiological regulators or pathways. It also needs to be considered, that many thyroid hormone effects are of permissive nature, where thyroid hormone provides a general basis or stage for other more specific agents such as hormones, metabolites or pharmaceuticals to exert their specific action. Serum T3 is a less reliable parameter as readout for thyroid function. Its serum half life is in the order of one day and thus much shorter than that of T4 both due to its lower binding affinity to serum binding proteins and also because its main origin is from extrathyroidal deiodination by 5’DI and 5’DII. T3 formed by the latter deiodinase enzyme probably does not appear in serum and is only formed at the local tissue or cellular level, therefore serum T3 mainly reflects hepatic and to less extent renal and thyroid source respectively alterations occurring in these tissues [17, 94]. In addition, 5’DI is subject to inhibition or decreased expression under conditions summarized as the euthyroid sick syndrome, where increased levels or proinflammatory cytokines, altered levels of growth factors, disturbed nutritional balance and accumulation of disease-related metabolites impairs hepatic (as well as renal and thyroid) 5’DI activity [105–107]. Among the most frequently used readouts for thyroid hormone action at the tissue level are the mitochondrial enzyme glyerolphosphate dehydrogenase (GPDH) (frequently dubbed as endpoint of action with unclear metabolic function for thyroidologists), the cytosolic hepatic malic enzyme, myosin heavy chains of the heart muscle, lipid parameters or several brain enzymes or proteins [20, 21, 108]. Alterations of these parameters should only be interpreted as indicator of altered tissue specific action of thyroid hormone in the context of altered serum TSH and/or thyroid hormone parameters. However, even the latter statement needs to be cautiously interpreted in the light of tissue and even cell-specific control of thyroid hormone uptake, metabolism and action especially due to the prominent potential of local deiodinase activity which efficiently controls ligand availability to the T3-receptor family and thus acts as ‘guardian to the gate’ of thyroid hormone action exerted by the T3 receptors connected in series beyond the deiodinases.

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7.6 Risk Assessment for Compounds Interfering with Thyroid Function or Components of the Thyroid Hormone Axis The notion that the thyroid hormone axis and the thyroid hormone network is regulated in a rather complex, redundant and fail-safe system of mutually connected circuits makes it both difficult to identify the molecular or systemic point of interference. On the other hand this network warrants a huge potential to compensate for disturbances of this essential regulatory network required for adequate foetal and neonatal development and differentiation not only of the brain but also of the whole organism. As thyroid hormones also control most anabolic and catabolic reactions, as well as many pathways of the energy and structural metabolism risk assessment for compounds interfering with this network is not a trivial task. Nevertheless some traditional animal models as well as modern sensitive laboratory techniques provide the necessary tools both for global screening approaches and sophisticated analysis of mode and mechanism of action of compounds interfering with this regulatory permissive hormone system. One of the most sensitive readout system for global action of thyroid hormones is the amphibian metamorphosis from tadpoles to frogs or the related process in the axolotl [109]. Goitrogenesis in many species including man and sensitive laboratory parameters are indicative of disturbed thyroid hormone network [52], The recent development of transgenic and knockout mice now will also enable the generation of biosensor mice [110] for thyroid hormones as already developed and used for the steroid hormones or xenobiotics . Isolated cell lines are and will be of rather limited use in understanding and analysing complex hormonal feedback networks which require an intact vertebrate of even higher mammalian organism in order to extrapolate from controlled laboratory conditions to risks of daily or life long exposure of nutritive, environmental or pharmaceutical agents with potential effects on hormonal axis. Unfortunately the current awareness, public, private, commercial, political and administrative interest appears too much focussed on popular aspects such as mammary cancer, optimal reproductive performance, myths such as declining sperm quality, but tends to almost completely neglect potential hazards such as impaired brain and intellectual as well as global physical development which are under prominent control by the thyroid hormone axis, during early development by that of the pregnant mother but from the second trimester of pregnancy to the end of life by the thyroid of the individual human or animal. Impaired thyroid function still affects millions of people in our country and almost one third of the human population world wide, which is highly vulnerable to interference in the thyroid hormone axis due to still insufficient nutritive iodine supply [111–118].

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Summary Medicinal and nutritional plant constituents, containing various potent polyphenolic compounds, such as flavonoids and isoflavonoids, affect the pituitary-thyroid-periphery axis. They might thus interfere with thyroid hormone feedback regulation, thyroid hormone synthesis and secretion, serum and tissue binding as well as intracellular metabolism of thyroid hormones and their metabolites, resulting in altered thyroid hormone action at the cellular level. Phenolic secondary metabolites of plants, including flavonoids, are active antithyroid ingredients, formerly used in folk medicine to treat thyroid hormone related disorders. Synthetic flavonoids, such as F 21388 have been used as lead compounds to analyse the molecular mode of action. In vitro as well as in vivo studies using animal models revealed that F21388 not only interferes with thyroid hormone synthesis, secretion, binding and metabolism but also crosses the placenta and reaches foetal brain. The main targets identified so far in the thyroid hormone network are thyroperoxidase, the serum distribution protein transthyretin and the key enzyme for thyroid hormone activation, the type 1 5’-deiodinase. Several of these models have been evaluated for in vitro and in vivo screening of polyphenols and endpoints have been identified for target identification and risk assessment of polyphenols and flavonoids as potential “thyroid disrupters”.

Acknowledgment Supported by DFG (Ko-922/7-1,2, Ko-922/8-1,2, Ko-922/12-1)

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25. Middleton, E. Jr.; Kandaswami, C.; Theoharides, T. C. (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 52, 673–751. 26. Köhrle, J.; Fang, S. L.; Yang, Y.; Irmscher, K.; Hesch, R. D.; Pino, S.; Alex, S.; Braverman, L. E. (1989) Rapid Effects of the Flavonoid EMD 21388 on Serum Thyroid Hormone Binding and Thyrotropin Regulation in the Rat. Endocrinology 125, 532–537. 27. Schröder-van der Elst, J. P.; Van der Heide, D.; Rokos, H.; Morreale de Escobar, G.; Köhrle, J. (1998) Synthetic flavonoids cross the placenta in the rat and are found in fetal brain. Am. J. Physiol. 274, E253–E256. 28. Schröder-van der Elst, J. P.; Van der Heide, D.; Rokos, H.; Köhrle, J.; Morreale de Escobar, G. (1997) Different tissue distribution, elimination, and kinetics of thyroxine and its conformational analog, the synthetic flavonoid EMD 49209 in the rat. Endocrinology 138, 79–84. 29. Southwell, B. R.; Duan, W.; Alcorn, D.; Brack, C.; Richardson, S. J.; Köhrle, J.; Schreiber, G. (1993) Thyroxine transport to the brain: Role of protein synthesis by the choroid plexus. Endocrinology 133, 2116–2126. 30. Mendel, C. M.; Cavalieri, R. R.; Köhrle, J. (1992) Thyroxine (T4) transport and distribution in rats treated with EMD 21388, a synthetic flavonoid that displaces T4 from transthyretin. Endocrinology 130, 1525–1532. 31. Schröder-van der Elst, J. P.; Van der Heide, D.; Köhrle, J. (1991) In vivo effects of flavonoid EMD 21388 on thyroid hormone secretion and metabolism in the rat. Am. J. Physiol. 261, E227–E232. 32. Spanka, M.; Hesch, R.-D.; Irmscher, K.; Köhrle, J. (1990) 5l-Deiodination in rat hepatocytes: Effects of specific flavonoid inhibitors. Endocrinology 126, 1660–1667. 33. Köhrle, J.; Fang, S. L.; Yang, Y.; Irmscher, K.; Hesch, R. D.; Pino, S.; Alex, S.; Braverman, L. E. (1989) Rapid effects of the flavonoid EMD 21388 on serum thyroid hormone binding and thyrotropin regulation in the rat. Endocrinology 125, 532–537. 34. Köhrle, J.; Spanka, M.; Irmscher, K.; Hesch, R. D. (1988) Flavonoid Effects on Transport, Metabolism and Action of Thyroid Hormones. In: Cody, V.; Middleton, E.; Harborne, J. B.; Beretz, A. (Ed.) 1, Alan R. Liss, New York, 323–340. 35. Cody, V.; Köhrle, J.; Auflmkolk, M.; Hesch, R. D. (1986) Structure-activity relationships of flavonoid deiodinase inhibitors and enzyme active site models. In: Cody, V.; Middleton, E.; Harborne, J. B. (Ed.) 1, Alan R. Liss, New York, 373–382. 36. Köhrle, J.; Auflmkolk, M.; Spanka, M.; Irmscher, K.; Cody, V.; Hesch, R. D. (1986) Iodothyronine deiodinase is inhibited by plant flavonoids. In: Cody, V.; Middleton, E.; Harborne, J. B. (Ed.) 1, Alan R. Liss, New York, 359–371. 37. Lueprasitsakul, W.; Alex, S.; Fang, S.-L.; Pino, S.; Irmscher, K.; Köhrle, J.; Braverman, L. E. (1990) Flavonoid administration immediately displaces thyroxine (t4) from serum transthyretin, increases serum free T4, and decreases serum thyrotropin in the rat. Endocrinology 126, 2890–2895. 38. Winterhoff, H.; Sourgens, H.; Kemper, F. H. (1983) Antihormonal effects of plant extracts – Pharmacodynamic effects of Lithospermum officinale on the thyroid gland or rats; comparison with the effects of iodide. Horm. Metab. Res. 15, 503–507. 39. Breneman, W. R.; Zeller, F. J.; Carmack, M.; Kelley, C. J. (1976) In vivo inhibition of gonadotropins and thyrotropin in the chick by extracts of lithospermum ruderale. Gen. Comp. Endocrinol. 28, 24–32. 40. Köhrle, J.; Schomburg, L.; Drescher, S.; Fekete, E.; Bauer, K. (1995) Rapid stimulation of type I 5l-deiodinase in rat pituitaries by 3,3l,5-triiodo-L-thyronine. Mol. Cell. Endocrinol. 108, 17–21.

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91. Safran, M. ; Leonard, J. L. (1991) Comparison of the physicochemical properties of type I and type II iodothyronine 5ldeiodinase. J. Biol. Chem. 266, 3233–3238. 92. Leonard, J. L.; Visser, T. J.; Leonard, D. M. (2001) Characterization of the subunit structure of the catalytically active type I iodothyronine deiodinase. J. Biol. Chem. 276 (4), 2600–2607. 93. Leonard, J. L.; Ekenbarger, D. M.; Frank, S. J.; Farwell, A. P.; Köhrle, J. (1991) Localization of type I iodothyronine 5ldeiodinase to the basolateral plasma membrane of rat kidney and LLC-PK1 renal cortical cells. J. Biol. Chem. 266, 11262– 11269. 94. Köhrle, J. (2000) The selenoenzyme family of deiodinase isozymes controls local thyroid hormone availability. Reviews in endocrine & metabolic disorders 1, 49–58. 95. Visser, T. J. (1996) Pathways of thyroid hormone metabolism. Acta Med. Austriaca 23, 10–16. 96. Burger, A. G.; Engler, D.; Buergi, U.; Weissel, M.; Steiger, G.; Ingbar, S. H.; Rosin, R. E.; Babior, B. M. (1983) Ether link cleavage is the major pathway of iodothyronine metabolism in the phagocytosing human leukocyte and also occurs in vivo in the rat. J. Clin. Invest. 71, 935–949. 97. Balsam, A.; Sexton, F.; Borges, M.; Ingbar, S. H. (1983) Formation of diiodotyrosine from thyroxine. Ether-link cleavage, an alternate pathway of thyroxine metabolism. J. Clin. Invest. 72, 1234–1245. 98. Meinhold, H.; Gramm, H.-J.; Meissner, W.; Zimmermann, J.; Schwander, J.; Dennhardt, R.; Voigt, K. (1991) Elevated serum diiodotyrosine (DIT) in severe infections and sepsis: DIT, a possible new marker of leukocyte activity. J. Clin. Endocrinol. Metab. 72, 945–953. 99. Derwahl, M. ; Studer, H. (2001) Nodular goiter and goiter nodules: Where iodine deficiency falls short of explaining the facts. Exp. Clin. Endocrinol. Diabetes 109, 250–260. 100. Dumont, J. E.; Jauniaux, J. C.; Roger, P. P. (1989) The cyclic AMP-mediated stimulation of cell proliferation. TIBS 14, 67–71. 101. Dumont, J. E. (1971) The Action of Thyrotropin on Thyroid Hormone Metabolism. Vitam. Horm. 29, 287ff. 102. Köhrle, J. (1990) Thyrotropin (TSH) action on thyroid hormone deiodination and secretion: One aspect of thyrotropin regulation of thyroid cell biology. Horm. Metab. Res. 23 Suppl., 18–28. 103. Köhrle, J.; Jakob, F.; Schmutzler, C.; Schütze, N. (1999) Angiogenesis and Vascular Remodelling in the Human Thyroid. In: Nawroth, P.; Seibel, M.; Ziegler, R. (Ed.) Berliner Medizinische Verlagsanstalt GmbH, Berlin, 29–39. 104. Flores-Morales, A.; Gullberg, H.; Fernandez, L.; Stahlberg, N.; Lee, N. H.; Vennstrom, B.; Norstedt, G. (2002) Patterns of Liver Gene Expression Governed by TRbeta. Mol. Endocrinol. 16, 1257–1268. 105. Jakobs, T. C.; Mentrup, B.; Schmutzler, C.; Dreher, I.; Kohrle, J. (2002) Proinflammatory cytokines inhibit the expression and function of human type I 5l-deiodinase in HepG2 hepatocarcinoma cells. Eur. J. Endocrinol. 146, 559–566. 106. Papanicolaou, D. A. (2000) Euthyroid sick syndrome and the role of cytokines. Reviews in endocrine & metabolic disorders 1, 43–48. 107. Bartalena, L.; Bogazzi, F.; Brogioni, S.; Grasso, L.; Martino, E. (1998) Role of cytokines in the pathogenesis of the euthyroid sick syndrome. Eur. J. Endocrinol. 138, 603–614. 108. Dümmler, K.; Müller, S.; Seitz, H. J. (1996) Regulation of adenine nucleotide translocase and glycerol 3-phosphate dehydrogenase expression by thyroid hormones in different rat tissues. Biochem. J. 317, 913–918.

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109. Tata, J. R. (1999) Amphibian metamorphosis as a model for studying the developmental actions of thyroid hormone. Biochimie 81, 359–366. 110. Ciana, P.; Di Luccio, G.; Belcredito, S.; Pollio, G.; Vegeto, E.; Tatangelo, L.; Tiveron, C.; Maggi, A. (2001) Engineering of a mouse for the in vivo profiling of estrogen receptor activity. Mol. Endocrinol. 15, 1104–1113. 111. Delange, F. (2002) Iodine deficiency in Europe and its consequences: an update. Eur. J Nucl. Med Mol. Imaging 29, 404-416. 112. Delange, F.; de Benoist, B.; Pretell, E.; Dunn, J. T. (2001) Iodine deficiency in the world: where do we stand at the turn of the century? Thyroid 11, 437–447. 113. Delange, F. (2001) Iodine deficiency as a cause of brain damage. Postgrad. Med. J. 77, 217–220. 114. Morreale, d. E.; Obregon, M. J.; Escobar, d. R. (2000) Is neuropsychological development related to maternal hypothyroidism or to maternal hypothyroxinemia? J. Clin. Endocrinol. Metab 85, 3975–3987. 115. Soriguer, F.; Millon, M. C.; Munoz, R.; Mancha, I.; Lopez Siguero, J. P.; Martinez Aedo, M. J.; Gomez-Huelga, R.; Garriga, M. J.; Rojo-Martinez, G.; Esteva, I.; Tinahones, F. J. (2000) The auditory threshold in a school-age population is related to iodine intake and thyroid function. Thyroid 10, 991–999. 116. Hetzel, B. S. (2000) Iodine and neuropsychological development. J. Nutr. 130, 493–495. 117. Dobson, J. E. (1998) The iodine factor in health and evolution. The Geographical Review 88, 1–28. 118. Glinoer, D. (1997) Maternal and Fetal Impact of Chronic Iodine Deficiency. Clin. Obstet. Gynecol. 40, 102–116.

8 Host Factors Relevant for Bioavailabilty: Transporters, Metabolizing Enzymes and Genetic Polymorphisms Dieter Schrenk*

8.1 Introduction After administration/intake of drugs, chemicals and food contaminants or certain food constituents these are metabolized by proteins/enzymes of drug metabolism. In most higher organisms the predominant portion of the organism’s xenobiotic-metabolizing capacity is localized in the liver or the gut wall, while most other organs also exhibit certain activities. When, e. g., a secondary plant metabolite present in food reaches the intestinal mucosa or the liver (via the portal blood stream), it has a certain chance to enter an epithelial cell (enter-

* Food Chemistry and Environmental Toxicology, University of Kaiserslautern, 67663 Kaiserslautern, Germany

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ocyte or hepatocyte). This can occur either by passive diffusion, depending on the physicochemical properties of the molecule, or by active transport. A variety of uptake transporters reside at the outer membrane of a variety of cell types such as the enterocyte or the hepatocyte taking up substrates with certain structural/functional properties. For example, many organic anions are taken up by members of the organic anion transporter (OATP) family. After having entered the enterocyte (or hepatocyte), certain enzymes introduce functional groups (if required) into the molecule. The usual mechanism of introducing those groups (e. g. hydroxyl-, keto-, carboxy-functions) is by activation of oxygen and subsequent oxygenation of the substrate carried out by members of the cytochrome P450 (CYP) superfamily (‘phase I of drug metabolism‘). The reduction of quinones to hydroquinones is catalyzed by NAD(P)H quinone oxidoreductases (NQOs). Those molecules/metabolites bearing functional groups are conjugated with bulky, highly polar groups such as glucuronate, sulfate or glutathione (‘phase II of drug metabolism‘). The conjugations usually are catalyzed by members of enzyme superfamilies of UDP-glucuronosyltransferases (UDPGTs), sulfotransferases (STs), glutathione-S-transferases (GSTs) or Nacetyltransferases (NATs). The conjugates thus formed usually cannot readily leave the cell but have to be pumped out by members of the multidrug resistance protein (MRP) family of conjugate export pumps. Apical localization of export pumps allows the export into the intestinal lumen or into bile whereas basolateral localization results in export into the blood circulation. In addition, non-conjugated xenobiotics (or bile acid analogues) can be eliminated into the intestinal lumen or into bile by the transmembrane export pumps MDR1/P-glycoprotein or the bile salt export pump (BSEP), respectively.

8.2 Modulating Factors 8.2.1

Induction of Drug Metabolizing Proteins

A variety of endogenous and exogenous factors can modulate the sequence of events in drug/xenobiotic metabolism. Prominent examples are the action of inducers and inhibitors. Well-known examples of inducers of phase I and II of drug metabolism are the hypnotic drug phenobarbital, the anti-hyperlipidemic drug clofibrate, the antibiotic rifampin, and the contaminant TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) which induce certain isozymes of phase I and II of drug metabolism. Most inducers act via complex signal tranduction pathways which comprise a receptor/sensor, signal transduction, and finally activation/action of/as a transcription factor resulting in the enhanced transcription of the induced genes. A variety of receptors and other factors involved in these scenarios have been discovered in recent years (Tab. 8.1). 138

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Table 8.1. Inducible genes, inducers, and signal transduction pathways of induction of drug/xenobiotic-metabolizing proteins. Inducible gene/protein

Prototype inducer(s)

receptor/transcription factor – consensus element; or other mechanism

CYP1A1, 1A2, 1B1, GSTYa, UGT1A6, NQO1

TCDD, non-ortho-subst. PCBs, PAHs

Ah receptor/ARNT complex – XRE

CYP2B1, 2B2, 2B6, UGT2B1, GST5-5

Phenobarbital, DDT, TCPOBOP, rifampin, ‘nondioxinlike‘ PCBs

CAR/RXR – PBREM

CYP3A(4), 3A11, 7A1, OATP2, MRP2, MDR1/Pgp

Clotrimazole, lithocholic acid, pregnenolone-16acarbonitrile, rifampin,

PXR/RXR –

CYP4A GSTYa, GSTM4, GSTP1, NQO1

Clofibrate t-butylated hydroquinone

PPARa/RXR – PPRE Nrf2 – ARE

CYP2E1

Isoniazid, ethanol

Blocking of cAMP-dependent phosphorylation of Ser129 (stabilization of the enzyme)

‘Monofunctional’ inducers which predominantly induce GSTs and related conjugating enzymes are t-butylhydroquinone and related redoxactive compounds. A number of them act via an antioxidant-responsive element in the 5’-flanking region of responsive genes. Furthermore, MDR1/Pglycoprotein and certain MRPs such as MRP2 are also inducible by xenobiotics (Fig. 8.1). However, recent results suggest that the inducibility can differ markedly between species and cell types [1].

Figure 8.1. Induction of MRP2 gene expression in rat hepatocytes in primary culture treated with by quercetin. Total RNA was prepared from hepatocytes 48 h after adition of quercetin to the medium. Expression was analyzed by semiquantitative RT-PCR.

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Table 8.2. Inducible genes of drug/xenobiotic-metabolizing proteins and inducing food constituents/natural compounds/factors. Inducible gene/protein

Inducing food constituent/natural compound/factor

CYP1A1, 1A2, 1B1, GSTYa, UGT1A6, NQO1

Smoking, charcoal-broiled beef, flavone, furocoumarins*, indole-3-carbinol (indole-[2,3b]carbazole) BHA, BHT, flavone St.-John’s wort

CYP2B1, 2B2, 2B6, UGT2B1, GST5-5 CYP3A(4), 3A11, 7A1, OATP2, MRP2, MDR1/Pgp CYP4A GSTYa, GSTM4, GSTP1, NQO1 CYP2E1

? Broccoli, ellagic acid, diallyl sulfide, sulphoraphane Ethanol

*see Ref. [7]

Our knowledge on the inducing properties of natural compounds present in food is rather small with the exception of ‘monofunctional’ inducers which act by the Nrf2/ARE-pathway. A variety of plant constituents present in cruciferous vegetables such as broccoli, in garlic, onions, rosemary, green tea, fruits etc. were described to act in this way. Their consumption was recommended in order to induce ‘detoxifying’ enzymes of drug metabolism which may reduce or prevent deleterious effects of chemical carcinogens and/or other potentially harmful compounds (‘chemoprevention’). In some

Figure 8.2. Metabolic activation of 4-aminobiphenyl after ‘dioxin-type’ induction [2]. Rat hepatocytes in primary culture were incubated with 50 mM 4-aminobiphenyl. 4-Aminobiphenyl metabolites and post-labeled modified nucleotides derived from nuclear DNA were analyzed.

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instances, however, the effects of a massive consumption of such inducers on other pathways of drug metabolism and/or other events in cellular regulation have not been studied sufficiently. In particular chemical carcinogens such as PAH, aromatic and heterocyclic amines present in tobacco smoke may be activated to a larger extent when induction of drug metabolism has occured. Likewise, metabolic activation of 4-aminobiphenyl, a carcinogen present in tobacco smoke, was significantly enhanced when rat hepatocytes were pretreated with ‘dioxinlike’ inducers (Fig. 8.2). Under these conditions modified nucleotides derived from nuclear hepatocyte DNA and N-hydroxylated, metabolically activated, metabolites were increased significantly [2]. Decreased levels in circulating thyroid hormones were also reported as a consequence of ‘dioxin-type’ induction (reviewed in Ref. [3]).

8.2.2 Genetic Polymorphisms Genetic polymorphisms of drug-metabolizing enzymes have been discovered, e. g., following clinical observations that treatment with certain drugs result in abnormally low or high blood levels and/or metabolite levels in blood in certain individuals. Molecular gene analysis revealed that in most of these cases changes/mutations in the genes encoding certain drug-metabolizing enzymes are responsible for these effects. These include single nucleotide polymorphisms (SNPs) leading to amino acid exchanges and/or truncated mRNA, intronic changes with shown or suggested effects on mRNA processing (stability, splicing etc.), and changes in genomic structure (gene amplifications etc.). Examples for the effects of these alterations and corresponding substrates are listed in Tab. 8.3. An example for a genetic polymorphism of an ABC export pump possibly relevant for the kinetics/blood levels of dietary constituents is the Arg433 ser mutation in the human MRP1 gene [4]. Transport studies in vesicles showed that the mutant exhibits a ca. 50 % reduced transport activity towards the standard substrate LTC4, a GSH conjugate, and that of estrogen-3-sulfate in the presence of 2 mM GSH. However, activity towards the substrate estradiol-17ß-glucuronide was not affected in the absence of GSH by the mutation suggesting that the mutation is critical for the transport of substrates bearing/requiring a GSH moiety. Analysis of transport kinetics in vesicles isolated from cells stably transfected human embryo kidney cells revealed that apparent vmax but not KM for LTC4 was affected. Examples for ABC export pumps relevant for the kinetics of food constituents are ABCG5 and ABCG8 which regulate the bioavailability of dietary ß-sitosterol. Mutation in this genes lead to sitosterolemia, an autosomal recessive disorder resulting in hyperabsorption of a variety of sterols including ß-sitosterol [5, 6]. 141

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Table 8.3. Genetic polymorphisms of drug-metabolizing enzymes (examples). Gene/enzyme

Type of mutation/effect

Substrate(s); examples

CYP2C9

Exonic (CYP2C9*2, CYP2C9*3)

CYP2C19

Exonic (CYP2C19*2 – CYP219*8) Exonic (various), ‘poor metabolizer’ Amplification (CYP2D6*2), increased activity ‘Null phenotype’

Diclofenac, phenytoin, THC, S-warfarin Diazepam, imipramine, propranolol i 150 drugs

CYP2D6 CYP2D6 GSTM1 GSTT1 NAT1 NAT2 MDR1/Pgp

MRP1 MRP2

‘Slow acetylator’ (various) Intronic (decreased expression in the duodenum) Exonic (Arg433Ser)*; reduced activity towards LTC4 Exonic (various); Dubin-Johnson syndrome

i 150 drugs Dichloroethane, dichloromethane, hexachlorobutadiene, various electrophiles Aromatic and heterocyclic (PhIP, MeIQ etc.) amines, isoniazid Lipophilic, uncharged or cationic organic molecules with a ’bulky’ structure LTC4, conjugates, cotransport with GSH LTC4, bilirubin glucuronide, conjugates, cotransport with GSH

* see Ref. [4]

8.3 Summary and Conclusive Recommendations Drug metabolizing enzymes present in the liver, intestine and many other tissues represent an important physiological defense system towards contaminants and non-nutritive constituents in the diet. A major function of the drug-metabolizing system is the metabolic conversion of certain xenobiotics/exogenous compounds into water-soluble, biologically inactive metabolites which can be excreted rapidly by the organism. A possible influence of dietary factors on drug-metabolizing enzymes/ proteins is induction. This effect can result, e. g., in an accumulation or decrease in endogenous substrates of those enzymes and/or in an accumulation/enhanced clearance of other exogenous substrates such as drugs or dietary constituents. Desired effects include the induction of conjugating phase II enzymes which can catalyze the detoxification of metabolites of certain chemical carcinogens. Since a variety of inducers act simultaneously as inhibitors of the inducible enzymes, it is rather difficult to make any predictions on the consequences for the organism. These consequences clearly depend on a number of parameters such as frequency of intake, dosage, bioavailability etc. 142

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The consequences of a number of genetic polymorphisms of drug-metabolizing proteins for the metabolism of certain drugs have been investigated in recent years. In contrast, our knowledge on the possible effects of these polymorphisms on the fate of food constituents including possible beneficial or adverse effects is just at the beginning.

Abbreviations ABC, ATP-binding cassette; AhR, aryl hydrocarbon receptor; ARE, antioxidant-responsive element; ARNT, AhR nuclear translocator; BHA, butylated hydroxyanisol; BHT, butylated hydroxytoluene; CAR, constitutively active or constitutive androstane receptor; CYP, cytochrome P450; EROD, 7-ethoxyresorufin O-deethylase; GSH, glutathione; LTC4, leukotriene C4; NQO, NAD(P)H quinone-oxidoreductase; GST, glutathione S-transferase; MDR/ Pgp; multidrug resistance/P-glycoprotein; MRP, multidrug resistance protein; NAT, N-acetyl transferase; OATP, organic anion transporter; Nrf, Nuclear factor-erythroid 2(NF-E2) related factor; PBREM, phenobarbital-responsive enhancer element; PCB polychlorinated biphenyl; PPR, peroxisome proliferator receptor; PPRE, peroxisome proliferator-responsive element; PROD, 7-pentoxyreorufin O-dealkylase; PXR, pregnane X-receptor; RXR, retinoid X-receptor; SNP single-nucleotide polymorphism; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene; THC, D9-tetrahydrocannabinol; UDPGT, uridine-diphosphate glucuronosyl transferase; XRE, xenobiotic-responsive element.

Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Bonn, the Federal Ministry of Education and Research (BMBF), Bonn, and the Government of the State of Rhineland-Palatinate, Mainz, Germany.

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References 1. Kauffmann H-M, Pfannschmidt S, Zöller H, Benz A, Vordestemann B, Webster JI, Schrenk D (2002): Influence of redox-active compounds and PXR-activators on human MRP1 and MRP2 gene expression. Toxicology 171, 137–146. 2. Orzechowski A, Schrenk D, Schut HAJ, Bock KW (1994) Consequences of 3 methylcholanthrene-type induction for the metabolism of 4-aminobiphenyl in isolated rat hepatocytes. Carcinogenesis 15, 489–494. 3. Schrenk D (1998): Consequences of ‘dioxin-type‘ induction of drug-metabolizing enzymes for the metabolism of endo- and xenobiotics – a commentary. Biochem Pharmacol 55, 1155–162. 4. Conrad S, Kauffmann H-M, Ito K-I, Leslie EM, Deeley RG, Schrenk D, Cole SPC (2002): Mutation of Arg433 in the multidrug resistance protein 1 (MRP1/ABCC1) results in a selective decrease in organic anion transport and in altered drug resistance. Pharmacogenetics, in press. 5. Hubacek JA, Berge KE, Cohen JC, Hobbs HH (2001): Mutations in ATP-cassette binding proteins G5 (ABCG5) and G8 (ABCG8) causing sitosterolemia. Hum Mutat 18, 359–360. 6. Schmitz G, Langmann T, Heimerl S (2001): Role of ABCG1 and other ABCG family members in lipid metabolism. J Lipid Res 42, 1513–1520. 7. Baumgart et al., in preparation.

9 Food Matrix and Related Factors Affecting Bioavailability Karin H. van het Hof* and Johan M. M. van Amelsvoort

Abstract Bioavailability can be defined as the proportion of a nutrient from a diet or food that becomes available after intestinal absorption for utilization in physiologic functions. With respect to the safety and efficacy of functional foods, there are two important aspects to consider: 1. the availability of a functional ingredient from the carrier; 2. the impact of an (added) functional ingredient on the bioavailability of other nutrients present in the food or diet. In this chapter, the importance of both aspects will be discussed by using carotenoids and folate as examples.

* Unilever Health Institute, Unilever Research & Development, Vlaardingen, The Netherlands

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9.1 Introduction Bioavailability can be defined as the proportion of a nutrient from a diet or food that becomes available after intestinal absorption for utilization in physiological functions. This definition indicates the importance of bioavailability for health: if a nutrient present in the diet or a (functional) food, does not become available to express its function in the body, the presence of that nutrient in the diet has no nutritional relevance. With respect to the safety and efficacy of functional foods, there are two important aspects to consider: 1. the availability of a functional ingredient from the carrier; 2. the impact of an (added) functional ingredient on the bioavailability of other nutrients present in the food or diet. In this chapter, the importance of both aspects will be discussed by using carotenoids and folate as examples. In humans, some carotenoids (a-carotene, b-carotene and b-cryptoxanthin) can be converted into vitamin A. In addition, carotenoids are potent antioxidants [1] and they have been shown to enhance intercellular communication [2] and interfere with the immune system [3]. Therefore, they are thought to contribute to the inverse relationship between fruit and vegetable consumption and the risk of coronary heart disease and some types of cancer [4, 5]. However, currently, the role of carotenoids in the prevention of chronic diseases is still a hypothesis and not yet confirmed by intervention trials on the effect of carotenoid supplementation at dietary intake levels [6, 7]. Folate is a B-vitamin which is involved in various metabolic processes. Supplementation with folic acid around conception decreases the risk of women having offspring with neural tube defects [8, 9]. In addition, increased folate intake decreases elevated plasma levels of homocysteine, which is considered to be a risk factor for cardiovascular disease [10]. Most studies on the effect of different food-related factors have determined the (relative) bioavailability in humans by measurement of plasma responses of carotenoids and folate and comparing these responses with each other. For folate, Brouwer et al. [11] introduced the term ‘bioefficacy’, comparing the reduction of plasma homocysteine concentrations following folate or folic acid supplementation.

9.2 Absorption of Carotenoids and Folate Carotenoids are fat-soluble compounds and are absorbed in the small intestine along with dietary fat in the mixed micelles as described previously [12]. Folate is a water-soluble vitamin and is primarily transported across the brush-border membrane by a saturable carrier system. At high intake levels, folate can be taken up by diffusion. 145

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Folate refers to the form of folate naturally present in foods and folic acid is the synthetic variant which is used for fortification and in dietary supplements. Approximately 80 % of all food folate exists as polyglutamates which have to be cleaved into folate monoglutamates before absorption. This is an enzymatic process, mediated by pteroylpolyglutamate hydrolase (conjugase) which is present in the intestinal brush border.

9.3 Bioavailability of Carotenoids and Folate from the Carrier Food Various studies have shown that the matrix in which carotenoids are present largely determines their bioavailability. The plasma responses of b-carotene following supplementation with purified b-carotene are much higher than those induced by vegetables [13]. Fig. 9.1 shows the plasma responses of b-carotene following four weeks supplementation with either vegetables or b-carotene added to oil, both providing approximately the same amount of b-carotene. In the same study, a higher plasma response was found also for lutein, following the diet supplemented with purified lutein added to oil than following the diet supplemented with vegetables, although the relative differences were smaller than those found for b-carotene. The relative bioavailability of b-carotene from vegetables compared with the added b-carotene was 14 % and that of lutein 67 % [13].

Figure 9.1. Carotenoids are more bioavailable from oil than from vegetables. Mean baseline plasma concentrations of carotenoids and changes after four weeks consumption of diets providing low or high amounts of vegetables or a low vegetable diet supplemented with b-carotene and lutein added to oil (n = 10–22 healthy adults per group; amount of carotenoids provided shown between brackets (mg/d)). Based on [13].

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Also for folate, significantly higher responses of folate concentrations in plasma and red blood cells have been found after supplementation with folic acid vs consumption of vegetables. Sauberlich et al. [14] found a relative bioavailability of I 50 % from vegetables. Based on this study and the study by Cuskelly et al. [15], the US Food and Nutrition Board [16] has defined ‘dietary folate equivalents’ stating that 1 mg food folate equals 0.5 mg folic acid (on an empty stomach). More recently, Brouwer et al. [17] showed somewhat smaller differences in responses following vegetables and citrus fruit vs a supplement (relative bioavailability of 78 % based on plasma response and 98 % based on red blood cell folate). However, in this study, the folic acid supplement of 250 mg/d was given as a 500 mg supplement every other day. The authors suggest that at this high level, the absorption and metabolism of folic acid may have been less efficient, thus overestimating the bioavailability from vegetables and fruits. In addition, also between different types of vegetables, major differences have been found in their ability to increase plasma responses of carotenoids in particular [18]. Broccoli and green peas were better sources of b-carotene than spinach, despite a 10-fold lower b-carotene content. Although less pronounced, a similar phenomenon was found for lutein. Further, De Pee et al. [19] showed that fruits were twice as good a source of b-carotene than a mix of spinach and carrots, based on an assessment of the changes in serum retinol concentrations in anemic children. For both carotenoids and folate, a disruption of the vegetable matrix by pureeing and/or heating improves the plasma responses of these nutrients (10–210 %). For carotenoids, this has been shown for b-carotene from spinach as well as from carrots [18, 20–22] and also for lycopene from tomatoes [12, 23, 24]. For folate, the effect has only been determined for spinach [18, 25]. In summary, when considering the safety and efficacy of a functional ingredient in a food, the carrier in which it is present or to which it has been added may largely impact its bioavailability and thus its efficacy. For example, when adding carotenoids to a fat-matrix, such as an oil or fat spread, the dosage required to be effective is likely to be much lower than that present in vegetables. With respect to the safety, Paracelsus (1493– 1541) already noted that “All substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy”. If the bioavailability from a certain (food) matrix is superior, it should be realised that toxicity may occur at much lower dosages than envisaged from the habitual food sources. This may for instance be one of the reasons for the findings in the long term intervention studies on the effect of b-carotene supplementation on the risk of lung cancer, which found an increased risk in the supplemented group [26, 27]. The dosages used in these studies (20–30 mg/d) were, when translated to ‘natural foods’, ca. 150–210 mg/d, when taking into account the superior (relative) bioavailability of pure b-carotene. This equals to 50–70 times the average normal dietary intake from natural sources of ca. 3 mg b-carotene per day [28–30].

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9.4 Impact of Functional Ingredients on the Bioavailability of Carotenoids and Folate When adding a functional ingredient to the diet, its impact on the bioavailability of other nutrients should be considered in the evaluation of the nutritional implications of such an ingredient. This is not only important for novel ingredients, normally not present in the diet, but also for ‘natural’ compounds, such as for example dietary fibre. Riedl et al. [31] showed that addition of different types of fibre to a single dose of purified carotenoids significantly reduced the subsequent increase of the plasma carotenoid concentrations by between 18 % and 73 % as compared to a supplement without added dietary fibre. Dietary fibre may interfere with the formation and uptake of mixed micelles containing the carotenoids into the mucosa of the small intestine. For folic acid, the bioavailability seems reduced when ingested in conjunction with other foods, such as maize, rice and bread [32]. However, wheat bran and beans did not decrease the folic acid responses in plasma following a single dose. In contrast, the wheat bran tended to show an enhancing effect [33]. Dietary fibre may interact with enhancing as well as inhibiting factors of folate absorption, although the authors of the latter study do not exclude the possibility that the wheat bran itself contained additional folate. As carotenoids are absorbed along with dietary fat in the mixed micelles, indigestible fat, like sucrose polyesters has been shown to reduce the bioavailability of carotenoids. Four weeks consumption of a spread with the fat-replacer sucrose polyester (12.4 g/d), reduced plasma carotenoid concentrations by 20–52 %, with the largest impact on the most lipophillic carotenoids, b-carotene and lycopene [34]. Also plant sterols, which effectively reduce blood cholesterol by inhibition of cholesterol absorption, reduce plasma carotenoid concentrations. However, the effect shown is smaller than that of sucrose polyester (i. e. 15–19 % with 3 g/d of plant sterols) [35]. These data indicate that various dietary factors may interfere with the absorption and bioavailability of carotenoids and folate and that when designing functional foods, the impact of the added active ingredient should be considered.

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9.5 Conclusion and Implications In order to determine the nutritional impact of a functional ingredient and the change in bioavailability of other nutrients, the natural variation in bioavailability as well as the natural variation in the body’s status should be taken into account. In particular for carotenoids, a large variation in bioavailability among different foods exists. Even among different vegetable types and between fruits and vegetables, substantial differences have been found in relative bioavailability of carotenoids (2–10-fold differences) [18, 19]. Further, plasma levels of carotenoids vary during the year with coefficients of variation within individuals mounting to 22–46 % [36, 37]. In conclusion, the impact of a functional ingredient on the bioavailability of other nutrients as well as the bioavailability of the functional ingredient itself should be taken into account in the evaluation of the safety and efficacy of a functional food. However, the nutritional relevance of a possible negative impact should be rated against the natural variation in blood levels of the compounds, which can be substantial in the case of carotenoids.

References 1. Sies H, Stahl W (1995) Vitamins E and C, b-carotene and other carotenoids as antioxidants. Am. J. Clin. Nutr. 62, 1315–1321. 2. Zhang L-X, Cooney RV, Bertram JS (1991) Carotenoids enhance gap junctional communication and inhibit lipid peroxidation in C3H/10T1/2 cells: relationship to their cancer chemopreventive action. Carcinogenesis 12, 2109–2114. 3. Santos MS, Meydani SN, Leka L, Wu D, Fotouhi N, Meydani M, Hennekens CH, Gaziano JM (1996) Natural killer cell activity in elderly men is enhanced by b-carotene supplementation. Am. J. Clin. Nutr. 64, 772–777. 4. Peto R, Doll R, Buckley JD, Sporn MB (1981) Can dietary beta-carotene materially reduce human cancer rates? Nature 290, 201–208. 5. Van Poppel G (1996) Epidemiological evidence for b-carotene in prevention of cancer and cardiovascular disease. Eur. J. Clin. Nutr. 50, 57–61. 6. IARC (1998) Handbooks of cancer prevention. Volume 2 Carotenoids. Lyon, France. 7. Institute of Medicine (2000), Standing Committee on the Scientific Evaluation of Dietary Reference Intakes. Dietary reference intakes for vitamin C, vitamin E, selenium and carotenoids. National Academy Press, Washington DC, 2000. 8. Czeizel AE, Duda`s, I (1992) Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N. Engl. J. Med. 327, 1832–1835. 9. Medical Research Council Vitamin Study Research Group. (1991) Prevention of neural tube defects: Results of the Medical Research Council Vitamin Study. Lancet 338, 131–137. 10. Boushey CJ, Beresford SA, Omenn GS, Motulsky AG (1995) A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. J. Am. Med. Assoc. 274, 1049–1057.

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11. Brouwer IA, Van Dusseldorp M, West CE, Steegers-Theunissen RPM (2001) Bioavailability and bioefficacy of folate and folic acid in man. Nutr. Res. Rev. 14, 267–293. 12. Van het Hof KH, West CE, Weststrate JA, Hautvast JGAJ (2000) Dietary factors that affect the bioavailability of carotenoids. J. Nutr. 130, 503–506. 13. Van het Hof KH, Brouwer IA, West CE, Haddeman E, Steegers-Theunissen RPM, Van Dusseldorp M, Weststrate JA, Eskes TKAB, Hautvast JGAJ (1999) Bioavailability of lutein from vegetables is five times higher than that of b-carotene. Am. J. Clin. Nutr. 70, 261–268. 14. Sauberlich HE, Kretsch MJ, Skala JH, Hornson HL, Taylor PC (1987) Folate requirement and metabolism in non-pregnant women. Am. J. Clin. Nutr. 46, 1016–1028. 15. Cuskelly GJ, McNulty H, Scott JM (1996) Effect of increasing dietary folate on red-cell folate: implications for prevention of neural tube defects. Lancet 347, 657–659. 16. Institute of Medicine (1998) Standing Committee on the Scientific Evaluation of Dietary Reference Intakes. Dietary reference intakes for thiamin, riboflavin, nicacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin and choline. National Academy Press, Washington DC, 1998. 17. Brouwer IA, Van Dusseldorp M, West CE, Meyboom S, Thomas CMG, Duran M, Van het Hof KH, Eskes TKAB, Hautvast JGAJ, Steegers-Theunissen RPM (1999) Dietary folate from vegetables and citrus fruit decreases plasma homocysteine concentrations in humans in a dietary controlled trial. J. Nutr. 129, 1135–1139. 18. Van het Hof KH, Tijburg LBM, Pietrzik K, Weststrate JA (1999) Bioavailability of carotenoids and folate from different vegetables. Effect of disruption of the vegetable matrix. Br. J. Nutr. 82, 203–212. 19. De Pee S, West CE, Permaesih D, Martuti S, Muhilal, Hautvast JGAJ (1998) Orange fruit is more effective than are dark-green, leafy vegetables in increasing serum concentrations of retinol and b-carotene in schoolchildren in Indonesia. Am. J. Clin. Nutr. 68, 1058–1067. 20. Castenmiller JJM, West CE, Linssen JPH, Van het Hof KH, Voragen AGJ (1999) The food matrix of spinach is a limiting factor in determining the bioavailability of b-carotene and to a lesser extent of lutein in humans. J. Nutr. 129, 349–355. 21. Rock CL, Lovalvo JL, Emenhiser C, Ruffin MT, Flatt SW, Schwartz SJ (1998) Bioavailability of b-carotene is lower in raw than in processed carrots and spinach in women. J. Nutr. 128, 913–916. 22. Edwards AJ, Nguyen CH, You C-S, Swanson JE, Emenhiser C, Parker RS (2002) a- and b-carotene from a commercial carrot puree are more bioavailable to humans than from boiled-mashed carrots, as determined using an extrinsic stable isotope reference method. J. Nutr. 132, 159–167. 23. Gärtner C, Stahl W, Sies H (1997) Lycopene is more bioavailable from tomato paste than from fresh tomatoes. Am. J. Clin. Nutr. 66, 116–122. 24. Porrini M, Riso P, Testolin G (1998) Absorption of lycopene from single or daily portions of raw and processed tomato. Br. J. Nutr. 80, 353–361. 25. Castenmiller, JJM, Van der Poll CJ, West CE, Brouwer IA, Thomas CMG, Van Dusseldorp M (2000) Bioavailability of folate from processed spinach in humans. Ann. Nutr. Metab. 44, 163–169. 26. ATBC Cancer Prevention Study Group (1994) The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N. Engl. J. Med. 330, 1029–1035. 27. Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, Keogh JP, Meyskens FL, Valanis B, Williams JH, Barnhart S, Hammar S (1996) Effects of a

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combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. N. Engl. J. Med. 334, 1150–1155. Chug-Ahuja JK, Holden JM, Forman MR, Mangels R, Beecher GR, Lanza E (1993) The development and application of a carotenoid database for fruits, vegetables and selected multicomponent foods. J. Am. Diet Assoc. 93, 318–323. Scott KJ, Turnham DI, Hart DJ, Bingham SA, Day, K. (1996) The correlation between the intake of lutein, lycopene and beta-carotene from vegetables and fruits, and blood plasma concentrations in a group of women aged 50–65 years in the UK. Br J Nutr 75, 409–418. Goldbohm RA, Brants HAM, Hulshof KFAM, Van den Brandt PA (1998) The contribution of various foods to intake of vitamin A and carotenoids in the Netherlands. Internat. J. Vit. Nutr. Res. 68, 378–383. Riedl J, Linseisen J, Hoffmann J, Wolfram G (1999) Some dietary fibres reduce the absorption of carotenoids in women. J. Nutr. 129, 2170–2176. Colman N, Green R, Metz J (1975) Prevention of folate deficiency by food fortification. II. Absorption of folic acid from fortified staple foods. Am. J. Clin. Nutr. 28, 459–464. Keagy PM, Shane B, Oace SM (1988) Folate bioavailability in humans: effects of wheat bran and beans. Am. J. Clin. Nutr. 47, 80–88. Weststrate JA, Van het Hof KH (1995) Sucrose polyester and plasma carotenoid concentrations in healthy subjects. Am. J. Clin. Nutr. 62, 591–597. Weststrate JA, Meijer GW (1998) Plant sterol-enriched margarines and reduction of plasma total- and LDL-cholesterol concentrations in normocholesterolaemic and mildly hypercholestrolaemic subjects. Eur. J. Clin. Nutr. 52, 334–343. Olmedilla B, Granado F, Blanco I, Rojas-Hidalgo E (1994) Seasonal and sex-related variations in six serum carotenoids, retinol and a-tocopherol. Am. J. Clin. Nutr. 60, 106–110. Saintot M, Astre C, Scali J, Gerber M (1995). Within-subjects seasonal variation and determination of inter-individual variations of plasma beta carotene. Int. J. Vit. Nutr. Res. 65, 169–174.

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10 Toxicokinetics/Toxicodynamics Ron Walker*

The contribution of Prof. Dr. Ron Walker was not infull. However, since the slides provided represent the essentials in a highly condensed and clear cut form, it was decided to include them into the proceedings.

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11 Biomarkers to Assess Safety Aspects and Functional Effects of Food Beatrice L. Pool-Zobel*

Summary Functional foods contain ingredients with diverse biological activities, some of which may not be beneficial at high concentrations; therefore it is necessary to discriminate between dietary risk and dietary chemoprevention when evaluating novel foods with high levels of functional ingredients. Current biomarkers of toxicology (e. g. detection of genetic damage in peripheral blood lymphocytes) are being used to assess how dietary intervention with the foods reduces base line levels of toxic damage. Accordingly, if the genetic damage is not modulated in a favourable way, they will equally indicate potential risks. Biomarkers using faecal water and cells from colon biopsies are being developed to assess how food intake relates to enhancing or reducing risks specifically for the colon. Chemical analysis of the faeces reveals the overall load of genotoxins or of chemoprotective components in the gut. The balance of the opposing factors can be detected by biological analysis of the faecal water. An example is to study genotoxic effects of faecal water in colonocytes. Methods to measure specific gene sequences in DNA of exfoliated cells hold promise to yield non-invasive biomarkers of susceptibility that can be used routinely in wide scale monitoring. In addition to the techniques of toxicological assessment and exposure monitoring, new end points which indicate the chemoprotective potential of dietary intervention are needed to judge how functional foods actually fulfill the claims of being beneficial and how they counteract the process of carcinogenesis or development of other diseases. Examples are e. g. to monitor the induction of toxicological defence systems or to study proliferation and apoptosis in biopsies. It is expected that new parameters will be identified by the future use of high throughput methods of genome, transcriptome and proteome analysis. However, in order to develop these parameters into biomarkers for population monitoring, it is necessary to subject them to a number of additional and rigorous validation steps, especially to understand their predictive value and reliability.

* Department of Nutritional Toxicology, Institute for Nutrition, Friedrich-SchillerUniversity of Jena, Dornburger Str. 25, 07743 Jena, Germany

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11.1 Introduction Biomarkers may be used in human trials to study associations of dietary exposure and cancer risks. A representative definition describes biomarkers as “parameters which can be evaluated quantitatively, semi-quantitatively or qualitatively and which provide information on exposure to a xenobiotic or on the actual or potential effects of that exposure in an individual or in a group” [1]. In the context of diet and cancer, however, an extension of this definition should also include “exposure to chemopreventive compounds”, since the balance of both damaging and protective factors will be decisive for both risk and benefits from diet. Chemoprevention is the “use of pharmacological or natural agents that inhibit the development of invasive cancer either by blocking the DNA damage that initiates carcinogenesis or by arresting or reversing the progression of premalignant cells in which such damage has already occurred” [2]. Since this is also one of the final expected benefits of a healthy diet, dietary prevention is a complex form of chemoprevention. In order to assess efficacy of dietary intervention, appropriate biomarkers will be a combination of techniques to determine two types of effect (reduction of damage and induction of protective processes), two types of exposure (increase in protective factors and decrease in risk factors) and several types of susceptibility properties (age, sex, predisposing diseases, immunological status, predetermining and predisposing genetic alterations). The measurements can be specific for target individual tumor types, or they can reveal systemic effects by food, depending on the tissue analysed. In the following, a general overview of cancer, diet and of biomarker approaches to study associations of diet and colon cancer is presented. Examples are described of potential biomarkers, which we are presently employing to study for colon cancer the relationships between dietary risks, dietary protection and the balance favouring chemoprotection.

11.2 Associations of Cancer and Diet The 10 most frequent cancers in males and females from European countries include neoplasms of the lung, in tissues of the gastrointestinal tract and in hormone dependent tissues [3]. The most important avoidable life style factors are tobacco smoke and diet, each causing approximately 30–35 % of all human tumors in western countries [4]. Individual heterogeneous dietary factors may contribute to enhancing risks of developing the various cancers. These are largely specific for different tissues. In general, however, diets high in total fat or in saturated/animal fat are considered possibly causative whereas vegetables and fruits act protective. The numerous findings are exemplified by studies of Block et al. [5], or Steinmetz [6] reviewed by Hill 165

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[7]. The associations have been identified with different levels of certainty, and are therefore described as being “convincing, probable, or possible” [8]. The solidity of evidence varies for specific tissues and for individual food groups. Very rarely, if at all, has it been possible to identify single dietary compounds as being actually able to induce or prevent cancer in humans. The future application of biomarkers promises to improve the evidence base on the role of nutrition in cancer and to identify more precisely causative and preventive diets and foods.

11.3 Colorectal Cancer Cancers of the intestines (mainly colon and rectum) rank on place 2 (behind lung) in terms of frequency and incidence for men and women. Age-standardised death certification rates in Europe are 21.5/100.000 for males and 14.3/ 100.000 for females with mean incidence rates ( % of all tumors) of 11.9 (males) and 11.3 (females). The major types of diseases are mainly adenomatosis-polyposis carcinoma, hereditary non-polyposis colon cancer, rectal adenocarcinoma and epidermoid carcinoma of the anal region [9]. The majority of colorectal cancers, sporadic cancers develop with age from benign adenomas. The process involves mutations in APC (5q), DNA hypomethylation and the acquisition of multiple additional alterations, especially in KRAS2 (12p), DCC (18q), P53 (17p) [10]. In addition, mechanisms initiated by mutational inactivation of genes encoding proteins of the mismatch repair system cause important early lesions that are followed by genetic instability [1]. The alterations accumulate over a number of years and it may take decades for the development of a malignant carcinoma [11]. This increases the possibility that intervention mechanisms could prevent or delay the onset of such genetic alterations. Colorectal cancers are not only among the most frequent cancers with high mortality rates, but they are probably the tumor types most strongly linked to diet. Both factors of increasing risk and of reducing risk have been identified. Epidemiological evidence suggests that high meat and high alcohol intake increases the risk. Some experimental evidence that supports the role of meat and fats are findings on the link to probable exposures with heterocyclic amines, polycyclic aromatic hydrocarbons, and nitroso compounds when ingesting processed or heated meat products. Iron may lead to an increase in the production of reactive oxygen species from peroxides via the Fenton Reaction and oxidative stress leads to cell damage including mutations [12–17]. Some of these carcinogens and oxidants will be formed in the colon; they may arrive there directly with the ingested food or after first pass metabolism by the liver via bladder or bile. They may be retained there for hours, depending on the stool transit time. Thus, a special exposure situation in terms of time, source and quality (preactivated carcinogens) is 166

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present in the colon. Especially these factors may be utilised as biomarkers to assess the exposure situation arising from components of the gut lumen. The efficacy of faecal components to induce genotoxicity and oxidative damage has been utilised for some time as a biomarker of effect [18, 19]. Alternatively, epidemiological evidence is also available for protective diets. Especially vegetables (and physical activity) have been shown to reduce risk in cross sectional comparisons [20]. Cruciferous vegetables, non-starch polysaccharides (fibre), starch and carotenoids possibly decrease risk, whereas the evidence for resistant starch is insufficient [8]. The experimental evidence for protective properties of dietary factors in the colon include findings showing that the in vivo supplementation or in vitro treatment with calcium has led to a decreased proliferation of colonocytes [21]. More basically, beneficial functions of specific bacterial strains of the gut flora, and processes resulting from the fermentation of dietary fibre indicate how plant foods may in some way reduce cancer risks in the colon [22, 23]. Altogether a positive profile of various aspects of fermentation could reduce exposure to risk factors by several different mechanisms [8]. The anticarcinogenic success of dietary chemoprotection in the colon is discussed controversially, since it has hardly been shown in humans that the potential mechanisms actually result in a reduced recurrence of the disease or of precursors [24–28]. Since many novel foods are processed with functional ingredients, especially, for the purpose of enhancing intestinal well being and reducing exposure to risk factors in the gut, it will be important to use biomarkers to experimentally assess the intervention-effects, also in terms of putative prevention of colon carcinogenesis [29, 30].

11.4 Biomarkers to Study Associations of Colon Cancer and Diet Several dietary intervention studies have been performed in which biomarkers have been more or less accurate in predicting or confirming epidemiological-defined associations between nutrition and cancer [30, 31]. The techniques are especially useful to obtain knowledge on protective mechanisms of food components during nutritional intervention studies.

11.4.1

Biomarkers of Exposure (Risk and Protective Factors)

The detection of compounds associated with risk (e. g. carcinogens in food, reactive oxygen species, and products of lipid peroxidation) or with cancer prevention (e. g. antioxidants, fermentation products of the gut flora) is usually based on the causal associations derived from epidemiological data or on data from experimental systems (in vivo chronic animal bioassays, mutation assays with cultivated cells). Although it is difficult to actually pin167

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point single compounds as „biomarkers of exposure“, the chemical detection of groups of food-associated compounds, and their relative concentrations, can be evaluated as exposures which may increase or decrease risk. This detection can be performed in the faeces. Examples are e. g., the determination of bile acids [32], short chain fatty acids [33, 34], gut flora composition [35], dietary mutagens and oxidants [36, 37], nitroso compounds [14, 38], activities of bacterial enzymes (b-glucuronidase, b-glycosidase, azoreductase and nitro reductases) [39]. An interesting newer approach is the determination of oxidative stress in the faeces [40]. Next to the chemical analysis it is also possible to study functional consequences of faecal ingredients by analysing the genotoxicity and other parameters of faecal water in colon cells in vitro [41]. An example of one of our own studies was described recently: Faecal water from the subjects, on a normal healthy diet e intervention with a functional food was isolated by centrifugation of the faeces at 25000 x g for 2 h at 4 hC. 50 ml portions were incubated with human colon cells and DNA damage was quantitated using the “comet-assay“. Faecal water induced DNA damage and oxidised DNA bases in HT29clone19A cells [42]. There is considerable interindividual variability that was not lower in subjects consuming identical diets. However the results of a pilot study with bread enriched with a linseed fraction (rich in lignans) did show an intervention effect which was significant on an individual basis [42]. Another recent study has revealed that the intervention with low fat, high starch diets reduces the excretion of genotoxins in the faeces [43].

11.4.2

Biomarkers of Effect

DNA breaks, oxidative DNA damage, micronuclei, DNA adducts (e. g. in blood lymphocytes) may be indicative of an increased risk, based on the assumption that increased DNA damage will enhance the probability of mutations occurring in critical target genes and cells, and/or that increased DNA damage is the result of a higher load of genotoxic agents which will enhance the process of carcinogenesis (by inducing DNA damage as well as other molecular processes of carcinogenesis). For this we have e. g. developed the single cell microgel electrophoresis (Comet) assay to detect DNA damage and oxidised DNA bases in peripheral lymphocytes and in colon cells. We have now demonstrated that nutritional intervention with carotinoid- or flavonoid-containing juices results in a 3-fold decrease of oxidised DNA bases in peripheral lymphocytes of volunteers [44, 45]. To our knowledge, this was the first time that whole foods had been analysed for their impact on genetic damage. It followed up on the previous investigation of effects on intervention with dietary supplements [46]. The degree of reduction of genetic damage by the vegetable juices was just as clear-cut as was found for the supplements. Meanwhile other foods [47] have been shown to be as effective and the method of detecting genetic damage, including oxidative base damage, is now one of the better characterized biomarker systems 168

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with high potential for future regular assessment of novel functional foods [46, 48, 49] The colon is a well accessible tissue of the body. Biopsies can be taken relatively painlessly during medical diagnostic procedures. The biopsies can be worked up to yield isolated macromolecules (DNA, proteins, and lipids), single cell suspensions, individual colon crypts or whole tissue specimens for histological investigations. With these specimens proliferation can be determined in colon crypt cells [50]. The analysis of crypts allows the localisation of cells with more or less proliferative activity, thus enabling not only a tissue specific but also a cell specific detection. Similarly, the immunhistological detection of altered DNA or other cellular parameters [51], GST- [52] and CYP1A2- [53] activities, rate of apoptosis, KRAS amplification or mutations, [54], P53 deletions [55] or microsatellite instability [56] in tissue sections [57] may be used. Genetic lesions can now be monitored even in the small quantity of cells that are available from human colon biopsies [58]. Thus we have shown that considerable levels of damage, including oxidized bases, occur in colon cells. In combination with other methods on enhanced expression of genes encoding deactivating phase II enzymes [59], the biomarkers are gaining more and more relevance to show protective properties of individual foods or of nutritional regimens in humans [60]. Moreover, they are of value for increasing our understanding of cancer preventive mechanisms by nutrition. Future developments can focus on identifying parameters of reduced risks and of enhanced chemoprotection [30]. Presently we are studying the impact of intervention with a synbiotic schedule (combination of prebiotic and probiotic) in humans [61]. The analysis of genetic damage and induction of the phase II enzyme glutathione S-transferase in peripheral blood cells, human colon cells and in cultured cells treated with the faecal water of the patients, before and after intervention will give us more information on the possibility of measuring a reduced exposure to risk factors using the various methods.

11.4.3

Biomarkers of Susceptibility

Genetic factors: Another alternative way of detecting effect biomarkers in colon cells is to use exfoliated cells. One example is the detection of predetermining mutations in KRAS [62] or total DNA content [63] in these cells. Predetermining germline alterations, which may lead to colon cancer, affect the genes APC, DCC, hMLH2, hMSH1. Additionally, somatic alterations arise in KRAS2, P53 and others (s. genetic predisposition). KRAS2 mutations and amplifications have been not only detected in colon cells, but also in effluents of the colon and in serum. The non-invasive biomarker holds great value for dietary prevention studies. Recently, it has been reported that high consumption of olive oil is associated with a decrease in the risk of cancer in wild type K-ras genotypes. Vice versa, high calcium intake is associated with a decreased risk of K-ras mutated tumors but not with a 169

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decrease of K-ras wild type tumors [64]. This may indicate, that calcium and olive oil, respectively, protect against different type of colon cancer risk factors. Mutations in hMLH2, hMSH1 may be reflected by diminished DNA repair capacity. Diminished repair is being developed as a parameter of susceptibility biomarkers [65, 66]. Genetic polymorphisms for foreign compound metabolism: Several case control studies have been performed to reveal associations between GST polymorphisms and colon cancer risk. Some of these studies showed no significant associations e. g. for GSTM1*0 [67], GSTT1*0 [68], or only trends were observed which were, however, not significant, e. g. for GSTM1*0 [69], whilst others showed significant associations, e. g. for GSTT1*0 [70], for CYP1A1-MSP1 mutation in Exon 7 for Japanese [71], for GSTM1*0 [72], or for patients with Ulcerative colitis [73]. The meta analysis [74] of 6 further case control studies in colorectal cancer patients, reported an odds ratio of 1.67 in Caucasians and 0.84 in Asians for rapid versus slow acetylators (the slow phenotype was associated with a protective effect). Carcinogenic heterocyclic amines from individual heavily cooked meats are metabolised by the enzymes N-acetyl transferase (NAT) and CYP1A2. Mutations in the NAT2 gene lead to lower activity. The enzyme has been shown to acetylate hydroxylated aromatic heterocyclic amines, the products of which are DNA adducts. Thus lower capacity to produce active intermediates can explain a lower risk. A recent study has shown a large risk for the simultaneous occurrence of several factors, including the described genetic polymorphisms but also life style factors such as smoking (which induces the enzymes that activate the heterocyclic amines) and the habit of eating thoroughly fried meat (which increases exposure to heterocyclic amines) [75]. Gender: No differences between men and women have been found for associations of decreased colon cancer risk and increased vegetable and fruit consumption (reviewed in [20]). However, in one study, women on a low fat/high fibre diet had a reduced risk for neoplastic polyp recurrence and reduced concentrations of faecal bile acid concentrations, whereas men did not [76]. This was explained by a potential higher compatibility of women in comparison to men to keep to the dietary schedule. In cells isolated from colon biopsies males have significantly more oxidised bases than females [77]. Accordingly, it should be interesting now to utilise the biomarker of measuring oxidised bases in colon cells of men who receive dietary intervention with functional foods containing antioxidant ingredients.

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11.5 Further Research Needs 11.5.1

Validation and Understanding Predictivity

Biomarkers used in cancer prevention strategies are mainly based on the assumption that the reduction of exposure to risk factors will decrease the probability of tumor development in the colon. There has been no adequate evaluation on whether this assumption is actually valid, or more over with which impact an observed exposure-reduction actually relates to tumor prevention. Clearly, the biomarkers need to be validated in many ways, most obviously in respect to their predictivity. Several intervention studies have been performed to investigate the ability of fat, red meat, fibre, fruit and vegetables or alcohol to modulate colorectal carcinogenesis. It is, however, still unresolved which degree risk factors and/or their metabolites from these foods will actually contribute to the carcinogenesis process – and how. Moreover, only little is known on potential interactions of different dietary factors (e. g. impact of simultaneous vegetable and meat consumption) and how they will modulate biomarker responses. According to a review of recent, in part still ongoing, human studies, folate, selenium and v-3 fatty acids were emerging as important agents in nutrition chemoprevention, whereas antioxidant vitamins and calcium were not giving equivocal results [78]. In these studies the biomarker end points were recurrence of adenoma or colorectal cancer, both of which are invasive and require very large numbers of subjects (several hundreds, thousands) for statistical evaluation. Other intermediate biomarkers directed at earlier stages of carcinogenesis are needed to evaluate dietary chemoprevention for the general public. The methods described above are a few examples of techniques, which on the whole are expected to reduce the number of subjects and the duration of the studies. However, their utilisation has yet to be accurately validated in clinical studies. Moreover, individual parameters need to be conclusively identified as leading to reduction of cancer risk. Also new specific, but non-invasive, markers need to be developed. Some novel approaches are addressing these needs and are assessing how the modulation of genes involved in the biotransformation of chemical carcinogens can be utilised as methods to indicate chemoprotective effects by dietary compounds. One of these mechanisms could be the induction of phase II deactivation systems which impairs the bioactivation of risk factors and thus leads to a lower vulnerability of cells toward genotoxins [38, 79]. Whether or not this parameter is reliable and whether actual benefits arise for human health is presently being assessed for the parameter of glutathione S-transferase induction [80]. Its enhanced expression caused by the drug oltipraz has meanwhile been associated with a mode of metabolism favouring deactivation of the environmental carcinogen aflatoxin B1 in humans. The follow up studies will include additional biomarker analysis, and finally the assessment of cancer incidence in treated or placebo groups of the monitored population [80]. The study pro171

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mises to yield very important results on which further biomarker analysis of functional effects can be based.

11.5.2

Toxicogenomics/Pharmacogenomics

As gene discovery accelerates, toxicogenomics/pharmacogenomics will be increasingly important to carefully assess the relative benefit/risk relationships of targeted intervention strategies and to understand individual susceptibilities on the basis of genetic polymorphisms. In contrast to learning how food can affect individual genes or compounds, the type of newly developed methods of the “genomics” era allow us to study a very high number of cellular macromolecules. The technologies also provide a new horizon in the pursuit of human genes relevant to health risks/benefits from environmental toxicants and from life style factors. Based on the use of high throughput expressed gene analysis or microarray technology, a rapid analysis of hundreds to ten thousands of genes is possible. The employment of the methods can be a complete reversion of the traditional approaches based on descriptive, hypothesis-driven surveys. The logical way to increase our knowledge base for developing biomarkers to assess exposure is therefore to use good in vitro models or animal experiments to study the physiological response to exposures and identify key targets. On this knowledge basis it will then be a prerequisite to integrate the methodology into sophisticated strategies of clinical, occupational or environmental exposure settings. In this context we need to study (1) how dietary responses vary on the basis gene expression patterns or genetic polymorphisms and how the response varies among individuals with different genotypes, (2) what the prevalence of relevant genotypes is in the population and in relevant subpopulations, and (3) whether and to what degree life style factors (such as other drugs and diet) interact with genetic factors to influence response of the dietary exposures we are monitoring. For assessing functional foods, we need to study impacts of the products on specific groups of genes (Genomic and Genetic Toxicology) and to enhance the understanding for the basis of individual responses to chemicals (Pharmacogenetics). Biomarker approaches in the long run, should be then used as reliable methods to study functional food in humans and to perform the final assessment on whether they actually improve patterns of gene expression, reduce individual sensitivities without enhancing risks in any known manner (Chemoprevention).

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11.6 Conclusions We will need accurate and reliable methods to study functional foods in the future. For one it is necessary to consider safety aspects. For the other, it is important to prove functionality before defining a food as functional. Finally we need to enhance our understanding of how food can modulate risks for developing cancer on an individual basis. Biomarkers will be important methods of the overall evaluation procedures. Presently, a large variety of biomarkers are anticipated that could be used to assess positive and negative effects of functional foods. The established parameters, however, need to be validated for their applicability, reliability and predictivity in human dietary studies. None of the methods have actually been evaluated to the degree, which shows that the outcome of the biomarker result is actually related to the tumor yield. Another set of new techniques targeting expression of genes, such as those involved in the biotransformation of risk factors, hold potential to serve as indicators of protective effects during dietary intervention, since they indicate a type of toxicological defence, e. g. a reduced exposure to risk factors. This type of assessment of chemoprotective properties by functional foods in humans can be considered to be a new field of human toxicology, which deserves intensive development and scientific analysis in the future.

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45. Bub, A.; Watzl, B.; Blockhaus, M.; Briviba, K.; Liegibel, U. M.; Müller, H.; PoolZobel, B. L.; Rechkemmer, G. Fruit juice consumption modulates antioxidative status, immune status and DNA damage. Nutritional Biochemistry 06/2002. 46. Duthie, S. J., Ma, A., Ross, M. A., and Collins, A. R. Antioxidant Supplementation Decreases Oxidative DNA Damage in Human Lymphocytes. Cancer Research 1996; 56: 1291–5. 47. Mitchell, J. H. and Collins, A. R. Effects of a Soy Milk Supplement on Plasma Cholesterol Levels and Oxidative DNA Damage in Men – a Pilot Study. European Journal of Nutrition 1999; 38: 143–8. 48. Collins, A. R., Duthie, S. J., and Dobson, V. L. Direct Enzymic Detection of Endogenous Oxidative Base Damage in Human Lymphocyted DNA. Carcinogenesis 1995; 14(9): 1733–5. 49. Collins, A. R., Dusinska´, M., Gedik, C. M., and Stetina, R. Oxidative Damage to DNA: Do We Have a Reliable Biomarker? Environmental Health Perspectives 1996; 104 (Suppl 3): 465–9. 50. Lipkin, M. Biomarkers of Increased Susceptibility to Gastrointestinal Cancer. Gastroenterology 1987; 92: 1083–6. 51. Albaugh, G. P., Iyengar, V., Lohani, A., Malayeri, M., Bala, S., and Nair, P. P. Isolation of Exfoliated Colonic Epithelial Cells. A Novel Non-Invasive Approach to the Study of Cellular Markers. International Journal of Cancer 1992; 52: 347–50. 52. Nijhoff, W. A., Mulder, T. P. J., Verhagen, H., van Poppel, G., and Peters, W. H. M. Effects of Consumption of Brussels Sprouts on Plasma and Urinary Glutathione S-Transferase Class-a and -p in Humans. Carcinogenesis 1995; 16: 955–7. 53. Sinha, R., Rothman, N., Brown, E. D., Mark, S. D., Hoover, R. N., Caporaso, N. E., Levander, O. A., Knize, M. G., Lang, N. P., and Kadlubar, F. F. Pan-Fried Meat Containing High Levels of Heterocyclic Aromatic Amines but Low Levels of Polycyclic Aromatic Hydrocarbons Induces Cytochrome P4501A2 Activity in Humans. Cancer Research 1994; 54: 6154–9. 54. Burmer, G. C., Rabinovitch, P. S., and Loeb, L. A. Frequency and Spectrum of C-Ki-Ras Mutations in Human Sporadic Colon Carcinoma, Carcinomas Arising in Ulcerative Colitis and Pancreatic Adenocarcinoma. Environmental Health Perspectives 1991; 93: 27–31. 55. Nelson, E. Laboratory Probing of Oncogenes From Human Liquid and Solid Specimens As Markers of Exposure to Toxicants. Critical Reviews in Toxicology 1997; 26: 483–549. 56. Loeb, L. A. Microsatellite Instability: Marker of a Mutator Phenotype in Cancer. Cancer Research 1994; 54: 5059–63. 57. Anker, P., Lefort, F., Visioukhin, V., Lyautey, J., Lederrey, C., Chen, X. Q., Stroun, M., Mulcahy, H. E., and Farthing, M. J. G. K-Ras Mutations Are Found in DNA Extracted From the Plasma of Patients With Colorectal Cancer. Gastroenterology 1997; 112: 1114–20. 58. Pool-Zobel, B. L. and Leucht, U. Induction of DNA Damage in Human Colon Cells Derived From Biopsies by Suggested Risk Factors of Colon Cancer. Mutation Research 1997; 375: 105–16. 59. Pool-Zobel, B. L., Bub, A., Liegibel, U. M., Treptow-van Lishaut, S., and Rechkemmer, G. Mechanisms by Which Vegetable Consumption Reduces Genetic Damage in Humans. Cancer Epidemiology, Biomarkers & Prevention 1998; 7: 891–9. 60. Klinder, A.; Pool-Zobel, B. L. Funktionelle Biomarker für die Untersuchung der Beziehungen zwischen Ernährungsfaktoren und Risiken für die Entwicklung von Krebserkrankungen. Lebensmittelchemische Gesellschaft, Fachgruppe in der GDCh and Deutsche Gesellschaft für Ernährung. Funktionelle Lebensmittel –

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75. Marchand, L. L., Hankin, J. H., Wilkens, L. R., Pierce, L. M., Franke, A., Kolonel, L. N., Seifried, A., Custer, L. J., Chang, W., Lum-Jones, A., and Donlon, T. Combined Effects of Well-Done Red Meat, Smoking, and Rapid N-Acetyltransferase 2 and CYP1A2 Phenotypes in Increasing Colorectal Cancer Risk. Cancer Epidemiology, Biomarkers & Prevention 2002; 10: 1259–66. 76. McKeown-Eyssen, G. E., Bight-See, E., Bruce, W. R., Jazmaji, V., and The Toronto Polyp Prevention Group. A Ranomized Trial of a Low Fat High Fibre Diet in the Recurrence of Colorectal Polyps. Journal of Clinical Epidemiology 1994; 47: 525–36. 77. Pool-Zobel, B. L., Abrahamse, S. L., Collins, A. R., Kark, W., Gugler, R., Oberreuther, D., Siegel, E. G., Treptow-van Lishaut, S., and Rechkemmer, G. Analysis of DNA Strand Breaks, Oxidized Bases and Glutathione S-Transferase P1 in Human Colon Cells. Cancer Epidemiology, Biomarkers & Prevention 1999; 8: 609–14. 78. Kim, Y. I. and Mason, J. B. Nutrition Chemoprevention of Gastrointestinal Cancers: a Critical Review. Nutrition Reviews 1996; 54: 259–79. 79. Clapper, M. L. and Szarka, C. E. Glutathione S-Transferases-Biomarkers of Cancer Risk and Chemopreventive Response. Chemical and Biological Interactions 1998; 111–112: 377–88. 80. Szarka, C. E., Yao, K. S., Pfeiffer, G. R., Balshem, A. M., Litwin, S., Frucht, H., Goosenberg, E. B., Engstrom, P. F., Clapper, M. L., and O’Dwyer, P. J. Chronic Dosing of Oltipraz in People at Increased Risk for Colorectal Cancer. Cancer Detection and Prevention 2002; 25: 352–61.

12 Biomarkers Relevant to Cardiovascular Disease Margreet R. Olthof*, Peter L. Zock*, Petra Verhoef*, Martijn B. Katan*

A biomarker is an indicator of disease risk. Formal proof for efficacy of dietary supplements or functional foods requires clinical trials with hard endpoints, such as morbidity or death. However, it is not always feasible to measure hard endpoints. Therefore, a biomarker is often used as a ‘predictor’ of the actual effect of diet on disease in the following way:

Biomarker

Diet

Disease

* Wageningen University, Wageningen, The Netherlands

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A prerequisite for a biomarker is its validity: a change in the value of a biomarker should reflect a change in disease risk. For research purposes, a biomarker should be easily measurable and potentially modifiable within the timeframe of a study. Use of biomarkers offers the advantages that much smaller and shorter studies are required than when ‘hard’ endpoints are used. Effects on biomarkers have been a basis for dietary recommendations to the public, and for supporting health claims on (functional) foods. However for evaluating efficacy and safety it is important to combine evidence from biomarker studies with other types of evidence, such as that from epidemiologic and animal studies, e. g. to evaluate safety aspects. Several biomarkers for cardiovascular disease will be discussed during the presentation.

13 Biomarkers of Effect on the Immune System Raymond Pieters*

13.1 Introduction Nutritional effects on immune status are studied either in relation to nutrient deficiencies that may occur in case of malnutrition or undernourishment or in relation to supra-physiological intakes in order to increase health status [1–6]. Among the nutrients that are most frequently studied are vitamins (e. g. vitamins A and E) and certain trace elements (e. g. zinc, selenium) [7–10], essential polyunsaturated fatty acids [1] and probiotics [11, 12]. In particular in animal models, some of these nutrients have been shown to profoundly modulate certain immunological parameters. Also in man, immunological effects of nutrient supplementation have been documented. However, in case of normal healthy individuals but also in elderly immunological effects of supra-physiological intake levels are not always very clear and therefore equivocal. The most important reason for this is that the final clinical or health outcome of an immune response is regulated by a complex interplay of multiple inherent and environmental factors (i. e. markers of susceptibility). As a corollary, variation in immune responsiveness in a group of individuals and sometimes also in groups of inbred strains of

* Immunotoxicology group, Institute of Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands

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animals can be very high and subtle immunomodulatory effects of nutrients are easily missed. In addition many studies measure a limited number of isolated immune parameters, that often cannot easily be translated to immune function. This brief report does not attempt to give an overview of immunomodulatory effects of food constituents (see for reviews on the subject [1, 3, 6]. Rather, it aims to provide a conceptual immunological framework that may help to further define and interpret biomarkers of immunological effect or safety. Such a framework may also be of use to identify future research needs, such as further characterization of susceptibility factors. A recent animal study [13] and a somewhat older human study [14] on the immunomodulatory effects of n-3 polyunsaturated fatty acids (n-3PUFA) will be compared as to illustrate existing difficulties and limitations in the assessment of effectiveness of nutrients and functional foods on immune status.

13.2 A Bird’s Eye View on the Immune System The main function of the immune system is to protect the body against a diversity of parasites (e. g. bacteria, viruses etc). In addition, the immune system is capable of killing tumor cells (for instance by means of NK cells) and ridding dead cells. The immune system has developed to do so in a fast and flexible manner, meanwhile ensuring that it does not react against harmless antigens and trigger allergic responses or destroy self by means of autoimmune reactions. In order to respond fast, a number of first-line innate mechanisms have evolved that are composed of a variety of cellular and non-cellular components (see Fig. 13.1). Although the innate system has receptors that can recognize certain patterns of molecules as signs of danger, it is not capable of adapting its recognition pattern. The latter is a unique property of T lymphocytes and antibody-producing B lymphocytes that together form the adaptive arm of the immune system. T and B lymphocytes specifically recognize antigens, and memorize previously encountered antigens. The adaptive immune system receives activating signals from the innate immune system, and vice versa, antibodies and cytokines produced by the adaptive immune system help effector mechanisms (often components of the innate immune system) to react more efficiently and effectively. Together, the innate and adaptive immune system co-operate as to assure homeostasis. Activation of T cells depends on antigen presentation by dendritic cells (DCs), which are the professional antigen presenting cells (APC) of the myeloid lineage. DCs engulf antigens, and present peptides derived from these antigens in the context of major histocompatibility complex class II (MHC class II)-molecules to naive T cells (T helper 0, or Th0 cells). In addition, adju180

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Figure 13.1. A bird’s eye view on the immune system. When triggered by for instance intruding parasites innate immune components may become activated and pick up antigens. Part of antigens will be presented to Th0 cells by dendritic cells (DC) and together with a plethora of proinflammatory signals this may lead to activation of these naive Th0 cells. Th0 cells differentiate into more specialized Th1 and Th2 cells that control type-1 and type-2 responses, respectively. Th1 and Th2 cells keep each other in balance by regulatory cytokines (there are indications for the existence of a third category of Th cells, Th3 cells, which may be regulatory and of mucosal origin). APC, antigen presenting cell; MHC, major histocompatability complex; NK, natural killer; M2, macrophage.

vant signals as for instance induced by the proinflammatory cytokine TNFa, activate DCs to upregulate certain costimulatory receptors and produce cytokines that are stimulatory for Th0 cells. Together, MHCII-peptide complexes and costimulatory molecules may cause activation of Th0 cells which under influence of regulatory cytokines differentiate into more specialized Th1 or Th2 cells. These Th cells drive type-1 and type-2 immune responses, respectively, and eventually determine the balance between cellular and humoral immunity. In turn, the balance between cellular and humoral immunity eventually determines which innate effector mechanisms are activated and hence contributes to the ensuing clinical effects. Typical type-1-driven clinical outcomes are for instance immune-dependent diabetes mellitus (IDDM) and contact allergy, whereas typical type-2-driven adverse clinical phenomena include food allergy and asthma. Important to note, however, is that immunomodulation, either stimulatory or inhibitory, does not necessarily lead to changes in immune function or clinical effects. The reason for this is that further development of the type and outcome of an immune response is not only influenced by regula181

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Figure 13.2. Schematic representation of factors that are important in initiation of stimulation and sensitization of lymphocytes (antigen plus adjuvant-like signals) and factors that control and regulate further development (activation of effector mechanisms, balance of immune response) of immune responses.

tory cytokines but also by a vast number of additional internal (predisposing partly inherent factors, such as MHC haplotype, neuroendocrine factors, gender, and metabolic polymorphism) and also external factors (e. g. ongoing infections, intake of certain chemicals, including drugs and pollutants, and of course food constituents) (see Fig. 13.2). Also age (e. g. at young age the immune system is more vulnerable because it is in development, and old age is often accompanied by declined and changed, more type-2 like immune responsiveness), pregnancy and stress (neuro-endocrine influences) are known to have profound impacts on immune status. As already mentioned before, these susceptibility factors are probably the main reason for extreme heterogeneous responses in human populations.

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13.3 Biomarkers of Effect Adjustability or adaptation to new situations is an important prerequisite for optimum functioning of the immune system, and it is at the same time the reason why immune responses can be modulated by external factors, such as immunotoxic chemicals or functional foods. Immunotoxicity is only one aspect of immunomodulation, and particularly refers to adverse immunomodulation. Immunotoxicity may lead to either depression of the immune system (mostly referred to as immunosuppression, with the eventual risk of increased or prolonged infections, and decreased protection against tumor formation) or stimulation of innate and/or adaptive immune responses (occasionally resulting in allergies or autoimmune diseases in susceptible persons). Certain food constituents may induce either one of these immunotoxic adverse effects, but these constituents are mostly impurities or contaminants (for instance dioxins that may possibly induce immunosuppression, or olive oil contaminated with rapeseed-oil that caused an auto-immune like toxic oil syndrome) [15] or (genetically modified) allergenic proteins [16]. This report will not deal with immune effects of such agents. Benificial immunomodulation due to functional foods obviously aims at improvement of health. For instance diet enforcement may be directed to increased protection against tumors (increases of innate immunity, in particular NK cells), reduction of inflammation, or immune deviation (change in the balance between type-1 and type-2 immunity). Together, it is important to realize that optimal immunity depends on a balanced immune system and that adverse immunomodulation (e. g. immunotoxicity) and benificial immunomodulation are flip-sides of the same coin. In extreme situations immumodulation that aims at improvement of health may eventually have an opposite effect. For instance, suppression of inflammation may relieve joint aches in rheumatoid arthritis patients but at the same time increase the vulnerability to opportunistic infections. Likewise, a new setting of the balance between type-1 and type-2 responses with the goal to shift the balance from type-2 towards type-1 may suppress a person’s asthmatic allergy but burden this person with a Th1-driven contact allergy. This dualism is of course an important aspect to consider with respect to effectiveness and safety of immunopotent functional foods. For evaluation of immunotoxic potential of chemicals requirements have been laid down in various guidelines (i.e OECD and CPMP guidelines in Europe) [17]. These guidelines are set up for assessment of unintended immunotoxic effects, and because of huge costs the number of tests that are required are often limited as much as possible without the risk of unsafety. But as in case of functional foods assessment of immunological effects is not only done for safety reasons but also for detection of effectiveness, a much broader picture of the status of the immune system is required. This 183

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can only be gained by an integrated approach, taking into account biomarkers of innate as well as of adaptive immunity. Tab 13.1 gives an overview of relevant parameters that could be used in animal tests or human studies. As it is often not feasible to detect all parameters, choices need to made based on relevance for the particular situation. Of course, these choices need to comply with important prerequisites for biomarkers, i. e. they should clearly relate to functionality of the immune system, and they need to be responsive to modulations. Important to keep in mind is that the parameters of choice should cover a broad spectrum of the immune system. Furthermore for proper determination of its functionality, the immune system needs to be challenged with an antigenic stimulus. In case of human studies, biomarkers also need to be ethically feasible and thus as less invasive as possible. Immune parameters that can relate immunomodulatory effects found in animals to those observed in man are then of additional interest. Examples of such parameters are: differential cell counting in blood (including distribution of blood lymphocyte subsets),

Table 13.1. List of immunological parameters recommended as biomarkers for effects on the immune system (choice is made as to be applicable in animal and in man, except when indicated). General x Complete blood count with differential count and clinical chemistry screen Innate immune response x Complement and acute phase proteins x Pro-inflammatory cytokines (e. g. systemic TNFa and produced by in vitro LPSstimulated blood cells) and mediators (e. g. prostaglandins) x NK cell enumerations and activity (cytolytic activity against NK-target cell) x Phagocytic activity of, oxidative burst in white blood cells (polymorphic neutrophils) Adaptive immune response T cell (-mediated) functions x Enumerations of CD4 and CD8 subsets and of regulatory T cells. x T cell activation: proliferation (DNA synthesis) and/or activation (cytokine production) to mitogens Con A or PHA, recall antigens or CD3/CD28) x Delayed Type Hypersensitivity (DTH) to sets of recall antigens B cell (-mediated) functions x Enumerations of B cells (CD19, CD20) x Antibody titers and production (differentiated by isotype (IgM, IgG, IgE, IgA), preferably antigen-specific and both primary and secondary responses. (in man; responses directed to vaccines or model antigens like keyhole limpet hemacyonin, KLH) Immune disease-specific responses x Hematocrit (indication of inflammation) x Auto-antibody titers to nuclei (predefined sets, e. g. antinuclear/nucleolar antibodies) x IgE to allergens x Mast cell products in serum (in man; skin prick tests for allergy)

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acute phase proteins, complement activity, phagocytic activity and oxidative burst in blood leukocytes, NK cell activity in blood, mitogen- or antigenstimulated lymphocyte proliferative and cytokine responses, and antigenand isotype-specific antibody titers. In addition to these parameters that can be measured in blood samples, delayed type hypersensitivity (DTH) responses to standard antigens can be measured as function test [18]. In animals, DTH responses can be measured in sensitization models with well characterized model antigens. In some human studies, vaccine responses (antibody titers, DTH responses as well as in vitro T cell responses) have been used as indicators of immune function. The difficulty with vaccineinduced immune responses is that they may differ substantially dependent on the vaccine that is used [18]. So, although immune responses are very complex, the battery of immune function biomarkers mentioned here may give an integrated, and interpretable view of the immune status. Until now, however, only few human and animal studies have been performed showing such a broad spectrum evaluation of the immune status in relation to functional foods. Studies on the immunological effect of dietary fats may be a positive exception, albeit that scientific proof of health benifit is still lacking. Nevertheless, data of “dietary fat-research“ serves as a good example of the possibilities and limitations of assessment of immune responsiveness with the help of biomarkers.

13.4 Dietary Fat-related Immune Alterations: an Example Dietary fat may influence immune function and is one of the nutrients that has been studied more intensively than others [2–4]. The reason for studying the impact of dietary fat on the immune status is based on the observation that in communities where the intake of n-3PUFA, present in high quantities in fish, is high the incidence of inflammatory and autoimmune disorders is low. Hence, it was speculated that high intake of n-3PUFA is benificial for human health. But, although the influence of fatty acid composition of the diet on the immune system has been a subject of research for many years, results were not always in agreement (see for discussion on this [2, 19]). Some studies appear to indicate anti-inflammatory effects of n-3PUFA whereas others show proinflammatory potential. Similarly, increased n-3PUFA-intake has been shown to reduce lymphoproliferation in some studies and to be stimulatory in others. Duration of experiments, species differences (man vs mouse or rat) and health status of subjects are among the many factors that may account for these contradictory results. Recently, we have used a murine sensitization model to evaluate the immunological effects of diets enriched in n-3PUFA combined with a suffi185

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cient or high level of vitamin E [13]. Briefly, female BALB/c mice were fed experimental diets for 8 weeks, and were sensitized in the fifth week by smearing DNCB on the dorsum of both ears. After another two weeks, mice were challenged with DNCB on the ears and ear thickness was determined after 24 hrs (as measure for DTH). Five days after challenge mice were dissected. Both innate as well as adaptive immune parameters were measured. Among the innate parameters were phagocytosis and oxidative burst by white blood cells, LPS-induced production of TNFa and the n-3PUFA-derived product, PGE2. Adaptive parameters apart from DTH included DNP(immunologically recognizable hapten of DNCB)-specific antibodies, and responses of lymphocytes taken from the ear-draining lymph node and from the spleen to ConA (proliferation and cytokine production). In this mouse study it was found that only in combination with low vitamin E intake n-3PUFA was able to cause increased oxidative burst activity, decreased PGE2 production, increased TNFa production, decreased IFNg/ IL4 ratio decreased serum levels of IgG2a and IgG2b antibodies (in the mouse indicative of type-1 responses) and increased DNP-specific IgE antibodies (type-2 isotype). At high vitamin E levels, TNFa and PGE2 production were similarly altered, whereas type-1 vs type-2 responses remained as in controls. Apparently, according to results in this murine sensitization model, the modulatory effects of n-3PUFA indicate an activation of innate immunity, combined with a decrease of some (IgG2a levels, IFNg/IL4 ratio) but not all (DTH) type-1 immune responses and increased type-2 responses. Moreover, changes of adaptive immune responses but not of innate responses depend on the levels of vitamin E. Results indicate a change in the balance of type-1 vs type-2 responsiveness resulting in more type-2-like phenotype. For comparison, results of a 24 wks human study on the immune effects of n-3PUFA in the form of a low fat, high fish diet showed a decrease of TNFa production and an increased percentage of CD8 cells [14]. As CD8 cell activation depends on IFNg the latter finding may indicate an increased Th1 dependent immunity. So, presented murine data seem not in agreement with previous human data. The reason for this is unknown, but can possibly be found in the earlier mentioned array of predisposing and environmental factors that influence the immune response. For instance, the mice used in the study mentioned here were of the BALB/c strain, and although both type-1 and type-2 responses can be easily stimulated in these mice [20], they are mostly referred to as typical type-2 responding strain. It can be speculated from this that type-1 responses in these BALB/c mice are less strong and thus more prone to dietary effects than type-2 responses. In addition, the time period of nutrient intake may have a profound influence on the immune effectiveness of n-3PUFA (as well as other nutrients).

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13.5 Concluding Remarks According to literature, nutrients can have profound effects on immune status and function. Benificial immunomodulation is of course most clear in immunodeficient situation as for instance in elderly. In normal healthy persons, and probably also in healthy animals, effects are often too subtle to detect. Also, changes in immune status will often only become visible in case immune function is tested, which is mostly not done. Susceptibility factors and animal-to-human extrapolations (see above example of n-3PUFA) form additional complications in immune function studies. Thus, although biomarkers as such can be identified based on the available immunological knowledge, sensitization/vaccination models and protocols (including time schedules of intake) as well as susceptibility factors are not well-defined. Evidently, better definition and (mechanistic) characterization of predictive animal models may help to solve inconsistency of findings. This characterization process should of course include the influence of susceptibility factors. Acquired knowledge on the role of these factors can be used to adopt and/or develop case-specific test models that better mimic specific conditions in man. Eventually, it may turn out that various test models need to be used in parallel to get a good idea of immune function effects. With regard to human studies, further characterization, standardization and validation of protocols for biomarker analyses should be pursued. This especially accounts for vaccination protocols as these indicate true immune function. Conceivably, only comparable study designs will allow evaluation of immunological effectiveness and safety of functional foods against the background of the huge number of strongly influential susceptibility factors.

References 1. Calder PC. n-3 polyunsaturated fatty acids and cytokine production in health and disease. Ann. Nutr. Metab. 1997; 41: 203–234. 2. Calder PC. Fat chance of immunomodulation. Immunol Today. 1998; 19: 244–247. 3. Harbige LS. Nutrition and immunity with emphasis on infection and autoimmune disease. Nutr. Health. 1996; 10: 285–312. 4. Harbige LS. Dietary n-6 and n-3 fatty acids in immunity and autoimmune disease. Proc. Nutr. Soc. 1998; 57: 555–562. 5. Daudu PA, Kelley DS, Taylor PC, Burri BJ, Wu MM. Effects of low b-carotene diet on the immune functions of adult women. Am. J. Clin. Nutr. 1994; 60: 969–972. 6. High KP. Micronutrient supplementation and immune function in the elderly. Clin. Infect. Dis. 1999; 28: 717–722. 7. Chandra RK. Effect of vitamin and trace-element supplementation on immune responses and infection in elderly subjects. Lancet. 1992; 340: 1124–1127.

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8. Provinciali M, Montenovo A, Di Stefano G, Colombo M, Daghetta L, Cairati M, Veroni C, Cassino R, Della Torre F, Fabris N. Effect of zinc or zinc plus arginine supplementation on antibody titre and lymphocyte subsets after influenza vaccination in elderly subjects: a randomized controlled trial. Age Ageing. 1998; 27: 715–722. 9. Ravaglia G, Forti P, Maioli F, Bastagli L, Facchini A, Mariani E, Savarino L, Sassi S, Cucinotta D, Lenaz G. Effect of micronutrient status on natural killer cell immune function in healthy free-living subjects aged i/=90 y. Am. J. Clin. Nutr. 2000; 71: 590–598. 10. Gardner EM, Bernstein ED, Popoff KA, Abrutyn E, Gross P, Murasko DM. Immune response to influenza vaccine in healthy elderly: lack of association with plasma beta-carotene, retinol, alpha-tocopherol, or zinc. Mech Ageing Dev. 2000; 117: 29–45. 11. Fang H, Elina T, Heikki A, Seppo S. Modulation of humoral immune response through probiotic intake. FEMS Immunol. Med. Microbiol. 2000; 29: 47–52. 12. Matricardi PM. Probiotics against allergy: data, doubts, and perspectives. Allergy. 2002; 57: 185–187. 13. Albers R, Bol M, Bleumink R, Willems A, Blonk C, Pieters R. Effects of dietary lipids on immune function in a murine sensitisation model. Br. J. Nutr. 2002; 88: 291–299. 14. Meydani SN, Lichtenstein AH, Cornwall S, Meydani M, Goldin BR, Rasmussen H, Dinarello CA, Schaefer EJ. Immunologic effects of national cholesterol education panel step-2 diets with and without fish-derived N-3 fatty acid enrichment. J. Clin. Invest. 1993; 92: 105–113. 15. Powell JJ, Van de Water J, Gershwin ME. Evidence for the role of environmental agents in the initiation or progression of autoimmune conditions. Environ. Health Perspect. 1999, 107 suppl 5: 667–672. 16. Taylor SL, Hefle SL. Will genetically modified foods be allergenic? J. Allergy Clin. Immunol. 2001; 107: 765–771. 17. Vos JG, De Waal EJ, Van Loveren H, Albers R, Pieters RHH. Immunotoxicology. In Principles of immunopharmacology, ed. FP Nijkamp and MJ Parnham. (Birkhauser Verlag Basel/Switzerland) 1999, 407–441. 18. Meydani SN, Meydani M, Blumberg JB, Leka LS, Siber G, Loszewski R, Thompson C, Pedrosa MC, Diamond RD, Stollar BD. Vitamin E supplementation and in vivo immune response in healthy elderly subjects: a randomized control trial. JAMA 1997; 277: 1380–1386. 19. Netea MG, Kullberg BJ, Blok WL, Van der Meer JW. Immunomodulation by n-3 polyunsaturated fatty acids. Immunol Today. 1999; 20: 103. 20. Albers R, de Heer C, Bol M, Bleumink R, Seinen W, Pieters R. Selective immunomodulation by the autoimmunity-inducing xenobiotics streptozotocin and HgCl2. Eur. J. Immunol. 1998, 28: 1233–1242. 21. Van Loveren H, Van Amsterdam JG, Vandebriel RJ, Kimman TG, Rumke HC, Steerenberg PS, Vos JG. Vaccine-induced antibody responses as parameters of the influence of endogenous and environmental factors. Environ. Health Perspect. 2001, 109: 757–764.

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14 Mechanistic Understanding of Potential Adverse Effects of b-Carotene Supplementation Xiang-Dong Wang*

Abstract A number of epidemiological studies have consistently demonstrated the beneficial effects of fruits and vegetables rich in carotenoids on risk reduction of cancer. In contrast, clinical intervention trials conducted to determine the effect of b-carotene supplementation on the incidence of lung cancer in smokers found either no protective effect or a negative effect. However, supporting evidence for a protective role of fruits and vegetables rich in carotenoids in cancer prevention continues to be reported in human epidemiological and interventional studies. A number of animal and laboratory studies have also shown that carotenoids can block certain carcinogenic processes and inhibit specific tumor cell growth. Understanding the anticarcinogenic/procarcinogenic response to carotenoids supplementation is of importance due to ongoing interest of both research scientists and the public in carotenoid’s potential as a chemopreventive agent. The failure of the intervention studies to demonstrate a protective effect of supplemental b-carotene could be due to many factors. Recent in vivo and in vitro studies have provided useful information on the controversy regarding the chemopreventive vs the carcinogenic activity of b-carotene. One biologic basis for the harmful effects of high dose b-carotene supplementation in smokers is associated with the dosage used and free radical-rich atmosphere in lungs of cigarette smokers. This environment alters b-carotene metabolism, and produces undesirable oxidative metabolites, which can facilitate the binding of metabolites of benz[a]pyrene to DNA, down-regulate retinoic acid receptor beta, up-regulate AP-1 (c-Jun and c-Fos) activity, induce carcinogen-activating enzymes, interfere with retinoid metabolism and enhance the induction of BALB/c 3T3 cell transformation by benz[a]pyrene. Thus, the carcinogenic response to high dose b-carotene supplementation reported in the human intervention trials and animal studies highlights the critical importance 1) of dose, 2) of metabolism, 3) of animal models, 4) of interaction with exogenous exposures such as tobacco and alcohol, and 5) of genetic signaling pathways leading to tumor – in considering the role of nutrients in cancer prevention. * Associate Professor and Director, Laboratory of Nutrition and Cancer Biology, JMUSDA Human Nutrition Research Center on Aging, Tufts University, 711 Washington Street, Boston, USA

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14.1 Introduction Cigarette smoke contains both chemical carcinogens and free radicals, which can interact with DNA and cause damage and mutations in oncogenes (e. g., Kirsten-ras) and tumor suppressor genes (e. g., p53, retinoic acid receptorbeta) [1–3]. If such gene damage cannot be repaired, epithelial cells (e. g., lung, oral mucosa, stomach, etc.) can escape the normal proliferative controls, and ultimately result in a tumor. Lung cancer is the most common type of cancer worldwide. Although the best protection against lung cancer is the avoidance of tobacco smoke, the number of current smokers remains high. Quitting smoke can only decrease the risk of lung cancer but does not nessessarily prevent lung carcinogenesis. In addition, passive smokers (environmental cigarette smoke-exposed, smoker’s spouse and childen) are at risk because more free radicals are generated from smoke as the smoke becomes aged. Nutritional intervention to protect tissues against smoke-borned chemical carcinogens and oxidative free radical damage is an appropriate way to rationally modify cigarette smoke-induced cancer risk. A number of epidemiological studies have consistently demonstrated the beneficial effects of fruits and vegetables rich in carotenoids on risk reduction of cancer, especially for lung cancer [4]. More than 560 carotenoids have been isolated from nature. The most active of them that has been extensively studied is all-trans-b-carotene, which is one of the most important food components for which there is strong evidence for an anticancer role. b-carotene is a symmetrical molecule with several conjugated double bonds on its polyene chain (Fig. 14.1). Vitamin A activity is an important function of b-carotene. b-carotene also has a variety of other functions, including 1) function as an antioxidant [5, 6], 2) be a precursor of retinoic acid [7, 8], 3) enhance gap junction communication [9, 10], 4) increase immunologic function [11, 12], and 5) induce carcinogen-metabolizing enzymes [13]. There remains a remarkable discordance between the results of the observational epidemiologic studies and the intervention trials using b-carotene as a potential chemopreventive agent. Clinical intervention trials conducted to determine the effect of b-carotene supplementation on the incidence of lung cancer in smokers found either no protective effect or a negative effect [14–18]. However, supporting evidence for a protective role of fruits and vegetables rich in carotenoids in cancer prevention continues to be reported in human epidemiological studies [19–21] and intervention studies [22, 23]. A number of animal and laboratory studies have also shown that carotenoids can block certain carcinogenic processes and inhibit specific tumor cell growth [24–28]. In this review, we will focus on the understanding of potential adverse effects of b-carotene supplementation in both the human smoker and the ferret model to mimic human intervention studies. Some important new information related to b-carotene metabolism and interaction will also be reviewed. 190

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Figure 14.1. Structures of metabolites in the metabolic pathway of b-carotene (Reproduced with permission from [8]).

14.2 Understanding of Potential Adverse Effects of b-Carotene Supplemention in Human Using Animal Models It is highly unlikely that another human intervention study will be conducted in smokers receiving b-carotene supplements to address key mechanistic questions of the promoting vs inhibiting actions of b-carotene in lung carcinogenesis. An appropriate animal model can be used to resolve the mechanistic issues. However, any experimental studies on the anticancer or procancer activity of b-carotene have been confounded by the poor absorption and low tissue levels of carotenoids in the rodent models used for such studies [29]. For example, rats, mice, and hamsters absorb carotenoids poorly from the diet. Thus, the epidemiologic data suggesting a protective effect of dietary carotenoids against cancer have been difficult to confirm in an experimental model. Similarly, the harmful effects of b-carotene in the b-carotene intervention trials in smokers have been difficult to reproduce in an experimental model. The important point is that humans can absorb small but significant amounts of unchanged b-carotene and accumulate very high concentrations of b-carotene in the peripheral tissues, while most species (e. g., rat, 191

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rabbit, chick, pig, and sheep) break down b-carotene in their intestinal tracts and, except at high doses, virtually no b-carotene is absorbed in its intact form [30]. It is also possible that the beneficial or adverse effect of b-carotene in rodent models is due to its metabolism into various b-carotene metabolites. It should be mentioned that, even in animals, there are species differences in the ability to convert b-carotene to vitamin A. Some animals, such as cats, are unable to convert b-carotene to vitamin A, although they do accumulate appreciable tissue b-carotene. An accepted animal model to mimic human b-carotene metabolism and function should meet the following criteria: (a) absorption of intact b-carotene since humans are able to absorb unchanged dietary b-carotene into the lymph and the blood; (b) ability to convert b-carotene into retinoids in the intestine since human intestine can convert dietary b-carotene into retinoids; (c) accumulation of intact b-carotene in tissue since humans deposit b-carotene in the adipose tissue, adrenal cortex, liver, kidney, testes, and skin; (d) pathophysiologic relevance to human conditions and diseases because of the differences between human and animal species; and (e) homology, expression and function of target genes which be similar to human. In 1939, Jensen and With [31] reported measurable carotenoid values in the liver of ferrets (Mustela putorius furo). Fox [32] showed that gastrointestinal tracts of ferrets have many anatomic and physiologic features that are similar to those of humans. We and other investigators conducted a series of experiments to study b-carotene metabolism and to establish the ferret as a model to mimic human b-carotene metabolism [33–38]. From these studies, we showed that ferrets appear to be appropriate for studying the intricacies of b-carotene metabolism both in vitro and in vivo. The ferret offers an excellent model for use in mimicking the conditions of the human b-carotene intervention studies, considering the similarities between ferret and human in a) absorption of intact b-carotene [34], b) accumulation of b-carotene in lung tissue [39], c) appearance of oxidative metabolites of b-carotene in the lung [40], d) lung architecture [32], and e) cigarette smoke-induced lung pathology [41, 42]. The ferret might be a better model if we consider that the ferret is a tractable, inexpensive and easily maintained laboratory animal as compared to the rhesus monkey [43] and preruminant calf [44]. However, high concentrations of retinyl esters in sera of members of the mustelidae families has been reported previously [45]. That is, compared with humans, ferrets have 10–38 times higher retinyl ester concentrations both in blood and peripheral tissues (liver, kidney, adrenal and adipose tissue) [46]. We found that the serum retinol level in both ferret and human are nearly identical; however, retinyl esters represent 93 % of total vitamin A level in ferret serum. In the human serum retinyl esters represents I 3 % of total vitamin A [47]. This property of the ferret has to be considered when using ferret as a model, although high retinyl ester levels in blood are a general phenomenon in carnivores [45, 48].

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14.2.1 Absorption and Accumulation of b-Carotene Serum b-carotene concentration may be affected by dietary factors, efficiency of absorption, rate of tissue uptake, and the rate of b-carotene metabolism. The uptake and absorption of b-carotene take place in the intestinal mucosa in both animals and humans [49]. The rate of absorption of b-carotene is affected by a number of conditions, e. g., the amount of lipid, protein and calories in the diet. Once inside the cell, b-carotene can either travel to the lateral surface of the cell and be incorporated into the chylomicrons or be cleaved at the central or peripheral double bonds of the molecule by b-carotene cleavage enzyme(s) [50]. In humans, approximately 9–17 % of orally administered labeled b-carotene is absorbed via the lymphatic system and 60–70 % of the absorbed radioactivity is found as retinyl esters, whereas 15 % remains as intact b-carotene [51]. In the fasting state, low-density lipoproteins and high-density lipoproteins were the major carriers of plasma bcarotene, containing 65 and 27 %, respectively, of the total plasma b-carotene [52]. In humans, the major sites of carotenoid storage are the adipose tissue and the liver, besides the high concentration of b-carotene in adrenals and testes [53, 54]. To determine whether the absorption of b-carotene in the lymph of ferrets and humans is of similar magnitude, Wang et al., [34] perfused 14C-bcarotene micellar solution in the ferret intestine. The maximal absorption rate of 0.054 mg/h/100 cm intestine in ferrets was compared with that of unchanged b-carotene in human lymph derived from Goodman and Blomstrand’s work [51, 55]. In their human subjects, the absorption rates were equivalent to 0.038 and 0.12 mg/h/100 cm intestine. These values bracket our ferret absorption rate of 0.054 mg/h/100 cm intestine, indicating a close similarity between humans and ferrets with respect to b-carotene absorption. Of total uptake/absorbed radioactivity, only 3.2 % was found in the lymph after 4 hours, whereas 28 % was transported via the portal vein. This may be due to the fact that the differential flow rate between lymph and portal vein is very large. It is noted that in previous studies only 3–6 % in rat [56, 57] and 8.7–16.8 % in human [55] of the absorbed radioactivity could be found in the lymph even after prolonged lymph collection. We found that b-carotene, retinyl esters, retinol, and the less-polar metabolites are absorbed into lymph, whereas the more polar metabolites, which include b-apo-carotenals, retinoyl-b-glucuronide, retinyl-b-glucuronide, and retinoic acid, are absorbed directly into the portal blood [34]. These studies indicate that portal vein plays an important role in b-carotene metabolism and that the differential absorption of b-carotene and its metabolites into lymph or into portal blood is dependent on the polarities of the metabolites involved. This aspect should be considered particularly when high dose of b-carotene was given in both animal study and human intervention study. We have investigated whether ferrets accumulate significant amounts of b-carotene in certain target organs (e. g., lung) after chronic feeding either 193

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physiologic or pharmacologic doses of b-carotene dietary b-carotene and cigarette smoke exposure [39, 28]. We have conducted chronic feeding experiments in the ferrets using [28]. In our study, the daily food intake of ferrets (1.0–1.2 kg ferret) is about 200 g/day, the average intake of b-carotene from the basal diet during the experimental period was 0.16 mg/kg/day (0.8 mg/g x 200 g/day). Since the total length of small intestine in ferrets is 1/5 of that of human and total absorption of b-carotene by the ferret is about 5 times less than human [34], this b-carotene intake in the ferret is equivalent to a 2.3 mg/day intake in a 70 Kg man. Physiologic b-carotene feeding group (0.43 mg b-carotene/Kg/day including the b-carotene in the basal diet): This dose of b-carotene in the ferret is three times higher than is contained in their basal diet and is equivalent to 6 mg b-carotene per day in humans. This amount of b-carotene is easily achievable in humans eating a high fruit and vegetable diet. Pharmacologic b-carotene feeding group (2.4 mg b-carotene/Kg/day including the b-carotene in the basal diet): To mimic human trials, we will feed 2.4 mg b-carotene/Kg/day to ferrets which is 15 times higher than the 0.16 mg b-carotene/Kg/day in the ferret’s basal intake. This dose of b-carotene in the ferret is equivalent to a 30 mg b-carotene per day intake in a 70 Kg man. The level of lung b-carotene in ferrets increased as the level of b-carotene in the diet increased (Tab. 14.1). However, the levels of lung b-carotene decreased dramatically in the smoke-exposed ferrets with high dose b-carotene supplementation, compared with the high dose b-carotene feeding alone group (Tab. 14.1). This decrease is related to the free radical-rich atmosphere in lungs of cigarette smokeTable 14.1. Concentrations of b-carotene and retinoids in the six groups of ferrets after six months of treatment. Control Plasma (nmol/L) b-Carotene 7e Retinol 749 e Retinyl 618 e palmitate Retinoic acid 1.38 e Lung Tissue (pmol/100 b-Carotene 11 e Retinol 39 e Retinyl 524 e palmitate Retinoic acid 1.75 e

SM

LBC

3a 55 146

2 e 1b 64 e 66 551 e 178

25 e 5c 736 e 71 684 e 108

0.19 mg) 2a 12 120

1.24 e 0.24 ND 33 e 7 506 e 148

SM LBC

SM HBC

6 e 2a 669 e 58 649 e 114

43 e 11e 729 e 82 708 e 143

1.42 e 0.28

1.48 e 0.31 1.33 e 0.14

1.28 e 0.26

283 e 37b 37 e 9 567 e 139

2230 e 224c 42 e 13 676 e 185

155 e 24e 35 e 10 532 e 119

0.33a 0.09 e 0.05b 1.81 e 0.45a

HBC 119 e 22d 798 e 90 737 e 220

21 e 5d 34 e 5 487 e 101

0.31 e 0.12c 0.46 e 0.16c ND

Value = Means e standard deviation (n = 6). ND = not detected. For a given tissue, different superscript letters for a given compound indicate that those values are significantly different from each other (PI0.05). SM = Smoke-exposed; LBC = Low-dose bcarotene; HBC = High-dose b-carotene; SM LBC = Smoke-exposed plus low-dose bcarotene; SM HBC = Smoke-exposed plus high-dose b-carotene. (Reproduced with permission from [28]).

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exposed ferrets which enhances oxidative cleavage of b-carotene and produces increased amounts of b-carotene oxidative metabolites in the ferret lung tissue [40]. Interestingly, the concentration of b-carotene in lung tissue (171 pmol/100 mg) from the smoke-exposed and high dose b-carotene supplemented group of ferrets was comparable to the b-carotene level in the lung (76 pmol/100 mg) of two active participants from CARET intervention study [58]. These studies indicated that, under certain condition, e. g., high dose b-carotene supplementation, the smoke-exposure alters b-carotene metabolism and produces more oxidative metabolites, which may facilitate carcinogenesis as discussed below.

14.2.2 Metabolism of b-Carotene 14.2.2.1 Central Cleavage Pathway b-carotene is the most active precursor of vitamin A. It has been widely accepted that b-carotene is converted to vitamin A by a soluble 15,15l-dioxygenase enzyme in the intestinal mucosa. This is based on the work of Goodman and Olson in the early 1960s [51, 59]. Both groups demonstrated the enzymatic synthesis of retinal from b-carotene in rat intestine and suggested that a dioxygenase reaction occurs. In this reaction molecular oxygen reacts with the central two carbon atoms of the carotene molecule, after which the central bond 15,15’-double bond of the carotene is cleaved yielding two molecules of retinal. Retinal could be subsequently reduced to retinol or oxidized further to form retinoic acid (Fig. 14.1). This observation was extended by recent cloning of the central cleavage enzyme of b-carotene, b-carotene 15,15l-dioxygenase, by several research groups using different species [60, 61]. However, recent studies have shown that the mechanism of the central cleavage in b-carotene is a non-heme iron monooxygenase [62, 63]. Regardless of the cleavage mechanism of b-carotene, the central cleavage is a major pathway leading to vitamin A formation [50]. 14.2.2.2 Excentric Cleavage Pathway The existence of excentric cleavage pathways in mammals has been a controversial issue among scientists for several decades [50]. Glover in 1960 [64] proposed an excentric cleavage (or random cleavage) pathway. According to this mechanism oxidative cleavage of b-carotene starts at either end of chain with equal probability; the oxidative breakdown continues with removal of two chain carbon units until the further oxidation is blocked by the methyl group at the C13 position, which is at the b-position with respect to central carbon atom of b-carotene. Thus, b-carotene can only yield one molecule of retinal via a series of b-apo-carotenals of different chain lengths. This hypothesis was partially supported by Ganguly and co-workers in 1977 [65, 66]. They isolated significant amounts of b-apo-8l-, 10l-, and 12l-carote195

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nal from the intestine of chickens given b-carotene and provided evidence for a random cleavage of the b-carotene molecule, and suggested that excentric cleavage may play an important role in b-carotene metabolism. Later, Wang et al. [8] demonstrate that the b-apo-carotenals could be oxidized to the corresponding b-apo-carotenoic acid, which might be further oxidized through a process analogous to b-oxidation yielding retinoic acid (Fig. 14.1). Considering the fact that the location of excentric cleavage enzyme(s) is unknown, our investigator used the intestinal mucosa homogenates after low speed centrifugation in in vitro studies. We demonstrated that a series of homologous carbonyl cleavage products are produced including retinal, b-apo-14l, 12l, 10l, and 8l-carotenals and b-apo-13-carotenone [33, 67]. We have observed that the production of b-apo-carotenals from b-carotene in rat intestinal mucosal homogenates continued for 60 minutes, whereas the appearance of retinal and retinoic acid was delayed, suggesting that b-apocarotenals may be intermediate compounds in the production of retinoids from b-carotene. In contrast, disulfiram, an inhibitor of sulfhydryl-containing enzymes, appears to be a nonspecific inhibitor because the formation of both b-apo-carotenals and retinoids from b-carotene was completely inhibited by 2 mM disulfiram when incubated with rat intestinal homogenates. Further evidence for excentric cleavage of b-carotene is that we have isolated b-apo-12lcarotenal and b-apo-10l-carotenal as well as retinoids from ferret intestinal mucosa after perfusion of b-carotene in vivo [29, 36]. This was strongly supported by recent publications on the molecular identification of b-carotene9’,10’-dioxgenase, catalyzing the excentric cleavage of b-carotene at the 9’,10’ double bond, and forming b-apo-10’-carotenal [68]. Based on these observations, we concluded that an excentric cleavage mechanism is involved in b-carotene metabolism, in addition to a central cleavage mechanism. Although the role of the excentric cleavage pathway of b-carotene in either physiological or pathological states needs more investigation, recently, we demonstrate that the biologic basis for the harmful effect of b-carotene supplementation in smokers is associated with the excentric cleavage metabolites of b-carotene and free-radical-rich atmosphere in the lungs of cigarette smokers [39, 28]. With the pharmacological (high) dose b-carotene supplementation, the environment of the lungs of cigarette smokers enhances b-carotene metabolism to produce oxidative by-products, such as, b-apo12’-, 10’-, and 8’-carotenals and b-carotene 5,6,-epoxide (Fig. 14.2). These metabolites of b-carotene can facilitate the binding of metabolites of benz[a]pyrene to DNA, down-regulate retinoic acid receptor b, up-regulate AP-1 (c-Jun and c-Fos) activity, induce carcinogen-activating enzymes, and interfere with retinoid metabolism, as disccussed below.

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Figure 14.2. HPLC analysis of carotenoids in ferret lung post-nuclear fraction after incubation with 10 mM b-carotene. Upper panel with normal lung post-nuclear fraction. Lower panel with smoke-exposed lung post-nuclear fraction. (Adapted from [39]).

14.2.2.3 Biosynthesis of Retinoic Acid Retinoic acid is a well known regulator of cell differentiation, proliferation, and apoptosis, particularly in certain carcinoma cell lines, and this property has been used in the successful treatment of acute promyelocytic leukemia. Therefore, retinoic acid produced from b-carotene metabolism might explain an anticancer effect of b-carotene, particularly in species, such as humans, that are capable of absorbing small but significant amounts of intact b-carotene and accumulating b-carotene in peripheral tissues. We conducted several experiments by either incubating intestinal homogenates with b-carotene in vitro [33, 29] or perfusing b-carotene micellar solution in the ferret intestine 197

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[35, 69] to determine whether b-carotene could be a precursor of retinoic acid. We observed that retinoic acid was produced from b-carotene. To ascertain if the production of retinoic acid occurred via the central cleavage pathway, involving direct oxidation of retinal to retinoic acid, or via the excentric pathway, involving oxidation of b-apo-carotenals to retinoic acid, we used the inhibitor, citral, which could block the oxidation of retinal in human intestinal mucosa in these experiments [29, 70]. We demonstrated that 1 mM citral did not block the production of various b-apo-carotenals and retinoic acid from the metabolism of b-carotene. Therefore, retinoic acid and b-apo-carotenals were both formed from incubation of intestinal homogenate with b-carotene in both the presence and absence of citral, which proves the existence of an excentric cleavage pathway of b-carotene. It is interesting that, similar to the small intestine, peripheral tissues (such as the lung, kidney, liver and fat) of ferret, rat, monkey and human tissue can also convert b-carotene into retinoic acid [7, 33]. Thus, it is possible that increased dietary b-carotene or other provitamin A carotenoids may affect the steady-state concentration of retinoic acid in body fluids or tissues. The very rapid metabolic clearance of retinoic acid from body fluids and tissue may increase the significance of local tissue conversion. 14.2.2.4 Perturbations in Retinoic Acid Metabolism by High Dose b-Carotene Supplementation in Cigarette Smoker and Alcohol Drinker Contribute to Lung Carcinogenesis It has been reported that b-apo-8l-carotenal, an excentric cleavage product of b-carotene, but not b-carotene itself, is a strong inducer of CYP1A1 in rats [71]. This is particularly interesting to us because the formation of b-apo-8lcarotenal from b-carotene was 2.5 fold higher in lung extracts of smokeexposed ferrets than from non-smoke exposed ferrets [39]. Therefore, induction of CYPs by either b-carotene oxidative cleavage products or by cigarette smoke has two possible detrimental actions in lung tissue: 1) bioactivating carcinogens and 2) destroying retinoic acid, thereby enhancing lung carcinogenesis (Fig. 14.3). In our study, the concentration of retinoic acid in lung tissue was significantly lower in the animals treated with smoke alone or with high dose bcarotene (with or without smoke-exposure), but was not lower in ferrets treated with low dose b-carotene alone [28]. Recently, Paolini et al. [72], showed a significant increase in several cytochrome P450 (CYP) enzymes (CYP1A1/2, CYP2A1, CYP2B1, and CYP3A1/2) in the lungs of rats supplemented with very high doses of b-carotene (500 mg/per kg body weight). We hypothesize that the destruction of retinoic acid in the ferret lung after smoke alone or after high dose b-carotene (with or without smoke) treatment is due to CYP induction, and this is supported by our data that retinoic acid levels are lower in the lungs of smoke-exposed, high dose b-carotene supplemented ferrets [39]. We have carried out a study to test if CYPs are involved in the destructtion of retinoic acid. Using retinoic acid as the substrate, we found 198

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Figure 14.3. Possible biochemical mechanism(s) by which high dose b-carotene supplementation increased lung cancer risk in smokers, whereas low-dose b-carotene with or without combined vitamins E and C may provide protection against lung cancer risk (Reproduced with permission from [40]).

that the formation of the polar metabolites including 18-hydroxy-retinoic acid and 4-oxo-retinoic acid increased 6 to 10 fold after incubation with smokeexposed, high dose b-carotene supplemented, or both treated ferret lung microsomes, as compared with the controls [73]. Furthermore, this enhancement of microsomal retinoic acid catabolism can be inhibited by liarozole, a non-specific inhibitor for CYPs. This observation provides a possible explanation for why retinoic acid level was lower in the lung of smoke-exposed, high dose b-carotene supplemented ferrets. The interaction between alcohol and b-carotene in the ATBC study has been observed, such that the b-carotene effect was more harmful among the men who had claimed to consume larger amounts of alcohol [14, 15]. In human, b-carotene beadlets potentiate hepatotoxicity of alcohol with increased leakage of liver enzymes in the plasma and an inflammatory response in the liver [74]. Recent study demonstrate that b-carotene potentiates the CYP2E1 induction by ethanol in rat liver [75], which may, at least in part, explain the associated adverse effects. Recently, we have shown that 199

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cytochrome P4502E1 is the major CYP responsible for the ethanol-enhanced catabolism of retinoic acid in hepatic tissue after treatment with alcohol [76]. Although the mechanism of interaction between alcohol and b-carotene needs more study, it is likely that heavy alcohol intake may interfere with b-carotene and retinoic acid metabolism similar to that of cigrarett smoke exposure. This hypothesis was supported by our recent observation that chronic alcohol intake greatly interfere with retinoid biosynthesis and catabolism, particularly resulting in a lowering of retinoic acid levels, producing an environment which may contribute to hepatocellular cell proliferation and carcinogenesis [77–79]. It seems that chronic and excessive alcohol intake is a risk not only for hepatic but also for extra-hepatic cell proliferation and carcinogenesis, since it has been reported that CYP2E1 is also present and inducible by alcohol in the esophagus, forestomach, and surface epithelium of the proximal colon [80].

14.2.3 Instability of the b-Carotene Molecule in the Antioxidant-poor Environment of the Lungs of Cigarette Smokers The possible mechanism to explain the instability of the b-carotene molecule is that exposure of lung cells to smoke results in increased lung cell oxidative stress and thereby causes a decrease in other antioxidants, such as ascorbate and a-tocopherol (Fig. 14.3), which normally have a stabilizing effect on the unoxidized form of b-carotene [81–84]. The failure of the b-carotene intervention trials to show a benefit against lung carcinogenesis in smokers, has shift interest from the use of single nutrients to some of the nutrient combinations found in foods (particularly in fruits and vegetables) for chemoprevention. 14.2.3.1 b-Carotene and Vitamin E Several lines of evidence demonstrate that interactions occur between b-carotene and tocopherols. Pretreating human lung cells with both vitamin E and b-carotene provides significant protection on DNA strand breakage induced by tobacco-specific nitrosamines [85]. Furthermore, a recent study suggests that b-carotene is capable of regenerating a-tocopherol from its radical [84]. Conversely, vitamin E protect carotenoids from autoxidation; the combination of b-carotene and a-tocopherol results in inhibition of free radical induced lipid peroxidation which is significantly greater than the sum of the individual inhibitions [86]. This synergistic interaction was accompanied by an increased loss of a-tocopherol. These data suggest that tocopherol may limit the prooxidant effects of carotenoids in biologic systems. Moreover, as in the human, we have shown that there is no antagonistic effect between the absorption of fat-soluble b-carotene and vitamin E [87]. Rather, vitamin E enhances lymphatic transport of b-carotene and central cleavage of b-carotene to form vitamin A (rather than oxidative byproducts) [87].

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14.2.3.2 b-Carotene and Vitamin C Vitamin C is a strong reducing agent known to act as an antioxidant in vitro and in vivo [88]. It has been reported that vitamin C is able to convert the b-carotene radical back to b-carotene and can help to maintain b-carotene in its unoxidized form [81, 82]. Epidemiologic evidence consistently and strongly indicates that vitamin C has a protective effect against a variety of human tumors. Diets high in fruit and vegetables, and hence high in vitamin C, have been found to be associated with a lower risk for cancers of the lung, oral cavity, esophagus, stomach, and colon [89]. Also vitamin C protects lipids in human plasma against peroxidative damage by scavenging oxygenderived free radicals [90]. Epidemiologic studies have shown that smokers have significantly lower plasma levels of vitamin C compared with nonsmokers [91]. Similarly, passive smokers have reduced ascorbic acid concentrations in their plasma [92]. Several studies have reported that low intake of vitamin C is associated with an increased risk of cervical cancer [93], which is especially apparent in smokers. However, it has been reported that in vitro vitamin C could induce the decomposition of lipid hydroperoxides which have the capacity to damage DNA [94]. Thus, it will be also important to examine whether vitamin C at varying doses could affect smoke-induced lung lesions (protective vs harmful). 14.2.3.3 Combination of b-Carotene with Vitamins E and C b-Carotene, vitamin E, and vitamin C act together in a network fashion to provide broad antioxidant protection, and should be studied for possible chemopreventive effects. Although pre-treatment of human lung cells with both vitamin E and b-carotene has been shown to provide protection against DNA strand breaks induced by tobacco-specific nitrosamines [85], the combination of b-carotene (20 mg/day) and vitamin E (50 mg/day) were not found to be protective against smoke-related lung cancer in the ATBC study. However, vitamin C, which would facilitate both vitamin E recycling and b-carotene stability, was not used in the ATBC study. It is particularly important to have broad antioxidant protection when using high doses of b-carotene in order to prevent the production of carotene excentric cleavage products and the subsequent cascade of events that may result from them (i. e. bioactivating carcinogens and destroying retinoic acid via induction of CYP enzymes, or facilitating the binding of benzo[a]pyrene metabolites to DNA). Both vitamins E and C can inhibit cytochrome P-450 mediated lipid peroxidation and carcinogen activation. The combination of b-carotene, a-tocopherol and vitamin C has been shown by Böhm et al. [82] to provide synergistic protection against free radical damage in an in vitro cell system. Moreover, possible protective effects of combined antioxidant supplementation in humans exposed to environmental tobacco smoke has been reported [95, 96]. Howard et al. [95] demonstrated that oxidative stress induced by environmental tobacco smoke can be reduced in humans by supplementation 201

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of 3 mg of b-carotene, 60 mg of vitamin C, and 30 IU of a-tocopherol plus minerals. Duthie et al [97] carried out a double-blind antioxidant supplementation study with vitamin C (100 mg/day), vitamin E (280 mg/day) and b-carotene (25 mg/day) in 50 smokers and 50 non-smokers for 20 weeks. These investigators reported that supplementation of the combined antioxidants resulted in a significant (p I 0.002) decrease in endogenous oxidative base damage in lymphocyte DNA in both smokers and non-smokers. These studies and the known biochemical interactions of b-carotene, vitamin E and vitamin C suggest that this combination of nutrients would be an effective chemopreventive strategy against lung cancer in smokers (Fig. 14.3). However, the use of an animal model and in vitro experiments to see if low and high dose b-carotene, used in combination with other dietary antioxidants, may have a possible chemopreventive effects against lung cancer, are the justifiable approaches for both possible mechanisms and safety before any initiation of huamn intervention study using the combination.

14.2.4 The Carcinogenic Response to b-Carotene Supplementation may be Related to the Dosage Used One of the important questions, which remain to be answered, is whether an anticarcinogenic or procarcinogenic effect of carotenoids is related to the carotenoid dose, which is administered in vivo (Tab. 14.2). More specifically, what dose is safe? Would a smaller dose of carotenoids provide optimal antioxidant protection, while not giving risk to undesirable metabolic byproducts – and thus provide protection against carcinogenesis? Recently, Lowe et al. [98] demonstrated that b-carotene and lycopene protect against oxidative DNA damage (induced by xanthine/xanthine oxidase) in HT29 cells at relatively low concentrations (1–3 mM), but loses this capacity at higher concentrations (4–10 mM). Yeh and Hu [99] demonstrated Table 14.2. Comparison of b-carotene doses, plasma b-carotene levels and risk of lung cancer in smokers and smoke-exposed ferrets.

Observational studies (5-9 servings of fruits and vegetables) Intervention studies ATBC1 CARET2 PHS3 (11 % smoker) Ferret studies4

b-Carotene Intake (mg/day)

Plasma b-Carotene increase (fold)

Risk of Lung Cancer

Z6

1.5 – 3

Decrease

20 30 50/other day 30 6

18 14 4 21 3

Increase Increase No effect Increase Decrease

Data: 1Ref: [15]; 2Ref: [17]; 3Ref:[18]; 4Ref: [39, 28, 100]

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that b-carotene and lycopene at a high concentration (20 mM) signifycantly enhanced levels of lipid peroxidation induced by a lipid-soluble radical generator (2,2l-azobis[2,4-dimethylvaleronitrile]. Recently, we have determined if there is a true hazard associated with high dose b-carotene supplementation and smoking in an animal model. We evaluated the effects of b-carotene at low-dose (equivalent to an intake of 6 mg of b-C/day/70 kg human) vs high-dose (equivalent to an intake of 30 mg of b-C/day/70 kg human) supplementation on proliferating cellular nuclear antigen (PCNA) expression, and histopathological changes in the lungs of cigarette smoke exposed ferrets. Cell proliferation and squamous metaplasia were assessed by examining proliferating cellular nuclear antigen expression and histopathology. The concentration of b-carotene and retinoids in the lung and plasma were analyzed by high performance liquid chromatography. Our results showed that alveolar cell proliferation and keratinized squamous metaplasia were observed in the lung tissue of high dose b-carotene supplemented group with or without smoke exposure, but not in the low dose b-carotene supplemented groups with or without smoke exposure (Fig. 14.4, [28, 40, 100]). PCNA expression

Figure 14.4. Pathologic changes in the lung tissue of 3 groups of ferrets after six months of treatment (smoke exposure (SM)); combination of smoke exposure with low-dose b-carotene (SM LBC); combination of smoke exposure with high-dose b-carotene (SM HBC). Hematoxylin-eosin-stained (HE) sections at original magnification of x5 are indicated by HE x5; HE x50 for magnification of x50; and IM for immunohistochemical staining for keratin (at magnification of x50). Any keratinized squamous metaplasia lesion (histological examination with confirmation by immunohistochemistry by anti-keratin antibody) in the right upper lobe of lung was defined as positive. The squamous metaplasia lesions were observed in the groups of SM HBC as well as the SM group compared with the groups of SM LBC (no positive animal identified). (Reproduced with permission from [28]).

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was correlated with histopathological changes in the lung tissue among the groups. In contrast to the animals receiving high dose b-carotene supplementation, neither the level of retinoic acid nor the expression of RARb in the lung was affected in the animals treated with low dose b-carotene, as compared with the control group. In fact, compared with the smoke-exposed group, low dose b-carotene supplementation in the smoke-exposed group resulted in a significantly lower decrease of lung retinoic acid levels. This supports the possibility, that b-carotene, when given at a low dose, could act to supply adequate retinoic acid to the lung tissue of smoke-exposed ferrets (Fig. 14.6). However, when given at a high dose, oxidative by-products of b-carotene (e. g., b-apo-8’-carotenal) could induce cytochrome P450 enzymes,

Figure 14.5. Simplified schematic illustration of possible molecular mechanism(s) of cigarette smoke and high dose b-carotene supplementation on lung cell proliferation and carcinogenesis.

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thus destroying retinoic acid (Fig. 14.5). The dosages of b-carotene used in the ATBC and CARET studies were 20 to 30 mg per day for 2-8 years, and these doses are 10 to 15 fold higher than the average intake (2 mg) of b-carotene in a typical American diet. Such a pharmacological dose of b-carotene in humans could result in an accumulation of a relative high b-carotene level in lung tissue, especially after long periods of supplementation, which could also lead to a decrease in lung retinoic acid concentration [100]. In all of these studies, the dose of b-carotene appears to be an important factor (Tab. 14.2). Thus, the carcinogenic response to high dose b-carotene supplementation reported in the human intervention trials may be related to the dosage used and/or the instability of the b-carotene molecule in the free radicalrich environment of the lungs of cigarette smokers. These data suggest that, in contrast with high dose b-C supplementation, low-dose b-C supplementation in smoke exposed ferrets has no harmful effects and may afford protection against lung damage induced by cigarette smoke. This hypothesis

Figure 14.6. Simplified schematic illustration of possible molecular mechanism(s) of cigarette smoke and low dose b-carotene supplementation on lung cell proliferation and carcinogenesis.

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was also supported by recent b-carotene intervention studies in human, which indicate that b-carotene may prevent gastric carcinogenesis and oral precancerous lesions [23, 22]. Unlike lung tissue, eliminating b-carotene though epithelial cells can prevent accumulation of excessive b-carotene in oral and gastric mucosa. This hypothesis was also supported by recent animal study reported that both dietary and topical b-carotene prevents skin carcinoma formation in mice and induces retinoic acid receptor expression, while failing to act as tumor promoter in the two-stage model of skin tumorigenesis [25].

14.2.5 Action of b-Carotene on Genetic Signaling Pathway and Carcinogenic Process 14.2.5.1 MAP Kinase Pathway Recent evidence has accumulated, which supports a role for reactive oxygen species in the activation of Jun N-terminal kinase (JNK) activity and the regulation of AP-1 gene expression [101]. We observed that AP-1(c-Jun and c-Fos) expression is up-regulated in the high dose b-carotene-supplemented ferrets with or without smoke-exposure as compared with the control animals (Fig. 14.5). This overexpression of AP-1 was positively associated with increased PCNA expression and with squamous metaplasia in the lungs of the high dose b-carotene supplemented animals, with or without smokeexposure. Recently, Lee et al. [102–104] provided the first evidence that alltrans retinoic acid suppresses Jun N-terminal kinase (JNK) activity by inhibiting JNK phosphorylation and inducing MAP kinase phosphatase 1 (MKP-1). As AP-1 sites are found in a number of genes that are important in the control of cell proliferation, this type of interaction is almost certainly responsible for certain of the anti-proliferative and anti-cancer properties attributed to retinoic acid. It has been reported that c-Jun is required for progression through the G1 phase of the cell cycle by a mechanism that involves direct transcriptional control of the cyclin D1 gene [105]. It is conceivable that the overexpression of c-Jun by cigarette smoke or chronic excess b-carotene intake may cause abnormal cell cycle regulation and drive cells into premature S phase, resulting in an aberrant mitotic process, thereby causing cell proliferation and promoting carcinogenesis. This hypothesis is supported by the increase of expression of both c-Jun and cyclin D1 in the lungs of the high dose b-carotene supplemented animals with or without smoke-exposure [28, 39, 73]. 14.2.5.2 p53 Tumor Suppressor Since the multistep process of carcinogenesis requires an accumulation of multiple genetic alterations in order for normal cells to progress to cancer, the question arises if any genes (such as p53 tumor suppressor gene and its 206

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target genes) can be affected by b-carotene supplementation. The role of p53 tumor suppressor gene mutations in human cancer has been well established. Overexpression of p53 protein is associated with cigarette consumption [106] and there is a dose-response relationship between the quantity of tobacco consumed and the frequency of p53 mutations among lung cancer patients [107, 108]. Sozzi et al. [109] have detected p53 mutations in severe dysplasia of the bronchial mucosa adjacent to lung cancer, and indicated that p53 mutation occurs at an early stage of lung carcinogenesis. By using both Western blot analysis and immunohistochemical staining, we observed that p53 gene expression in the lung was significantly increased in smoke-exposed ferrets with or without high dose b-carotene supplementation, compared with the control group [73]. Since the detection of p53 protein expression by immunohistochemistry is used as an indirect indicator of p53 gene malfunction, these data suggest that lung damage by both cigarette smoking and high dose b-carotene supplementation may occur via a DNA damage or phopharylation mechanism of the p53 gene in the ferret. p53 may function as a dual action gene: tumor suppressor in its wild type form, and dominant oncogene in some of its mutant forms. Since the loss of function of p53 by mutation might be expected to result in enhanced proliferative activity and tumor progression, elevated levels of p53 may be a useful indicator for monitoring various types of genotoxic effects and tumorigenesis (for example over-expression of p53 protein correlated with carcinogenesis in the lung). 14.2.5.3 Retinoid Signaling Pathway Retinoic acid derived from either vitamin A or b-carotene acts on normal bronchial epithelium by inducing mucous and blocking squamous differentiation [110, 111]. Since squamous metaplasia occurs during the early stages of lung carcinogenesis, perturbations in retinoid signaling may contribute to lung carcinogenesis [2, 112]. A role for RARb (which is induced by retinoic acid) as a tumor suppressor gene has been proposed [113]. RARb is abnormally expressed in human lung cancer cell lines and in a number of primary tumors [114]. Recent in situ hybridization studies show that up to 50 % of primary lung tumors lack RARb expression and that loss of expression is an early event in lung carcinogenesis [112], although the molecular mechanism through which RARb expression is lost is uncertain [115]. Conversely, restoration of RARb2 in a RARb-negative lung cancer cell line has been reported to inhibit tumorigenicity in nude mice [116]. The retinoic acid receptors gene expressions in the lungs of the ferrets were examined. The results showed that the concentration of retinoic acid in the lung tissue was significantly lower in ferrets exposed to high dose b-carotene alone or b-carotene plus smoke as compared with the control group. Similarly, RARb gene expression, but not RAR-a and RAR-g, was down-regulated between 18 % to 73 % in the three treatment groups, compared with the control group; whereas, no differences in RAR-b gene expression were observed in low dose b-carotene group [28]. We concluded that diminished retinoid signaling by the down-regula207

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tion of RARb expression could be a mechanism for enhancement of lung tumorigenesis after high-dose b-carotene supplementation and cigarette smoke exposure (Fig. 14.5). 14.2.5.3 b-Carotene Metabolites Can Act as co-Carcinogens Several reports have appeared pertaining to the question of whether intact b-carotene or its metabolites can act as co-carcinogens. Salgo et al. [117] reported that b-carotene decreases the binding of metabolites of benzo[a]pyrene (one of the most important smoke-borne carcinogens) to DNA, whereas the HPLC fractions containing b-carotene oxidative metabolites facilitate the binding of metabolites of benzo[a]pyrene to DNA. Although the oxidative metabolites were not identified, their study [117] provided a basis for the possible mechanism(s) for a harmful effect of the combination of smoking and b-carotene supplementation on initiation of carcinogenesis. Perocco et al. [118] showed that induction of BALB/c 3T3 cell transformation by benzo[a]pyrene was markedly enhanced by the presence of b-carotene, although it is not clear whether the enhancement of cell transforming activity was due to b-carotene itself or to its metabolites. In general, these studies indicate that b-carotene itself can act as an anticarcinogen, but that its oxidized products may facilitate carcinogenesis (Fig. 14.3).

Summary To better understand the potential function of b-carotene in the prevention of cancer, greater knowledge of b-carotene metabolism and a suitable animal model to mimic human b-carotene metabolism are necessary. The close similarity between humans and ferrets with respect to intact b-carotene absorption/metabolism and biological function indicates that the ferret affords a model to study b-carotene metabolism that is highly analogous to the process in humans. The use of appropriate in vitro experiments, and/or animal models are the most justifiable approaches to resolve mechanistic issues regarding protective or harmful effects of carotenoids. These information are critically needed for future human studies involving carotenoid for prevention of lung cancer and cancers at other tissue sites. Furthermore, the failure of the b-carotene intervention trials to show a benefit against lung carcinogenesis in smokers, has shifted interest to use of other carotenoids found in fruits and vegetables for chemoprevention (e. g., lycopene). However, the metabolism and molecular biological properties of a number of carotenoids remain poorly understood. Especially, knowledge of dose effects, tissue specific and possible adverse effects with tobacco and alcohol are lacking. In order to avoid similar problems in the use of potential chemopreventive agents, such as was found in the b-carotene trials, it is important to conduct animal 208

References experiments using varying carotenoid doses, with varying organs and with and without exposure to exogenous factors such as tobacco smoking and alcohol drinking. The results from the animal studies will guide the cell culture studies and subsequent human translational studies.

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48. Wilson DE, Hejazi J, Elstad NL, Chan IF, Gleeson JM, Iverius PH. (1987) Novel aspects of vitamin A metabolism in the dog: distribution of lipoprotein retinyl esters in vitamin A-deprived and cholesterol-fed animals. Biochim. Biophys. Acta 922, 247–258. 49. Wang X-D. (1994) Absorption and metabolism of b-carotene. J. Am. Coll. Nutr. 13, 314–325. 50. Wang X-D, Krinsky NI. (1998) The bioconversion of b-carotene into retinoids. In Biochemistry of lipid-soluble vitamins. Subcellular Biochemistry 27, 159–180. 51a. Goodman DS, Huang HS. (1965) Biosynthesis of vitamin A with rat intestinal enzymes. Science 149, 879–880. 51b. Goodman DS, Blomstrand R, Werner B, Huang HS, Shirator T. (1966) The intestinal absorption and metabolism of vitamin A and b-carotene in man. J. Clin. Invest. 45, 1615–1623. 52. Johnson EJ, Russell RM. (1992) Distribution of orally administered b-carotene among lipoproteins in healthy men. Am. J. Clin. Nutr. 56, 128–135. 53. Parker RS. (1988) Carotenoid and tocopherol composition of human adipose tissue. Am. J. Clin. Nutr. 47, 33–36. 54. Kaplan LA, Lau JM, Stein EA. (1990) Carotenoid composition, concentrations, and relationships in various human organs. Clin. Physiol. Biochem. 8, 1–10. 55. Blomstrand R, Werner B. (1967) Studies on the intestinal absorption of radioactive b-carotene and vitamin A in man. Scand. J. Clin. Lab. Invest. 19, 339–345. 56. Huang HS, Goodman DS. (1965) Vitamin A and carotenoids I. Intestinal absorption and metabolism of 14C-labeled vitamin A alcohol and b-carotene in the rat. J. Biol. Chem. 240, 2839–2844. 57. Lakshman MR, Asher KA, Attlesey MG, Satchithanandam S, Mychkovsky I, Coutlakis PJ. (1989) Absorption, storage, and distribution of beta-carotene in normal and beta-carotene-fed rats: roles of parenchymal and stellate cells. J. Lipid Res. Oct; 30 10, 1545–50. 58. Redlich CA, Blaner WS, Van Bennekum AM, Chung JS, Clever SL, Holm CT, Cullen MR. (1998) Effect of supplementation with b-carotene and vitamin A on lung nutrient levels. Cancer Epidemiol. Biomarkers Prev. 7, 211–4. 59. Olson JA, Hayaishi O. (1965) The enzymatic cleavage of b-carotene into vitamin A by soluble enzymes of rat liver and intestine. Proc. Natl. Acad. Sci. USA 54, 1364– 1370. 60. von Lintig J, Vogt K. (2000) Filling the gap in vitamin A research. Molecular identification of an enzyme cleaving b-carotene to retinal. J. Biol. Chem. 275, 11915–20. 61. Wyss A, Wirtz G, Woggon W, Brugger R, Wyss M, Friedlein A, Bachmann H, Hunziker W. (2000) Cloning and expression of b, b-carotene 15,15l-dioxygenase. Biochem. Biophys. Res. Commun. 271, 334–336. 62. Leuenberger MG, Engeloch-Jarret C, Woggon WD. (2001) The Reaction Mechanism of the Enzyme-Catalyzed Central Cleavage of beta-Carotene to Retinal. Angew. Chem. Int. Ed. Jul 16, 40 (14), 2613–2617. 63. Lindqvist A, Andersson S. (2002) Biochemical properties of purified recombinant human beta-carotene 15,15l-monooxygenase. J. Biol. Chem. Jun 28, 277 (26), 23942–8. 64. Glover J. (1960) The conversion of b-carotene into vitamin A. Vitam. Horm. 18, 371–386. 65. Sharma RV, Mathur SN, Ganguly J. (1976) Studies on the relative biopotencies and intestinal absorption of different apo-b-carotenoids in rats and chickens. Biochem. J. 158, 377–383.

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References 66. Sharma RV, Mathur SN, Dmitrovskii AA, Das RC, Ganguly J. (1977) Studies on the metabolism of b-carotene and apo-b-carotenoids in rats and chickens. Biochim. Biophys. Acta 486, 183–194. 67. Tang G, Wang XD, Russell RM, Krinsky NI. (1991) Characterization of b-apo-13carotenone and b-apo-14l-carotenal as enzymatic products of the excentric cleavage of b-carotene. Biochemistry 30, 9829–9834. 68. Kiefer C, Hessel S, Lampert JM, Vogt K, Lederer MO, Breithaupt DE, von Lintig J. (2001) Identification and characterization of a mammalian enzyme catalyzing the asymmetric oxidative cleavage of provitamin A. J. Biol. Chem. 276, 14110–6. 69. Hebuterne X, Wang X-D, Smith DEH, Tang G, Russell RM. (1996) In vivo biosynthesis of retinoic acid from b-carotene involves an eccentric cleavage pathway in ferret intestine. J. Lipid Res. 31, 482–492. 70. Liu C, Wang X-D, Russell RM. (1997) Biosynthesis of retinoic acid from b-apo-14lcarotenal in ferret in vivo. J. Nutr. Biochem. 8, 157–161. 71. Gradelet S, Leclerc J, Siess MH, Astorg P. (1996) b-Apo-8l-carotenal, but not b-carotene, is a strong inducer of liver cytochromes P4501A1 and 1A2 in rats. Xenobiotica 26, 909–919. 72. Paolini M, Cantelli-Forti G, Perocco P et al. (1999) Co-carcinogenic effect of b-carotene. Nature 398, 760–1. 73. Liu C, Russell RM, Wang XD. (2003) Exposing ferrets to cigarette smoke and a pharmacological dose of beta-carotene supplementation enhance in vitro retinoic acid catabolism in lungs via induction of cytochrome P450 enzymes. J. Nutr. 133 (1), 173–9. 74. Leo MA, Lieber CS. (1999) Alcohol, vitamin A, and b-carotene: adverse interactions, including hepatotoxicity and carcinogenicity. Am. J. Clin. Nutr. 69, 1071–85. 75. Kessova IG, Leo MA, Lieber CS. (2001) Effect of beta-carotene on hepatic cytochrome P-450 in ethanol-fed rats. Alcohol Clin. Exp. Res. 25 (9), 1368–72. 76. Liu C, Russell RM, Seitz HK, Wang X-D. (2001) Ethanol enhances retinoic acid metabolism into polar metabolites in rat liver via induction of cytochrome P4502E1. Gastroenterology 120, 179–189. 77. Wang X-D. (2001) Retinoids and alcoholic liver disease. In: Agarwal DP, Seitz HK (ed.) Alcohol in Health and Disease. Marcel Dekker Inc: New York, 427–452. 78. Chung J, Liu C, Smith DE, Seitz HK, Russell RM, Wang X-D. (2001) Restoration of retinoic acid concentration suppresses ethanol-enhanced c-Jun expression and hepatocyte proliferation in rat liver. Carcinogenesis 22, 1213–1219. 79. Chung J, Chavez RG, Russell RM, Wang X-D. (2002) Retinoic acid inhibits hepatic Jun N-terminal kinase-dependent signaling pathway in ethanol-fed rats. Oncogene 21, 1539–1547. 80. Shimizu M, Lasker JM, Tsutsumi M, Lieber CS. (1990) Immunohistochemical localization of ethanol-inducible P450IIE1 in the rat alimentary tract. Gastroenterology 99, 1044–1053. 81. Black HS. (1998) Radical interception by carotenoids and effects on UV carcinogenesis. Nutr. Cancer 31, 212–217. 82. Böhm F, Edge R, McGarvey DJ, Truscott TG. (1998) b-Carotene with vitamins E and C offers synergistic cell protection against NOx. FEBS Lett. 436, 387–389. 83. Handelman GJ, Packer L, Cross CE. (1996) Destruction of tocopherols, carotenoids, and retinol in human plasma by cigarette smoke. Am. J. Clin. Nutr. 63 (4), 559-65. 84. Böhm F, Edge R, Land EJ, McGarvey DJ, and Truscott TG. (1997) Carotenoids enhance vitamin E antioxidant efficiency. J. Am. Chem. Soc. 119, 621–622. 85. Weitberg AB, Corvese D. (1997) Effect of vitamin E and b-carotene on DNA strand breakage induced by tobacco-specific nitrosamines and stimulated human phagocytes. Journal of Experimental and Clinical Cancer Research 16, 11–4.

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86. Palozza P, Krinsky NI. (1992) b-Carotene and a-tocopherol are synergistic antioxidants. Arch. Biochem. Biophys. 297, 184–7. 87. Wang X-D, Marini RP, Hebuterne X, Fox J, Krinsky NI, Russell RM. (1995) Vitamin E can enhance lymphatic absorption of b-carotene and its conversion into vitamin A in the ferret. Gastroenterology 108, 719–726. 88. Frei B. (1994) Reactive oxygen species and antioxidant vitamins: mechanisms of action. Am. J. Med. 97, 5–13. 89. Byers T, Guerrero N. (1995) Epidemiologic evidence for vitamin C and vitamin E in cancer prevention. Am. J. Clin. Nutr. 62, 1385–1392. 90. Frei B, Forte TM, Ames BN, Cross CE. (1991) Gas phase oxidants of cigarette smoke induce lipid peroxidation and changes in lipoprotein properties in human blood plasma: protective effects of ascorbic acid. Biochem. J. 277, 133–138. 91. Schectman G, Byrd JC, Gruchow HW. (1989) The influence of smoking on vitamin C status in adults. Am. J. Public Health 79, 158–162. 92. Tribble DL, Giuliana LJ, Fortmann SP. (1993) Reduced plasma ascorbic acid concentrations in nonsmokers regularly exposed to environmental tobacco smoke. Am. J. Clin. Nutr. 58, 886–90. 93. Sauberlich HE. (1994) Pharmacology of vitamin C. Annu. Rev. Nutr. 14, 371–391. 94. Lee SH, Oe T, Blair IA. (2001) Vitamin C-induced decomposition of lipid hydroperoxides to endogenous genotoxins. Science 292 (5524), 2083–6. 95. Howard DJ, Ota RB, Briggs LA, Hampton M, Pritsos CA. (1998) Oxidative stress induced by environmental tobacco smoke in the workplace is mitigated by antioxidant supplementation. Cancer Epidemiol. Biomark. Prev. 7, 981–988. 96. Yong L-C, Brown CC, Shatzkin A, Dresser CM, Slesinski MJ, Cox CS, Taylor PR. (1997) Intake of vitamins E, C, and A and risk of lung cancer. Am. J. Epidemiol. 146, 231–43. 97. Duthie JS, Ma A, Ross MA, Collins AR. (1996) Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res. 56, 1291– 1295. 98. Lowe GM, Booth LA, Young AJ, Bilton RF. (1999) Lycopene and b-carotene protect against oxidative damage in HT29 cells at low concentrations but rapidly lose this capacity at higher doses. Free Radiol. Biol. Med. 30, 141–51. 99. Yeh SL, Hu ML. (2001) Induction of oxidative DNA damage in human foreskin fibroblast Hs68 cells by oxidized beta-Carotene and lycopene. Free Radic. Res. 35 (2), 203–13. 100. Liu C, Lian F, Smith DE, Russell RM, Wang XD. (2003) Lycopene supplementation inhibits lung squamous metaplasia and induces apoptosis via up-regulating insulin-like growth factor-binding protein 3 in cigarette smoke-exposed ferrets. Cancer Res. 63 (12), 3138–44. 101. Palmer HJ, Paulson KE. (1997) Reactive oxygen species and antioxidants in signal transduction and gene expression. Nutr. Rev. 55 (10), 353–61. 102. Lee H-Y, Dawson MI, Walsh GL, Nesbitt JC, Eckert RL, Fuchs E, Hong WK, Lotan R, Kurie JM. (1996) Retinoic acid receptor- and retinoid X receptor-selective retinoids activate signalling pathways that converge on AP-1 and inhibit squamous differentiation in human bronchial epithelial cells. Cell Growth & Differentiation 8, 997–1004. 103. Lee H-Y, Dawson MI, Claret FX, Chen JD, Walsh GL, Hong WK, et al. (1997) Evidence of a retinoid signaling alteration involving the activator protein 1 complex in tumorigenic human bronchial epithelial cells and non-small cell lung cancer cells. Cell Growth & Differentiation 8, 283–91.

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References 104. Lee H-Y, Sueoka N, Hong W-K, Mangelsdorf DJ, Claret FX, Kurie JM. (1999) Alltrans-retinoic acid inhibits Jun N-terminal kinase by increasing dual-specificity phosphatase activity. Mol. Cel. Biol. 19, 1973–1980. 105. Wisdom R, Johnson RS, Moore C. (1999) c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms. EMBO J. 18, 188–197. 106. Gosney JR, Butt SA, Gosney MA, Field JK. (1993) Exposure to cigarette smoke and expression of the protein encoded by the p53 gene in bronchial carcinoma. Ann. NY Acad. Sci. 28, 243–247. 107. Suzuki H, Takahashi T, Kuroishi T et al. (1992) p53 mutations in non-small cell lung cancer in Japan: Association between mutations and smoking. Cancer Res. 52, 734–736. 108. Kondo K, Tsuzuki H, Sasa M, Sumitomo M, Uyama T, Monden Y. (1996) A doseresponse relationship between the frequency of p53 mutations and tabacco consumption in lung cancer patients. J. Surg. Oncol. 61, 20–26. 109. Sozzi G, Miozzo M, Donghi R et al. (1992) Deletions of 17p and p53 mutations in preneoplastic lesions of the lung. Cancer Res. 52, 6079–6082. 110. Jetten AM, George AM, Pettit GR, Rearick JI. (1989) Effects of bryostains and retinoic acid on phorbol ester- and diacylglycerol-induced squamous differentiation in human tracheobronchial epithelial cells. Cancer Res. 49, 3990–3995. 111. Kim YH, Dohi DF, Han GR, Zou CP, Oridate N, Walsh GL, Nesbitt JC, Xu XC, Hong WK, Lotan R, Kuri JM. (1995) Retinoid refractoriness occurs during lung carcinogenesis despite functional retinoid receptors. Cancer Res. 55, 5603–5610. 112. Zhang XK, Liu Y, Lee MO. (1996) Retinoid receptors in human lung cancer and breast cancer. Mutat. Res. 350, 267–77. 113. Houle B, Rochette-Egly C, Bradley WEC. (1993) Tumor-suppressive effect of the retinoic acid receptor-b in human epidermoid lung cancer cells. Proc. Natl. Acad. Sci. USA 90, 985–989. 114. Xu XC, Lee JS, Lee JJ, Morice RC, Liu X, Lippman SM, Hong WK, Lotan R. (1999) Nuclear retinoid acid receptor b in bronchial epithelium of smokers before and during chemoprevention. J. Natl. Cancer Inst. 91, 1317–21. 115. Altucci L, Gronemeyer H. (2001) The promise of retinoids to fight against cancer. Nature Rev. Cancer 1, 181–193. 116. Houle B, Pelletier M, Wu J, Goodyer C, Bradley WEC. (1994) Fetal isoform of human retinoic acid receptor b expressed in small cell lung cancer lines. Cancer Research 54, 365–369. 117. Salgo MG, Cueto R, Winston GQ, Pryor WA. (1999) b carotene and its oxidation products have different effects on microsome mediated binding of benzo[a]pyrene to DNA. Free Radiol. Biol. Med. 26, 162–173. 118. Perocco P, Paolini M, Mazzullo M, Biagi GL, Cantelli-Forti G. (1999) b-carotene as enhancer of cell transforming activity of powerful carcinogens and cigarettesmoke condensate on BALB/c 3T3 cells in vitro. Mutat. Res. 440, 83–90.

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15 Potential Adverse Mechanisms of Antioxidants During Cancer Therapy Hans Konrad Biesalski*, Jürgen Frank*, Debra K. Kelleher**, Christine Lambert*, Oliver Thews**, and Peter Vaupel**

The aim of conservative treatment of cancer with chemotherapy or high energy radiation is either the induction of apoptosis or the direct fatal damage to tumor cells. Both forms of treatment exert their effects on induction of apoptosis or necrosis in part via formation of free radicals. From recent experiments we have strong evidence that two increasingly used treatment strategies, low energy radiation (photo dynamic therapy – PDT) and hyperthermia (HT) exert their therapeutic effects also via formation of free radicals. We carried out a couple of animal experiments to elucidate whether HT- or PDT-treatment results in formation of reactive oxygen species (ROS) and whether the effect of ROS formation may explain the effects of theses treatments on neoplastic cells. During hyperthermia superoxide anions are formed due to specific oxygenation conditions of the tumor tissue. PDT alone produces severe damage of endothelial cells with the result of impaired oxygenation and blood flow in the tumor. To optimize this approach both treatment methods were combined. Oxidative stress-related changes in tumors (in vivo) upon localized hyperthermia (HT), 5-aminolevulinic acidbased photodynamic therapy (ALA-PDT) and their combination were examined following the observation that antitumor effects of ALA-PDT were significantly enhanced upon simultaneous application of HT. HT alone induces formation of free radicals with subsequent protein- and lipid-peroxidation and apoptosis, an event which is potentiated if oxygen is delivered via the mask (induction of ischemia/reperfusion). In comparison to HT alone, an increase in the number of cells undergoing apoptosis was seen with ALAPDT and ALA-PDT HT (TUNEL-assay, DNA-fragmentation). Increased caspase-3 and -8 activities were found after HT, ALA-PDT, or ALA-PDT HT. Immunohistological detection of protein nitration was used to localize reactive nitrogen-related damage. Increased peroxynitrite formation was most prominent after ALA-PDT HT preferably in endothelial cells. The formation of cytotoxic peroxynitrate however, strongly depends on the presence of superoxide anions. Taken together the formation of reactive oxygen species with subsequent oxidation/nitration of proteins and induction of apoptotic signaling pathways is a major event during HT and PDT. Antioxidant compounds, which can accumulate in tumor tissues might counteract the effects

* Inst. Biol. Chemistry & Nutrition, University of Hohenheim, 70593 Stuttgart, Germany ** Inst. Physiol. & Pathophysiol., University of Mainz, 55099 Mainz, Germany

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of therapy induced ROS in tumor tissues. In addition there is evidence that therapy resistant tumor cells express higher levels of antioxidant enzymes than sensitive cells. Treatment of resistant cells with either inhibitors of SOD (2-ME) or transfection of radical generating systems (e. g. amino acid oxidases) results in an increased sensitivity. Our results, and the fact that radiation and chemotherapy exert their effects via the formation of free radicals raises the critical question whether antioxidants should be avoided during conservative tumor therapy.

References 1. Frank J, Biesalski HK, Dominici S, Pompella A. The visualization of oxidant stress in tissues and isolated cells. Histol Histopathol. 2000; 15: 173–84. 2. Frank J, Kelleher DK, Pompella A, Thews O, Biesalski HK, Vaupel P. Enhancement of oxidative cell injury and antitumor effects of localized 44 degrees C hyperthermia upon combination with respiratory hyperoxia and xanthine oxidase. Cancer Res. 1998; 58: 2693–8. 3. Frank J, Pompella A, Biesalski HK. Histochemical visualization of oxidant stress. Free Radic. Biol. Med. 2000; 29: 1096–105.

16 Cholesterol Lowering Vegetable Oil Spreads: Results of a Post Launch Monitoring Programme Linda J. Lea* and Paul A. Hepburn

Abstract Phytosterol-esters (PE) have been approved under regulation (EC) No 258/97 on Novel Foods and Food Ingredients as a novel food ingredient for use in vegetable oil spreads. PE enhances the blood cholesterol lowering activity of the spread, by reducing the absorption of cholesterol from the small intestine. The pre-market safety assessment of toxicological and clinical studies had established that there was a reasonable certainty of no harm resulting from consumption of PE. However, a requirement of the European Commis-

* Safety & Environmental Assurance Centre, Unilever, Colworth Hause, Sharnbrook, Bedfordshire, MK44 1 LQ, UK

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sion decision was to establish a post launch monitoring (PLM) programme to accompany the marketing of the product. The PLM scheme developed by Unilever for PE consisted of three components: a) Is the use as predicted/recommended? b) Are known effects and side effects as predicted? c) Does the product induce unknown side-effects? Part A (Is use as predicted/recommended?) A standard procedure for most manufacturers is to monitor the introduction of new food products into the market place. In this case, market place surveys were commissioned to check that consumer usage patterns were consistent with marketing expectations i. e. to establish which consumers are using the product and to estimate how much they are using. This continuous quantitative method of market research provides data direct from households, which are classified into demographic groups (age of primary shopper; size of household; socio-economic group; presence of children; geographical region), and allows a detailed estimate of intake. The results of the market research have demonstrated that the product is being bought by the target population and is predominantly being used by one person in each household. Intake of the product is lower than the original assumptions even for regular/established users, with median intakes of the spread being less than 20 g/day (or I 1.6 g phytosterols/day). Part B (Are known effects and side effects as predicted?) The known effects of PE have been consistently demonstrated in a large number of clinical studies i. e. a beneficial reduction in serum total and LDL-cholesterol levels. The only observed side-effect has been a small reduction in the serum levels of the most lipophilic carotenoids (e. g. b-carotene). A long-term study has confirmed that an intake of 20 g/day of a spread containing PE is not associated with a biologically significant lowering of serum carotenoids [1], being less than both the individual variation and the seasonal variation in carotenoid levels. It has also been shown that carotenoid levels can be maintained by incorporating the use of PE-enriched spreads into a healthy diet rich in fruit and vegetables [2]. Therefore, it is highly unlikely that current intakes of spreads containing PE will lead to reductions in carotenoids that have biological significance to humans. Part C (Does the product induce unknown side-effects?) Unilever operating companies have telephone care lines in place for their products, in which consumer comments and complaints are answered and 218

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monitored. The care line number is available on all packs and Unilever is taking advantage of this well established consumer contact mechanism to collect information on potential consumer reactions. This information is being gathered in all countries where Unilever is marketing spreads containing PE. This has enabled the monitoring of health-related calls and detection of any trends in unexpected side effects on a global basis and thereby maximised the potential for identifying possible adverse events. Information on all health-related calls made to consumer telephone care lines regarding the use of spreads containing PE has been collected and assessed centrally using a well-defined scheme. Given the extensive exposure to spreads containing PE in Europe the number of health-related calls is very small. Complaints including health related calls were highest during the first few months after introduction to the market, but have now slowed considerably despite a significant rise in sales. These reports cover a broad range of self-reported conditions that are well within the normal occurrence of such conditions in the general population with no clear patterns emerging. The reports to date reveal no adverse health effects associated with PE and therefore no cause for concern. In summary, a structured approach to the Post Launch Monitoring of PEcontaining spreads is described. Data collected in the market place under “real life” conditions has been helpful to confirm the validity of the initial risk assessment and as a basis to check the assumptions used to derive it. In particular, i) the product is being bought by the target population, but the intakes are lower than the assumptions which were made in the original risk assessment; and ii) there is no evidence for the occurrence of adverse health effects providing further support that PE-containing spreads are both safe and effective.

16.1 Introduction Phytosterols (e. g. b-sitosterol, campesterol and stigmasterol) occur naturally as minor components of vegetable oils present in the unsaponifiable fraction. They occur in the free form, esterified with C12–C18 fatty acids and also conjugated as glucosides. Average phytosterol intakes are in the range 200– 400 mg/day, whilst individual phytosterol intake may be as high as 680 mg/ day. Vegetarians generally have a higher intake of phytosterols, which can be approximately 40 % higher than in non-vegetarians. Phytosterols lower serum cholesterol by decreasing cholesterol absorption in the small intestine. The principal mechanism of serum cholesterol lowering is considered to be competition between cholesterol and the phytosterols for micellar solubilisation in the small intestine. The first studies demonstrating the cholesterol-lowering effect of phytosterols in humans

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were reported by Pollak in the early 1950’s and since then a large number of clinical studies with phytosterols have been conducted [3]. A comprehensive investigation of the efficacy of phytosterol-esters has been conducted. To date, more than 2000 individuals have taken part in over 30 clinical trials at study sites in six continents. These studies confirm that the daily consumption of PE consistently lowers LDL cholesterol by 6–15 % depending on the daily intake level. The cholesterol-lowering effect is measurable after 2–2.5 weeks [4–6], reaching a steady state after approximately three weeks. The cholesterol-lowering effect of phytosterols is persistent at the steady state for periods measured up to one year as long as consumption of the spread is maintained [1]. Furthermore, the cholesterollowering effect has been shown to be independent of dietary or life style modification, baseline cholesterol level, dietary fat intake or fat level of the spread. The PE-enriched spread is a high (PUFA) spread and contains 14 % plant sterol-esters (maximum of 8 % free sterols compared with 0.3 % for typical PUFA spreads). The phytosterols have been obtained from vegetable oil sources and have been esterified with fatty acids from sunflower oil. There is a history of safe consumption of phytosterols within the normal dietary intake of between 200–400 mg/day. Normal use of the PE-containing spread increases phytosterol intake by between 5–10-fold. To support the increased intake of PE a comprehensive safety testing programme has been carried out. In summary, the conclusions from these studies were as follows: x x x

x x

No evidence of genotoxicity [7]. Absorption is very low [8]. No evidence of subchronic toxicity – NOAEL of 6.6 g phytosterol-esters/ kg/bodyweight/day in a 90 day rat feeding study (equal to the maximum dose tested) [9]. No effect on the reproductive system, and no oestrogenic activity [10, 11]. High doses produced no adverse physiological effects in humans [12, 13].

The safety assessment for the use of PE for use in a vegetable oil spread was based on a number of assumptions concerning the use of the spread. These were: x x x

typical daily intake of the product would be 20–30 g, upper (95th percentile) intake levels in the vicinity of 57 g (UK) and 70 g (NL), consumers would be mainly over 45 years old and concerned about their cholesterol level.

The typical daily intake and upper intake levels were based on published information on the consumption of vegetable oil spreads in the UK and the Netherlands and were consistent with product marketing information from 220

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commissioned trials. The assumption about the consumer profile was based on market research and the sales performance of similar products. This paper describes a programme of Post Launch Monitoring (PLM) which was conducted to confirm these assumptions. It is based on the normal procedures which are used by (large) food companies to monitor the introduction of new food products into the market place. Consumer feedback is typically monitored through consumer telephone care lines and the commissioning of market surveys to check that consumer usage patterns are consistent with predictions. There are three components to the scheme: A) Is use as predicted/recommended? B) Are known effects and side-effects as predicted? C) Does the product induce unknown side-effects?

16.2 Methodology 16.2.1

PLM Part A: Is Use as Predicted/Recommended?

Data has been obtained from independent market research organisations to establish which consumers are using the PE-enriched spread and to estimate how much they are using. This continuous quantitative method of market research supplies data direct from households. The number of registered households varied according to the size of the population in each country e. g. 3000 in Belgium to 12 000 in Germany. When members of the registered households go shopping they do so in the normal way, but on returning home they scan the barcodes of all their purchases into a special barcode reader unit, which then transfers the information to a centralised database at the market research company. The data provides information on what the households buy, when they buy it and what demographic group the purchaser belongs to. Registered households are classified by the following terms: Age of primary shopper; Size of household; Socio-economic group; Presence of children; Geographical region. The technique is non-invasive and as panel members are not aware of which purchases are being monitored, it is able to provide real purchase patterns and supply a link between purchase data and consumer type. It also allows a detailed estimate of intake. Data are continuously recorded by each household registered in the scheme. This information is then weighted to bring the panel in line with known population profiles for the country. Every nine months questionnaires are sent to the panellists. This provides an opportunity to ask attitudinal questions and also ensure that the demographic profile for the household is 221

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correct. In addition, regular checks are made to ensure purchases are being accurately recorded. Purchase level is also compared against standard for the household and against other households that are known to have high compliance. The information output can then be used to answer many questions, both about absolute quantities of products sold and about trends. This provides a powerful technique for monitoring multiple product and combined product intakes. By including information from an attitudinal questionnaire and combining it with the demographic profiles it is possible to ascertain more complex information.

16.2.2

PLM Part B: Are Known Effects and Side-Effects as Predicted?

Clinical studies have consistently demonstrated a beneficial reduction in the blood total and LDL-cholesterol levels – the known effects. The only sideeffect observed has been a small reduction in the absorption of the most lipophilic carotenoids (e. g. b-carotene). The approach taken to assess this known side-effect was to: x x x

monitor serum carotenoid levels following controlled intake of the product (20 g/day) over a one year period [1], assess whether this level of intake causes a biologically significant lowering in serum carotenoids, assess whether actual consumer intakes of the PE-enriched spread raise any cause for concern.

16.2.3

PLM Part C: Does the Product Induce Unknown Side-Effects?

A system has been developed using the telephone care lines, which are in place within the operating companies, to collect consumer comments and complaints. The care line telephone number is provided on all packs and the information is being gathered in all countries where the PE-enriched spread is marketed. This enables the monitoring of health-related calls and detection of any trends in unexpected side-effects on a national or global basis and thereby maximises the potential for identifying possible adverse events. An integrated network system has been established world-wide for the care line staff and they have undergone additional training in order for them to be able to identify and collect relevant information on unexpected side-effects from consumers in an unbiased manner. When a call is received by the care line, information is collected and then transferred to a central PLM team based at Unilever’s Safety and Environmental Assurance Centre (SEAC). The likelihood of a causal relationship between consumption of the product and the reported symptoms is then assessed by a clinician and 222

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a toxicologist. All calls and their classifications are monitored to allow the potential identification of signals indicating that unexpected side-effects are occurring.

16.3 Results 16.3.1

PLM Part A: Is Use as Predicted/Recommended?

Market research data up to 1 April 2001 have been analysed from approximately 2000 households in total. Households included are from the Netherlands, UK, France, Germany and Belgium which are the major EU markets for the PE-containing spread. Data are provided for both ‘regular’ purchasers and ‘all’ purchasers’ (includes those that have only bought the product once or infrequently during the monitoring period). Size of household Across the five main markets, between 62 and 82 % of all people purchasing the product came from one- or two-person households. Similar figures were seen for regular purchasers with 66 to 90 percent coming from one- or twoperson households. Use by households with children There were no children living in 78–91 % of the households purchasing the product. For regular purchasers, the numbers were slightly higher, with 87–96 % of households having no children living at home. In the Netherlands additional information was available regarding the age of children in the household. This showed where households buying the product had children these were over the age of five. Age of purchaser In the UK, Netherlands and Germany 83–91 % of all purchasers and 87–95 % of regular purchasers were over 45 years old. For Belgium and France slightly different age groups were assessed. In these countries, respectively, 75 and 77 % of all purchasers, and 79 and 83 % of regular purchasers were over 50 years old. Median daily intake of product per household The daily intake per household has been estimated based on the number of packs bought during a 12 or 13 week period. The monitoring period was 223

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deliberately chosen to be at the end of the period being reviewed (i. e. during Quarter 1, 2001) to allow the market to settle and regular purchase patterns to be established. The data for all users shows that the median daily intake of product per household was between 3 g/day (France) and 12 g/day (Netherlands/Germany). For the regular users a more consistent pattern of usage was estimated with median intakes per household between 15 and 18 g/day. Considering all users the upper (95th percentile) intake per household was between 21 g/day (France) and 33 g/day (Belgium). For regular users the upper intakes were between 27 g/day (France) and 45 g/day (Netherlands). Household panel data for the remaining EU markets have indicated a similar consumer profile to the five largest markets, but the number of households involved is insufficient for a rigorous statistical analysis.

16.3.2

PLM Part B: Are Known Effects and Side-Effects as Predicted?

Hendriks et al. [1] investigated the long-term safety and efficacy of PE using 185 volunteers. As part of their habitual diet, individuals consumed 20 g per day of either a spread containing PE (equivalent to 1.6 g sterols/day) or a control spread (without added PE) for a period of one year. As well as monitoring the cholesterol-lowering efficacy of PE a large number of additional parameters (including carotenoids) were also monitored in this trial. Total and LDL cholesterol were significantly lowered in the group receiving the PE-enriched spread when compared to the control group while HDL cholesterol, triglyceride and lipoprotein (a) remained unchanged. Mean carotenoid concentrations were not significantly different between the PE group and the control group after 26 weeks of the study. At week 52, a- and b-carotene and lycopene were significantly reduced in the PE-group when compared to the control group. However, after correction for LDL-cholesterol concentrations (carotenoids are transported in the plasma primarily with the LDL fraction), only the a-carotene reduction remained significant. In another study [2], serum carotenoid levels were monitored in volunteers consuming specified daily amounts of fruit and vegetables in addition to PE-enriched spreads over a three week period. This showed that the consumption of an additional daily serving of a fruit or vegetable that is rich in carotenoids is effective in maintaining carotenoid levels when consuming spreads containing PE. The level of reduction of serum carotenoids observed is less than that due to other dietary influences, person to person variation and seasonal variations (which are also linked to diet). The serum level of carotenoids can vary from season to season by up to 30 % depending on the main fruit and vegetables available at the time [14–18]. Thus any study of the longer-term effects of phytosterols needs to be seen in the context of this larger seasonal variation as well as the considerable inter-individual variation. Furthermore, PE-enriched spreads are recommended as part of a healthy diet rich in fruit 224

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and vegetables. As demonstrated by Noakes et al. [2], this can have a significant influence on serum carotenoid levels. Current evidence does not indicate that a small reduction in the serum levels of carotenoids poses a health risk. In particular, there is inadequate evidence to support b-carotene having cancer-preventative activity [19]. The one-year human study [1] has confirmed that intakes of 20 g/day PE-enriched spread are not associated with biologically significant lowering of serum carotenoid levels. Part A of the PLM scheme established that average intake levels are below the predicted 20 g spread per day. Therefore, it is highly unlikely that current intakes of the PE-enriched spread will lead to reductions of serum carotenoids that have biological significance to humans.

16.3.3

PLM Part C: Does the Product Induce Unknown Side-Effects?

The data presented are for the period from when the product was first introduced to the EU market, through to the end of August 2001. The date of introduction to the market varied between countries, but for the majority of countries this represents a period of slightly more than one year. An initial assessment of the data collected was made at the end of March 2001 (approximately seven months of data) and then again at the end of August 2001. It is known that complaints for all consumer products are highest when they are first introduced. Calls to the care lines For the first one-year period nearly 84 000 contacts to the care lines had been made. More than 53 000 of these were during the first seven-month period. Of these contacts 1673 were complaints, 1168 during the period August 2000 to March 2001 and 505 from April to August 2001. The majority of contacts were requests for product information including availability, positive feedback and general enquiries. Health-related calls Included in the 1673 complaints were a total of 227 calls related to health issues. These were unevenly distributed between the two time periods reviewed with 146 being received in the period up to April 2001 and 81 being received between April and August 2001. These calls were unevenly distributed between the different countries and are not consistently related to the volume of sales in that country. In particular, health-related calls are heavily biased towards the UK and Netherlands with 173 of the total 227 calls coming from these two countries.

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Caller demographics Health-related calls were evenly distributed between men and women. Not all callers were willing to provide details of their age but of those who did the majority were over 45 years old. No health-related calls involved children or pregnant women. Where information was provided the majority of these callers reported using 20 g or less per day. Classification of health-related calls After the assessment of these calls by a clinician and a toxicologist, 11 were dismissed as irrelevant (i. e. non-health-related). For a further 70 there was no association between product use and onset of symptoms. For the remaining 148 calls a causal relationship between product use and onset of symptoms could not be excluded. Types of health-related calls All the health-related calls were made by consumers. No reports from health care professionals on behalf of their patients were received. The majority (i 80 %) of reports have been mainly related to gastro-intestinal (GI) effects and skin conditions. The GI reports cover a wide range of self-diagnosed conditions such as diarrhoea and constipation. The skin calls related to urticaria, rashes, pimples and itching on various parts of the body. The remaining calls covered a range of miscellaneous self-reported conditions such as tingling, headache, dizziness, shortness of breath, rapid heartbeat and pain in joints. Most of these were single occurrences.

16.4 Discussion The household panel data has shown that the product is being bought by the target population (i 45 years and cholesterol concerned), but the intakes are lower than the assumptions made in the original risk assessment, with regular users consuming 15–18 g/day. Furthermore, the estimated 95th percentile intakes of between 27 g and 45 g/day were also lower than the 57 g (UK) and 70 g (Netherlands) per day assumed in the risk assessment. The main feature that this household panel data cannot provide is which member of the household actually uses the product (apart from one-person households). However, the similarity between the intakes for one person households and larger households indicates that in general use is predominantly by one person per household. The non-invasive nature of the data collection (panel members are unaware of which of their purchases are being monitored) means that realistic estimates can be obtained without the inher226

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ent bias of supervised surveys of individuals purchasing and consumption behaviour. Another advantage is that intakes are being monitored over a longer period of time so they can provide a more accurate picture of consumer use in the longer term. Furthermore, by assuming that only one person per household is consuming the product a worst case scenario is being considered. For these reasons the use of the household intakes as an indicator of individual intakes is therefore considered to be a valid approach. Clinical studies have consistently demonstrated a beneficial cholesterollowering effect. Long-term use of PE-enriched spreads results in a slight reduction in serum levels of the most lipophilic carotenoids. This is not biologically significant and less than either the individual variation or the seasonal variation in carotenoid levels. Carotenoid levels can be maintained by incorporating the use of PE-enriched spreads into a healthy diet rich in fruit and vegetables [2]. It is well known that for all consumer products, complaints are at the highest when the product is first introduced to the market. At this time point sales are still being established so complaints are disproportionately high compared to sales. For this reason the data was assessed at two time points, the first after seven months when usage patterns were still being established, and then after 12 months when it is anticipated that consumer use will have stabilised. As anticipated, more consumer contacts were made during the first seven months of consumer use, and around 70 % of complaints were made during this period despite a significant increase in sales during the shorter second time period. Health-related calls were also higher in the first seven months of consumer use. Public awareness of the product (new type of food, cost) may influence consumers to associate an illness with use of the product. In general, health-related calls were received from consumers in the target population (over 45 years old) and no calls related to the use of the product by pregnant women or children. There was also no indication that callers reporting health-related complaints had been consuming more than the recommended amounts of spread. These findings are consistent with our findings from the household panel data. Health-related calls were also heavily biased to two countries, the UK and the Netherlands that together accounted for over 75 % of the healthrelated calls. The reason for this is not entirely clear. The UK is one of the largest European markets for the PE-enriched spread and also has the highest number of consumer contacts. For the other countries there is no correlation between the size of the market, which reflects the exposure of consumers to the spread and the number of health-related calls. The figures may also reflect cultural differences. Although this provides an important perspective on the data, the main purpose of this part of the scheme was to evaluate the calls for possible safety alerts. Given the extensive exposure to the PE-enriched spread in Europe the number of health-related calls has been small. Of the 145 calls where an association had been made between use of PE-enriched spread and onset 227

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of symptoms, the calls fell into two main types: GI and skin conditions. Both the GI and skin complaints were broad in nature with no clear patterns emerging. The remaining calls related to a wide range of other miscellaneous clinical conditions. For all health calls the conditions reported were self-diagnosed although some consumers had sought medical advice for their condition. However, there were no adverse health reports received through health care professionals and no reports of consumers requiring hospital treatment for the complaints indicating that the complaints were mild in nature. These adverse health reports are well within the ‘normal’ occurrence of such complaints in the general population and are not unexpected for a product that has been ingested. The types and occurrence of the conditions reported are similar to those seen in control groups in clinical trials, in particular the one-year study [1] and, based on the information available, do not give any cause for concern. It is considered that the adverse health reports received are not due to the phytosterol-esters in the product but we cannot rule out the possibility of another explanation such as a reaction to a change in diet or a reaction to one of the other ingredients e. g. lactose or starch.

References 1. Hendriks HFJ, Ntanios FY, Brink EJ, Princen HMG, Buytenhek R, Meijer GW, (2001). One-year follow-up study on the use of a low fat spread enriched with plant sterol-esters. Annals of Nutrition and Metabolism, 45 (supplement 1), 100. (Proceedings of the 17th International Congress of Nutrition, August 27–30 2001, Vienna, Austria. Abstract (full study report made available to the SCF, March 2000 as document D00-015)). 2. Noakes M, Clifton P, Ntanios F, Shrapnel W, Record I, McInerney J, (2002). An increase in dietary carotenoids when consuming plant sterols or stanols is effective in maintaining plasma carotenoid levels. American Journal of Clinical Nutrition, 75, 79–86. 3. Pollak OJ, (1985). Effect of plant sterols on serum lipids and atherosclerosis. Pharmacology and Therapeutics, 31, 177–208. 4. Jones PJ, Ntanios FY, Raeini-Sarjaz M, Vanstone CA, (1999). Cholesterol-lowering efficacy of a sitostanol-containing phytosterol mixture with a prudent diet in hyperlipidemic men. American Journal of Clinical Nutrition, 69, 1144–50. 5. Maki KC, Davidson MH, Umporowicz DM, Schaefer EJ, Dicklin MR, Ingram KA, Chen S, McNamara JR, Gebhart BW, Ribaya-Mercado JD, Perrone G, Robins SJ, Franke WC, (2001). Lipid responses to plant sterol enriched reduced fat spreads incorporated into a National Cholesterol Education Program Step I diet. American Journal of Clinical Nutrition, 74, 33–43. 6. Weststrate JA, Meijer GW, (1998). Plant sterol-enriched margarines and reduction of plasma total- and LDL-cholesterol concentrations in normocholesterolaemic and mildly hypercholesterolaemic subjects. European Journal of Clinical Nutrition, 52, 334–343.

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References 7. Wolfreys AM, Hepburn PA, (2002). Safety Evaluation of Phytosterol Esters Part 7. Assessment of mutagenic activity of phytosterols, phytosterol-esters and the cholesterol derivative, 4-cholesten-3-one. Food and Chemical Toxicology, 40, 461–470. 8. Sanders DJ, Minter HJ, Howes D, Hepburn PA, (2000). Safety Evaluation of Phytosterol Esters. Part 6. The Comparative Absorption and Tissue Distribution of Phytosterols in the Rat. Food and Chemical Toxicology, 38, 485–491. 9. Hepburn PA, Horner SA, Smith M, (1999). Safety Evaluation of Phytosterol Esters. Part 2. Sub-chronic 90-day Oral Toxicity Study on Phytosterol Esters – A Novel Functional Food. Food and Chemical Toxicology, 37, 521–532. 10. Baker VA, Hepburn PA, Kennedy SJ, Jones PA, Lea LJ, Sumpter JP, Ashby J, (1999). Safety Evaluation of Phytosterol Esters Part 1. Assessment of Oestrogenicity using a Combination of In vivo and In vitro assays. Food and Chemical Toxicology, 37, 13–22. 11. Waalkens-Berendsen DH, Wolterbeek APM, Wijnands MVW, Richold M, Hepburn PA, (1999). Safety Evaluation of Phytosterol Esters. Part 3. Two Generation Reproduction Study in Rats with Phytosterol Esters - A Novel Functional Food. Food and Chemical Toxicology, 37, 683–696. 12. Weststrate JA, Ayesh R, Bauer-Plank C, Drewitt PN, (1999). Safety Evaluation of Phytosterol Esters. Part 4. Faecal Concentrations of Bile Acids and Neutral Sterols in Healthy Normolipidaemic Volunteers Consuming a Controlled Diet either with or without a Phytosterol Ester-enriched Margarine. Food and Chemical Toxicology, 37, 1063–1071. 13. Ayesh R, Weststrate JA, Drewitt PN, Hepburn PA, (1999). Safety Evaluation of Phytosterol Esters. Part 5. Faecal Short-chain Fatty Acid and Microflora Content, Faecal Bacterial Enzyme Activity and Serum Female Sex Hormones in Healthy Normolipidaemic Volunteers Consuming a Controlled Diet Either with or without a Phytosterol Ester-enriched Margarine. Food and Chemical Toxicology, 37, 1127– 1138. 14. van het Hof KH, Tijburg LB, Pietrzik K, Weststrate JA, (1999). Influence of feeding different vegetables on plasma levels of carotenoids, folate and vitamin C. Effect of disruption of the vegetable matrix. British Journal of Nutrition, 82, 203–12. 15. Lux O, Naidoo D, (1994). Biological Variation Of Beta-Carotene. Nutrition Research, 14, 693–698. 16. Olmedilla B, Granado F, Blanco I, Rojas-Hidalgo E, (1994). Seasonal and sexrelated variations in six serum carotenoids, retinol, and alpha-tocopherol. American Journal of Clinical Nutrition, 60, 106–10. 17. Saintot M, Astre C, Scali J, Gerber M, (1995). Within-subjects seasonal variation and determinants of inter-individual variations of plasma beta-carotene. International Journal for Vitamin and Nutrition Research, 65, 169–74. 18. Scott KJ, Finglas PM, Seale R, Hart DJ, de Froidmont-Gortz I, (1996). Interlaboratory studies of HPLC procedures for the analysis of carotenoids in foods. Food Chemistry, 57, 85–90. 19. IARC, (1998). International Agency for Research on Cancer. Handbooks of Cancer Prevention, volume 2, Carotenoids IARC Press, Washington D. C. 20. Albanes D, Virtamo J, Taylor PR, Rautalahati M, Pietinen P, Heinonen OP, (1997). Effects of supplemental b-carotene, cigarette smoking, and alcohol smoking on serum carotenoids in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. American Journal of Clinical Nutrition, 66, 366–372. 21. Amundsen ÅL, Ose L, Ntanios FY, (2002). Plant sterol ester-enriched spread lowers plasma total- and LDL-cholesterol in children with familial hypercholesterolemia. American Journal of Clinical Nutrition, 76, 338–44.

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22. Amundsen ÅL, Ose L, Ntanios FY, (2001). Effects of plant sterol ester-enriched spread on plasma lipids and safety parameters in children with familial hypercholesterolemia (FH) in controlled and follow-up periods. Abstract in Annals of Nutrition and Metabolism, 45 (supplement 1), 125. (Proceedings of the 17th International Congress of Nutrition, August 27–30 2001, Vienna, Austria). 23. Alpha-Tocopherol, Beta-Carotene (ATBC) Cancer Prevention Study Group (1994). The effect of vitamin E and Beta-carotene on the incidence of lung cancer and other cancers in male smokers. New England Journal of Medicine, 330, 1029–1035. 24. Greenberg ER, Baron JA, Tosteson TD, Freeman DH, Beck GJ, Bond JH, Colacchio TA, Coller JA, Frankl HD, Haile RW, Mandel JS, Nierenberg DW, Rothstein R, Snover DC, Stevens MM, Summers RW, van Stolk RU, for the Polyp Prevention Study Group, (1994). A clinical trial of antioxidant vitamins to prevent colorectal adenomas. New England Journal of Medicine, 331, 141–147. 25. Heinonen OP, Albanes D, Virtamo J, Taylor PR, Huttunen JK, Hartman AM, Haapakoski J, Malila N, Rautalahti M, Ripatti S, Maenpaa H, Teerenhovi L, Koss L, Virolainen M, Edwards BK, (1998). Prostate cancer and supplementation with a-tocopherol and b-carotene: Incidence and mortality in a controlled trial. Journal-National Cancer Institute, 90, 440–446. 26. Hennekens CH, Buring JE, Manson JE, Stampfer M, Rosner B, Cook NR, Belanger C, LaMotte F, Gaziano JM, Ridker PM, Willett W, Peto R, (1996). Lack of effect of long-term supplementation with beta-carotene on the incidence of malignant neoplasma and cardiovascular disease. New England Journal of Medicine, 334, 1145–1149. 27. Law MR, Morris JK, (1998). By how much does fruit and vegetable consumption reduce the risk of ischaemic heart disease? European Journal of Clinical Nutrition, 52, 549–556. 28. MacLennan R, Macrae F, Bain C, Battistutta D, Chapuis P, Gratten H, Lambert J, Newland RC, Ngu M, Russell A, Ward M, Wahlqvist ML, the Australian Polyp Prevention Project, (1995). Randomized trial of intake of fat, fibre and beta-carotene to prevent colorectal adenomas. Journal of the National Cancer Institute, 87, 1760–1766. 29. Neil HAW, Meijer GW, Roe LS, (2001). Randomised controlled trial of use by hypercholesterolaemic patients of a vegetable oil sterol-enriched fat spread. Atherosclerosis, 156, 329–37. 30. Ness AR, Powles JW, (1997). Fruit and vegetables, and cardiovascular disease: a review. International Journal of Epidemiology, 26, 1–13. 31. Nigon F, Serfaty-Lacrosnie`re C, Beucler I, Chauvois D, Neveu C, Giral P, Chapman MJ, Bruckert E, (2001). Plant sterol-enriched margarine lowers plasma LDL in hyperlipidemic subjects with low cholesterol intake: effect of fibrate treatment. Clinical Chemistry and Laboratory Medicine, 39, 634–40. 32. Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, Keogh JP, Meyskens FL, Valanis B, Williams JH, Barnhart S, Hammar S, (1996). Effects of a combination of beta-carotene and vitamin A on lung cancer and cardiovascular disease. New England Journal of Medicine, 334, 1150–1155. 33. Rapola JM, Virtamo J, Haukka JK, Heinonen OP, Albanes D, Taylor PR, Huttunen JK, (1996). Effect of vitamin E and beta-carotene on the incidence of angina pectoris. A randomized, double-blind, controlled trial. Journal of the American Medical Association, 275, 693–698. 34. Rapola JM, Virtamo J, Ripatti S, Huttunen JK, Albanes D, Taylor PR, Heinonen OP, (1997). Randomised trial of a-tocopherol and b-carotene supplements on incidence of major coronary events in men with previous myocardial infarction. Lancet, 349, 1715–1720.

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35. Steinmetz KA, Potter JD, (1996). Vegetables, fruit and cancer prevention: A review. Journal of the American Dietetic Association, 96, 1027–1039. 36. Vainio H, Rautalahti M, (1998). An international evaluation of the cancer preventive potential of carotenoids. Cancer Epidemiology, Biomarkers and Prevention, 7, 725–728. 37. Varis K, Taylor PR, Sipponen P, Samloff IM, Heinonen OP, Albanes D, Harkonen M, Huttunen JK, Laxen F, Virtamo J and the Helsinki Gastritis Study Group, (1998). Gastric cancer and pre-malignant lesions in atrophic gastritis: A controlled trial on the effect of supplementation with alpha-tocopherol and beta-carotene. Scandinavian Journal of Gastroenterology, 33, 294–300.

17 Effect of Flavonoids on Human Topoisomerases Doris Marko*

Abstract Many flavonoids have been reported to target human topoisomerases. As a result, depending on the mode of interaction, DNA integrity might be affected. Several flavonoids, such as quercetin, kaempferol, apigenin, EGCG or genistein are known to stabilize the DNA-topoisomerase II intermediate, acting as topoisomerase II poisons. Recent studies indicate, that depending on the structure, site-specific DNA cleavage might be induced by these compounds. Several flavonoids have been reported to induce specific cleavage in the human myeloid-lymphoid leukemia gene (MLL), an effect which is discussed to contribute to the onset of infant leukemia.

17.1 Introduction DNA topology is regulated and controlled by topoisomerases, ubiquitous enzymes, breaking and resealing the polyphosphate backbone of the DNA, thus enabling the passage of other DNA strands through the transient gap [1–5]. In mammals, two major classes of topoisomerases have been characterized: topoisomerase I and II. Topoisomerase I alters DNA topology, transiently breaking one strand by the formation of a covalent DNA-topoisome* Department of Chemistry, Division of Food Chemistry and Environmental Toxicology, Universitiy of Kaiserslautern, Erwin-Schrödinger-Str. 52, 67663 Kaiserslautern, Germany

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rase-intermediate (cleavable complex), allowing the passage of the other strand through the break, followed by a religation step [1]. In contrast, topoisomerase II, an ATP-dependent enzyme, inserts a transient double strandbreak, through which a second DNA double-helix is passed [3]. During the DNA-cleavage step, topoisomerase II is covalently linked to the 5l-phosphoryl end of the broken DNA. Functional topoisomerases are required for central processes of DNA metabolism, such as replication, transcription [6–7], recombination [8], DNA-repair [9] or (de)condensation of chromosomes [10].

17.2 Compounds Targeting Topoisomerases Topoisomerases have been identified as the target of a number of potent anticancer drugs such as camptothecin (topoisomerase I) or the epipodophyllotoxin etoposide (topoisomerase II). As a consequence of topoisomerase inhibition, cells are arrested in the G2-phase of the cell cycle and undergo apoptosis [11]. Many flavonoids of different classes have been reported to interfere with human topoisomerases. The green tea catechin (–)epigallocatechin-3-gallate (EGCG), the soy bean isoflavone genistein and the flavonols quercetin, myricetin, morin and fisetin target topoisomerase I and II [12]. We could recently show that also delphinidin and cyanidin, the aglycons of the most abundant anthocyanins in food, represent potent inhibitors of both topoisomerase subclasses (manuscript in preparation). In contrast, the dihydrochalcone phloretin and the flavone kaempferol preferentially inhibit topoisomerase II [12–14]. Structural requirements for effective topoisomerase I and II inhibition by flavonoids are hydroxy groups at the C-3, C-7, C-3l and C-4l positions together with a keto group at C-4 and a C-2, C-3 double bond. Additional B-ring hydroxylation enhances topoisomerase I inhibitory activity [12]. For effective topoisomerase II inhibition, the C-2, C-3 double bond was found to be essential together with hydroxyl groups at the 5, 7, 3l and 4l positions. Additional hydroxy groups are tolerated at the 3, 6 and 5l positions [14].

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17.3 Mode of Interaction with the Target Enzyme: Inhibitor or Poison? Consequences of the interference with the different topoisomerases for DNAintegrity strongly depend on the mode of interaction. The catalytic cycle of both classes of topoisomerases can be subdivided in 3 main steps: DNAbinding, cleavage of the DNA-topoisomerase-intermediate and religation. Depending on the compound, interference at different stages of the catalytic cycle has been reported. As a result, compounds targeting topoisomerases are divided into two different classes. Topoisomerase inhibitors block the catalytic activity of the enzymes, preventing the generation of the covalent DNA-topoisomerase-intermediate (Fig. 17.2A). As a consequence, no induction of DNA strand breaks is observed with pure topoisomerase inhibitors.

(A)

O N N O H5C2 O

OH

H (B)

H3 C

O

O

O HO

OH

O

O O O O

MeO

OMe OH

Figure 17.1. (A) Camptothecin, (B) Etoposide.

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(A) Topoisomerase inhibitors: Topo I Inhibitor

P

P

(B) Topoisomerase poisons: Topo I

Binding Topo I

P

Cleavage P

P

Inhibitor

Religation P

P

Figure 17.2. Topoisomerase inhibitors and poisons: model of selective drug interactions with topoisomerase I reactions (according to [17], modified).

However, the majority of the compounds known to target topoisomerases interfere with the already established cleavable complex, trapping the covalent DNA-topoisomerase-intermediate in a ternary complex (Fig. 17.2B). As a result of the stabilisation of the inserted DNA strandbreak, DNA damage might occur. Especially the collision with the replication fork might result in DNA double strandbreaks [11, 15]. Compounds stabilizing the cleavable complex are therefore classified as topoisomerase poisons [16]. This mechanism is characteristic for compounds like camptothecin or epipodophyllotoxins. In contrast to camptothecin, quercetin and the related flavones apigenin, kaempferol and morin do not stabilize the covalent DNA-topoisomerase-I-intermediate, but inhibit topoisomerase I-catalyzed DNA religation [17]. However, many flavonoids, such as quercetin, kaempferol, apigenin, EGCG or genistein are known to stabilize the DNA-topoisomerase II intermediate. Topoisomerase II-mediated DNA cleavage has been shown for most of these compounds [14].

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17.4 Physiological Relevance In nearly 80 % of all cases of infant leukemia an abnormality in the myeloidlymphoid leukemia gene MLL at 11q23 is diagnosed. Secondary acute myeloid leukemias (AML), occuring as a result of the treatment with epipodophyllotoxins during chemotherapy, often manifest the same MLL abnormalities [18]. These findings led to the hypothesis that maternal exposure to DNA topoisomerase II inhibitors during pregnancy, leading to an enhanced in utero exposure, could be associated with an increased risk of infant leukemia [19]. Indeed, preliminary epidemiological studies suggest that increased maternal consumption during pregnancy of foods containing dietary topoisomerase II inhibitors is positively associated with infant AML only [19]. MLL is rearranged with partner genes in 40 different translocations, with the translocation breakpoints in an 8.3 kb breakpoint cluster region (BCR). Interestingly the MLL breakpoints of both, the therapy-related and infant leukemia patients, occur in the same region of the BCR, suggesting a similar mechanism of DNA damage [20]. Strick et al. [21] demonstrated that several flavonoids available in the diet can induce cleavage of the MLL gene in human myeloid and lymphoid progenitor cells and cell lines. Quercetin and fisetin exhibited the same effectiveness like etoposide. But also genistein, luteolin, and apigenin were found to induce cleavage in the MLL gene. Interestingly, the site of cleavage colocalizes with cleavage sites induced by etoposide [21–22].

17.5 Conclusion Although flavonoids in general are usually regarded to be of health benefit, effects on human topoisomerases appear to represent potentially adverse substance properties, which need to be taken into account with respect to food safety. Several flavonoids present in the diet are known to act as topoisomerase II poisons in vitro. Enhancement of dietary uptake of such flavonoids for example in the context of functional food, raises the question whether concentrations might be achieved at which topoisomerase-mediated DNA damage might occur. At present it can not be excluded that in utero exposure with dietary topoisomerase II inhibitors might represent a risk factor for infant leukemia. Factors like timing of exposure and the impact of genetic susceptibility of individuals need to be further investigated.

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References 1. Gupta, M.; Fujimori, A.; Pommier, Y. (1995) Eukaryotic DNA topoisomerases I. Biochim. Biophys. Acta 1262, 1–14. 2. Osheroff, N. (1989) Biochemical basis for the interactions of type I and type II topoisomerases with DNA. Pharmacol. Ther. 41, 223–41. 3. Osheroff, N.; Zechiedrich, E. L.; Gale, K. C. (1991) Catalytic function of DNA topoisomerase II. Bioessays 13, 269–73. 4. Wang, J. C. (1985) DNA topoisomerases. Annu. Rev. Biochem. 54, 665–97. 5. Wang, J. C. (1987) Recent studies of DNA topoisomerases. Biochim. Biophys. Acta 909, 1–9. 6. Kretzschmar, M.; Meisterernst, M.; Roeder, R. G. (1993) Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II. Proc. Natl. Acad. Sci. USA 90, 11508–12. 7. Zhang, H.; Wang, J. C.; Liu, L. F. (1988) Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes. Proc. Natl. Acad. Sci. USA 85, 1060–4. 8. Lim, M.; Liu, L. F.; Jacobson, K. D.; Williams J. R. (1986). Induction of sister chromatide exchanges by inhibitors of topoisomerases. Cell. Biol. Toxicol. 2, 485–494. 9. Stevnsner, T.; Bohr, V. A. (1993) Studies on the role of topoisomerases in general, gene- and strand-specific DNA repair. Carcinogenesis 14, 1841–50. 10. Adachi, Y.; Luke, M.; Laemmli, U. K. (1991) Chromosome assembly in vitro: topoisomerase II is required for condensation. Cell 64, 137–48. 11. Hsiang, Y. H.; Lihou, M. G.; Liu, L. F. (1989) Arrest of replication forks by drugstabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin. Cancer Res. 49, 5077–82. 12. Constantinou, A.; Mehta, R.; Runyan, C.; Rao, K.; Vaughan, A.; Moon, R. (1995) Flavonoids as DNA topoisomerase antagonists and poisons: structure-activity relationships. J. Nat. Prod. 58, 217–25. 13. Markovits, J.; Linassier, C.; Fosse, P.; Couprie, J.; Pierre, J.; Jacquemin-Sablon, A.; Saucier, J. M.; Le Pecq, J. B.; Larsen, A. K. (1989) Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res. 49, 5111–7. 14. Austin, C. A.; Patel, S.; Ono, K.; Nakane, H.; Fisher, L. M. (1992) Site-specific DNA cleavage by mammalian DNA topoisomerase II induced by novel flavone and catechin derivatives. Biochem. J. 282, 883–9. 15. Hertzberg, R. P.; Caranfa, M. J.; Hecht, S. M. (1989) On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex. Biochemistry 28, 4629–38. 16. Froelich-Ammon, S.; Osheroff N. (1996) Topoisomerase poisons: harnessing the dark side of enzyme mechanism. J. Biol. Chem. 270, 21429–21432. 17. Boege, F.; Straub, T.; Kehr, A.; Boesenberg, C.; Christiansen, K.; Andersen, A.; Jakob, F.; Kohrle, J. (1996) Selected novel flavones inhibit the DNA binding or the DNA religation step of eukaryotic topoisomerase I. J. Biol. Chem. 271, 2262–70. 18. Ross, J. A. (2000) Dietary flavonoids and the MLL gene: A pathway to infant leukemia? Proc. Natl. Acad. Sci. USA 97, 4411–3. 19. Ross, J. A.; Potter, J. D.; Reaman, G. H.; Pendergrass, T. W.; Robison, L. L. (1996) Maternal exposure to potential inhibitors of DNA topoisomerase II and infant leukemia (United States): a report from the Children’s Cancer Group. Cancer Causes Control 7, 581–90.

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20. Cimino, G.; Rapanotti, M. C.; Biondi, A.; Elia, L.; Lo Coco, F.; Price, C.; Rossi, V.; Rivolta, A.; Canaani, E.; Croce, C. M.; Mandelli, F.; Greaves, M. (1997) Infant acute leukemias show the same biased distribution of ALL1 gene breaks as topoisomerase II related secondary acute leukemias. Cancer Res. 57, 2879–83. 21. Strick, R.; Strissel, P. L.; Borgers, S.; Smith, S. L.; Rowley, J. D. (2000) Dietary bioflavonoids induce cleavage in the MLL gene and may contribute to infant leukemia. Proc. Natl. Acad. Sci. USA 97, 4790–5. 22. Strissel, P. L.; Strick, R.; Rowley, J. D.; Zeleznik-Le, N. J. (1998) An in vivo topoisomerase II cleavage site and a DNase I hypersensitive site colocalize near exon 9 in the MLL breakpoint cluster region. Blood 92, 3793–803.

18 Risk/Benefit Aspects of Phytoestrogen Consumption Catherine Boyle

Phytoestrogens are naturally occurring compounds found in plant-based foods such as soya. They have been shown to exhibit estrogenic activity in wildlife, experimental animals and humans. In 1996, the UK government’s scientific advisory committee, the Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment (COT) were asked for a view on the public health implications of dietary phytoestrogens. The COT

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expressed concern over the potential for phytoestrogens to cause adverse effects, particularly in infants fed soya-based infant formula. However, at that time the COT was unable to reach definitive conclusions due to the paucity of information. The COT made a number of research recommendations to address the data gaps and in 1997 the UK Government set up a wide ranging research programme at a cost of 1 million/year. In 2000, the COT established a working group to review the data from the research programme and the scientific literature to assess the risks and benefits of dietary phytoestrogens to human health. The COT aim to publish a report of the group’s findings in autumn 2002. This presentation will outline the working group’s review and the problems faced when interpreting the data. Projects from the FSA phytoestrogen research programme will also be highlighted. Information on the COT phytoestrogen working group is available from the Food Standards Agency web site: http://www.food.gov.uk/science/ouradvisors/toxicity/COTwg/wg_phyto/ Note: The COT published a report of its working group’s findings in May 2003. The report is available at the following website address: http://www.food.gov.uk/news/newsarchive/phytoreport0403news

19 Influence of Phytoestrogens on the Biotransformation of Endogenous Estrogens Manfred Metzler*, Erika Pfeiffer, and Leane Lehmann

19.1 Introduction Although it has been known since the mid-1940s that some plants used for animal and human nutrition contain substances with hormonal activity, the scientific and public interest in phytoestrogens has surged only during recent years. This is mainly due to the increasing awareness that bioactive nonnutrient compounds present in plants and in the corresponding food items may have beneficial health effects. The observation that diseases prevalent in Western societies, e. g. cardiovascular problems, breast cancer in women and prostate cancer, are much rarer in Asian populations has been linked to the high consumption of soy-based food [1–4]. Soybeans and all soyprotein products contain high concentrations of isoflavone phytoestrogens,

* Institute of Food Chemistry and Toxicology, University of Karlsruhe, P. O. Box 6980, 76128 Karlsruhe, Germany

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Figure 19.1. Phytoestrogens associated with health benefits.

in particular daidzein and genistein (Fig. 19.1). Likewise, epidemiological evidence suggests that a diet containing high levels of plant lignans such as secoisolariciresinol and matairesinol reduces the risk for breast and prostate cancer [1, 4]. Plant lignans are common in cereals and oilseeds, and particularly high in flaxseed. It is known that secoisolariciresinol and matairesinol are converted to the weakly estrogenic mammalian lignans enterodiol and enterolactone by intestinal bacteria. The putative anticarcinogenic effects of isoflavones and lignans, together with their purported alleviation of menopausal symptoms such as hot flashes and osteoporosis, have not only helped to promote phytoestrogen-rich food items but have also stimulated the use of dietary supplements. The mechanisms of the putative cancer protective effects of phytoestrogens are still a matter of debate [1–4]. Although some isoflavones, e. g. genistein, have been shown to exert non-hormonal effects such as inhibition of protein tyrosine kinase and interference with cell signal transduction pathways, it is widely believed that hormonal mechanisms mediated by the estrogen receptors a and b play a key role. However, other biochemical mechanisms may also be involved. In the present communication, we focus on the hypothesis that isoflavones (and possibly lignans) may interfere with the biosynthesis and metabolism of the endogenous steroid estrogens, and advocate this potential mechanism for further investigation. 239

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19.2 Biosynthesis and Metabolism of Steroid Estrogens The endogenous estrogens in mammals, estrone (E1) and 17b-estradiol (E2), are biosynthesized from cholesterol through a series of intermediates. The last steps comprise the conversion of androstendione to E1 catalyzed by the enzyme aromatase, and the reversible reduction of the less estrogenic E1 to the potent estrogen E2 mediated by 17b-hydroxysteroid dehydrogenase (17b-HSD, Fig. 19.2). E2 is primarily biosynthesized in the maturing follicle in premenopausal women but to a much lower extent also in other tissues, e. g. the adrenal, brain and fat. The major metabolic routes comprise conjugation with glucuronic acid and sulfate, leading to highly polar products excreted in urine and bile, but also hydroxylation at the aromatic ring and other sites of the steroid molecule prior to conjugation (Fig. 19.2). Aromatic hydroxylation at position 2 or 4 next to the 3-hydroxyl group gives rise to the catechol estrogens (Fig. 19.3), and these oxidative metabolites together with 16a-hydroxy-E1 are believed to play a role in the biochemical mechanism of carcinogenesis mediated by steroid estrogens [5–7]. There is increasing evidence that the semiquinone and quinone metabolites of catechol estrogens, in particular of 4-hydroxy-E, are capable of inducing various kinds of DNA damage. Therefore, the methylation of catechol estrogens, catalyzed by catechol-O-methyltransferases (COMT), is considered an important metabolic step for the inactivation of catechol estrogens [8]. This notion is supported by several observations. For example, exposure of MCF-7 human breast cancer cells to the specific COMT inhibitor Ro 41,0960 increases the ratio of catechol estrogens to methylated catechols and enhances oxidative DNA damage [9], and treatment of Syrian golden hamsters with the COMT

Figure 19.2. Biosynthesis and metabolism of endogenous steroid estrogens.

240

Figure 19.3. Metabolic activation and inactivation of steroid estrogens.

19 Influence of Phytoestrogens on the Biotransformation

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inhibitor quercetin increases the level of catechol estrogens in the kidney and enhances E2-mediated renal carcinogenesis [10]. Furthermore, susceptibility to breast cancer in women has been associated with the COMT genotype [11].

19.3 Metabolism of Isoflavone and Lignan Phytoestrogens The isoflavones daidzein and genistein occur as glycosides, malonylglycosides and acetylglycosides in soybeans and in non-fermented soy products. Upon ingestion, the glycosides are hydrolyzed by intestinal bacteria and the absorbed aglycons conjugated in the intestinal epithelium and the liver predominantly to glucuronides and to a lesser extent to sulfates (Fig. 19.4). Like steroid estrogens, these conjugates are excreted with the urine and bile and undergo enterohepatic circulation. Several reductive metabolites, e. g. equol and O-desmethylangolensin are also formed from free isoflavones by the gut flora. Whereas the hydrolytic and reductive metabolism of soy isoflavones has been known for many years, oxidative biotransformation of daidzein and genistein has been reported only recently. In studies with rat and human hepatic microsomes, several mono- and dihydroxylated metabolites of both isoflavones were observed and their chemical structures identified [12]. The major hydroxylation products, e. g. 6-hydroxy-, 8-hydroxy- and 3’-hydroxydaidzein and -genistein represent catechols and were also found, together with some of their methyl ethers, in the urine of humans fed a soy diet [13]. The plant lignans secoisolariciresinol and matairesinol are also ingested as glycosides. Intestinal bacteria liberate the aglycons and reduce them to enterodiol and enterolactone, which are absorbed, conjugated with glucuro-

Figure 19.4. Pathways in isoflavone metabolism.

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nic acid and sulfate, and subjected to enterohepatic circulation much like the isoflavones. Recently, it has been reported that enterodiol and enterolactone also undergo oxidative biotransformation [14]. With rat and human liver microsomes, various products of aliphatic and aromatic hydroxylation were demonstrated. Several of the oxidative lignan metabolites were also detected in the urine of humans fed a flaxseed diet [15].

19.4 Effects of Phytoestrogens on the Metabolism of Steroid Estrogens Since endogenous steroid estrogens and dietary phytoestrogens share metabolic pathways of conjugation and hydroxylation, it is conceivable that isoflavones and lignans interfere with the disposition of E2. Moreover, effects of phytoestrogens on the enzymes involved in the biosynthesis of steroid estrogens are possible. Intake of a diet rich in phytoestrogens such as soy or flaxseed is known to lead to micromolar concentrations of isoflavones or lignans in serum and tissues [1], which exceed the levels of steroid estrogens by at least two orders of magnitude. Several phytoestrogens and other phytochemicals, mostly flavonoids, have been studied for inhibitory effects on aromatase and 17b-HSD, two key enzymes of E2 biosynthesis. These data have recently been summarized by Kirk et al. [16], and the most active compounds are listed in Table 19.1. It

Table 19.1. Reported inhibitory effects, expressed as micromolar concentration causing 50 % inhibition, of phytoestrogens and other phytochemical on the biosynthesis and conjugation of steroid estrogens, according to [16]. Aromatase

17b-HSD

7-HO-flavone

0.2

Coumestrol

Chrysen

0.5

Apigenin

Apigenin Luteolin Enterolactone

1.2 4.8 6

Genistein Biochanin A Daidzein

Quercetin

12

Coumestrol Equol Daidzein

25 150 i1000

Sulfatase 0.12 0.12 1.0 4.9 10

Daidzein-4’,7di-O-sulfate Daidzein(4’-O-)sulfate Quercetin Kaempferol Naringenin

SULT1A1 1.0

Quercetin

0.1

5.9

Genistein

0.2

I 10 z 10 z 10

Daidzein Equol Daidzein-4’O-sulfate Daidzein4’,7- diO-sulfate

0.3 0.5 i 100 i 100

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appears that the food-borne isoflavones and lignans tested so far are rather poor inhibitors of aromatase and 17b-HSD. Because circulating E1 sulfate contributes significantly to the level of steroid estrogens in the breast, the activity of steroid sulfatase and of sulfotransferases (SULT) is important. Whereas the mono- and disulfate of daidzein exhibit a modest inhibitory effect on steroid sulfatase, a more pronounced inhibition of SULT1A1was observed for some dietary isoflavones (Table 19.1). SULT1A1 sulfates steroid estrogens and numerous environmental and dietary phenolic compounds. Whereas inhibition of enzymes involved in biosynthesis and conjugation may affect the local concentrations of steroid estrogens, inhibition of certain cytochrome P450 (CYP) isoforms may have a bearing on the metabolic activation of E2. Several CYP isoforms are known to catalyze the aromatic hydroxylation of E2 in rat and human tissues (Table 2). Whereas most CYP isoforms favor 2-hydroxylation over 4-hydroxylation, CYP1B1 leads preferentially to the genotoxic and carcinogenic 4-hydroxy-E2 [5, 7]. There is very little information to date on the CYP isoforms mediating the hydroxylation of isoflavone and lignan phytoestrogens. In studies using microsomes from the liver of rats pretreated with various inducers and in experiments with recombinant human CYP enzymes, the isoforms listed in Table 19.2 were demonstrated to form various catechol metabolites of genistein [17]. Based on these limited data it appears that steroid estrogens and isoflavones are hydroxylated, at least in part, by the same CYP isoforms. High concentrations of genistein and daidzein may therefore affect the hydroxylation of E2. In fact, a few recent studies reported decreased urinary steroid estrogen excretion and increased ratios of urinary 2-hydroxy- to 4-hydroxy- and 2-hydroxy- to 16a-hydroxy-estrogen in premenopausal women on a high isoflavone soy diet as compared to a low isoflavone soy diet [18, 19]. A slight shift of E2 metabolism away from the potentially carcinogenic 4-hydroxy- and 16a-hydroxyestrogens was also observed in postmenopausal women on a isoflavone-rich soy diet [20].

Table 19.2. Cytochrome P450 isoforms capable of hydroxylating steroid estrogens and isoflavones. Steroid estrogens [7]

Genistein [17]

Rat

1A2, 2B1/2, 2C6/11, 3A4/5 (mostly 2-hydroxylation) 1B1 (mostly 4-hydroxylation)

1A, 3A

Human

1A2, 3A4/5 (mostly 2-hydroxylation) 1B1 (mostly 4-hydroxylation)

1A1, 1A2, 1B1, 3A4

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19.5 Conclusion High dietary intake of soy isoflavones and flaxseed lignans appears to protect against cancer of the breast and prostate, but the biochemical mechanisms underlying this beneficial health effect of phytoestrogens remain elusive. In addition to estrogen receptor-mediated inhibition of cell proliferation, modulation of signal transduction pathways, induction of sex hormone binding globulin and antioxidant action, the interference of isoflavones and lignans with the biosynthesis and metabolism of the endogenous steroid estrogens should be explored. Steroid estrogens and phytoestrogens share metabolic pathways of conjugation and hydroxylation, and interaction at the metabolic level may have beneficial but also adverse effects. The limited number of in vitro and in vivo studies published to date support this notion, and further research must be performed now to study the effect of individual phytoestrogens on E2 metabolism in suitable in vitro systems and eventually in animals and humans in vivo.

References 1. Adlercreutz, H. (1995) Phytoestrogens: epidemiology and a possible role in cancer protection. Environ. Health Perspect. 103 (Suppl. 7), 103–112. 2. Messina, M. J.; Persky, V.; Setchell, K. D. R.; Barnes, S. (1995) Soy intake and cancer risk: a review of the in vitro and in vivo data. Nutr. Cancer 21, 113–131. 3. Setchell, K. D. R.; Cassidy, A. (1999) Dietary isoflavones: biological effects and relevance to human health. J. Nutr. 129, 758–767. 4. Ward, W. E.; Thompson, L. U. (2001) Dietary estrogens of plant and fungal origin: occurrence and exposure. In: Metzler, M. (Ed.) Endocrine Disruptors, Part I. The Handbook of Environmental Chemistry, Vol 3/L, Springer-Verlag Berlin Heidelberg, 101–128. 5. Yager, J. D.; Liehr, J. G. (1996) Molecular mechanisms of estrogen carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 36, 203–232. 6. Metzler, M. (2002) Genotoxic potential of natural and synthetic endocrine active compounds. In: Metzler, M. (Ed.) Endocrine Disruptors, Part II. The Handbook of Environmental Chemistry, Vol 3/M, Springer-Verlag Berlin Heidelberg, 187–207. 7. Zhu, B. T.; Conney, A. H. (1998) Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis 19, 1–27. 8. Zhu, B. T; Conney, A. H. (1998) Is 2-methoxyestradiol an endogenous estrogen metabolite that inhibits mammary carcinogenesis? Cancer Res. 58, 2269–2277. 9. Lavigne, J. A.; Goodman, J. E.; Fonong, T.; Odwin, S.; He, P.; Roberts, D. W.; Yager, J. D. (2001) The effect of catechol-O-methyltransferase inhibition on estrogen metabolite and oxidative DNA damage levels in estradiol-treated MCF-7 cells. Cancer Res. 61, 7488–7494.

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10. Zhu, B. T.; Liehr, J. G. (1996) Inhibition of catechol O-methyltransferase-catalyzed O-methylation of 2- and 4-hydroxyestradiol by quercetin. J. Biol. Chem. 271, 1357– 1363. 11. Lavigne, J. A.; Helzlsouer, K. J.; Huang, H. Y.; Strickland, P. T.; Bell, D. A.; Selmin, O.; Watson, M. A.; Hoffman, S.; Comstock, G. W.; Yager, J. D. (1997) An association between the allele coding for a low activity variant of catechol-O-methyltransferase and the risk for breast cancer. Cancer Res. 57, 5493–5497. 12. Kulling, S. E.; Honig, D.; Simat, T. J.; Metzler, M. (2000) Oxidative in vitro metabolism of the soy phytoestrogens daidzein and genistein. J. Agric. Food Chem. 48, 2910–2919. 13. Kulling, S. E.; Honig, D.; Metzler, M. (2001) Oxidative metabolism of the soy isoflavones daidzein and genistein in humans in vitro and in vivo. J. Agric. Food Chem. 49, 3024–3033. 14. Jacobs, E.; Metzler, M. (1999) Oxidative metabolism of the mammalian lignans enterolactone and enterodiol by rat, pig and humans microsomes. J. Agric. Food Chem. 47, 1071–1077. 15. Jacobs, E.; Kulling, S. E.; Metzler, M. (1999) Novel metabolites of the mammalian lignans enterolactone and enterodiol in human urine. J. Steroid Biochem. Molec. Biol. 68, 211–218. 16. Kirk, C. J.; Harris, R. M.; Wood, D. M.; Waring, R. H.; Hughes, P. J. (2001) Do dietary phytoestrogens influence susceptibility to hormone-dependent cancer by disrupting the metabolism of endogenous oestrogens? Biochem. Soc. Transact. 29, 209–216. 17. Roberts-Kirchhoff, E. S.; Crowley, J. R.; Hollenberg, P. F.; Kim, H. (1999) Metabolism of genistein by rat and human cytochrome P450s. Chem. Res. Toxicol. 12, 610–616. 18. Xu, X.; Duncan, A. M.; Merz, B. E.; Kurzer, M. S. (1998) Effects of soy isoflavone consumption on estrogen and phytoestrogen metabolism in premenopausal women. Cancer Epidemiol. Biomarkers Prevent. 7, 1101–1108. 19. Lu, L.-J. W.; Cree, M.; Josyula, S.; Nagamani, M.; Grady, J. J.; Anderson, K. E. (2000) Increased urinary excretion of 2-hydroxyestrone but not 16a-hydroxyestrone in premenopausal women during a soya diet containing isoflavones. Cancer Res. 60, 1299–1305. 20. Xu, X.; Duncan, A. M.; Wangen, K. E.; Kurzer, M. S. (2000) Soy consumption alters endogenous estrogen metabolism in postmenopausal women. Cancer Epidemiol. Biomarkers Prevent. 9, 781–786.

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How Selective are Prebiotics?

20 How Selective are Prebiotics?

Walter P. Hammes* and Fabio Dal Bello

Summary Growth as well as the performance of living organisms are affected by ecological factors to which the individual strain is adapted. With regard to the intestinal flora (IF), substrate factors, pH, redox potential, implicit factors (i. e. interaction within the microbial populations resting on synergistic or antagonistic principles) are of importance. In addition, poorly known factors exerted by the human host play an important role, as it can be concluded from the specific interrelationship between intestinal strains and a human individual. The substrates of the IF originate from the host (e. g. mucus, epithelial cells, enzymes) as well as the human diet. In the large bowel, compounds not digested and resorbed in the small intestine can become nutrients for the IF. Storage compounds and plant cell walls are the major compounds thus reaching the IF and serve as substrates for a multitude of species present therein. Therefore true selective substrate do not really exist. However some groups acquire a selective advantage by an efficient fermentation of the substrate and increase their numbers over those that do not, or not so efficiently, utilise these compounds or products thereof. Bifidobacteria and to some extend Lactobacilli increase their numbers in the described ways when defined carbohydrates, such as fructooligosaccharide (FOS), inulin and galactooligosaccharides (GOS) are consumed. As certain health promoting effects are attributed to the increase of the share of lactic acid bacteria, these carbohydrates are used as so called prebiotic which is defined as: “non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon that can improve host health” [16]. The results of in vivo and in vitro studies on prebiotics are consistent with the above description of the ecological factors acting on the intestinal flora and based on the analyses of the populations using culture techniques as well as methods targeting the genotype. As only 50–70 % of the species in the intestinal flora are culturable, culture techniques provide only limited information on the composition of the intestinal flora and, thus, of changes therein. The sum of the efforts in the area of prebiotic research allows to con-

* Institute of Food Technology, University of Hohenheim, Garbenstr. 28, 70599 Stuttgart, Germany

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clude that in the average, but not with all human individuals, bifidogenic effects can be identified. The efficiency is not clearly dose-dependent, and different species and strains, including also those of other genera, especially of Lactobacillus, can increase their numbers in the population. A clear prediction of the effect of a prebiotic on the selective growth of a defined species or even group of microorganisms covering all human individuals cannot be given.

20.1 The Microbial Community of the Gastro-Intestinal Tract The interrelationship between humans and their intestinal microflora is presently under heavy investigation but still numerous gaps exist in our knowledge concerning the microbial ecology of the intestinal flora (IF), the taxonomy of the species contained therein and their impact on human physiology. It is remarkable that just those microorganisms remained for so long, and still are little known that live in the closest possible contact with our body, in spite of the great progress achieved in microbiology. It is characteristic for that situation that only 10–50 % of the bacteria present in the large bowl are culturable. In Fig. 20.1, the human digestive tract is depicted and the microflora in the various sections is compiled showing the most important pathogens and human commensal genera. It can be recognised that by far the greatest microbial diversity and highest cell counts are found in the colon. The 15 listed genera represent ca. 99 % of the total counts which mount up to > 1011 bacteria per gram in the faeces, i. e. their mass constitutes up to ca. 60 % of that excrement. The organisms are anaerobic, to some extent facultative anaerobic, and exhibit a fermentative metabolism. The high cell counts indicate that these bacteria are well supplied with fermentable nutrients in their habitat. Our knowledge of the intestinal flora rests largely on the analysis of the faecal flora, as the access to the large intestines of a healthy person is extremely difficult and even automatic capsule systems do not deliver samples that are representative for a defined section at a defined time. Within the multitude of microbial groups ca. 40 species are commonly isolated and at least ca. 400 have been isolated from human faeces [1]. Finally numerous pathogenic bacteria, viruses and parasites may invade orally the intestines, frequently via contaminated foods including water. These pathogens are a challenge for all food hygiene measures as it is estimated that ca. 30 % of the population suffer once a year from a food borne infection, and globally 2.2 million persons, including 1.8 million of children, die every year of diarrhoetic infections [2]. This unsatisfactory situation together with the intention to improve the status of human health provides the background for the development of stra248

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Figure 20.1. Major groups of microorganisms present in the human gastrointestinal tract.

tegies to influence the intestinal flora to a more healthy composition. A prerequisite to achieve that aim is the knowledge of 1. the IF including their contribution to health or disease, 2. the factors controlling the growth and performance of the IF, and 3. ways to affect or even to control the IF composition and/or metabolism.

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20.2 The Metabolic Achievements of the Intestinal Microbial Community Starting with Metchnikoff [3], followed by studies of Mitsuoka [4], it became evident that the active metabolism of the human commensal intestinal flora may affect human health adversely as well as beneficially, and the respective effects had been attributed to certain species or groups of bacteria as it is shown in Fig. 20.2. Bacterial metabolites that may be involved in colonic tumorigenesis were compiled by Gibson & MacFarlane [5] (Tab. 20.1). As depicted in Fig. 20.3 the products of intestinal putrefaction included in that compilation are derived from bacterial fermentative conversion of amino acids and can be detected in the faeces. On the other hand, the products of microbial fermentation of carbohydrates are available as nutrients for the human body, unless they are not further metabolised by the microbial com-

Figure 20.2. Contributions of the intestinal microflora to human health.

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Figure 20.3. Metabolic conversion of polysaccharides and proteins by the intestinal microflora.

Table 20.1. Examples of gut bacterial metabolites that may be involved in colonic tumorigenesis [5, modified]. Metabolite

Method of involvement

Fecapentaenes

Plasmalogens are converted to mutagenic fecapentaenes by Bacteroides species Reaction of nitrite (product of microbial reduction of nitrate) with a secondary amine Bacterial transformation into co-carcinogens Mutagenic after activation by certain bacterial enzymes Co-carcinogens produced from tyrosine metabolism Microbial b-glycosidase activity Potentially carcinogenic after reduction by gut bacteria A tumour promoter microbiologically formed from certain dietary lipids Bacterial deamination of amino acids and nitrate reduction Bacterial nitro reduction causes carcinogenesis in laboratory animals Decarboxylated aromatic amino acids Co-carcinogens produced from tryptophan metabolism

Nitrosamines Bile salts Heterocyclic amines Phenolic compounds Aglycones Azo compounds Diacyglycerol Ammonia Nitrated polycyclic aromatic hydrocarbons Amines Indolic compounds

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munity. Thus gases, microbial biomass and break down products of amino acid metabolism are excreted from the body upon fermentation of carbohydrates and proteins in the large bowel.

20.3 The Prebiotic Concept The fate of these products of carbohydrate fermentation in human physiology is summarised in Tab. 20.2, showing that with regard to propionate and butyrate health supporting effects are discussed that are beyond their energy providing functions. The organisms of the IF use individual metabolic pathways and are specialized in their utilization of fermentation substrates. As shown in Tab. 20.3, several of the main groups of the IF ferment exclusively carbohydrates as energy sources and thus do not contribute to “intestinal putrefaction”, whereas others utilize amino acids and may be considered as potentially exerting adverse health effects. Based on this observation, concepts were developed aiming at shifting the IF or their metabolism to a preponderance of the carbohydrate fermentation by beneficial bacteria. In Fig. 20.4 the probiotic, the prebiotic and the synbiotic concepts are described. They differ in their ways to achieve the desired aim. With probiotics the beneficial microorganisms are orally introduced into the intestine whereas the prebiotics support the selective growth and/or activity of beneficial bacteria. Finally in synbiotics the beneficial organisms are ingested together with the

Figure 20.4. Concepts to shift the intestinal flora to a preponderance of a carbohydrate fermenting and more beneficial community.

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prebiotic and act in combination beneficially in the intestines. Inherent to the definition of a prebiotic are properties such as 1. not to become hydrolysed or absorbed in the upper part of the GI, 2. acting as a selective substrate for one or a limited number of potentially beneficial commensal bacteria in the colon, thus stimulating the bacteria to grow, become metabolically activated, or both;3. possessing the capability as a consequence to alter the colonic microflora toward a healthier composition and 4. exerting luminal or systemic effects that are beneficial to the health of the host. Table 20.2. Metabolic fate of products of carbohydrate metabolism in the human colon [14, modified]. Product

Fate

Acetate Propionate

Metabolised in muscle, kidney, heart and brain Cleared by the liver, possible glucogenic precursor, suppresses cholesterol synthesis Metabolised by the colonic epithelium, regulator of cell growth and differentiation Absorbed, electron sink products, further fermented to shortchain fatty acids Partially excreted in breath, metabolised by hydrogenotrophic bacteria

Butyrate Ethanol, succinate, lactate, pyruvate Hydrogen

Table 20.3. Fermentation substrates and products of the main groups of the human gut microbiota. Bacteria

Fermentation Substrate

Fermentation products

Bacteroides Bifidobacteria Eubacteria Peptococci Methanobrevibacter Ruminococci

Carbohydrates Carbohydrates Carbohydrates/amino acid Amino acids H2 (Chemolithotrophic)

Acetate, Acetate, Acetate, Acetate, CH4

Carbohydrates

Enterococci Actinomyces Clostridia

Carbohydrates Carbohydrates Carbohydrates/amino acid

Peptostreptococci Propionibacteria Lactobacilli Escherichia

Carbohydrates/amino acid Carbohydrates, lactate Carbohydrates Carbohydrates

Fusobacteria Desulfovibrio

Amino acids/carbohydrates H2, lactate, SO4–2

Acetate, Lactate, Succinate, Formate Lactate, Acetate Acetate, Lactate, Succinate Acetate, Propionate, Butyrate, Lactate, Ethanol/AFP* Acetate, Lactate/AFP* Acetate, Propionate Lactate, Acetate, Ethanol Lactate, Acetate, Succinate, Formate, Ethanol Butyrate, Acetate/AFP* Acetate, H2S

Propionate, Succinate Lactate, Formate, Ethanol Butyrate, Lactate/AFP* Butyrate, Lactate/AFP*

*AFP, amino-acid fermentation products include: H2S, NH3, amines, phenols, indole

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Table 20.4. Fermentable substrates in the human colon [17, modified]. Substrate

Origin*

Amount (g/day)

Total dietary residues Carbohydrates Resistant starch (RS) Non-Starch-Polysaccharides (NSP) (e. g. cellulose, hemicellulose, pectin, inulin) Not absorbable sugars and sugar-alcohols Chitin and amino-sugars Synthetic carbohydrates (Lactulose, Lactitol, Polydextrose) N-compounds Proteins Pancreatic enzymes N2, nitrate Others Mucus Bacterial lysis products Epithelial cells

D

70

D D

8–40 8–18

D D D

2–10 1–2 **

D Hd D

3–12 4–6 0.5

Hd D/Hd Hd

2–3 ? ?

* D, dietary origin; Hd, Human derived origin ** specific to an individual, but generally low

Table 20.5. Some factors affecting fermentation of carbohydrates in the human large intestine [6, modified]. Ecological factors (pH, rH) including competitive and cooperative interactions among bacteria Chemical composition of the substrate Amount of substrate available for the fermentation Physical form of the substrate including particle size, solubility, and association with undigestible complexes such as lignins, tannins and silica Colonic transit time Composition of the gut microbiota with respect to species diversity and relative numbers of different types of bacteria Rates of depolymerisation of substrates Substrate specificities and catabolite regulatory mechanisms of individual gut species Fermentation strategies of individual substrate utilizing bacteria Availability of inorganic electron acceptors pH of gut contents Antibiotic therapy Host specific factors secretion of antagonistic compounds (e. g. defensins, lysozyme) cellular and humoral immunity (mucosal tolerance) bile acids and pancreatic secretion (hormonal) effects derived from stress, age and sex peristalsis enzymes adherence sites

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20.4 The Selective Fermentation of Probiotics The human diet contains plenty of undigestible components but only a limited number that might fulfil all essential properties of a prebiotic. Clearly, carbohydrates are candidates to select appropriate prebiotics as they end up in health supporting products and originate mainly from dietary fiber, plant storage compounds and undigestible starch. However, as shown in Tab. 20.4, the IF of the large bowel will always receive fermentable substrates from our food and endogenous sources that support the growth of the microbial community. In addition, as pointed out by Gibson et al. [6], numerous factors (Tab. 20.5) affect the fermentation of carbohydrates in the colon and especially human host factors have decisive effects which are still poorly known. In spite of all limitations, prebiotics have been developed and are used as functional food ingredients as it can be concluded from the production numbers of the year 1995 (Tab. 20.6). These compounds pass through the small intestines and are fermented in the colon. Their composition is shown in Tab. 20.7, and numerous studies (Tab. 20.8) have been performed in vitro and in vivo to determine the efficiency of potential prebiotics. As described by Simmering and Blaut [7] evidence for the positive effects shown in Tab. 20.9 has been obtained. The evidence for health supporting effects of some carbohydrates is increasing and bifidogenic effects have apparently been shown in vitro and in vivo. Therefore, a role in controlling the growth and metabolic activity of defined groups of the IF as well as reducing these vital parameters in other groups appears to be consistent with the definition of a prebiotic. On the other hand, the conclusions drawn from microbial counts determined in the faeces may be misleading when basing on culture techniques. As shown in Fig. 20.5, the fermentation process in the colon is characterised by local specialities. The carbohydrate fermentation is most active in the proximal colon

Table 20.6. Production of oligosaccharides [18]. Oligosaccharide

Tons/Year

Lactulose Galacto-oligosaccharide Fructo-oligosaccharide Isomalto-oligosaccharide Malto-oligosaccharide Palatinose-oligosaccharide Cyclodextrine Soy-oligosaccharides Lactosucrose Gentio-oligosaccharides Xylo-oligosaccharides

20 000 15 000 12 000 11 000 10 000 5000 4000 2000 1600 400 300

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Table 20.7. Chemical composition of common prebiotics. Compound

Chemical structure

Inulin type fructans

a-D-Glcp- (1p2)-[b-D-Fruf-(2p1)]n-1-D-Fruf and b-D-Frup[b-D-Fruf]n-1-D-Fruf), with n varying from 2 to i 70 synthetic disaccharide, b-D-Galp-(1p4)-D-Frup galactose-containing oligosaccharides, e. g. a-D-Glcp(1p4)-[b-D-Galp-(1p6)]n, with n = 2–5. Produced from lactose with the aid of transgalactosylase predominant oligosaccharides are raffinose and stachyose

Lactulose Transgalactooligosaccharides (TOS) Soybean oligosaccharides (SOE) Isomalto-oligosaccharides (IMO) Lactosucrose

Gluco-oligosaccharides (GOS) Xylo-oligosaccharides (XOS) Gentio-oligosaccharides

glucose monomers linked by a-(1p6) glucosidic linkages produced from a mixture of lactose and sucrose using the enzyme b-fructosidase, b-D-Galp-(1p4)-a-D-Glcp-(1m2)b-D-Frup produced using the glucosyl-transferase from Leuconostoc mesenteroides to transfer glucose molecules from a sucrose donor to a maltose acceptor mainly consisting of xylobiose, xylotriose and xylo-tetrose, linked by b-(1p4) bonds glucose polymers linked b-D-Glcp-(1p6)-[b-D-Glcp(1p6)]n, with n = 1-5

where the water content as well as the substrate concentrations are still high. On the way of the fermenting mass to the anus the concentration of fermentable carbohydrates becomes reduced, products may accumulate and finally the environment of the microorganisms in the distal colon is quite different from that in the proximal part, for example, with regard to substrates products, pH and water content. The microorganisms present in the faeces may have thus undergone a stress which reduces their recovery by culturing. As pointed out by Hartemink [8] “the increase of bifidobacteria may be a combined effect of growth by these bacteria as well as a reduced transit time with subsequent better recovery of bifidobacteria in the faeces”. The same author has investigated the fermentation by intestinal bacteria of 19 non digestible oligosaccharides including the most used prebiotics. 31 species belonging to 18 genera were studied and it was revealed that none of the compounds was fermented by just one species or even genus. For example, FOS and TOS were fermented by 30 and 28 species, respectively, of divers genera. Thus, the non-digestible oligosaccharides are no selective substrates in the narrow sense of selectivity. Nevertheless, in an ecosystem the strains with the highest adaptation to the environment and utilisation of a substrate will perform better than less adapted competitors. It is thus conceivable that an increase in numbers and metabolic activity will result, as it is detected especially by in vitro studies. To overcome the limitations of the faecal analysis by use of culture dependent methods, additional approaches are used to support the prebiotic 256

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Table 20.8. Examples of in vitro and in vivo studies designed to determine the efficiency of candidate prebiotics [6, 19]. Candidate prebiotic

In vitro study

In vivo study

In vivo observed effects*

Lactulose

[20]

Fructo-oligosaccharides

[9] [24] [25] [26] [27] [13]

Bo Bo, Bdq, Cq, Sq, Ebq Bo, So, Lo, Cq, Bdq, Coq, Euq Bo Bo, Lo Bo Bo, Bdq, Cq, Fq Ao, Ebo, Bo Bo, Ecq, Bdq, Ebq Bo Bo

Galacto-oligosaccharides

[37] [38] [39]

Soybean oligosaccharides

[45] [46] [47] [49] [27] [51]

[21] [22] [23] [28] [29] [30] [31] [32] [33] [34] [35] [36] [40] [41] [42] [43] [39] [33] [48] [46]

SCFAo, H2o Bo, Lo Bo, Lo, Ebq Bo Bm Bo, Bdq, Cq Bo, Lo, Cq, Pq

[50]

Bo, Bdo

[51]

Bo, Bdo, Cq

[53]

Bo, Bdq

Isomalto-oligosaccharides (a1-6 linked) Gluco-oligosaccharides (b1-6 linked) Xylo-oligosaccharides Exopolysaccharidesfrom lactobacilli

[52] [13]

* o, increase; q, decrease; m stable; A, Aerobes; B, Bifidobacteria; Bd, Bacteroides; C, Clostridia; Co, Coliforms; E, Enterobacteria; Eu, Eubacteria; L, Lactobacilli; S, Streptococci

Table 20.9. Evidence for positive effects of prebiotics: a summary of the results obtained [7]. Effect

References

Increase in biomass and stool bulking Increased production of SCFA Stimulation of the growth and/or activity of lactic acid bacteria Improvement of the bioavailability of minerals Lowering of serum triacylglycerols and serum cholesterol Decrease in the risk of cancer

30 54, 55 9, 24, 32, 35, 26 56–60 61, 62 63, 64

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Figure 20.5. Fermentation events in the human colon.

effect of a compound. Faecal samples of volunteers are used to inoculate media containing the potential prebiotic (Fig. 20.6). The propagation of the bacteria as well as the analysis of changes in substrates and product formation are investigated in fermentor systems that simulate the conditions in the colon or sections thereof. Under these conditions the formation of key metabolites such as butyrate, propionate and gas versus lactate is, as introduced in table 20.3, to some extent indicative for groups of bacteria that turnover a given substrate. For example, bifidobacteria produce mainly lactate and acetate but no gas or butyrate and propionate. In in vitro fermentations with mixed faecal inocula these calculations have been made [8], and the data were consistent with, for example, the preferential use of FOS by lactic acid bacteria, most likely bifidobacteria. On the other hand, xyloglucan and 258

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Figure 20.6. Methods available to study the prebiotic properties of non-digestible carbohydrates in the small intestine.

guar gum yielded mainly gas and butyrate together with propionate. These latter compounds indicate the preferential use of the substrates by clostridia. The fermentor studies indicated also great variations within individual persons, i. e. with the inocula of some individuals the pattern of products from TOS did not change upon addition of the substrate whereas in one out of four volunteers a large change was observed. These observations confirm the strong effect of host specific factors on the performance of the intestinal bacteria. In a fermentor system the SCFA accumulate and affect clearly the pH, quite different from the in vivo situation. In the intestine a higher buffer capacity and absorption of SCFA will prevent a strong drop of the pH. As the pH is a dominant ecological factor the selective conditions of low pH values will be less strong. This conclusion is consistent with the observation [9–11] that FOS increase gas production in vivo (and may cause flatulence). This reaction can not be performed by bifidobacteria which may nevertheless increase their numbers, especially in persons harbouring low numbers of bifidobacteria constitutively. Thus, in addition, animal models with different species as well as human flora associated (HFA) rodents have been studied. Of special value in the investigation of complex microbial associations in general and especially of the intestinal flora are culture independent microbial analyses. These methods aim at unique DNA sequences that are characteristic for a taxon. The most used target sequences are those of the 16S RNA to which probes and primers are designed. Techniques such as “Fluorescence in situ Hybridization” (FISH), dot blot hybridization and PCR-D/TGGE have proven their power as analytical tools in analysis of the intestinal flora [12]. An example of the application of in vitro fermentation of a potential prebiotic with faecal inocula in combination with a microbial analysis using PCR-DGGE was provided by Dal Bello et al. [13]. It was observed that levan exopolysaccharides formed by Lactobacillus isolates from sourdough 259

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led upon 4 propagation steps repeatedly to the accumulation of bifidobacteria in the culture. The species were characteristic for the specific levan source (see poster presentation Dal Bello et al., this volume).

20.5 Conclusions The study of the human intestinal microflora has revealed that carbohydrates which are non digestible in the human small intestine can exert certain health promoting effects. In addition, certain groups of intestinal bacteria, especially bifidobacteria, can increase their numbers upon ingestion of the prebiotic or adding the compound to in vitro fermenting cultures of faecal inocula. As a consequence of multiple factors exerting effects on the complex microbial communities, great variations can occur within different individuals. A selective effect does neither mean that the prebiotic compound is selectively utilised by the accumulating bacterial groups, nor that a bifidogenic effect is truly correlated to supporting the health of the consumer.

References 1. Moore, W. E.; Holdeman, L. V. (1974) Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Appl. Microbiol. 27, 961–79. 2. WHO (2000) Food safety and food-born infections. WHO Implication Fact Sheets Nh237. http://www.who.int/inf-fs/en/fact237.html. 3. Metchnikoff, E. (1907) The prolongation of life. G. P. Putnam and Sons, The Knickerbocker Press, New York, USA. 4. Mitsuoka, T. (1992) The human gastrointestinal tract. In: Wood, B. J.B, (Ed.) The lactic acid bacteria in health and disease, Elsevier Science Publishers, Barking, UK, 69–114. 5. Gibson, G. R.; MacFarlane, G. T. (1994) Intestinal bacteria and disease. In: Gibson, S. A. W. (Ed.) Human health – The contribution of microorganisms, Springer-Verlag, London, UK, 53–62. 6. Gibson, G. R.; Ottaway, P. B.; Rastall, R. A. (2000) Prebiotics, new developments in functional foods, Chandos Publishing, Oxford, UK. 7. Simmering R.; Blaut, M. (2001) Pro- and prebiotics – the tasty guardian angels? Appl. Microbiol. Biotechnol. 55, 19–28. 8. Hartemink, R. (1999) Prebiotic effects of non-digestible oligo- and polysaccharides, Phd thesis, Landbouwuniversiteit Wageningen, Ponsen and Looijen, Wageningen, Nl. 9. Wang, X.; Gibson, G. R. (1993) Effects of the in vitro fermentation of oligofructose and inulin by bacteria growing in the human large intestine. J. Appl. Bacteriol. 75, 373–380.

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29. Williams, C. H.; Witherly, S. A.; Buddington, R. K. (1994) Influence of dietary neosugar on selected bacterial groups of the human faecal microbiota. Microb. Ecol. Health Dis. 7, 91–97. 30. Gibson, G. R.; Beatty, E. R.; Wang, X.; Cummings, J. H. (1995) Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin. Gastroenterology. 108, 975–982. 31. Buddington, R. K.; Williams, C. H.; Chen, S.; Witherly, S. A. (1996) Dietary supplement of neosugar alters the fecal flora and decreases activities of some reductive enzymes in human subjects. Am. J. Clin. Nutr. 63, 709–716. 32. Kleesen, B.; Sykura, B.; Zunft, H.-J.; Blaut, M. (1997) Effects of inulin and lactose on fecal microflora, microbial activity and bowel habit in elderly constipated persons. Am. J. Clin. Nutr. 65, 1397–1402. 33. Bouhnik, Y; Bornet, F. (1998) Effects of an enteral nutritional formula (ENF) administration containing or not containing supplemental fructooligosaccharides (FOS) in healthy human adults. Food Chem. Toxicol. 36, 1031–1032. 34. Roberfroid, M. B.; Van Loo, J.; Gibson, G. R. (1998) The bifidogenic nature of chicory inulin and its hydrolysis products. J. Nutr. 128, 11–18. 35. Kruse, H. P.; Kleessen, B.; Blaut, M. (1999) Effects of inulin on faecal bifidobacteria in human subjects. Br. J. Nutr. 82, 375–382. 36. Tuohy, K. M.; Kolida, S.; Lustenberger, A. M.; Gibson, G. R. (2001) The prebiotic effects of biscuits containing partially hydrolysed guar gum and fructo-oligosaccharides – a human volunteer study. Br. J. Nutr. 86, 341–348. 37. Tanaka, R.; Takayama, H.; Morotomi, M.; Kuroshima, T.; Ueyama, S.; Matsumoto, K.; Kuroda, A.; Mutai, M. (1983) Effects of administration of TOS and Bifidobacterium breve 4006 on the human fecal flora. Bifid. Microflora. 2, 17–24. 38. Durand, M.; Cordelet, C.; Hannequart, G.; Beaumatin, P. (1992) In vitro fermentation of a galacto-oligosaccharide by human bacteria in continuous culture. Proc. Nutr. Soc. 51, 6A. 39. Bouhnik, Y.; Flourie, B.; D’Agay-Abensour, L.; Pochart, P.; Gramet, G.; Durand, M.; Rambaud, J. C. (1997) Administration of transgalacto-oligosaccharides increases fecal bifidobacteria and modifies colonic fermentation metabolism in healthy humans. J. Nutr. 127, 444–448. 40. Andrieux, C.; Szylit, O. (1992). Effects of galacto-oligosaccharides (TOS) on bacterial enzyme activities and metabolite production in rats associated with a human faecal flora. Proc. Nutr. Soc. 51, 7A. 41. Ito, M.; Deguchi, Y.; Miyamori, A.; Matsumoto, K.; Kikuchi, H.; Kobayashi, Y.; Yajima, T.; Kan, T. (1990) Effects of administration of galactooligosaccharides on the human fecal microflora, stool weight and abdominal sensation. Microb. Ecol. Health Dis. 3, 285–292. 42. Ito, M.; Kimura, M.; Deguchi, Y.; Myajima, A.; Kann, T. (1993) Effects of transgalactosylated disaccharides on the human intestinal microflora and their metabolism. J. Nutr. Sci. Vitaminol. 39, 279–288. 43. Rowland, I. R.; Tanaka, R. (1993) The effects of transgalactosylated oligosaccharides on gut flora metabolism in rats associated with a human faecal microflora. J. Appl. Bacteriol. 74, 667–674. 44. Satokari, R. M.; Vaughan, E. E.; Akkermans, A. D.; Saarela, M.; De Vos, W. M. (2001) Polymerase chain reaction and denaturing gradient gel electrophoresis monitoring of fecal bifidobacterium populations in a prebiotic and probiotic feeding trial. Syst. Appl. Microbiol. 24, 227–231. 45. Tamura, Z. (1983) Nutriology of bifidobacteria. Bifid. Microflora. 2, 3–16.

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21 Pathogen or Probiotic – Where is the Boundary? Maria Saarela*, Jaana Mättö, and Tiina Mattila-Sandolm

Abstract Probiotics are live microbial preparations that benefit the health of the consumers by maintaining or improving their intestinal microbiota balance. Established probiotic effects include improved lactose digestion, modulation of gut microbiota, immune modulation, reduced duration of rotavirus diarrhoea, changes in biomarkers such as harmful faecal enzyme activities, alleviation of atopic dermatitis symptoms in babies, and positive effects against superficial bladder cancer and cervical cancer. Most bacteria that have probiotic properties belong to the genera Lactobacillus and Bifidobacterium. Since probiotic consumption involves ingestion of large numbers of viable bacterial cells (daily dosage between log9-log11 CFU) safety aspects of probiotic consumption are of utmost importance. Knowledge on the survival of probiotics in the GI-tract, their translocation and colonisation properties and the fate of probiotic-derived active components is important for the evaluation of possible negative and positive effects of probiotic consumption. Assessing the risks of probiotic consumption can be a very expensive and time-consuming task. While considering the risk of probiotic consumption we have to keep in mind that lactic acid bacteria have been globally consumed in a myriad of fermented food varieties (milk, meat, vegetable and cereal products) for a very long time without an indication that they could be generally harmful to the consumers’ health. In Finland Lactobacillus strains isolated from bacteremia have been systematically characterized for 10 years. Although the yearly consumption of probiotic products containing lactobacilli has increased during this time, the incidence of lactobacillemia has not increased. However, there is one local infection case with an undistinguishable isolate to L. rhamnosus GG. The extensive follow-up period of 10 years indicates that the risk of serious infection by one single probiotic strain is very low. Simultaneously, it is difficult to estimate all the health benefits the same probiotic strain has implemented, also in immunocompromised patients. No live bacterium can be guaranteed for a zero risk in each individual host since the outcome of bacterial ingestion (passage through GI-tract, colonisation, infection) is determined both by the host and the bacterium.

* VTT Biotechnology, P. O. Box 1500, 02044 VTT, Finland

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21.1 Human Gastrointestinal (GI) Tract as a Source for Probiotic Lactobacilli and Bifidobacteria Human large intestine has a rich and dynamic microbiota consisting of at least 400–500 bacterial species [1]. Maintaining a balanced microbial ecosystem is essential for the normal functions of the GI-tract, especially in preventing infections and stimulating the host’s immune response. Several factors including stress, antibiotic treatments and other medications can alter the GI-tract microbiota predisposing the host to various diseases [2, 3]. Overprescription and misuse of antibiotics has led to a situation where increasing numbers of pathogens are becoming resistant to antibiotics [4, 5]. The World Health Organisation (WHO) has indicated that alternative disease control strategies (including the use of probiotics) in the prevention-treatment of certain infections may be needed in the future [6]. Several attributes of probiotic bacteria should be considered before clinical trials are performed. These include origin (preferably human), safety, viability/activity in delivery vehicles (e. g. food), resistance to acid and bile, adherence to gut epithelial tissue, persistence in the GI-tract, production of antimicrobial substances, ability to stimulate host’s immune response, and the ability to influence metabolic activities [7]. The earlier clinical studies on probiotic efficacy generally suffered from ambiguities in probiotic product identification, stability and dosage, and from the small number of test subjects and controls recruited. Considerable efforts have been made to correct these deficiencies and evidence on the health-promoting effects of probiotics is currently accumulating from randomised and placebo-controlled doubleblinded studies. Most bacteria with probiotic properties belong to the genera Lactobacillus and Bifidobacterium that are common but non-dominant members of the mature indigenous microbiota of the human GI-tract [8, 9]. Their probiotic potential has been discussed in numerous reviews, and include well-documented management of intestinal disorders such as lactose intolerance, infant gastroenteritis and rotavirus-associated diarrhoea, antibiotic-associated intestinal symptoms (mainly diarrhoea), and food allergy in babies [10–14]. These disorders and diseases are associated with intestinal microbiota imbalance and increased gut permeability [15]. In addition to above beneficial effects on disturbed intestinal microbiota probiotics can modulate immune responses, lower biomarkers such as harmful faecal enzyme activities, and present positive effects against superficial bladder cancer and cervical cancer [16]. Still other potential areas of probiotic nutritional management include alleviation of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) symptoms, mucosal vaccines and immunomodulation, infection control and eradication of multidrug-resistant microbes, treatment of candidal vaginitis, prevention of transmission of AIDS and sexually transmitted diseases, cholestrol and blood pressure lowering, and antimutagenic/anticarcinogenic activity [13, 14, 17, 18]. 265

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Table 21.1. Probiotic health-effects. Note that not all effects can be attributed to the same probiotic strain (Saarela et al. [94] and this paper). Established effects

Potential effects

alleviation of lactose intolerance symptoms treatment of viral (rotavirus) diarrhea treatment of infant gastronteritis treatment of antibiotic associated diarrhea immune modulation modulation of intestinal microbiota

alleviation of IBD symptoms

lowering biomarkers (harmful fecal enzymes) alleviation of atopic dermatitis symptoms in babies positive effects on superficial bladder cancer and cervical cancer

alleviation of IBS symptoms improvement of constipation antimutagenic/anticarcinogenic activity cholestrol and blood pressure lowering eradication of multidrug-resistant microbes treatment of candidal and bacterial vaginitis infection control prevention of transmission of AIDS and sexually transmitted diseases

21.2 Infections Caused by Indigenous Lactobacilli and Bifidobacteria Both lactobacilli and bifidobacteria are common members of the indigenous microbiota of the human and, like other members of the indigenous microbiota of the oro-gastrointestinal tract, they occasionally cause opportunistic infections. Bifidobacteria and lactobacilli have been connected to certain dental infections, bacteremia, endocarditis and to rare cases of other infections [19].

21.2.1

Bacteremia

Although bacteremias caused by lactobacilli and bifidobacteria have been reported, their numbers are low compared to other bacteremia cases (incidence of lactobacilli 0.1–0.2 %, gram-negative organisms 38 %, coagulasenegative staphylococci 33 %, Staphylococcus aureus 16 %, streptococci 7 %, enterococci 5–15 %, for bifidobacteria there are no figures available) [19–22]. Predisposing factors for lactobacillemia include neutropenia, prior surgery, malignancy, diabetes, and prior treatment with antibiotics inactive against Lactobacillus (e. g. vancomycin) [23, 24]. The most common portals of entry for lactobacillemia are oropharynx, gastrointestinal tract and female reproductive tract [23]. In their review on 45 lactobacillemia cases Husni et al. [23] noted that cancer, recent surgery, and diabetes mellitus were the most common underlying conditions in the patients (40, 38 and 27 % of the patients, respectively). Several of the patients were on immunosuppressive 266

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therapy (N 11), receiving total parenteral nutriation (N 11) or had received antibiotics inactive against lactobacilli (N 23). Sixty percent of the bacteremia cases were polymicrobial (including e. g. enterococci, streptococci, Candida spp. and enteric gram-negative bacilli). Husni et al. [23] concluded that “Lactobacilli are relatively avirulent pathogens that produce bacteremia in patients with serious underlying illnesses, many of whom have received prior antibiotic therapy that may select out for the organisms”. Lactobacillus species identified from bacteremia cases often belong to the L. casei group (including also L. rhamnosus and L. paracasei) [23, 25– 28]. This reflects the common finding of these species in the oro-gastrointestinal tract samples [29]. The more serious form of bacteremia, septicemia, caused by lactobacilli seems to be very rare and only diagnosed in patients with serious underlying conditions [30–38]. Bacteremia due to Bifidobacterium has been rarely reported. In an early report of Bourne et al. [39] bifidobacteria were obtained from 9 patients during the 6-year period of the study (1972–1977). Underlying conditions of the patients included complications with pregnancy (the most common condition), bowel obstruction, cholecystectomy, and systemic lupus erythematosus [39]. Five of the cases were caused by “B. eriksonii”, currently reclassified as B. dentium, which is regarded a dental pathogen. There are only few reports on septicemia due to Bifidobacterium [40, 41]. In the report of Guillard et al. [40] the isolate was identified as “gram-positive rod similar to Corynebacterium group E (aerotolerant Bifidobacterium adolescentis)”. However, due to the limitations of the methodologies used this identification must be considered doubtful. In the case report of Ha et al. [41] the causative agent of septic infection was identified as Bifidobacterium longum.

21.2.2

Endocarditis

Dental operations causing transient bacteremias seem to be important starting points for endocarditis. When a portal of entry is created by dental operation/surgery (e. g. periodontal probing, scaling and root planing, extractions, gingival surgery) oral bacteria can reach alveolar blood and blood circulation. Host defences usually destroy these bacteria but in patients with predisposing factors that have damaged heart tissue or heart valve the bacteria can colonise the tissue/valve and cause endocarditis. Sometimes bacterial aggregates and emboli break away from the heart tissue and lodge in other organs leading to other systemic infections [42]. Streptococcus, Staphylococcus and gram-negative bacteria account for the majority (over 80 %) of all endocarditis cases [20]. According to Gasser [20] and Brouqui and Raoult [43] Lactobacillus (a common member of oral microbiota) is only rarely causing endocarditis (0.05-0.4 % of all endocarditis cases). Common factors in Lactobacillus endocarditis were dental operations (75 %) and systemic emboli (42 %) as portals of entry. In the majority (83 %) of Lactobacillus endocarditis cases the patients have had a serious underlying 267

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heart disease (e. g. a valvular prosthesis) [43, 44]. Lactobacillus endocarditis involved a substantial risk of relapse after medical therapy, and the occasional need for valve-replacement surgery to achieve cure [23, 44]. As in bacteremia cases L. casei group and also Lactobacillus plantarum were common findings in endocarditis [20, 23, 30, 44–51]. Like in the case of bacteremia, this reflects the common finding among lactobacilli main sources of infection, rather than greater virulence [52]. To our knowledge there are no reports on Bifidobacterium endocarditis.

21.2.3.

Other Infections

Lactobacilli have been rarely isolated from other human infections but they have been reportted to be involved in dental caries, urinary tract infections, endometritis, meningitis, intraabdominal, liver, splenic and reproductive tract abscesses, and chest and wound infections [23, 53]. Lactobacilli have been isolated from polymicrobial (90 % of cases) abscesses, aspiration pneumonia, and from miscellaneous other infections in children (N 40) [54]. Most (about 75 %) of the lactobacilli isolates were not identified to the species level [53, 54]. Bifidobacteria have been isolated from otitis media, abscesses, peritonitis, and from miscellaneous other infections in children (N 55) [54]. Only in two cases (4 %) was Bifidobacterium isolated in pure culture (B. adolescentis from otitis media and Bifidobacterium sp. from cervical adenitis). Isolated cases of B. dentium (previously B. eriksonii) infections have been reported in adults [55, 56]. Furthermore, Bifidobacterium sp. has been isolated from abdominal and reproductive tract abscesses [53]. Three Bifidobacterium species, B. dentium, B. inopinatum and B. denticolens, are associated with human dental caries [57]. We have to keep in mind that the isolation of lactobacilli or bifidobacteria in infection sites is not a solid evidence for any cause-effect relationship. Especially in situations when the infection is polymicrobial and antibiotic treatment had preceded the isolation of lactobacilli/bifidobacteria, drawing any conclusions on the role of these bacteria in infections is hardly possible.

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21.3 Safety Aspects of Ingested Probiotic Lactobacilli and Bifidobacteria Guidelines for the safety assessment of probiotics can be found in several articles [7, 19, 51, 58]. In the assessment of the probiotic safety several approaches, including studies on the intrinsic properties and pharmacokinetics of the probiotic strain and on interactions between the probiotic strain and the host, are possible. Data on survival of the probiotics within the GItract, their translocation and colonisation properties, and the fate of probiotic-derived active components, is important for the evaluation of the effects (both positive and negative) of probiotic ingestion. The survival of different probiotic strains in different parts of the GI-tract varies: Some strains are rapidly killed in the stomach while others can pass through the whole gut in high numbers [59]. The pharmacokinetics of probiotics in humans has been studied using intubation, perfusion and biopsy techniques [60–65]. Some enzymatic properties (which can be studied in vitro) including deconjugation of bile salts in the small intestine or degradation of mucus can potentially be detrimental [66, 67]. Platelet aggregation [68], adhesion to Table 21.2. Recommendations for safety of probiotic cultures and foods (the Probdemo approach, Salminen et al. [19]). 1. The producer that markets the food has the ultimate responsibility for supplying a safe food. Probiotic foods should be as safe as other foods. 2. When the probiotic food turns out to be a novel food it hence will be subject to the appropriate legal approval (EU directive for novel foods). 3. When a strain has a long history of safe use, it will be safe as a probiotic strain and will not result in a novel food. 4. The best test for food safety is a well documend history of safe human consumption. Thus when a strain belongs to a species for which no strains are known that are pathogenic and for which other strains have been described that have a long history of safe use, it is likely to be safe as a probiotic strain and will not result in a novel food. 5. When a strain belongs to a species for which no pathogenic strains are known but which do not have a history of safe use, it may be safe as a probiotic strain but will result in a novel food and hence should be treated as such. 6. When a new strain belongs to a species for which strains are known that are pathogenic, it will result in a novel food. 7. Proper state of the art taxonomy is required to describe a probiotic strain. Today it includes DNA-DNA hybridization and rRNA sequence determination. This reasoning specifically applies to mutants of a probiotic strain. 8. In line with recommendation 1, strains that carry transferable antibiotic resistance genes, i. e. genes encoding proteins that inactivate antibiotics should not be marketed. 9. Strains that have not been properly taxonomically described using the approaches as indicated above under 7 should not be marketed. Strains should also be deposited in an internationally recognised culture collection.

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human intestinal mucosa [69] and formation of unwanted metabolites can also be studied in vitro. However, in vitro platelet aggregation ability as well as adhesion to mucus, ileostomy glycoproteins or to Caco-2 cell line varies among bacteremia-associated lactobacilli [69], which undermines the value of these analyses in the safety assessment. O’Brien et al. [70] listed several metabolic activities that can be considered in probiotic safety evaluation. These included biogenic amine production, bile salt hydroxylase, D- vs. Llactate production, mucin degradation, cholyl glycine hydrolase, azoreducatse, nitroreductase, b-glucuronidase and b-glucosidase. All these properties can be studied in vitro. However, their in vivo relevance often remains speculative. The suitability of animal models for microbial risk assessment is considered inadequate [71]. This is mainly due to the high variability in species specific responses, which does not allow the extrapolation of the results to humans.

21.3.1

Antibiotic Resistance in Probiotic Lactobacilli and Bifidobacteria – Can Antibiotic Resistance be Transferred?

Several species of lactobacilli are intrinsically (naturally) resistant to vancomycin [72–74]. These species have peptidoglycan precursors terminating with D-lactate instead of the target precursor for vancomycin activity terminating with D-alanine [75]. Many intrinsically vancomycin resistant strains of lactobacilli have a long history of safe use as probiotics and there is no indication that vancomycin resistant lactobacilli could transfer the resistance to other bacteria. Tynkkynen et al. [76] and Klein et al. [77] have demonstrated that the vancomycin resistance factor of the probiotic strain L. rhamnosus GG is not closely related to those of enterococci. Furthermore, Tynkkynen et al. [76] could not observe the transfer of antibiotic resistances between L. rhamnosus GG and enterococci. Plasmid-linked antibiotic resistances seem to be rare among lactobacilli [78–80], but still their safety implications should be considered. Antibiotic susceptibility patterns vary greatly between different species of lactobacilli, and strains with atypical resistance to some clinically important antibiotics have been detected [74, 81], indicating the necessity for susceptibility testing of each probiotic strain. Bifidobacteria can be intrinsically resistant to nalidixic acid, neomycin, polymyxin B, kanamycin, gentamycin, streptomycin, metronidazole and vancomycin [82–84]. Several antibiotics that are used in treatment of anaerobic infections, such as penicillin, eryhtromycin and clindamycin, are highly active against bifidobacteria [83, 85, 86]. Susceptibility to vancomycin has been reported in several studies [82, 83, 85], however, Charteris et al. [84] observed vancomycin resistance as a general characteristic for bifidobacteria by using a disc diffusion method in susceptibility testing. To our knowledge there are no genetic studies on bifidobacterial antibiotic resistances. 270

21 21.3.2

Pathogen or Probiotic – Where is the Boundary?

Can ingested Probiotic Lactobacilli and Bifidobacteria Cause Infections?

To our knowledge there are up to date two reports of probiotic bacterium being found in an infection. One L. rhamnosus strain indistinguishable from L. rhamnosus GG has been isolated from a liver abscess from an elderly lady with a history of hypertension and diabetes mellitus [87]. In another case a probiotic L. rhamnosus strain (strain or product specifications were not given) was suggested to have caused endocarditis in an elderly male [88]. However, unlike in the liver abscess case, where strain identification was based on thorough genomic fingerprinting of the L. rhamnosus isolates, the endocarditis case study relied on conventional phenotypic strain identification methods and on pyrolysis mass spectrometry identification. The discriminatory power of conventional phenotypic identification methods in strain differentiation is usually poor and there is no data on the ability of pyrolysis mass spectrometry to differentiate between different lactobacilli strains. Therefore the data on the endocarditis case must be considered insufficient for proving that the ingested probiotic L. rhamnosus strain and not one of the indigenous L. rhamnosus strains is the causative agent. In the study of Presterl et al. [50] an ingested probiotic L. rhamnosus was suspected to be a causative agent of endocarditis. Phenotypic methods could not differentiate the endocarditis isolate from the probiotic strain. However, with molecular techniques the endocarditis isolate was shown to be different from the probiotic strain. This study underlines the importance of using proper molecular techniques in the strain-level identification in suspected cases of probiotic infection. Probiotic products have been safely consumed in large quantities for a long time in Europe and in Japan. However, the two above findings (one proved and one presumptive) indicate that sporadic localised infections in immunocompromised patients may occur, and show that no zero risk can be attributed to consumption of living microbes. It has to be noted that isolating a probiotic strain in infection site does not necessarily mean that there is a direct cause-effect relationship between the isolate and the disease since the growth of the probiotic strain in the site may be a secondary effect.

21.3.3

Case: L. rhamnosus GG and Finland

To our knowledge Finland is the only country where lactobacilli isolates from bacteremias have been systematically checked for identity with commercial starter and probiotic strains [21, 22]. During the years 1989–1994 the Department of Bacteriology and Immunology at the University of Helsinki collected 5192 blood culture isolates from Finns living in Southern Finland. Twelve (0.2 %) of the 5192 positive blood cultures were Lactobacillus-positive and none of the Lactobacillus isolates was identical with any known starter or probiotic strain [21, 22]. Since 1994 National Public Health Institute (NPHI) 271

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has registered bloodstream infections in Finland (population of appr. 5 million). In total 75 lactobacillemias were reported to NPHI between October 1994 and 1999 [89]. Lactobacilli represented about 0.2 % of all blood culture isolates during this time. Since 1990 17 isolates of L. rhamnosus have been characterized and 5 of them were genetically indistinguishable from L. rhamnosus GG. The current yearly consumption of Gefilus-products (with L. rhamnosus GG) in Finland is about 33 mill. L, about 4 L (2 x 1011 CFU) per capita. The reason why the undistinguishable isolates were detected in the blood samples is currently under investigation. (Dr. Maija Saxelin, personal communication).

21.4 Conclusions Common factors for Lactobacillus and Bifidobacterium infections are the following: rare occurrence but at all ages, indigenous source (oro-gastrointestinal tract), breakdown of host defences (immunosuppressed patients), and polymicrobial infection [20]. These features are hallmarks for opportunistic pathogens (pathogens produce disease in healthy subjects) and apply to all members of the indigenous microbiota. No live microbe, whether permanent or transient coloniser, can be regarded a priori as harmless organisms [20] and with live microbes there is no such thing as zero risk of infection. When considering the risk of probiotic consumption we have to keep in mind that lactic acid bacteria have been globally consumed in a myriad of fermented food varieties (milk, meat, vegetable and cereal products) for a very long time without an indication that they could be generally harmful to the consumers’ health. Thus, the long history of safe use of lactobacilli and bifidobacteria remains the best proof of their safety. However, since the number of elderly people (often with serious underlying diseases, clinical or subclinical) is increasing, also the predisposing factors for serious bacterial infections are becoming more common in the population. This sets great demands for the safety of probiotic products as well as for any other product containing living microbes. In seriously ill patient groups one possibility to minimise any safety risks could be the use of non-viable probiotic preparations. Non-viable cells do not have all the same properties as their living counterparts but they have proved promising in the treatment of certain disorders (e. g. lactose maldigestion and treatment of gastroenteritis) [90]. Assessing the risks of probiotic consumption can be very expensive and time-consuming. A low risk may have to be accepted when recommended to immunocompromised individuals, but the risk to benefit ratio needs to be clearly established in such cases. This requires relevant information on the efficacy and safety of the products. However, probiotics have been administered to various patient groups (Chrohn’s, diarrhoeal, AIDS, cancer), infants (allergic and healthy) and elderly without any reported adverse effects [91]. 272

References The systematic epidemiological research on lactobacillemias in Finland sets a very good example for other countries where probiotics are commonly used in foods. Only with these kind of post-marketing surveillance studies can we properly assess the risk related to probiotic (or fermented food) consumption. The research performed in Finland makes L. rhamnosus GG clearly the best safety assessed probiotic on the market (see also [91]). Safety evaluation of probiotic consumption is a difficult, but a very important task. Basis for this work comes from the following facts: Probiotic foods should be as safe as other foods, host factors are essential determinants of safety evaluation, and zero risk can never be applied to the ingestion of live microbes (whether probiotics, starters, or components of natural microbiota of fresh foods, e. g. fruits and vegetables). Suggesting direct causeeffect relationship should be based on critically assessed facts to rule out misjudgements. Therefore in the cases when a strain indistinguishable from an ingested strain is found in an infection site, several questions should be carefully considered: Was the sample representative (were all the bacteria, especially anaerobes, alive)? Was the microbiological methodology used appropriate? Was the infection polymicrobial? Was the patient treated with antibiotics prior sampling? Probiotics and their safety aspects have been important topics in EU research for years. After the finalisation of the programme “Demonstration of nutritional functionality of probiotic foods” in 2000 safety topic has been included as one of the relevant research sectors within the ongoing “Food, GI-tract functionality and Human health” cluster in the 5th framework programme [19, 92, 93].

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45. Shinar, E.; Leitersdorf, E.; Yevin, R. (1984) Lactobacillus plantarum endocarditis. Klin. Wochenschr. 62, 1173–1174. 46. Chong, Y.; Lim, H. S.; Lee, S. Y.; Cho, S. Y. (1991) Lactobacillus casei subspecies casei endocarditis. Yonsei Med. J. 32, 69–73. 47. Griffiths, J. K.; Daly, J. S.; Dodge, R. A. (1992) Two cases of endocarditis due to Lactobacillus species: Antimicrobial susceptibility, review, and discussion of therapy. Clin. Infect. Dis. 15, 250–255. 48. Aguirre, M.; Collins, M. D. (1993) Lactic acid bacteria and human clinical infection. J. Appl. Bacteriol. 75, 95–107. 49. Adams, M. R.; Marteau, P. (1995) On the safety of lactic acid bacteria from food. Int. J. Food. Microbiol. 27, 263–264. 50. Presterl, E.; Kneifel, W.; Mayer, H. K.; Zehetgruber, M.; Makristathis, A.; Graninger, W. (2001) Endocarditis by Lactobacillus rhamnosus due to yogurt ingestion? Scan. J. Infect. Dis. 33, 710–714. 51. Avlami, A.; Kordossis, T.; Vrizidis, N.; Sipsas, N. V. (2001). Lactobacillus rhamnosus endocarditis complicating colonoscopy. J. Infect. 42, 283–285. 52. Adams, M. R. (1999) Safety on industrial lactic acid bacteria. J. Biotech. 68, 171–178. 53. Brook, I.; Frazier, E. H. (1993) Significant recovery of nonsporulating anaerobic rods from clinical specimens. Clin. Infect. Dis. 16, 476–480. 54. Brook, I. (1996) Isolation of non-sporing anaerobic rods from infection in children. J. Med. Microbiol. 45, 21–26. 55. Thomas, A. V.; Sodeman, T. H.; Bentz, R. R. (1974) Bifidobacterium (Actinomyces) eriksonii infection. Am. Rev. Respirat. Dis. 110, 663–668. 56. Green, S. L. (1978) Case report: Fatal anaerobic pulmonary infection due to Bifidobacterium eriksonii. Postgrad. Med. 63, 187–192. 57. Crociani, F.; Biavati, B.; Alessandrini, A.; Chiarini, C.; Scardovi, V. (1996) Bifidobacterium inopinatum sp. nov. and Bifidobacterium denticolens sp. nov., two new species isolated from human dental caries. Int. J. Syst. Bacteriol. 46, 564–571. 58. Lee, Y.-K.; Salminen S. (1995) The coming of age of probiotics. Trends Food Sci. Technol. 6, 241–245. 59. Marteau, P.; Pochart, P.; Bouhnik, Y.; Rambaud, J. C. (1993) Fate and effects of some transiting micro-organisms in the human gastrointestinal tract. World Rev. Nutr. Diet. 74, 1–21. 60. Pochart, P.; Marteau, P.; Bouhnik, Y.; Goderel, I.; Bourlioux, P.; Rambaud, J.-C. (1992) Survival of bifidobacteria ingested via fermented milk during their passage through the human small intestine: An in vivo study using intestinal perfusion. Am. J. Clin. Nutr. 55, 78–80. 61. Johansson, M.-L.; Molin, G.; Jeppson, B.; Nobaek, S.; Ahrne´, S.; Bengmark, S. (1993) Administration of different Lactobacillus strains in fermented oatmeal soup. In vivo colonization of human intestinal mucosa and effect on the indigenous flora. Appl. Environ. Microbiol. 59, 15–20. 62. Nielsen, O. H.; Jorgensen, S.; Pedersen, K.; Justesen, T. (1994) Microbiological evaluation of jejunal aspirates and faecal samples after oral administration of bifidobacteria and lactic acid bacteria. J. Appl. Bacteriol. 76, 469–474. 63. Alander, M.; Korpela, R.; Saxelin, M.; Vilpponen-Salmela, T.; Mattila-Sandholm, T.; von Wright, A. (1997) Recovery of Lactobacillus rhamnosus GG from human colonic biopsies. Lett. Appl. Microbiol. 24, 361–364. 64. Alander, M.; Satokari, R.; Korpela, R.; Saxelin, M.; Vilpponen-Salmela, T.; MattilaSandholm, T.; von Wright, A. (1999) Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption. Appl. Environ. Microbiol. 65, 351–354.

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References 65. Marteau, P.; Vaerman, J. P.; Dehennin, J. P.; Bord, S.; Brassart, D.; Pochart, P.; Desjeux, J. F.; Rambaud, J. C. (1997) Effects of intrajejunal perfusion and chronic ingestion of Lactobacillus johnsonii strain La1 on serum concentrations and jejunal secretions of immunoglobulins and serum proteins in healthy humans. Gastroenterol. Clin. Biol. 21, 293–298. 66. Marteau, P.; Gerhardt, M. F.; Myara, A.; Bouvier, E.; Trivin, F.; Rambaud, J. C. (1995) Metabolism of bile salts by alimentary bacteria during transit in the human small intestine. Microb. Ecol. Health Dis. 8, 151–157. 67. Ruseler-van Embden, J. G. H.; van Lieshout, L. M. C.; Marteau, P. (1995) No degradation of intestinal mucus glycoproteins by Lactobacillus casei strain GG. Microecol. Ther. 25, 304–309. 68. Korpela, R.; Moilanen, E.; Saxelin, M.; Vapaasalo, H. (1997) Lactobacillus rhamnosus GG (ATCC 53013) and platelet aggregation in vitro. Int. J. Food Microbiol. 37, 83–86. 69. Kirjavainen, P. V.; Tuomola, E. M.; Crittenden, R. G.; Ouwehand, A. C.; Harty, D. W. S.; Morris, L. F.; Rautelin, H.; Playne, M. J.; Donohue, D. C.; Salminen, S. J. (1999) In vitro adhesion and platelet aggregation properties of bacteremia-associated lactobacilli. Infect. Immun. 67, 2653–2655. 70. O’Brien, J.; Crittenden, R.; Ouwehand, A. C.; Salminen, S. (1999) Safety evaluation of probiotics. Trends Food Sci. Technol. 10, 418–424. 71. ILSI Europe (1993) A scientific basis for regulation on pathogenic microorganisms in foods. Summary of a workshop held in May 1993 and organised by the Scientific Committee on Microbiology, ILSI Press. 72. Nicas, T. I.; Cole, C. T.; Preston, D. A.; Schabel, A. A.; Nagarajan, R. (1989) Activity of glycopeptides against vancomycin-resistant gram-positive bacteria. Antimicrob. Agents Chemother. 33, 1477–1481. 73. Swenson, J. M.; Facklam, R. R.; Thornsberry, C. (1990) Antimicrobial susceptibility of vancomycin-resistant Leuconostoc, Pediococcus, and Lactobacillus species. Antimicrobiol. Agents Chemother. 34, 543–549. 74. Charteris, W. P.; Kelly, P. M.; Morelli, L.; Collins, J. K. (1998) Antibiotic susceptibility of potentially probiotic Lactobacillus species. J. Food Prot. 61, 1636–1643. 75. Billot-Klein, D.; Gutmann, L.; Sable´, S.; Guittet, E.; van Heijenoort, J. (1994) Modification of peptidoglucan precursors is a common feature of the low-level vancomycin-resistant VANB-type Enterococcus D366 and of the naturally glycopeptideresistant species Lactobacillus casei, Pediococcus pentosaceus, Leuconostoc mesenteroides, and Enterococcus gallinarum. J. Bacteriol. 176, 2398–2405. 76. Tynkkynen, S.; Singh, K. V.; Varmanen, P. (1998) Vancomycin resistance factor of Lactobacillus rhamnosus GG in relation to enterococcal vancomycin resistance (van) genes. Int. J. Food Microbiol. 41, 195–204. 77. Klein, G.; Hallmann, C.; Casas, I. A.; Abad, J.; Louwers, J.; Reuter, G. (2000) Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods. J. Appl. Microbiol. 89, 815–824. 78. Ishiwa, H.; Iwata, M. (1980) Drug resistance plasmids in Lactobacillus fermentum. J. Gen. Appl. Microbiol. 26, 71–74. 79. Vescovo, M.; Morelli, L.; Bottazzi, V. (1982) Drug resistance plasmids in Lactobacillus acidophilus and Lactobacillus reuteri. Appl. Environ. Microbiol. 43, 50–56. 80. Rinckel, L. A.; Savage, D. C. (1990) Characterization of plasmids and plasmidborne macrolide resistance from Lactobacillus sp. strain 100–33. Plasmid 23, 119–125.

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81. Felten, A.; Barreau, C.; Bixet, C.; Lagrange, H.; Philippon, A. (1999) Lactobacillus species identification, H2O2 production, and antibiotic resistance and correlation with human clinical status. J. Clin. Microbiol. 37, 729–733. 82. Miller, L. G.; Finegold, S. M. (1967) Antimicrobial sensitivity of Bifidobacterium (Lactobacillus bifidus). J. Bacteriol. 93, 125–130. 83. Matteuzzi, D.; Crociani, F.; Brigidi, P. (1983) Antimicrobial susceptibility of Bifidobacterium. Ann. Microbiol. (Paris) 134A, 339–349. 84. Charteris, W. P.; Kelly, P. M.; Morelli, L., Collins, J. K. (1998) Antibiotic susceptibility of potentially probiotic Bifidobacterium isolates from the human gastrointestinal tract. Lett. Appl. Microbiol. 26, 333–337. 85. Lim, K. S.; Huh, C. S.; Baek, Y. J. (1993) Antimicrobial susceptibility of bifidobacteria. J. Dairy Sci. 76, 2168–2174. 86. Yazid, A. M.; Ali, A. M.; Shuhaimi, M.; Kalaivaani, V.; Rokiah, M. Y.; Reezal, A. (2000) Antimicrobial susceptibility of bifidobacteria. Lett. Appl. Microbiol. 31, 57–62. 87. Rautio, M.; Jousimies-Somer, H.; Kauma, H.; Pietarinen, I.; Saxelin, M.; Tynkkynen, S.; Koskela, M. (1999) Liver abscess due to a Lactobacillus rhamnosus strain indistinguishable from L. rhamnosus strain GG. Clin. Infect. Dis. 28, 1160–1161. 88. Mackay, A. D.; Taylor, M. B.; Kibbler, C. C.; Hamilton-Miller, J. M. T. (1999) Lactobacillus endocarditis caused by a probiotic organism. Clin. Microbiol. Infect. 6, 290–292. 89. Salminen, M. K.; Järvinen, A.; Saxelin, M.; Tynkkynen, S.; Rautelin, H.; Valtonen, V. (2001) Increasing consumption of Lactobacillus GG as a probiotic and the incidence of lactobacilli bacteraemia in Finland. Clin. Microbiol. Infect. 7 (suppl 1), 152. 90. Ouwehand, A. C.; Salminen, S. J. (1998) The health effects of cultured milk products with viable and non-viable bacteria. Int. Dairy J. 8, 749–758. 91. Salminen, S. J.; Donohue, D. C. (1996) Safety assessment of Lactobacillus strain GG (ATCC53013). Nutr. Today 31 (suppl.), 12–15. 92. Mattila-Sandholm, T.; Blum, S.; Collins, J. K.; Crittenden, R.; de Vos, W.; Dunne, C.; Fonden, R.; Grenov, B.; Isolauri, E.; Kiely, B.; Marteua, P.; Morelli, L.; Ouwehand, A.; Reniero, R.; Saarela, M.; Salminen, S.; Saxelin, M.; Schiffrin, E.; Shanahan, F.; Vaughan, E.; von Wright, A. (2000) Probiotics: towards demonstrating efficacy. Trends Food Sci. Technol. 10, 393–399. 93. Mattila-Sandholm, T.; Blaut, M.; Daly, C.; De Vuyst, L.; Dore, J.; Gibson, G.; Goossens, H.; Knorr, D.; Lucas, J.; Lähteenmaki, L.; Mercenier A.; Saarela, M.; Shahanan, F.; de Vos, W. M (2002) The food, GI-tract functionality and human health cluster. Microbial Ecol. Health Dis. 14, 65–74. 94. Saarela, M.; Mogensen, G.; Fonden, R.; Mättö, J.; Mattila-Sandholm, T. (2000) Probiotic bacteria: safety, functional and technological properties. J. Biotech. 84, 197–215.

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1 In vitro Study of Prebiotic Properties of Levan-Type Exopolysaccharides from Lactobacilli and nonDigestible Carbohydrates using Denaturing Gradient Gel Electrophoresis F. Dal Bello*, J. Walter, C. Hertel, and W. P. Hammes

In investigations of the effect of prebiotics on intestinal populations, bacteriological culture on media which are selective for a restricted group of gut bacteria have commonly been used. These methods are laborious, may deliver ambiguous results, and do not permit to recover the full diversity of the gut microflora. On the other hand, the application of culture-independent molecular techniques provides a more detailed view into the intestinal microflora. Especially, denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) of 16S rDNA amplicons have been demonstrated to be suitable tools for the analysis of microbial communities, because they permit a rapid detection of species and changes within a community structure. In this study batch cultures inoculated with human faeces were used to investigate the prebiotic properties of levan-type exopolysaccharides (EPS) from two strains of Lactobacillus sanfranciscensis, levan, inulin, and fructooligosaccharide (FOS). Denaturing gradient gel electrophoresis of 16S rDNA fragments generated by PCR with universal primers was used to analyse the cultures. The analyses revealed strong and characteristic changes in the composition of the bacteria during fermentation of the different carbohydrates. An enrichment of Bifidobacterium pseudocatenulatum and

* Institute of Food Technology, University of Hohenheim, Garbenstr. 28, 70599 Stuttgart, Germany; Tel. 0711-4594204; Fax 0711-4594199; e-Mail fabio_dal_bello@ hotmail.com

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2

Effects of Butyrate on Glutathione S-Transferase P1

B. ruminantium was found for the EPS and inulin but not for levan and FOS. The bifidogenic effect of the EPS was confirmed by culturing on selective medium. In addition, the use of EPS and FOS resulted in enhanced growth of Eubacterium biforme and Clostridium perfringens, respectively. In our fermentation studies the bifidogenic activity of inulin was confirmed, indicating that the use of PCR-DGGE in combination with batch culture enrichments over 4 days is a suitable tool to investigate prebiotic effects.

2 Effects of Butyrate on Glutathione S-Transferase P1 Expression in Human Colonic Adenoma Cells G. Festag*, N. Haag*, G. Beyer-Sehlmeyer*, M. N. Ebert*, B. Marian** and B. L. Pool-Zobel*

2.1 Introduction Glutathione S-transferases (GST) are important phase-II-enzymes, which play a major role in the detoxification of xenobiotics. Also, GSTP1-expression is enhanced during progression of colorectal carcinogenesis where it may serve as a predictive marker of overall survival [1, 2]. Recently, we have shown that butyrate, a gut fermentation product, induces GSTP1 and enhances chemoresistance in human colon tumour cells [3]. This could either mean that butyrate is beneficial and reduces exposure to cancer risk factors, if GST induction also occurs in non-transformed cells. Or the results could mean that, in a proportion of the treated cell population, butyrate counteracts its known beneficial and suppressing-agent activities of inhibiting proliferation and inducing apoptosis [3]. However, since butyrate-formation presumably is an important mechanism of beneficial health effects by dietary fiber in the normal population, it is important to assess its effects in less-transformed cells, representative of a “healthy” individual. So far, this type of work has been limited due to technical difficulties of cultivating non-transformed human colon cells in vitro. Recently, it has been possible to isolate a cell line from an adenoma [4]. The cells are representative of preneoplastic

* Department of Nutritional Toxicology, Institute for Nutrition, Friedrich Schiller University, Dornburger Str. 25, 07743 Jena ** Institute of Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna

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lesions that occur frequently at high incidence with increasing age, and are therefore relevant surrogate cells of the “non-diseased” human colon. Aim of the present study was therefore to analyse the butyrate-mediated effects in these more differentiated human colon cells. Here we report on the effect of this fermentation product on GSTP1 expression at mRNA- and proteinlevels and on the modulation of GST activity.

2.2 Material and Methods Following incubation, LT97 cells were harvested and RNA was isolated with TRIzol (Invitrogen) and GSTP1 mRNA expression was analysed by Northern Blotting according to [1]. Aliquot cell pellets were homogenised by sonication and the cytosol was prepared by ultracentrifugation at 4 hC. GST activity was determined towards 1-chloro-2,4-dinitrobenzol (CDNB) [5]. Total protein content was measured using the method by Bradford with bovine serum albumin as standard protein. GSTP1 protein level was quantified by Western Blotting using GST standard protein [6].

2.3 Results x

Data show that butyrate did not affect GSTP1 mRNA levels 72 h after addition of Na-butyrate in concentrations up to 1 mM.

x

GST activity (baseline level: 51.8 e 5.4 nmol x min–1 q 10–6 cells) was not modulated 72 h after addition of butyrate (0.25–2 mM) to the cell culture. Altogether the baseline activity was higher than in HT29 cells (36.9 e 5.9 nmol x min–1 q 10–6 cells).

x

Butyrate did not significantly inhibit GSTP1 protein level (untreated and 4 mM Butyrate, resp.: 425 e 209 mg q 10–6 cells to 332 e 180 mg q 10–6 cells). In contrast, in HT29 cells butyrate (4 mM) significantly increased GSTP1 protein level [3].

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References

2.4 Conclusion The butyrate-mediated effects on GSTP1 expression that are observed in HT29 cells [3] were not detectable in LT97 adenoma cells. Butyrate neither significantly modulated GSTP1 mRNA and GSTP1 protein expression, nor did it influence GST activity. This could mean that butyrate impairs the survival of adenoma cells by not giving them the benefit of developing the increased GSTP1 levels, which accompanies tumour development [1, 2]. Since butyrate is an important fermentation product, produced in the gut lumen after ingestion of high fibre diets, this mechanism could mean that tumour progression from these adenoma cells is retarded. In elderly carrying the adenoma, these results could imply that butyrogenic dietary fibres may help prolong the onset of disease.

Acknowledgements Supported by the BMELF (Grant No. 99HS039), Deutsche Krebshilfe (Grant No. 10-1572-Po1) and DFG (Grant No. PO 284/ 6-1).

References 1. Moorghen, M.; Cairns, J.; Forrester, L. M.; Hayes, J. D.; Hall, A.; Cattan, A. R.; Wolf, C. R.; Harris, A. L. (1991) Enhanced expression of glutathione S-transferases in colorectal carcinoma compared to non-neoplastic mucosa. Carcinogenesis 12, 13–17. 2. Mulder, T. P. J.; Verspaget, H. W.; Sier, C. F. M.; Roelofs, H. M. J.; Ganesh, S.; Griffioen, G.; Peters, W. H. M. (1995) Glutathione S-transferase p in colorectal tumors is predictive for overall survival. Cancer Research 55, 2696–2702. 3. Ebert, M. N.; Beyer-Sehlmeyer, G., Liegibel, U. M.; Kautenburger, T.; Becker, T. W., Pool-Zobel, B. L. (2001) Butyrate induces glutathione S-transferase in human colon cells and protects from genetic damage by 4-hydroxy-2-nonenal. Nutrition and Cancer 41, 156–164. 4. Richter, M.; Jurek, D.; Wrba, F.; Kaserer, K.; Wurzer, G.; Karner-Hanusch, J.; Marian, B. (2002). Cells obtained from colorectal microadenomas mirror early premalignant growth patterns in vitro. European Journal of Cancer (in press). 5 Habig, W. H.; Pabst, M. J.; Jakoby, W. B. (1974) Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. 6. Ebert et al. (in preparation).

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3 In vitro Fermentation Supernatants of Inulin-associated Prebiotics Modulate Proliferation and Glutathione S-Transferase Activity in Human Colon HT29 Cells Eva Gietl, Annett Klinder, Stella Pistoli*, Beatrice Pool-Zobel

3.1 Introduction Inulin is a functional prebiotic food ingredient, which may reduce the risk of colon carcinoma development. It is a substrate for selected bacteria of the intestinal flora, leading to an enhancement of lactic acid producing Bifidobacteria and Lactobacilli [1] and probably also of other saccharolytic species which produce short chain fatty acids. In rats it reduces yield and incidence of chemically induced preneoplastic lesions [2] and inhibits tumour formation in APC deficient mice.

3.2 Aim To elucidate some of the anti-carcinogenic mechanisms related to inulin intake and its gut fermentation products on a cellular level, we have now investigated samples of associated prebiotic/probiotic mixtures for biological effects in human colon tumour cells HT29.

3.3 Material and Methods The fermentation samples were produced by anaerobic fermentation of Synergy 1 (a mixture of inulin and oligofructose) with probiotics (pure bacteria cultures of Bifidobacteria lactis Bb12 and/or Lactobacillus rhamnosusGG) or with a faeces inoculum of different donors donor 1–4. Additionally, three-stage fermentation (“gut model” = GM) was used to simulate in vivo fermentation in the various colon segments (proximal V1, sigmoid V2, distal V3). HT 29 cells were incubated with sterile filtrated supernatants of the fer-

* Food Microbial Sciences Unit, School of Food Biosciences, University of Reading, Early Gate, Whitenights Road, Reading R6G 2EF, United Kingdom

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In vitro Fermentation Supernatants LGG in comparison

Proliferation (%)

120 100 80

*

60 40 20 0 0

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Concentration (% v/v) LGG+Syn1+FS1

LGG+Syn1+FS2

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LGG+Synergy1

Fecal slurry of donor 3 Proliferation (%)

120 100 80 60 40 20 0 0

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16

Concentration (% v/v) Bb12+Syn+FS3

LGG+Syn+FS3

Glucose+FS3

NaCl (0,9%)

Pure culture fermentation supernatants

Proliferation (%)

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*

80 60 40 20 0 0

2

4

6

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10

12

Concentration (% v/v) Bb12+Syn1

Bb12+Glu

LGG+Syn1

LGG+Glu

Bb12+LGG+Syn1

Bb12+LGG+Glu

Figure 3.1. Proliferation of HT29 cells was modified by the fermentation supernatants, with marked interindividual variability, depending on donor of faecal samples originally used to produce the samples. Also, the type of probiotic used, affected degrees of biological activities. Fermentation supernatants from Bifidobacteria lactis (Bb12) had a stronger anti-proliferating effect than those with Lactobacillus GG.

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GST-activity (n mol/min*ml) per 106 cells

Donor 3 125

*

100 75

**

*

50 25 0 Medium

Bb12+Syn1+FS3

NaCl

Glucose+FS3

LGG+Syn1+FS3

Butyrat

Gut model GST-activity (n mol/min*ml) per 106 cells

125 100

*

75 50 25 0 Medium

NaCl

GM1V3

GM1V1

GM1V2

BM+Syn1

Pure culture GST-activity (n mol/min*ml) per 106 cells

125 100 75 50 25 0 Medium LGG+Glu

NaCl

Bb12+Syn1 Bb12+LGG+Syn1

Bb12+Glu

LGG+Syn1

Bb12+LGG+Glu

Figure 3.2. When concentrations of 10 % supernatants (v/v) were used, the GST activity was only marginally affected by the pure culture fermentations, whereas the samples obtained from fermenting B. lactis Bb12 plus Synergy1 plus faecal slurry had a stronger effect.

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In vitro Fermentation Supernatants Donor 3

GST-activity (n mol/min*ml) per 106 cells

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75

* 50

25

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Medium

NaCl(4,5%)

NaCl(8,5%)

Bb12+Syn1+FS3(4,5%)

NaCl(14,5%)

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Glc + FS3 (8,5%)

Butyrat(4mM)

Gut model GST-activity (n mol/min*ml) per 106 cells

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**

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Medium

NaCl(13,5%)

NaCl(7%)

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GM1V2(8,5%)

GM1V3(7,5%)

Pure culture GST-activity (n mol/min*ml) per 106 cells

100

75

*

50

*

*

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Medium

NaCl(14%)

Bb12+Glu(14%) Bb+LGG+Syn1(13%)

NaCl(27%)

LGG+Syn1(15%)

Bb12+Syn1(12%) LGG+Glu(27%)

Bb+LGG+Glu(15%)

Figure 3.3. When the EC50-values (v/v) were used, the supernatant derived from stage 3 (V3) of the “gut model” (representative of products generated in the distal colon) was the most effective of the three gut model supernatants for inhibiting proliferation as well as for inducing GST.

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mentation mixtures (2.5 %; 5 %; 10 % and 15 % (v/v)) for 72 hours. Proliferation of cells was determined by quantifying fractions of remaining cells by fluorimetric measurement of DNA. The obtained EC50 values or concentrations of 10 % supernatants (v/v) were used to assess modulation of glutathione S-transferase (GST)-activity with 1-chloro-2,4-dinitrobenzene as substrate.

3.4 Results Figures 3.1–3.3 see side 285–287.

3.5 Conclusion Biological effects of inulin on colon cells may be mediated not only by the lactic acid-producing bacteria, but also by other bacteria of the gut lumen. The property of the supernatants to enhance GSTs is beneficial for cell populations in that it increases chemoresistance. However it can only be considered to contribute to the observed in vivo anticarcinogenic potentials if it is also inducible in the non transformed target cells of colon cancer. In contrast, these newly reported proliferation-inhibiting properties in HT29- tumour cells per se, are considered to be beneficial, since these types of suppressing activities contribute to the retardation of tumour progression.

References 1. Jenkins, D., et al. (1999). Inulin, Oligofructose and Intestinal Function. J. Nutr 129, 1431–1433. 2. Rowland, I. R., et al. (1998). Effect of bifidobacterium longum and inulin on gut bacterial metabolism and carcinogen-induced aberrant crypt foci in rats.Carcinogenesis 19, 281–285.

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b-Carotene Effects in a Tobacco Smoke

4 b-Carotene Effects in a Tobacco Smoke Carcinogeninduced Lung Cancer Model in vivo R. Goralczyk*, H. Bachmann, G. Riss, C.-P. Aebischer, B. Lenz, and K. Wertz

4.1 Introduction b-Carotene is the most important source for vitamin A in human nutrition. It has been estimated that up to 80 % of human vitamin A supply derives from the provitamin b-carotene, in particular in tropical countries, where meat consumption is low. b-Carotene also has become an important and safe food colorant since the 60s. Epidemiological studies [1–4] provide solid evidence for an association between high b-carotene uptake, or a high b-carotene plasma concentration, and a reduced risk for cancer, especially lung cancer. In addition, animal studies also demonstrate cancer-preventive activity for b-carotene [5, 6]. Unexpectedly, in three large clinical intervention trials, b-carotene supplementation either showed no effect (PHS) [7], or was associated with an increased incidence of lung cancer (ATBC) [8] (CARET) [9] in heavy smokers. The mechanism(s) by which b-carotene may be associated with an increased risk of lung cancer in heavy smokers is, as yet, unknown. As part of efforts to address this deficiency, a series of studies was initiated to investigate the influence of b-carotene intake on carcinogen-induced lung carcinogenesis in an appropriate in vivo model. An in vivo lung cancer model was selected and established allowing for the study of several hypotheses put forward to explain the unexpected outcome of the ATBC and CARET studies. With the A/J strain of mice, a model was chosen that is widely used in the search for chemo-preventive agents. The A/J mouse spontaneously develops lung tumors. Nearly exclusively adenomas and adenocarcinomas are found. The number of tumors formed is highly time-dependent, and can be increased by carcinogens in a dose-dependent manner. The model is sensitive to all known human carcinogens and most suspected carcinogens. To study the potential modulating effects of b-carotene on the development of lung tumors in the A/J mouse model, NNK (4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone) was selected as the initiating agent, since it is the preferred carcinogen in recent studies and it is one of the main carcinogens formed during the smoking of tobacco. As a nitrosamine derivative,

* Human Nutrition and Health, Roche Vitamins Ltd., Basel, Switzerland

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it is associated with known human carcinogens. The mechanism of action of nitrosamines (i. e., metabolic activation by cytochrome P450 and subsequent formation of a reactive radical capable of methylating DNA) is well understood. In the A/J mouse model, we investigated possible long-term interactions of b-carotene on spontaneous and NNK-initiated lung tumor formation. The effect of b-carotene at specific stages of lung carcinogenesis was addressed by supplementation during initiation, promotion or progression. In addition, a dose response trial was performed. b-carotene absorption was facilitated by feeding a 5 % cornoil, 0.25 % sodium cholate containing diet [10]. Since one proposed mechanism for b-carotene to exert its effect on smoke-induced lung carcinogenesis is that b-carotene could interfere with retinoid signaling pathways, RARb gene expression was determined.

4.2 Methods Male A/J mice were injected once with 3.5 (or 5) mg NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; ChemSyn Laboratories, Lenexa, Kansas, USA). b-carotene was supplemented as b-Carotene 10 % CWS beadlets (Roche Vitamins Ltd.) mixed with chow. The chow had been enriched with 5 % corn oil and 0.25 % Na cholate to facilitate absorption of intact b-carotene. Tumor multiplicity was determined by inspection under the dissection microscope. TaqManr real-time RT-PCR was applied to measure RARb expression. To establish dose relations, b-carotene was supplemented at 120 to 3000 ppm one month before initiation and over the whole trial period of 24 weeks. A possible effect of b-carotene on different stages of carcinogenesis was investigated by supplementing 600 ppm b-carotene around initiation (2 weeks before until one week after NNK injection), or starting from 4 weeks after NNK injection, or throughout the whole trial period of 28 weeks. b-Carotene and retinol were analyzed in plasma, lung and liver by HPLC.

4.3 Results With a modification of the standard rodent diet, it was possible to achieve plasma b-carotene concentrations in the range of those observed in both human studies (depending on duration of supplementation, peak plasma levels ranged between 3 and 6 mmol/L). Similarly, using this diet in the A/J mouse, tissue concentrations, particularly in the lung, were achieved that exceeded values reported in the CARET study (depending on duration of supplementation peak levels around 3–6 nmol/g). 290

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Since lung cancer formation in the A/J mouse model can be initiated and synchronized by a single dose of carcinogen, in this case NNK, the model was highly amenable to studying the effects of b-carotene during different stages of carcinogenesis (i. e., different times following NNK dosing). Tumor multiplicity, the functional endpoint in the A/J mouse model of lung tumorigenesis, was found to be reproducible over the course of 5 separate experiments and was in accordance with published values. Results from 5 separate experiments demonstrate that b-carotene did not increase the multiplicity of spontaneous tumors nor those induced by NNK. b-Carotene did not interfere with the metabolic activation of nitrosamines in vivo, a step necessary for the initiation of carcinogenesis. In two experiments, the effect of b-carotene on different stages of carcinogenesis was studied by starting the b-carotene supplementation before NNK treatment or at different times after NNK induction. There were no significant effects of b-carotene treatment on the multiplicity of tumors induced by NNK. Similarly, a dose-response study found no effect when b-carotene was administered at a dose range of 120 to 3,000 ppm in the diet for 24 weeks; 2 weeks prior to, and 22 weeks following, exposure to NNK. b-carotene is a precursor for retinoic acid (RA). Retinoic acid receptor b (RARb) has been suggested to be a tumor suppressor gene in the lung [11]. Therefore, b-carotene could hypothetically interfere with lung cancer development by downregulating RARb. The RARb protein is involved in transmitting the RA signal, while at the same time, the mRNA for the RARb gene is up-regulated by RA. We found that expression of all RARb isoforms was significantly reduced in lungs of A/J mice which had been treated with NNK 5 months before. b-Carotene supplementation did not influence the downregulation of RARb in NNK-treated animals. In contrast to this, b-carotene supplementation to non-initiated A/J-mice resulted in a mild, nonsignificant up-regulation of RARb2 and RARb4 transcripts. These RARb isoforms are known to be regulated in an RA-dependent manner. Thus, increased RARb2 and RARb4 expression indicates a physiological metabolization of b-carotene in the mice. No dose effect was observed for regulation of RARb expression by b-carotene.

4.4 Conclusion The A/J mouse is a reproducible animal model for investigation of tobacco smoke carcinogen-induced lung carcinogenesis. Relevant b-carotene exposure can be achieved, although high b-carotene doses are required. Administration of b-carotene had no significant effect on tumor multiplicity in the A/J mouse model, following NNK induction, at various time points and at various doses after initiation. I. e. neither initiation, promotion, progression nor spontaneous tumor rates were enhanced by b-carotene. RARb expression in lung 291

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was reduced in NNK-treated mice only, irrespective of b-carotene supplementation. These extensive studies provide no data to support a possible mechanism by which b-carotene supplementation was associated with an increased rate of lung tumor incidence or mortality in smokers in the ATBC and CARET intervention trials. Studies with cigarette smoke-exposed A/J mice will complement molecular-toxicological aspects.

References 1. Peto, R.; Doll, R.; Buckley, J. D.; Sporn, M. B. (1981): Can dietary b-carotene materially reduce human cancer rates? Nature 290, 201–9. 2. Block, G.; Patterson, B.; Subar, A., (1992): Fruit, vegetables, and cancer prevention: a review of the epidemiology evidence. Nutr. Cancer 18, 1–29. 3. Gerster, H. (1993): Beta-carotene, vitamin E and vitamin C in different stages of experimental carcinogenesis. Eur. J. Clin. Nutr. 49, 155–68. 4. Steinmetz, K. A.; Potter, J. D. (1991): Vegetables, fruit, and cancer. I. Epidemiology. Cancer causes control 2, 325–57. Steinmetz, K. A.; Potter, J. D. (1991): Vegetables, fruit, and cancer. II. Mechanisms. Cancer causes control 2, 427–42. 5. Krinsky, N. I. (1993): Actions of carotenoids in biological systems. Ann. Rev. Nutr. 13, 561–87. 6. Gerster, H. (1995): Anticarcinogenic effect of common carotenoids. Int. J. Vitam. Nutr. Res. 63, 93–121. 7. Hennekens, C. H.; Buring, J. E.; Manson, J. E., et al. Lack of long-term supplementation with beta-carotene on the incidence of malignant neoplasms and cardiovascular disease. (1996) New. Engl. J. Med. 334, 1145–1149. 8. ATBC Cancer Prevention Study Group (1994): The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. New Engl. J. Med. 330, 1029–1035. 9. Omenn, G. S.; Goodman, G. E.; Thornquist, M. D.; Balmes, J.; Cullen, M. R.; Glass, A.; Keogh, J. P.; Meyskens Jr., F. L.; Valanis, B.; Williams, J. H.; Barnhart, S.; Cherniak, M. G.; Brodkin, C. A.; Hammar, S. (1996) Risk factors for lung cancer and for intervention effects in CARET, the beta-carotene and retinol efficacy trial. J. Natl. Cancer Inst. 88, 1550–1559. 10. Umegaki, K., et al. (1995): Simultaneous dietary supplementation of sodium cholate and beta-carotene markedly enhances accumulation of beta-carotene in mice. J. Nutr. 125, 3081–6. 11. Xu, XC et al., (1997) Suppression of retinoic acid receptor beta in non-small-cell lung cancer in vivo: implications for lung cancer development. JNCI, 89 (9), 624–629.

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Influence of Daidzein and its Metabolites

5 Influence of Daidzein and its Metabolites on the Expression of Catalase in Rat Hepatoma Cells A. Kampkötter*, E. Röhrdanz**, K. Iwami*, S. Ohler*, W. Wätjen*, Y. Chovolou*, S. E. Kulling***, and R. Kahl1

5.1 Introduction Phytoestrogens, like daidzein, are phenolic compounds (Fig. 5.1) which are present in legumes, whole grains and vegetables. These dietary flavonoids are of high interest since they are suggested to benefit human health. These beneficial effects have been mainly attributed to their antioxidant properties. Flavonoids are able to scavenge free radicals and to chelate redox active metal ions and thus acting directly against oxidative stress [1, 2]. The antioxidant enzyme (AOE) system, present in all aerobic organisms, plays an important role in the defense against oxidative stress and in the protection against damage caused by reactive oxygen species [3–5]. The expression of the AOEs has been shown to be influenced by inflammatory mediators (lipopolysaccharides, cytokines) and by oxidative stress itself. In our work we want to examine the interaction of the daidzein and its metabolites with the AOE system on a molecular level. To investigate the effects of daidzein on rat hepatoma cells, H4IIE cells were treated with different concentrations of the flavonoid. The viability of the cells was reduced by about 20–30 % with 200–300 mM daidzein demonstrating cell toxiticity at these concentrations [6]. On the other hand treatment of H4IIE cells with the same concentrations of daidzein resulted in a 2–4 fold increase of catalase mRNA expression whereas the expression levels of other AOEs were not or only slightly (copper zinc superoxide dismutase, manganese superoxide dismutase, glutathione peroxidase) affected [6]. Since there is a strong and specific upregulation of the catalase mRNA level in response to daidzein exposure, we were interested to elucidate the mechanisms responsible for this transcriptional induction. Human liver hepatoma cells (HepG2) were transiently transfected with reporter plasmids con-

* Heinrich-Heine Universität, Institut für Toxikologie, P. O. Box 101007, 40001 Düsseldorf, Germany ** Bundesinstitut für Arzneimittel und Medizinprodukte, Friedrich-Ebert-Allee 38, D-53113 Bonn, Germany *** Bundesforschungsanstalt für Ernährung, Institut für Ernährungsphysiologie, Haid-und Neu-Str. 9, 76131 Karlsruhe, Germany

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taining different parts of the 1.1 kb upstream region (promoter) of the rat catalase gene and the effects of daidzein exposure to the activity of the variant promotor fragments were monitored by the activity of the reporter gene product (luciferase). In all tested promoter constructs, daidzein (50 and 100 mM) leads to a transcriptional induction [6]. Interestingly, the construct containing the complete 1.1 kb was less induced than the deletion constructs suggesting the presence of silencer elements in the more distant region of the catalase promoter [6]. In humans the phytoestrogen daidzein is metabolized both in a reductive and a oxidative manner. The reductive metabolism is mainly conferred by intestinal bacteria leading to equol and and O-desmethylangolesin. The oxidative metabolism takes place in the liver and generates several products including 6-hydroxy-daidzein (6,7,4l-trihydroxyisoflavone) and 3l-hydroxydadzein (7,3l,4l-trihydroxyisoflavone) [7]. In our present work we have started to investigate the influence of two daidzein metabolites (6-hydroxy-daidzein and 3l-hydroxy-dadzein; Fig. 5.1) on rat hepatoma cells and to compare them with effects of daidzein. Further on, we are interested in the elucidation of the mechanisms of the regulation of catalase expression.

Figure 5.1. Chemical structure of daidzein and of two of its metabolites.

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5.2 Results 5.2.1

Cytotoxicity of Daidzein, 3l-Hydroxy-Daidzein and 6-Hydroxy-Daidzein

The cytotoxicity of daidzein, 3l-hydroxy-daidzein and 6-hydroxy-daidzein in rat hepatoma H4IIE cells was determined (Fig. 5.2). The cytotoxicity curves of daidzein and 3l-hydroxy-daidzein are very similar whereas 6-hydroxy-daidzein treated cells were less viable. This means that the latter one is more toxic to the cells than the original compound and the other analysed metabolite.

Figure 5.2. Cytotoxicity of daidzein, 3l-hydroxy-daidzein and 6-hydroxy-daidzein. The cytotoxicity of daidzein, 3l-hydroxy-daidzein and 6-hydroxy-daidzein on rat hepatoma H4IIE cells was determined by the activity of the mitochondrial dehydrogenases in the MTT assay after a 24 h treatment of the cells with the given concentratrions of daidzein. Values are expressed as % of controls (means e SEM of four to twelve independent experiments).

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Activity of the Rat Catalase Gene Promoter

The inducibility of the 1.1 kb rat catalase gene promoter (Fig. 5.3) in response to exposure to daidzein, 3l-hydroxy-daidzein and 6-hydroxy-daidzein was determined by the luciferase reporter gene activity (Fig. 5.4).

Figure 5.3. Scheme of the promoter-reporter construct pLuc1. The 1.1 kb upstream region of the rat catalase gene was cloned in front of the luciferase reporter gene (pGL3 Basic; Promega). The location of putative silencer elements and of possible binding are indicated.

Figure 5.4. Activity of the rat catalase gene promoter in response to exposure to daidzein, 3l-hydroxy-daidzein and 6-hydroxy-daidzein. The promoter-reporter construct pLuc1 (Fig. 5.2) was transiently transfected in human (HepG2) and rat (H4IIE) hepatoma cells. After a 24 h treatment of the cells the activity of the promoter was measured with the “Dual Luciferase Assay Kit“ (Promega). Values are expressed as % of controls (means of two to six independent experiments).

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The induction of the 1.1 kb rat catalase gene promoter in response to daidzein in the human hepatoma HepG2 cell was higher than in the rat hepatoma H4IIE cells indicating species specific differences in the regulation of transcription (Fig. 5.4). Exposure to 50 mM daidzein led to induction in both cell types, whereas the induction after exposure to 100, 200 (only H4IIE) and 300 mM (only H4IIE) daidzein did not result in an induction. According to our (preliminary) data the two metabolites repressed the promoter activity.

5.2.3

Comparison of Rat Catalase mRNA Level and Promoter Activity

The catalase mRNA levels in H4IIE cells and the promoter activity in transiently transfected H4IIE cells in response to daidzein treatment were compared (Fig. 5.5). Our preliminary data for the activation of the 1.1 kb rat catalase promoter showed induction after treatment with 50 mM daidzein whereas the higher concentrations did not result in an induction. In contrast to this result the mRNA level increased with increasing concentrations of daidzein. This discrepancy between the steady state mRNA levels and the promoter inducibility may allow the speculation that the catalase expression in response to daidzein treatment is mainly regulated by posttranscriptional mechanisms (e. g. mRNA stability) and to a lower extent by transcriptional mechanisms.

Figure 5.5. Comparison of catalase mRNA level and catalase promoter activity. The catalase mRNA levels of H4IIE cells treated with daidzein for 24 h were determined by the densitometrical analysis of the signal intensities on hybridized northern blots (NB). For the curve of the promoter activity the same data as in figure 4 were used. Values are expressed as % of controls (means e SEM of two to six independent experiments).

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5.3 Summary x x x x

The daidzein metabolite 6-hydroxy-daidzein is more toxic than daidzein itself and its metabolite 3l-hydroxy-daidzein. The rat catalase gene promoter is stronger induced after daidzein treatment in transiently transfected human hepatoma cells than in rat hepatoma cells. Daidzein but not its metabolites led to induction of the catalase promoter in transiently transfected rat hepatoma cells. The rat catalase expression is presumably regulated mainly on a posttranscriptional level.

5.4 Outlook x x x

Refinement of the promoter analysis by the use of a longer (3,5 kb) upstream region and deletion constructs. Elucidation of the regulatory mechanisms of the catalase expression. Analysis of the influence of further metabolites of daidzein.

References 1. Robak, J. and Gryglewski, R. J. (1-3-1988). Flavonoids are scavengers of superoxide anions. Biochem.Pharmacol. 37, 837–841. 2. Morel, I., Lescoat, G., Cogrel, P., Sergent, O., Pasdeloup, N., Brissot, P., Cillard, P., and Cillard, J. (7-1-1993). Antioxidant and iron-chelating activities of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat hepatocyte cultures. Biochem. Pharmacol. 45, 13–19. 3. Rohrdanz, E. and Kahl, R. (1-1-1998). Alterations of antioxidant enzyme expression in response to hydrogen peroxide. Free Radic.Biol.Med. 24, 27–38. 4. Rohrdanz, E., Obertrifter, B., Ohler, S., Tran-Thi, Q. H., and Kahl, R. (2000). Influence of Adriamycin and paraquat on antioxidant enzyme expression in primary rat hepatocytes. Arch.Toxicol. 74, 231–237. 5. Visner, G. A., Dougall, W. C., Wilson, J. M., Burr, I. A., and Nick, H. S. (15-2-1990). Regulation of manganese superoxide dismutase by lipopolysaccharide, interleukin1, and tumor necrosis factor. Role in the acute inflammatory response. J.Biol.Chem. 265, 2856–2864. 6. Rohrdanz, E., Ohler, S., Tran-Thi, Q. H., and Kahl, R. (2002). The phytoestrogen daidzein affects the antioxidant enzyme system of rat hepatoma H4IIE cells. J.Nutr. 132, 370–375. 7. Kulling, S. E., Honig, D. M., and Metzler, M. (2001). Oxidative metabolism of the soy isoflavones daidzein and genistein in humans in vitro and in vivo. J.Agric.Food Chem. 49, 3024–3033.

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Flavonoids and the Central Nervous System

6 Flavonoids and the Central Nervous System: The Anxiolytic Flavone Hispidulin D. Kavvadias*, P. Sand**, P. Riederer**, E. Richling*, P. Schreier*

6.1 Introduction Effects of plant flavonoids on the central nervous system are known since 1990, when the existence of natural anxiolytic flavonoids was described for the first time. In the meantime, flavones were found to be the most active group. The major inhibitory system in the mammalian brain is mediated by the GABAA receptor complex, which plays an important role in the regulation of neurobiological mechanisms like anxiety, sleep and convulsions. The GABAA receptor is a membrane bound pentameric protein which can compose of 16 various subunits naturally found until now (6a, 4b, 3g, 1d und 1p). The most frequent isoform composes of two a-, two b- and one g-subunit. The binding of the amino acid GABA triggers the opening of the Cl–-ion channel. The increased Cl–-influx leads to membrane hyperpolarization and reduced neuronal excitability. In addition to the GABA binding site, the GABAA receptor complex possesses modulatory sites for other ligands that allosterically modify the frequency of the Cl–-channel openings. One of them is the benzodiazepine receptor (BzR), where the classical anxiolytics, the benzodiazepines, bind and exert their pharmacological effects. It has been shown that many other groups of compounds, especially flavones, can bind to the BzR with high affinity [1–3].

6.2 Results and Discussion Flavones are widely distributed in the plant kingdom. More than 4000 chemically unique flavonoids have been isolated, making them to one of the most important class of secondary metabolites in plants used, among others, as sedatives. In our studies, the methanol extract from sage (Salvia officinalis L.) showed remarkable activity in the radio receptor binding assay (RRA) for the benzodiazepine receptor. Using an RRA-guided purification and fractionation of the sage extract, we were able to isolate 5,7,4’-trihydroxyflavone

* Laboratory of Food Chemistry, University of Würzburg, Würzburg, Germany ** Laboratory of Neurochemistry, University of Würzburg, Würzburg, Germany

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(apigenin), 5,4’-dihydroxy-6,7-dimethoxyflavone (cirsimaritin) and 5,7,4’-trihydroxy-6-methoxyflavone (hispidulin) (Fig. 6.1). The binding activities of the isolated compounds were investigated in vitro using RRA by displacing of 3H-flumazenil bound to benzodiazepine receptors in human brain membrane preparations [4]. Hispidulin was found to be a potent benzodiazepine receptor ligand with an IC50 value of about 1 mM. Figure 6.2 shows the binding curve of hispidulin compared with that of whole sage extract and diazepam.

Figure 6.1. Chemical structures of BzR active flavones isolated from the methanol extract of sage leaves (Salvia officinalis).

Figure 6.2. Competitive binding curves of hispidulin, sage extract and diazepam resulting from RRA (inhibition of specific 3H-flumazenil binding to benzodiazepine receptors in human brain cortical membranes). Results are presented as triplicates. Error bars represent SEM.

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Figure 6.3. Binding affinities of flavone derivatives related to their substitution pattern. According to the “pharmacophore model of Cook” [5], H1 and H2 represent proton donating sites, L1 and L2 are lipophilic pockets, whereas S1 and S2 exhibit steric repulsive regions. IC50-values marked with 1, 2 and 3 are taken from the literature [2, 6, 7].

As shown in Fig. 6.3, the in vitro benzodiazepine receptor affinity of flavones strongly depends on their substitution pattern. Our structure-activity studies of various flavones exhibited remarkable increase of binding activity when a methoxy group was present in the 6-position. In contrast, methylation of hydroxy groups in the 7- and 3-position is accompanied by notably loss of biological activity at the benzodiazepine receptor.

6.3 Conclusions Like other 6-methoxyflavones, hispidulin was found to be an efficient in vitro benzodiazepine ligand. In vivo studies of its bioavailability and metabolism including safety aspects are required to evaluate its potential use as natural bioactive compound.

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Acknowledgements The Deutsche Forschungsgemeinschaft, Bonn, and the Fonds der Chemischen Industrie, Frankfurt, are thanked for financial support.

References 1. Paladini, A. C.; Marder, M.; Viola, H.; Wolfman, C.; Wasowski, C.; Medina, J. H. (1999) Flavonoids and the central nervous system: from forgotten factors to potent anxiolytic compounds. J. Pharm. Pharmacol. 51, 519–526. 2. Ai, J.; Dekermendjian, K.; Wang, X.; Nielsen, M.; Witt, M.-R. (1997) 6-Methylflavone, a benzodiazepine receptor ligand with antagonistic properties on rat brain and human recombinant GABAA receptors in vitro. Drug Dev. Res. 41, 99–106. 3. Medina, J. H.; Viola, H.; Wolfman, C.; Marder, M.; Wasowski, C.; Calvo, D.; Paladini, A. C. (1997) Overview-Flavonoids: a new family of benzodiazepine receptor ligands. Neurochem. Res. 22, 419–425. 4. Kavvadias, D.; Abou-Mandour, A. A.; Czygan, F.- C.; Beckmann, H.; Sand, P.; Riederer, P.; Schreier, P. (2000) Identification of benzodiazepines in Artemisia dracunculus and Solanum tuberosum rationalizing their endogenous formation in plant tissue. Biochem. Biophys. Res. Commun. 269, 290–295. 5. Zhang, W.; Köhler, K. F.; Zhang, P.; Cook, J. M. (1995) Development of a comprehensive pharmacophore model for the benzodiazepine receptor. Drug Des. Dis. 12, 193–248. 6. Marder, M.; Estiu, G.; Blanch, L. B.; Viola, H.; Wasowski, C.; Medina, J. H.; Paladini, A. C. (2001) Molecular modeling and QSAR analysis of the interaction of flavone derivatives with the benzodiazepine binding site of the GABAA receptor complex. Bioorg. Med. Chem. 9, 323–335. 7. Hui, K. M.; Wang, X. H.; Xue, H. (2000) Interaction of flavones from the roots of Scutellaria baicalensis with the benzodiazepine site. Planta Med. 66, 91–93.

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Comparative Bioavailability of Synthetic and Tomato-Based

7 Comparative Bioavailability of Synthetic and TomatoBased Lycopene in Humans? Peter P. Hoppe*, Klaus Kraemer*, Henk van den Berg**, Gery Steenge**, Trinette van Vliet**

Abstract The relative bioavailabilities of synthetic and tomato-based lycopene were determined in a single-blind, randomized, placebo-controlled, parallel trial in free living volunteers. Three groups (n 12/group) of healthy, normolipaemic male and female subjects with a mean baseline serum lycopene concentration of 0.36 mmol/L took a daily dose of 15 mg from either synthetic lycopene (LycoVitr 10 %, beadlets, BASF, Germany) or natural lycopene (LycO-Mato, beadlets, LycoRed Natural Products, Israel) or a placebo (without lycopene) together with the main meal. The increase in serum lycopene following 28 days of dosing was used as the parameter of bioavailability. Both lycopene sources resulted in marked and significant increases of serum total lycopene, total cis-lycopene and all-trans-lycopene whereas no such changes were found in the placebo treatment. Serum lycopene concentrations following synthetic and natural lycopene were not significantly different. Neither lycopene source affected the other serum carotenoids, viz. a-carotene, b-carotene, b-cryptoxanthin, zeaxanthin and lutein. We conclude that synthetic and natural lycopene are equivalent sources of lycopene and that there is no interaction with circulating carotenoids.

7.1 Introduction Lycopene is a prominent carotenoid in human blood and tissues, particularly in adrenal glands, lungs, testes, prostate gland, liver, and skin [1]. Epidemiological studies indicate that low intake of tomato products or low plasma lycopene concentration are associated with a higher risk of various cancers including prostate cancer [2]. In these studies, natural sources of lycopene were used including tomatoes and tomato-based products that also contain, inter alia, phytoene, phytofluene, z-carotene, neurosporene, g-carotene and

* BASF Aktiengesellschaft, 67056 Ludwigshafen, Germany ** TNO Nutrition and Food Research, Zeist, The Netherlands

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b-carotene [3]. Hence, it is not known if lycopene accounts for or contributes to the reported health benefits. Lycopene bioavailability from natural sources has been reviewed previously [4]. The sources included fresh tomatoes and tomato paste, juice, or oleoresin, beadlets from tomato oleoresin, and fruit and vegetable concentrate. Tomato juice failed to elevate plasma lycopene whereas heating of tomato juice in oil resulted in a plasma response [5]. A recent pilot study compared the plasma response to synthetic and tomato-based deuterated lycopene in two subjects each. Synthetic lycopene was found to be almost three times more bioavailable than lycopene from cooked and pureed tomatoes [6]. The aim of the present study was to compare the relative bioavailability of lycopene from a synthetic source and from tomato-oleoresin. Both sources were used as beadlet formulations.

7.2 Materials and Methods Lycopene sources used were (LycoVitr 10 %, BASF, Germany) containing synthetic lycopene as gelatine beadlets and Lyc-O-MatoTM Beads 5 % (LycoRed, Natural Products Industries Ltd, Israel), a beadlet preparation based on tomato oleoresin. LycoVit 10 % had an analyzed content of 11.45 % total lycopene (77 % all-trans-, 23 % total-cis-lycopene). Lyc-OMatoTM had an analyzed content of 4.3 % total lycopene (85 % all-transand 15 % total-cis-lycopene). Study substances were given in acid-soluble gelatine capsules that contained either 15 mg total lycopene or placebo. The study design was a singleblind, randomized, placebo-controlled, parallel trial in free living volunteers. Three groups (n 12/group) of healthy, normolipaemic male and female subjects participated. The study was done at TNO Nutrition and Food Research, Zeist, The Netherlands and the study protocol was approved by the TNO Medical Ethics Committee. All volunteers gave their written informed consent for participation in the study. Subjects were instructed to take one capsule per day during the main meal for 28 days, regardless whether this was at lunch or dinner time. Compliance was monitored by handing out a number of capsules exceeding the number to be taken during the study and by counting the capsules remaining at termination of the study. Blood was collected during the pre-study screening, on d 1 and at the end of the study (d 29) from an antecubital vein after an overnight fast. Serum samples were stored at –70 oC. All samples were analyzed for lycopene (total-, cis-, trans-), a-carotene, b-carotene, b-cryptoxanthin, zeaxanthin and lutein using reversed phase HPLC with UV-detection [7]. Phytoene and phytofluene were measured using a non-validated HPLC method.

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Differences in serum parameters between treatments were evaluated using analysis of variance (ANOVA) with gender and treatment as factors. Both values of day 29 and day 29 minus day 1 were used (data for the change are not presented). In case of an overall significant treatment effect, individual groups were compared using students t-test. In all statistical tests performed, the null hypothesis was rejected at the 0.05 level of probability.

7.3 Results All 36 subjects (12 males and 24 females) completed the study. Compliance was very good, only 1 % of the capsules was not consumed, equally distributed over the treatment groups. Intake of synthetic and natural lycopene resulted in marked increases of serum lycopene concentration without significant difference between sources (Tab. 7.1). The changes in serum lycopene (d 29 – d 1) were almost identical for both lycopene sources (0.58 vs. 0.57 mmol/L, LycoVitr 10 % vs Lyc-OMatoTM) and significantly greater than in the placebo group (0.1 mmol/L, p I 0.005). For both lycopene sources, the increase of total lycopene was due to a significant rise of total-cis- and all-trans-lycopene. On d 29, serum all-trans-lycopene concentration was slightly higher for natural than for synthetic lycopene (p I 0.02), however the change (d 29 – d 1) was not significantly different (data not shown). No significant differences between the three treatments were found for a-carotene, b-carotene, b-cryptoxanthin, lutein and zeaxanthin.

7.4 Discussion The objective of this study was to compare the relative bioavailability of a novel synthetic source and a common natural source of lycopene in humans. We aimed at detecting a possible difference between the lycopene sources of 30 %. To this end, normolipaemic subjects with plasma lycopene below 0.4 mmol/L were selected. In order to minimize the confounding effect of dietary fat, the dose was taken together with the main meal. This resulted in a coefficient of variation (CV) for mean serum total lycopene (n 36) of 28 % (pre-screening), 41 % (d 1) and 31 % (d 29), respectively. In epidemiologic studies, the CV is normally i 50 % [8]. In this study the synthetic preparation had the same bioavailability as the natural formulation. Thus, in terms of providing lycopene, both sources are equivalent. A marked increase in serum total lycopene was found in 23 out of 24 subjects. The highest serum concentration after 28 d supplementa305

306 e e e e e e e e 0.04 0.18 0.13 0.18 0.08 0.10 0.03 0.10

0.08 e 0.05 0.15 e 0.08

0.07 0.43 0.32 0.38 0.16 0.22 0.07 0.22

Natural lycopene e e e e e e e e 0.04 0.21 0.11 0.14 0.06 0.09 0.03 0.08

0.08 e 0.05 0.15 e 0.05

0.07 0.32 0.22 0.36 0.16 0.20 0.07 0.21

Placebo

Data are presented as treatment means e SD * Indicative values B P I 0.02 significantly different from synthetic lycopene C P I 0.001 significantly different from placebo

0.06 e 0.03 0.15 e 0.13

Phytoene* Phytofluene*

0.09 0.19 0.17 0.12 0.06 0.07 0.04 0.22

0.10 0.39 0.27 0.33 0.15 0.19 0.08 0.28

e e e e e e e e

Day 1 Synthetic lycopene

a-Carotene b-Carotene b-Cryptoxanthin Total lycopene Total-cis lycopene All-trans lycopene Zeaxanthin Lutein

Treatment e e e e e e e e 0.09 0.22 0.28 0.30C 0.13C 0.17C 0.04 0.20

0.06 e 0.05 0.14 e 0.09

0.09 0.42 0.30 0.91 0.39 0.53 0.09 0.29

Day 29 Synthetic lycopene e e e e e e e e 0.04 0.19 0.10 0.20C 0.07C 0.14BC 0.04 0.09

0.08 e 0.05 0.15 e 0.08

0.07 0.47 0.27 0.95 0.32 0.63 0.07 0.20

Natural lycopene e e e e e e e e

0.04 0.21 0.09 0.18 0.07 0.11 0.03 0.06 0.08 e 0.06 0.12 e 0.06

0.07 0.33 0.18 0.46 0.20 0.26 0.07 0.21

Placebo

Table 7.1. a-Carotene, b-carotene, b-cryptoxanthin, lycopene (total, total-cis, all-trans), zeaxanthin, lutein, phytoene and phytofluene (mmol/L) on day 1 and day 29 per treatment group.

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References tion was 1.42 mmol/L and the highest individual response (d 29 – d 1) was 1.05 mmol/L. This supports earlier reports that plasma concentrations of about 1 mmol/L appear to be the maximum attainable. We have no explanation for the single case of non-responder as his reported compliance was 100 % and none of the measured health parameters was outside the range. However, a low or no plasma response was also reported previously [9]. The proportion of trans- and cis-isomers in plasma is normally around 50 % each [1, 10, 11]. This was confirmed by the present study. A total of 7 geometrical isomers of lycopene were detected in human serum and tissues including 5-cis, 9-cis, 13-cis and 15-cis lycopene [11].

References 1. Clinton, S. K. (1998) Lycopene: Chemistry, biology, and implications for human health and disease. Nutr. Rev. 56, 35–51. 2. Giovannucci E. (1999) Tomatoes, tomato-based products, lycopene, and cancer: review of the epidemiologic literature. J. Natl. Cancer Inst. 91, 317–331. 3. Beecher, G. R. (1998) Nutrient content of tomatoes and tomato products. Proc. Soc. Exp. Biol. Med. 218, 98–100. 4. Sies, H.; Stahl, W. (1998) Lycopene: antioxidant and biological effects and its bioavailability in the human. Proc. Soc. Exp. Biol. Med. 218, 121–124. 5. Stahl, W.; Sies, H. (1992) Uptake of lycopene and its geometrical isomers is greater from heat-processed than from unprocessed tomato juice in humans. J. Nutr; 122, 2161–2166. 6. Ferreira, A. L. A.; Tang, G.; Grusak, M. A. et al. (2001) Absorption and metabolism of synthetic and natural (tomato) deuterated lycopene. International Symposium on the Role of Tomato Products and Carotenoids in Disease Prevention, New York April 19, 2001 [Abstract]. 7. Broekmans, W. M.R; Berendschot, T. T. J. M.; Klöpping-Ketelaars, I. A. A.; de Vries, A. J.; Goldbohm, R. A.; Tijburg, L. B. M.; Kardinaal A. F. M.; van Poppel, G. A. F. C. Macular pigment density in relation to serum and adipose tissue concentrations of lutein and serum concentrations of zeaxanthin. Am. J. Clin. Nutr. 76, 595–603. 8. Bowen, P. E.; Garg, V.; Stacewicz-Sapuntzakis, M.; Yelton, L.; Schreiner, R. S. (1993) Variability of serum carotenoids in response to controlled diets containing six servings of fruits and vegetables per day. Ann. NY Acad. Sci. 691, 241–243. 9. Micozzi, M. S.; Brown, E. D.; Edwards, B. K.; Bieri, J. G.; Taylor, P. R.; Khachik, F.; Beecher, G. R.; Smith, J. C. Jr. (1992) Plasma carotenoid response to chronic intake of selected foods and b-carotene supplements in men. Am. J. Clin. Nutr. 55, 1120– 1125. 10. Clinton, S. K.; Emenhiser, C.; Schwartz, S. J.; Bostwick, D. G.; Williams, A. W.; Moore, B. J.; Erdman, J. W. Jr. (1996) cis-trans-Lycopene isomers, carotenoids and retinol in the human prostate. Cancer Epidemiol. Biomarkers Prev. 5, 823–833. 11. Schierle, J.; Bretzel, W.; Bühler, I. (1997) Content and isomeric ratio of lycopene in food and human blood plasma. Food Chem. 59, 459–465.

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8 The Mycotoxin Ochratoxin A Induced DNA Damage in MDCK Cells and Primary Cultured Porcine Urinary Bladder Epithelial Celles (PUBEC) in vitro S. Lebrun*, H. Schulze**, and W. Föllmann*

8.1 Introduction Ochratoxin A (OTA) is a frequently found toxin produced by several mould fungi of the Aspergillus and Penecillium genera, under insufficient storing conditions of cereals. It was found in animal feeds and human foods and also in the blood of animals and humans after consumption of contaminated food all over the world. OTA accumulates in the kidney, liver and blood and it is possibly implicated in the aetiology of Balkan endemic nephropathy and in Danish porcine nephropathy. In laboratory animals OTA was cancerogenic, nephrotoxic and immunosuppressive. The mechanism which results in DNA-damage has not been resolved. It is still an open question whether metabolism of OTA is required to form reactive metabolites or if the accumulation of OTA modulates adverse secondary effects in target tissues such as kidney and urothelium. In this study the induction of DNA damage by OTA, the subsequent DNA repair, and the effect of inhibited OTA uptake by substrates of the hepatic organic anion transporter was investigated with the alkaline single cell gel electrophoresis (Comet) assay. The assays were performed with Mardin Darby canine kidney (MDCK) cell line and primary cultured porcine urinary bladder epithelial cells (PUBEC), two different model systems of potential target organs for OTA.

* Institute of Occupational Physiology, University of Dortmund, Dortmund, Germany ** Department of Urology, Städtische Kliniken Dortmund, Dortmund, Germany

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8.2 Materials and Methods 8.2.1

Isolation of Porcine Urinary Bladder Epithelial Cells (PUBEC)

The preparation procedure has been described in detail elsewhere [1]. Briefly, bladders were opened, the surface was washed with PBS buffer and the epithelial mucosa was carefully scraped from the underlying muscle layer with a sterile glass slide. The isolated cells were washed twice with culture medium (see below) by centrifugation at 50 g for 5 min. The cell number was determined and viability was judged by trypan blue exclusion. Viability ranged between 70–90 % and only cells from preparations with a viability of i 75 % were used for further experiments.

8.2.2

Cell Culture

1 q 105 PUBECs in q 3 ml culture medium (Ham’s F12 medium (146 mg/l glutamine) supplemented with 100 U/ml penicilin, 100 mg/ml streptomycin, 1.25 mg/ml amphotericin, 5 mg/ml human transferrin, 10 mg/ml bovine insulin, 2.7 mg/ml glucose, and 1 mg/ml hydrocortisone) or 7 q 104 MDCK cells in 3 ml culture medium (DMEM/ Ham’s F12 with Glutamax, 1:1, supplemented with 10 % fetal calf serum) per well were seeded in 6-well plastic culture dishes, and cultured in a humidified incubator (37 hC, 5 % CO2). Culture medium was changed after 24 hours to remove unattached cells and the incubation assay was performed on day three after seeding.

8.2.3

Incubation Procedure

After 3 days the culture medium was removed and replaced by fresh serumfree medium supplemented with the test compounds. Control cells were incubated only with the solvent (DMSO, final concentration I 0.3 %). DNA repair was inhibited with 10 nM hydroxyurea (HU) and 1.8 mM cytosine arabinoside (araC) according to Martin et al. [3]. After incubation the cells were washed with PBS buffer and detached from the substrate with trypsin/EDTA solution (0.05/0.02 %) to produce a single-cell suspension. The suspension was deluted with culture medium and centrifuged (50 g, 5 min). The pelleted cells were resuspended in medium and cell density was adjusted to contain 8 q l06 cells per ml medium. Then 25 ml containing 2 q 105 cells were added to 75 ml low melting point agarose (0.75 % in PBS).

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Alkaline Single Cell Gel Electrophoresis Assay (Comet Assay)

Two independent experiments were performed according to Singh et al. [6] and Tice et al. [7]. Frosted microscope slides were dipped into normal melting point agarose (1.5 % in Ca2 – and Mg2 – free PBS) at 60 hC. The bottom side of the slide was wiped off and the covered slide was dried at room temperature. The 100 ml cell suspension in low melting point agarose were transferred on the agarose-covered slide, covered with a glass coverslip and allowed to solidify on ice. The coverslips were removed and the slides were then placed in cold (4 hC) lysis buffer overnight [2.23 M NaCl, 0.09 M Na2EDTA, 0.25 M NaOH, pH 10. 1 % Na-laurylsarcosine, supplemented freshly with DMSO (final concentration 10 %) and Triton X-100 (final concentration 1 %)]. After lysis the slides were placed in a horizontal gel electrophoresis unit filled with fresh, chilled electrophoresis buffer (300 mM NaOH and 1 mM Na2EDTA in distilled water, pH 13.5) up to a level of about 2.5 mm above the slides and left for 30 min to allow the DNA to unwind completely. Electrophoresis was conducted for 30 min at 25 V and 300 mA. Slides were then transferred in neutralization buffer (0.4 M Tris in distilled water, pH 7.5) for 15 min, to remove alkali and detergents. The slides were then dehydrated in a series of ethyl alcohol (50 %, 75 % and 100 %) for 5 min each and then stained with 50 ml ethidium bromide (concentration: 20 mg/ml) and covered with a coverslip for immediate analysis.

8.2.5

Comet Analysis

A total of 50 cells from two slides of each concentration were analysed by image analysis using Image pro 4.0 software (Media cybernetics, Germany). Observations were made at 400 q magnification using an epifluorescent microscope DMRB (Leica, Germany) equipped with an excitation filter of 515–535 nm, a 100 W mercury lamp and a barrier filter at 590 nm and a videocamera (XC 300 P, Donpisha). Tail length (maximum tail minus half of maximum head) was the parameter used in this study. In the comet assay the tail length values were not normally distributed and violate the requirements for analysis by parametric statistics. To resolve this problem the median as the statistic parameter was used and the first and third quartile are shown. For the comparison of different incubation conditions the Mann-Whitney-U-Test using the statistic program SPSS Version 9.0 was applied for statistical analysis. Significance was given with p I 0.001.

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8.3 Results In MDCK cells OTA induced DNA damage in a concentration dependent manner with a maximum at 100 mM, whereas PUBEC cells showed only weak effects (Fig. 8.1). Higher OTA concentrations were cytotoxic. An incubation period of 3 hours was most effective for the induction of the adverse effects (here performed with 100 mM OTA) (Fig. 8.2). If DNA repair was inhibited by addition of cytosine arabinosid (araC) and hydroxyurea (HU), the tail length increased dramatically in both cell types (Fig. 8.3). A futher culture of these damaged cells in the absence of any supplement resulted in a complete repair of the DNA within three hours (results not shown). When a co-incubation with methotrexate, a substrate of the organic anion transporter in the liver [2], was performed the adverse effect of 100 mM OTA can be completely inhibited in MDCK cells (Fig. 8.4). In contrast, methotrexate itself induced DNA damage in PUBECs and the effect of OTA decreased only slightly. When the cells were co-incubated with the bile acid glucocholic acid, which did not induce DNA damage by itself in both cell types, a concentration dependent inhibition of the adverse effects of OTA occurred (Fig. 8.5).

80

PUBEC

MDCK

median of tail length in m

70

60

50

40

30

20

10

0 0,001

0,01

0,1

1

10

100

1000

OTA concentration [ M]

Figure 8.1. Concentration dependency of induction of DNA damage by OTA in MDCK and PUBEC cells. Median of tail length is given as the parameter for DNA damage and the bars represent the first and third quartile of the median.

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80

PUBEC

MDCK

70

median of tail length in m

60

50

40

30

20

10

0 0

50

100

150

200

250

300

350

400

incubation time [min]

Figure 8.2. Time course of induction of DNA damage by OTA in MDCK and PUBEC cells evaluated with the comet assay. Median of tail length is given as the parameter for DNA damage and the bars represent the first and third quartile of the median.

160

PUBEC

MDCK

median of tail length in m

140

120

100

80

60

40

20

0 0

50

100

150

200

250

300

350

400

incubation time [min]

Figure 8.3. Time course of induction of DNA damage by OTA in MDCK cells and PUBECs evaluated with the comet assay. Median of tail length is given as the parameter for DNA damage and bars represent the first and third quartile of the median. Cells were exposed to OTA supplemented with araC/HU.

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The Mycotoxin Ochratoxin A Induced DNA Damage

100

PUBEC

90

MDCK

median of tail length in m

80 70 60 50 40 30 20 10 0

Control

Control

Control

100 M

0,1 M MT

1 M MT

10 M MT

100 M MT

araC/HU

araC/HU

OTA

100 M

100 M

100 M

100 M

100 M MT

araC/HU

OTA

OTA

OTA

OTA

araC/HU

araC/HU

araC/HU

araC/HU

Figure 8.4. Reduction of the adverse effect of OTA in MDCK cells by co-incubation with methotrexate (MT). Cells 3 hours exposed to 100 mM OTA were compared with cells additionally exposed to methotrexate. Median of tail length is given as the parameter for DNA damage and the bars represent the first and third quartile of the median.

100

PUBEC

90

MDCK

Median of Tail length in m

80 70 60 50 40 30 20 10 0

Control

Control

Control

100 M

0,1 M GC

1 M GC

10 M GC

100 M GC

araC/HU

araC/HU

OTA

100 M

100 M

100 M

100 M

100 M GC

araC/HU

OTA

OTA

OTA

OTA

araC/HU

araC/HU

araC/HU

araC/HU

Figure 8.5. Reduction of the adverse effect of OTA in MDCK cells and PUBECs by co-incubation with glycocholic acid (GC). Cells 3 hours exposed to 100 mM OTA were compared with cells additionally exposed to GC. Median of tail length is given as the parameter for DNA damage and the bars represent the first and third quartile of the median.

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8.4 Conclusions The results demonstrated that OTA induced DNA damage in vitro. In the primary cultured epithelial cells (PUBECs) the damaged DNA can be repaired more effectively compared to cells of the cell line (MDCK cells). That DNA damage appeared also in PUBECs was demonstrated in the experiments where DNA repair was inhibited by araC and hydroxyurea and a dramatic increase in DNA damage was detected. This process was completely reversible shown by a further culture of the treated cells in OTA- and inhibitor-free medium (results not shown). From these results, it can be deduced that OTA may create strong adverse effects under specific conditions. It can be speculated that if the mechanism of DNA repair was impaired, e. g., during cell proliferation or exhausted repair capacity resulting from a high load of other toxic factors, the effect of OTA on DNA is much stronger and can persist more likely than in cells with intact and effective DNA repair systems. This may point to the involvement of further secondary mechanisms which can enhance the genotoxic effect of OTA. On the other hand a chronic exposure to OTA and/or its metabolites may possibly affect DNA repair mechanisms and would therefore be responsible for an enhanced adverse effect of OTA in the exposed tissues. Additionally, it was shown that a co-incubation of the cells with substrates of the hepatic organic anion transporter lead to a decreased DNA damage when these substrates did not induce DNA damage by itself. This demonstrated that by a competitive inhibition of OTA uptake the adverse OTA effect can be completely inhibited. This pointed to an involvement of such kind of transporters in the uptake of OTA and that uptake processes in the target organs play an important role in the toxicity of OTA. This was explained comprehensively by Petzinger and Ziegler [4] and also discussed by Schwerdt et al. [5].

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Genotoxic Potential of the Phytoestrogen Resveratrol

References 1. Guhe C, Degen GH, Schuhmacher U, Kiefer F, Föllmann W (1996) Drug metabolizing activities in porcine urinary bladder epithelial cell cultures (PUBEC). Arch. Toxicol. 70, 599–606. 2. Kontaxi M, Eckhardt B, Hagenbuch B, Stieger B, Meier PJ, and Petzinger, E (1996) Uptake of the mycotoxin ochratoxin A in liver cells occurs via the cloned organic anion transporting polypeptide. J. Pharmacol. Experim. Ther. 279, 1507–1513. 3. Martin FL, Cole KJ, Orme MH, Grover PL, Phillips DH, and Venitt S (1999) The DNA repair inhibitors hydroxyurea and cytosine arabinoside enhance the sensitivity of the alkaline single-cell gel electrophoresis (‘comet’) assay in metabolically-competent MCL-5 cells. Mutat. Res. 445, 21–43. 4. Petzinger E and Ziegler K (2000) Ochratoxin A from a toxicological perspective. J. Vet. Pharmacol. Ther. 23, 91–98. 5. Schwerdt G, Freudinger R, Silbernagl S, and Gekle M (1998) Apical uptake of radiolabelled ochratoxin A into Madin-Darby canine kidney cells. Toxicology 131, 193–202. 6. Singh NP, McCoy MT, Tice RR, and Schneider EL (1988) A simple technique for quantification of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191. 7. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, and Sasaki YS (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206–221.

9 Genotoxic Potential of the Phytoestrogen Resveratrol in Cultured V79 Chinese Hamster Fibroblasts Leane Lehmann*, Erika Pfeiffer, and Manfred Metzler

9.1 Introduction Resveratrol (Fig. 9.1) is a natural phytoalexin present in red wines and various human food items. Recently, resveratrol has been shown to exhibit estrogenic activity [1–4].The genotoxic potential of estrogenic agents is of interest because some estrogens are associated with cancer in humans and experimental animals [5, 6]. In the present study, the genotoxicity of resveratrol was assessed at five in vitro endpoints, i. e. (1) the induction of micronuclei

* University of Karlsruhe, Institute of Food Chemistry and Toxicology, 76128 Karlsruhe, Germany

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Figure 9.1. Chemical structure of resveratrol.

(MN), (2) the relative DNA content of nuclei, (3) the mitotic index, (4) the cell-free assembly of microtubules, and (5) the structure of the mitotic spindle and the cytoplasmic microtubule complex (CMTC).

9.2 Methods Using a single test protocol, the mitotic spindle and the CMTC were visualized by immunochemical staining of a-tubulin, and the induction of MN was assessed using immunochemical staining of kinetochore proteins to distinguish chromosome fragments and whole chromosomes [7]. Intercellular bridges exhibiting intense immunochemical staining of a-tubulin [8] indicated freshly divided cells. The relative DNA content per nucleus was determined after staining of nuclei with DAPI using a fluorescence-based cell counter. Fluorescence intensities were proportional to relative DNA content; thus, approximate proportions of cells in G1 phase could be calculated by integration of the gaussian fit of the G1 peak.

9.3 Results When V79 cells were treated with 100 mM resveratrol for 6 h and the numbers of MN and cell divisions were determined (Fig. 9.2) at various times thereafter, it was observed that resveratrol gave rise to a surge of freshly divided cells which peaked 11 h and 23 h after removing the compound from the medium, indicating a cell cycle arrest in G1 and/or S phase as long as cells were exposed to resveratrol. This is supported by comparison of the highresolution DNA histograms of cells treated with 100 mM resveratrol for 6 h and of untreated control cells (data not shown). Cell cycle was not delayed as indicated by the normal time span of 10 h to 11 h between the first and second accumulation of cell divisions after resveratrol treatment. The first 316

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surge of freshly divided cells was followed by a marked increase in the number of MN. The vast majority of MN did not exhibit kinetochore signals, indicating chromosome fragments, whereas the proportion of kinetochore-positive MN was within the range of untreated cells. Virtually no cells exhibiting apoptotic bodies and few karyorrhectic cells were observed until the second surge of freshly divided cells, in which karyorrhexis occurred more frequently. When the same experiment was carried out with 50 mM resveratrol, the surge of cell divisions peaked at postincubation time 8 h, 18 h, and about 28 h (Fig. 9.2). The number of micronucleated cells (MNC) were markedly increased from 8 h on with a MN rate about two third of that observed with 100 mM resveratrol. Resveratrol did not affect microtubules under cell-free conditions nor in cultured V79 fibroblasts (data not shown). However, cells with more than 2 spindle poles in all stages of mitosis were observed more frequently than in control cells. In addition, cells with additional nuclei which were too big to be classified as MN occurred within the recovery phase after resveratrol treatment. According to their morphological appearance, these cells were classified as polynucleated or karyorrhectic cells.

Figure 9.2. Time dependence of resveratrol-induced formation of micronuclei (MN) in cultured V79 fibroblasts. Cells were treated with 50 mM resveratrol (below) or 100 mM resveratrol (above) for 6 h, and subsequently incubated with fresh medium for various time periods up to 28 h.

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9.4 Conclusion V79 cells exposed to resveratrol for 6 h accumulate in G1/S phase of the cell cycle. After removal of the compound, they are released from the cell cycle arrest and a surge of freshly divided cells can be observed after 8 h (50 mM) or 11 h (100 mM). The synchronisation of the cells lasts for at least 3 (50 mM) or 2 (100 mM) periods of cell cycle, which are not prolonged. The surge of freshly divided cells is accompanied by kinetochore-negative MN, indicating a clastogenic potential of resveratrol which is compatible with the induction of the cell cycle arrest in G1 and/or S phase. In addition, resveratrol did not exhibit the potential to disturb the assembly of microtubules; however, it may affect the regulation of centrosome duplication. Further studies are needed to characterize the genotoxic potential of resveratrol.

References 1. Bhat, K. P.; Lantvit, D.; Christov, K.; Mehta, R. G.; Moon, R. C.; Pezzuto, J. M. (2001) Estrogenic and antiestrogenic properties of resveratrol in mammary tumor models. Cancer Res. 61, 7456–63. 2. Bhat, K. P.; Pezzuto, J. M. (2001) Resveratrol exhibits cytostatic and antiestrogenic properties with human endometrial adenocarcinoma (Ishikawa) cells. Cancer Res. 61, 6137–44. 3. Bowers, J. L.; Tyulmenkov, V. V.; Jernigan, S. C.; Klinge, C. M. (2000) Resveratrol acts as a mixed agonist/antagonist for estrogen receptors alpha and beta. Endocrinology 141, 3657–67. 4. Freyberger, A.; Hartmann, E.; Hildebrand, H.; Krotlinger, F. (2001) Differential response of immature rat uterine tissue to ethinylestradiol and the red wine constituent resveratrol. Arch. Toxicol. 74, 709–15. 5. Liehr, J. G. (2000) Is estradiol a genotoxic mutagenic carcinogen? Endocr. Rev. 21, 40–54. 6. Li, J. J.; Li, S. A.; Oberley, T. D.; Parsons, J. A. (1995) Carcinogenic activities of various steroidal and nonsteroidal estrogens in the hamster kidney: relation to hormonal activity and cell proliferation. Cancer Res. 55, 4347–51. 7. Pfeiffer, E.; Rosenberg, B.; Deuschel, S.; Metzler, M. (1997) Interference with microtubules and induction of micronuclei in vitro by various bisphenols. Mutat. Res. 390, 21–31. 8. Schultz, N.; Onfelt, A. (1994) Video time-lapse study of mitosis in binucleate V79 cells: chromosome segregation and cleavage. Mutagenesis 9, 117–23.

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Investigation of Oxidative Stress in H4IIE Cells

10 Investigation of Oxidative Stress in H4IIE Cells: Modulation by the Flavonoid Kaempferol P. Niering*, W. Wätjen, S. Ohler, I. Köhler, Y. Chovolou, A. Kampkötter, Q.-H. Tran-Thi, and R. Kahl

10.1 Introduction Oxidative stress is associated with diseases such as cancer, atherosclerosis and neurodegenerative processes. Compounds with antioxidative properties are of interest because of their protective potential against cellular consequences of oxidative damage. In this study we investigated the influence of the polyphenolic compound kaempferol (Fig. 10.1) on oxidative stress in rat H4IIE hepatoma cells. Kaempferol belongs to the substance class of flavonoids, which occur in abundance throughout the plant kingdom and therefore in a wide variety of human food and herbal medicines. Flavonoids are discussed for their broad spectrum of biochemical and pharmacological activities, e. g. antioxidative and radical scavenging abilities. A protective function against various stages of the cancer process was observed in epidemiological studies. On the other hand it was shown that some flavonoids are able to cause oxidative damage by themselves [1]. For the evaluation of a possible preventive use of these compounds, it is essential to examine whether both anti- and prooxidative actions occur in the same dose range. We investigated the influence of kaempferol on H2O2-induced oxidation processes, DNA strand breaks and apoptosis and the intrinsic effects of kaempferol on cellular integrity over a broad concentration range.

OH HO

O OH OH

O

Figure 10.1. Formula of kaempferol.

* Institute of Toxicology, Heinrich-Heine University, P. O. Box 101007, 40001 Düsseldorf, Germany

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10.2 Results 10.2.1

Cytotoxicity

The incubation of H4IIE cells with kaempferol resulted in a reduction of cell viability as measured by the neutral red accumulation assay [2]. The IC50 value for this process was 75 mM e 5 mM (24 h). The reduction in cell viability observed upon incubation of H4IIE cells with increasing concentrations of H2O2 could be reduced by preincubation for 0.5 h with a kaempferol concentration as low as 5 mM (data not shown). The gel electrophoresis of isolated DNA fragments from kaempferoltreated cells showed DNA ladder formation as a measure of oligonucleosomal DNA fragmentation due to apoptotic cell death at kaempferol concentrations from 50 mM up to 200 mM. At 350 mM, kaempferol led to a DNA smear due to necrotic cell death (Fig. 10.2).

Figure 10.2. DNA ladder formation by kaempferol. H4IIE rat hepatoma cells were grown in Dulbeccols modified Eaglels medium supplemented with 10 % fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin at 37 hC in a humidified atmosphere (5 % CO2). H4IIE cells were incubated with increasing concentrations of kaempferol for 24 h (0, 10, 50, 100, 200, 350 mM). After lysis of the treated cells, DNA was isolated and analyzed electrophoretically for oligonucleosomal DNA fragments (n i 3).

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Investigation of Oxidative Stress in H4IIE Cells

Lipid Peroxidation

Treatment of H4IIE cells with kaempferol resulted in the increased formation of malondialdehyde (MDA), a product of lipid peroxidation. At 500 mM kaempferol MDA formation increased by a factor of about 2 (Fig. 10.3). This result verifies an oxidative mode of action in kaempferol-mediated cell damage at higher concentrations. H2O2 leads to lipid peroxidation in cellular membranes. After the incubation of H4IIE cells with 500 mM H2O2 we detected a twofold increase of malondialdehyde production. A pre-incubation of the cells for 1 h with 50 mM kaempferol led to a decrease of MDA formation almost to the level of control cells (Fig. 10.4). This result demonstrates a suppression of cellular lipid peroxidation by kaempferol.

200

MDA (% of control)

175

150

125

100

75

50

0

100

200

300

400

500

Figure 10.3. MDA formation by kaempferol. H4IIE cells were incubated with increasing concentrations of kaempferol for 2 h. MDA concentration in the medium was measured by reaction with thiobarbituric acid followed by separation by HPLC [3, 4]. MDA formation is expressed as percentage of control value e SEM (n 3).

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1 0,9

pmol MDA/106 cells

0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 DMSO Kaempferol Figure 10.4. Inhibition of H2O2-induced MDA formation by kaempferol. H4IIE cells were incubated for 2 h with 500 mM H2O2 in the presence or absence of 50 mM kaempferol. MDA concentration in the medium was measured by reaction with thiobarbituric acid followed by separation by HPLC. MDA formation is expressed as percentage of control value e SEM (n 3).

10.2.3 Intracellular Oxidation Capacity The fluorescence of DCF resulting from oxidation of 2l,7l-dichlorodihydrofluorescein diacetate (H2DCF-DA) in untreated cells reflects endogenous oxidative stress. Incubation of cells with 50 mM kaempferol for 1 h diminished the DCF fluorescence by half, compared to control cells. After 1 h, 1 mM H2O2 was added in order to induce exogenous oxidative stress. In cells pretreated with 50 mM kaempferol the H2O2-induced DCF fluorescence was reduced by a factor of 1.5 (Fig. 10.5). These measurements show that oxidation by endogenous as well as exogenous reactive oxygen species (ROS) could be reduced by kaempferol in a time- and dose-dependent manner.

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relative fluorescent units

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Investigation of Oxidative Stress in H4IIE Cells

30000 25000 control

20000 15000 10000 5000 0 0

50

100

150

200

time (min.)

Figure 10.5. Effect of kaempferol on intracellular oxidation capacity. H4IIE cells were incubated for 1 h with increasing concentrations of kaempferol. After the addition of the fluorescence probe H2DCF/DA (50 mM, 0.5 h), fluorescence was followed for 1 h. H2O2 (1 mM) was then added and incubation was continued. H2DCF is not fluorescent until oxidized to DCF [5]. Increase of DCF fluorescence was measured using a Wallac Victor2 1420 fluorescence reader. Values are means e SEM (n 3)

10.2.4 H2O2-Mediated DNA Strand Breaks H2O2 produces a time- and dose-dependent enhancement of DNA strand breaks in H4IIE cells. The comet assay (single cell gel electrophoresis) showed a linear increase in average image length of the H2O2-treated cells from 13 mm (control) to 55 mm (500 mM H2O2). To investigate protective effects of kaempferol, the cells were pre-incubated with 50 mM kaempferol for 1 h followed by a treatment with 500 mM H2O2 for 2 h. In these experiments the group of kaempferol-incubated cells showed a slight reduction in comet formation (Fig. 10.6).

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70

image length (μm)

60 50 40 30 20 10 0 Figure 10.6. Protective effect of kaempferol on H2O2-mediated DNA strand breaks. H4IIE cells were pre-incubated with 50 mM kaempferol for 1 h followed by an incubation with 500 mM H2O2 for 24 h. DNA strand breaks were analyzed by single cell gel electrophoresis [6]. The average image length of 50 cells in mm representing the extent of H2O2-mediated DNA strand breaks is shown. Data are means e SEM (n 3).

10.3 Conclusion Application of H2O2 leads to oxidative damage in H4IIE cells and induces apoptotic cell death. Due to its antioxidative properties the flavonoid kaempferol at concentration of 5–50 mM shows a protective effect on H4IIE cells exposed to H2O2 as measured by a decrease in DCF fluorescence, malondialdehyde formation and DNA strand breaks. Kaempferol in higher concentrations exerts contrasting effects, causing apoptotic cell death with an IC50 for cytotoxicity of 75 mM and increasing malondialdehyde formation at concentrations exceeding 50 mM. We conclude that in H4IIE cells kaempferol exerts protective actions at low concentrations and adverse actions at high concentrations due to its dose-dependent ability to suppress or create oxidative stress.

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References 1. Sergediene, E.; Jonsson, K.; Szymusiak, H.; Tyrakowski, B.; Rietjens, IM.; Cenas, N. (1999) Prooxidant toxicity of polyphenolic antioxidants to HL-60 cells: description of quantitative structure-activity relationsships. FEBS Lett. 462, 392–396. 2. Borenfreund, E.; Puerner, J. A. (1985) Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24, 119–124. 3. Draper, H. H.; Sqires, E. J.; Mahmoodi, H.; Wu, J.; Agarwal, S.; Hadley, M. (1993) A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials. Free Rad. Biol. Med. 15, 353–363. 4. Chirico, S.; Smith, C.; Marchant, C.; Mitchinson, M. J.; Halliwell, B. (1993) Lipid peroxidation in hyperlipidaemic patients. A study of plasma using a HPLC-based thioarbituric acid test. Free Rad. Res. Comm. 19, 51–57. 5. Oyama, Y.; Hayashi, A.; Ueha, T.; Maekawa, K. (1994) Characterization of 2l,7ldichlorofluorescin fluorescence in dissociated mammalian brain neurons: estimation on intracellular content of hydrogen peroxide. Brain Res. 635, 113–117. 6. Singh, N. P.; Mc Coy, M. T.; Tice, R. R.; Schneider, E. L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191.

11 In Vitro Studies on the Estrogenic Activity and the Metabolism of Curcumin Erika Pfeiffer*, Harald L. Esch*, Simone Höhle*, Aniko M. Solyom**, Barbara N. Timmermann** and Manfred Metzler*

11.1 Introduction The powdered dry rhizome of the plant Curcuma longa, commonly called turmeric, is widely used as a coloring agent and spice in many food items. In several Asian countries, it has also been used for centuries as a traditional remedy for the treatment of inflammation and other diseases [1]. The yellow pigment of turmeric, which is composed of curcumin (CUR), mono-demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC) has been reported to possess anti-oxidative, anti-inflammatory and anti-carcinogenic proper-

* Institute of Food Chemistry and Toxicology, University of Karlsruhe, Karlsruhe, Germany ** Arizona Center for Phytomedicine Research, University of Arizona, Tucson, AZ, USA

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O

OH

R1

R2

HO

OH

cCUR

CUR F4

CUR

R1 = OCH3 ; R 2 = OCH3

71.5 %

38.7 %

DMC

R1 = OCH3 ; R 2 = H

19.4 %

25.7 %

BDMC

R1 = H ; R 2 = H

9.1 %

35.6 %

Figure 11.1. Chemical structures and composition of the curcuminoids.

ties [2]. Since these curcuminoids have a diphenolic structure similar to that of known phytoestrogens, we have studied the estrogenic activity of commercial curcumin (cCUR) and a fraction of turmeric extract (F4; Fig. 11.1) in Ishikawa cells, a cultured human endometrial adenocarcinoma cell line. Alkaline phosphatase (AP) activity in these cells is markedly stimulated by estrogens, and this enzyme can be easily quantified using p-nitrophenylphosphate as substrate [3]. Moreover, we have investigated the metabolism of curcumin in microsomes and precision-cut tissue slices (PCTS) from the liver of male Sprague Dawley rats.

11.2 Methods The assay for AP induction and cytotoxicity was carried out in 96-well-plates. 2 q 104 cells per well were plated and cultured in 200 mL medium (DMEM/ F12) containing 5 % CD-FCS (steroid-free fetal calf serum) at 37 hC for 24 h. The medium was then removed, replaced by medium containing the test compounds, and further incubated for 48 h. For measuring AP induction the medium was removed, the plates were placed on ice and 50 mL/well of an alkaline buffer containing 5 mM 4-nitrophenylphosphate was added. The production of 4-nitrophenol (4-NP) was monitored periodically at 405 nm. For the cytotoxicity assay 20 mL per well of a methylthiazoletetrazolium (MTT; 5 mg/mL) solution was added and incubated for 3 h. After removing 326

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the medium, isopropanol was added and the reduction of MTT was monitored at 570 nm. For the metabolism study in microsomes the curcuminoids were incubated in 0.1 M phosphate buffer pH 7.4 containing microsomal protein and the co-substrate NADPH or UDP glucuronic acid for 40 min. The PCTS were incubated with the curcuminoids for 24 h in culture medium [4]. The incubation mixtures were then extracted with ethylacetate prior to and after enzymatic cleavage of conjugated metabolites, and the extracts were analyzed by HPLC.

pmol 4-NP / min / well

A

Viable cells (%)

800

B

1nM

100

600 75

cCUR 400

F4

50

cCUR

200

25

F4 0

0 0

5

10

15

control

20

E2

0

5

pmol 4-NP / min / well 800

10

Concentration ( μg/mL)

Concentration (μg/mL) pmol 4-NP / min

C

1nM

D

800

cCUR plus 1 nM E2 600

F4 plus 1 nM E2

600

cCUR 400

400

200

200

0

F4

0 control

E2

0

5

Concentration (μg/mL)

10

0

5

10

Concentration (μg/mL)

Figure 11.2. Cytotoxicity of cCUR and turmeric fraction 4 (A) and effect of curcuminoids on AP activity in Ishikawa cells in the absence (B) and presence (C) of E2, as well as in incubations of human placental AP (D).

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11.3 Estrogenicity Studies In Ishikawa cells the number of viable cells decreased markedly at concentrations higher than 5 mg/mL of the curcuminoids (Fig. 11.2A). No enhanced AP activity was measured with the two samples (Fig. 11.2B). However, the curcuminoids markedly inhibited the AP activity stimulated with 1 nM E2 (Fig. 11.2C). This inhibition is not due to a direct effect on AP, since human placental AP was not affected by incubation with curcuminoids (Fig. 11.2D).

11.4 Metabolism Studies In microsomes fortified with NADPH, most of the curcumin disappeared but only small amounts of reductive metabolites and degradation products were found. In microsomes fortified with UDP glucuronic acid, the major metabolite formed was an extractable and chemically unstable glucuronide. In PCTS, no hydroxylated curcumin but reductive metabolites of curcumin were observed. The major one, identified as hexahydro-curcumin according to GC/MS, was conjugated to a large extent.

11.5 Conclusion Our studies show that curcumin is devoid of estrogenic activity in Ishikawa cells, but may inhibit the estrogenic effect of estradiol. In the rat liver, curcumin is biotransformed to reductive and conjugated metabolites. The glucuronide of curcumin is lipophilic and unstable, whereas the conjugates of the reductive metabolites are hydrophilic and stable.

Acknowledgement Supported by NIH Grant P50 AT00474 to the Arizona Center for Phytomedicine Research.

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Structural Studies of Sphingolipids

References 1. Ammon, H. P.; Wahl, M. A. (1991) Pharmacology of Curcuma longa. Planta Med. 57, 1–7. 2. Azuine, M. A.; Kayal, J. J.; Bhide, S. V. (1992) Protective role of aqueous turmeric extract against mutagenicity of direct-acting carcinogens as well as benzo [a] pyrene-induced genotoxicity and carcinogenicity. J. Cancer Res. Clin. Oncol. 118, 447–452. 3. Littlefield, B. A.; Gurpide, E.; Markiewicz, L.; McKinley, B.; Hochberg, R. B. (1990) A simple and sensitive microtiter plate estrogen bioassay based on stimulation of alkaline phosphatase in Ishikawa cells: estrogenic action of delta 5 adrenal steroids. Endocrinology 127, 2757–2762. 4. Fisher, R.; McCarthy, S.; Sipes, I. G.; Hanzlik, R. P.; Brendel, K. (1991) Metabolism of dichlorobenzenes in organ cultured liver slices. Adv. Exp. Med. Biol. 283, 717–723.

12 Structural Studies of Sphingolipids, a Class of Chemopreventive Compounds in Food W. Seefelder, N. Bartke, T. Gronauer, S. Fischer, H.-U. Humpf*

12.1 Introduction Sphingolipids are the most structurally diverse, as well as complex, class of lipids, which are found in all cellular membranes, lipoproteins and other lipid-rich sources. D-erythro-sphingosine is the prevalent sphingoid base of most mammalian sphingolipids, but also other sphingoid bases are known. The amino group of the sphingoid base is usually substituted with a longchain fatty acid to produce “ceramides”. More complex sphingolipids have polar headgroups at position 1, as shown by a few examples in Fig. 12.1. Sphingolipids are highly bioactive compounds and they are used by cells to regulate cell growth, differentiation, apoptosis and other cellular functions [1]. Studies with experimental animals clearly show that feeding sphingolipids inhibits colon carcinogenesis, reduces serum LDL cholesterol and elevates HDL, suggesting that sphingolipids represent “functional” constituents of food [2]. However, due to the difficult analysis of sphingolipids,

* Institut für Lebensmittelchemie, Westfälische Wilhelms-Universität Münster, Corrensstraße 45, D-48149 Münster, Germany, e-Mail: [email protected]

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Figure 12.1: General structure of sphingolipids.

little is known about the occurrence in food. Sphingolipid metabolism can also be modified by constituents of the diet, such as cholesterol, fatty acids and fumonisin mycotoxins [3], with consequences for cell regulation and disease. The analytical methods which are available for the identification of complex sphingolipids comprise hydrolysis and characterisation of the sphingoid bases, the fatty acid moieties as well as the headgroups (e. g. sugars) by highperformance liquid chromatography (HPLC) or gas chromatography (GC) after derivatisation. Since this procedures are time consuming and structural information is lost due to hydrolysis, we developed a high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLCESI-MS/MS) method for the analysis of intact sphingolipids.

12.2 Results and Discussion Under electrospray conditions sphingolipids could effectively be transformed into protonated [M H] or deprotonated [M–H]– molecular ions and almost no fragmentation was observed. In the MS/MS mode, low energy collisioninduced dissociation of the [M H] or [M–H]– molecular ions with argon as collision gas produced characteristic product ion spectra which can easily be used to identify sphingolipids. The fragmentation of the protonated molecular ions [M H] in the positive mode revealed product ions which were used for the identification of the sphingoid base backbone. In the case of ceramide C24:1 (Fig. 12.2) the sphingosine backbone was characterized by the product ion at m/z 264, which was formed due to the loss of the fatty acid 330

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Structural Studies of Sphingolipids

moiety and subsequent elimination of water. In the negative ESI mode product ions are characteristic for the fatty acid attached to the sphingoid base. As can be seen from Figure 12.2B, the product ions at m/z 390 and 365 are formed by the cleavage of sphingosine. Similar product ion spectra (not shown) were obtained for complex sphingolipids, e. g. galactosylceramide (simple glycosphingolipids are often termed “cerebrosides”). In addition to the typical fragment ions for the sphingoid base and the fatty acid, product ions formed by the loss of the sugar moiety were observed in the positive and the negative mode. The detection limit was determined using Nacetyl-sphingosine as model compound. In the single ion monitoring mode using the [M H] signal 50 pg (0.15 pmol) were detectable at a signal-tonoise ratio of 5:1. Based on measurements in the positive and the negative ESI mode HPLC-ESI-MS/MS provides highly structure specific data for the complete structural assignment of sphingolipids in the low picomole range in food such as potatoes. For sphingolipid analysis food sample were extracted with Chloroform/ Methanol according to the procedure by Bligh and Dyer [4]. The crude extracts were further purified using a silica-cartridge (1 g) and 2 % MeOH in CHCl3/1 % AcOH (“ceramide” fraction) followed by MeOH/CHCl3 (1:1) (“cerebroside” fraction) as solvent. HPLC separation of sphingolipids was achieved on a RP18 column (Waters Xterrar MS C18 100 q 2.1 mm, 3.5 mM) using a linear gradient of MeOH (2 mM NH4Ac) (solvent A) and

264.2

100

A

E+04 5.64

- H2O OH

OH

-fatty acid NH 11

C-24:1

- H2O

O

282

relative intensity %

630 80 60 40

630.4

20

264

252.3

282.2

365.9 431.4 470.2

200

B

m/z

390.5

N 11

C-24:1

OH

-sphingosine 390

O

365

relative intensity %

100

OH

612.7

E+04 2.12

80 60

646.8 365.0

40 237.2 20

268.6 281.4 200

406.4 420.8

616.8 599.7

647.7

m/z

Figure 12.2: Product ion spectra of ceramide C-24:1 in the positive ESI mode (A) (precursor ion m/z 648.5 [M H] ) and in the negative ESI mode (B) (precursor ion m/z 646.8 [M–H]-) obtained by collision induced dissociation (argon 2.2 mTorr, offset voltage 25 eV).

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R1:

OH O

4-Hydroxy-8-sphingenine

HO

OH

R1

O OH

OH OH

3

R2

NH

OH

O

R2:

OH 11-15

11-15

Figure 12.3: Structures of sphingolipids which were identified in potatoes by HPLCMS/MS.

t-butylmethylether (gradient: 0 min 95 % A, 1 min 95 % A, 15 min 70 % A, 25 min 70 % A). HPLC-MS/MS analysis of the ceramide and the cerebroside fraction of potatoes revealed several new sphingolipids (see Fig. 12.3). All ceramides isolated from potatoes are comprised of 4-hydroxy-8-sphingenine as sphingoid base with 22:0-26:0 fatty acids (including also a-hydroxy fatty acids) attached to the amino group. The cerebroside fraction of potatoes contained simple glycosphingolipids with a hexose attached to position 1. All compounds were detected in concentrations ranging from approximately 150 to 300 mg/kg. Furthermore, cell culture studies using immortalized human kidney epithelial cells were performed to demonstrate the inhibition of the sphingolipid metabolism by fumonisin, a class of mycotoxins which are found in corn containing foods [5, 6]. All tested fumonisins (FB1, FB2, HFB1, N-palmitoylHFB1) caused a significant increase of sphinganine compared to control cells, demonstrating that ceramide synthase was inhibited by all compopunds [7].

12.3 Conclusions HPLC-ESI-MS/MS provides highly structure specific data for the identification and characterisation of sphingolipids in the low picomole range in food. Using this method several new sphingolipids (range: 150–300 mg/kg) could be identified in potatoes. 332

References

References 1. Merrill, A. H.; Sweeley, C. C. (1996) Sphingolipids: metabolism and cell signalling. In: Vance, D. E.; Vance, J. E. (Eds.) Biochemistry of Lipids, Lipoproteins and Membranes, Elsevier, New York, NY, 43–73. 2. Vesper, H.; Schmelz, E. M.; Nikolova-Karakashian, M. N.; Dillehay, D. L.; Lynch, D. V.; Merrill, A. H. Jr. (2000) Sphingolipids in Food and the Emerging Importance of Sphingolipids to Nutrition. J. Nutr. 129, 1239–1250. 3. Humpf, H.-U.; Schmelz, E.-M.; Meredith, F. I.; Vesper, H.; Vales, T. R.; Wang, E.; Menaldino, D. S.; Liotta, D. C.; Merill, A. H. Jr. (1998) Acylation of Naturally Occurring and Synthetic 1-Deoxysphinganines by Ceramide Synthase. Formation of NPalmitoyl-Aminopentol Produces a Toxic Metabolite of Hydrolyzed Fumonisin, AP1, and a New Category of Ceramide Synthase Inhibitor. J. Biol. Chem. 273, 19060–19064. 4. Bligh, E.; Dyer, W. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. 37, 911–917. 5. Hartl, M.; Humpf, H.-U. (1999) Simultaneous Determination of Fumonisin B1 and Hydrolyzed Fumonisin B1 in Corn Products by Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS). J. Agric. Food Chem. 47, 5078–5083. 6. Seefelder, W.; Hartl, M.; Humpf, H.-U. (2001) Determination of N-(Carboxymethyl)fumonisin B1 in Corn Products by Liquid Chromatography/Electrospray IonizationMass Spectrometry. J. Agric. Food Chem. 49, 2146–2151. 7. Seefelder, W.; Humpf, H.-U.; Schwerdt, G.; Freudinger, R.; Gekle, M., Induction of Apoptosis in Cultured Human Proximal Tubule Cells by Fumonisins and Fumonisin Metabolites. Toxicol. Appl. Pharmacol., 2003, 192/2, 146–153.

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13 Modulation of (Oxidative) DNA Damage by Constituents of Carob Fibre S. Schäfer*, H. G. Kamp*, C. Müller*, B. Haber**, G. Eisenbrand*, C. Janzowski*

Abstract The aim of the present study was to characterize the protective and antioxidative potential of a fibre rich carob preparation against oxidative DNA damage induced by menadione in human colon cancer cells (Caco-2), using the Comet-Assay. Results: The aqueous extract of the carob preparation shows a distinct antioxidative potential in this test system. Fecal fermentation supernatants were also found to exhibit antioxidative efficacy, however, there was no difference between effects of fermentation supernatant with carob fibre compared to fermentation controls without carob.

13.1 Introduction It is widely accepted that diets rich in fruits and vegetables contribute to lower the risk for colon cancer [1]. Similar effects are attributed to certain food constituents such as antioxidants or dietary fibre. A carob fibre preparation obtained from the carob fruit pulp has been reported to contain substantial amounts of insoluble dietary fibre together with polyphenols, flavonoids, tannins and other phenolic components [2–5]. These compounds have been associated with protective biological effects including antioxidative effects, inhibition of cell growth and induction of detoxifying enzymes, especially of certain glutathione-S-transferases [6–9]. In this investigation the protective potential of the above carob preparation against oxidative DNA damage was studied in human colon cancer cells. Effects on menadione induced oxidative DNA damage were measured using an aqueous extract and a fecal fermentation supernatant of the carob fibre preparation [10]. * Division of Food Chemistry & Environmental Toxicology, Dept. of Chemistry, University, D-67663 Kaiserslautern ** Nutrinova Nutrition Specialties & Food Ingredients GmbH, Industriepark Höchst, 65926 Frankfurt/Main

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13.2 Methods Human colon adenocarcinoma cells (Caco-2) were cultured in DMEM/nutrient mix F-12 (1:1) with 1 % penicillin/streptomycin and 20 % fetal calf serum (FCS). Test materials were incubated in medium with 10 % FCS for 24 hours. Subsequently, cells were treated with menadione (2 mM, 37 hC) for 1 hour in serum-free medium. Appropiate amounts of menadione and quercetin were dissolved in DMSO to give a final concentration of 0.1 % (v/v) DMSO in the incubation medium, except for the fermentation supernatants. An aqueous extract was prepared from powdered carob fibre preparation by suspension in incubation medium, 4 min ultrasonic treatment, 20 min centrifugation and sterile filtration (filter 0,22m). Fermentation supernatants of carob fibre were prepared using an inoculum made from fresh feces collected from healthy human volunteers (fecal suspension: 10 g/l carob fibre, 1/10 diluted in a phosphate buffer, fermented 24 h under anaerobic conditions) and were immediately sterile filtered and diluted before use. A fermentation control (fecal suspension without substrate, blank) was included [11]. DNA damage was determined using single cell gel electrophoresis (comet-assay). The extent of DNA damage was expressed by percent tail intensity representing fluorescence intensity of the comet tail in percent of total fluorescence intensity [12, 13]. Damage resulting from oxidized purine bases was analyzed by additional treatment with the repair enzyme formamidopyrimidine-DNA-glycosylase (FPG) to screen for FPG-sensitive sites [13]. Quercetin (50 mM, 2 mM) served as reference antioxidant in comparison to aqueous carob fibre extracts [14]. Cell viability (number of viable cells in percent of total) and cell growth (number of viable cells in percent of control) were assayed by trypan blue exclusion.

13.3 Results and Discussion Menadione (1–5 mM, 1 h, 37 hC) induced (oxidative) DNA damage in a concentration dependent manner. Pre-incubation with quercetin (50 mM, 24 h) resulted in a 37 % reduction of DNA damage induced by 2 mM menadione. When samples were additionally treated with FPG, 42 %-protection of oxidative DNA-damage was obtained by quercetin. Aqueous extracts of carob fibre in concentrations of 0.01–1 % reduced menadione dependent DNA damage (Fig. 13.1). Maximal antioxidative effectiveness was observed at an extract concentration of 0.1 %. In contrast, at higher extract concentrations (3 %) an indication for prooxidative effects was observed. Fermentation supernatants (0.3–1 %) were found to protect against menadione induced DNA damage (Fig. 13.2), irrespective of whether carob 335

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control (C) menadione 2 M (Md) quercetin + Md aqueous extract + Md FPG

tail intensity (%)

30 25 20 15 10 5

3%

d 50 M 2 M 0. 00 1 0. % 01 % 0. 03 % 0. 1% 0. 3% 1%

M

C

0

Figure 13.1. Modulation of menadione induced (oxidative) DNA damage by 24 h preincubation with aqueous extracts of carob fibre and quercetin (as reference) n 3–7 (mean; sd)

tail intensity (%)

45 40 35 30

control (C) menadione 2 M (Md) blank-supernatant + Md fermentation-supernatant carob fibre + Md FPG

25 20 15 10 5 0 C Md

0.1%

0.3%

1%

3%

10%

Figure 13.2. Modulation of menadione induced (oxidative) DNA damage by 24 h preincubation with fermentation supernatants of carob fibre and fermentation control (blank) n 2 (mean; r except 0.3 %, Bl 10 %: n 1)

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References fibre extract was present in the fermentation incubations or not. Thus, the observed reduction of (oxidative) DNA damage in this test system obviously was brought about by fermentation products of the intestinal flora. Viability of cells was at least 90 % in all incubation conditions. Cell growth decreased during 24 h incubation with quercetin (50 mM: down to 80 %), aqueous fibre extract (3 %: down to 50 %) and with fermentation supernatant (10 %: down to 65 %). In conclusion, carob fibre extract contains water-soluble compounds with substantial antioxidative effectiveness as detected by the extent of protection against oxidative DNA damage induced by menadione in Caco-2 cells.

Acknowledgments The carob fibre Caromaxä was provided by Nutrinova, Frankfurt. The gift of FPG-protein by Dr. A. R. Collins (Rowett Res. Inst., Aberdeen, Scotland, UK) is gratefully acknowledged. The fermentation supernatants were kindly provided by Dr. M. Glei and Prof. Dr. B. L. Pool-Zobel, Institute for Nutrition and Environment, Friedrich Schiller University of Jena.

References 1. Steinmetz K. A. and Potter J. D. (1991): Vegetables, Fruit, and Cancer. I. Epidemiology; Cancer Causes and Control 2, 325–357. 2. Haber B. (2001): Personal communication on carob fibre; [Nutrinova, Frankfurt]. 3. Marakis S. (1996): Carob Bean in food and feet: current status and future potentials – a critical appraisal; J. Food Sci. Technol. 33, 365–383. 4. Baumgartner S., Genner-Ritzmann R., Haas J., Amado R. and Neukom H. (1986): Isolation and identification of cyclitols in carob pods (Ceratonia siliqua L.); J. Agric. Food Chem. 34, 827–829. 5. Zunft H. J. F., Lueder W., Harde A., Haber B., Graubaum H.-J. and Gruenwald J. (2001): Carob pulp preparation for treatment of hypercholesterolemia; Advances in Therapy 18, 230–236. 6. Gee J. M. and Johnson I. T. (2001): Polyphenolic compounds: Interactions with the gut and implications for human health; Current Medicinal Chemistry 8, 1245–1255. 7. Lamson D. W. and Brignall M. S. (2000): Antioxidants and cancer III: Quercetin; Alternative Medicine Reviews 5 (3) 196–208. 8. Abrahamse S. L., Pool-Zobel B. L. and Rechkemmer G. (1999): Potential of short chain fatty acids to modulate the induction of DNA damage and changes in the intracellular calcium concentration by oxidative stress in isolated rat distal colon cells; Carcinogenesis 20 (4) 629–634.

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9. Waladkhani A. and Clemens M. R. (1998): Effect of dietary phytochemicals in cancer development (Review); International Journal of Molecular Medicine 1, 747–753. 10. Chiou T.-J. and Tzeng W.-F. (2000): The roles of glutathione and antioxidant enzymes in menadione-induced oxidative stress; Toxicology 154, 75–84. 11. Barry B. L., Hoebler C., Macfarlane G. T., Macfarlane S., Mathers J. C., Reed K. A., Mortensen P. B., Nordgaard I., Rowland I. R. and Rumney C. J. (1995): Estimation of the fermentability of dietary fibre in vitro: a European interlaboratory study; British Journal of Nutrition 74, 303–322. 12. Collins A. R., Olmedilla B., Southon S., Granado F. and Duthie S. J. (1998): Serum carotenoids and oxidative DNA damage in human lymphocytes; Carcinogenesis 19 (12), 2159–2162. 13. Glaab V., Collins A. R., Eisenbrand G., Janzowski C. (2001): DNA-damaging potential and glutathion depletion of 2-cyclohexene-1-one in mammalian cells, compared to food relevant 2-alkenals; Mutation Research 497, 185–197. 14. Aherne S. A. and O’Brien N. M. (2000): Protection by the flavonoids myricetin, quercetin, and rutin against tert-butylhydroperoxide- and menadione-induced DNA single strand breaks in Caco2 cells; Free Radical Biology & Medicine 29 (6), 507–514.

14 Pro- and Antiapoptotic Effects of Flavonoids in H4IIE-Cells: Implication of Oxidative Stress W. Wätjen*, Y. Chovolou, P. Niering, A. Kampkötter, Q.-H. Tran-Thi, and R. Kahl

14.1 Introduction Flavonoids are polyphenolic compounds that occur ubiquitously in foods of plant origin, over 4000 flavonoids have been identified in plant sources. Flavonoids possess a remarkable spectrum of biochemical and pharmacological activities [1]. They affect cell functions such as growth, differentiation and apoptosis. Epidemiological studies have suggested that naturally occurring plant compounds such as flavonoids may inhibit and protect against various stages of the cancer process and are associated with a reduced incidence of coronary heart disease. As possible mechanisms by which flavonoids may affect tumorigenesis are discussed: antioxidative activities, scavenging effects on activated mutagens and carcinogens and altered gene expression. Flavonoids have been shown to be potent antioxidants because of their radi-

* Heinrich-Heine-University, Institute of Toxicology, P. O. Box 101007, 40001 Düsseldorf, Germany, E-mail: [email protected]

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Figure 14.1. Structures of selected flavonoids: 1: quercetin, 2: rutin, 3: myricetin, 4: morin, 5: taxifolin, 6: fisetin, 7: catechin

cal scavenging activity and their ability to complex iron. Although flavonoids may be powerful antioxidants they were also shown to be able to generate reactive oxygen species and induce apoptotic cell death. Hydroxyl radicals generated by the autoxidation and redox cycling of the polyphenolic flavonoids may initiate apoptotic processes. It is difficult to predict whether proor antioxidative properties of flavonoids are pre-dominant in a cellular system. The importance of the chemical structure of the flavonoids in relation to their antioxidant activity in biological systems has been controversely discussed. The aim of this study was to analyse the pro/antioxidative and pro/antiapoptotic activity of seven selected flavonoids in relationship to their chemical structure.

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14.2 Results and Discussion 14.2.1

Protection against H2O2-mediated DNA-Strand Breaks

In H4IIE cells H2O2 causes a time- and concentration-dependent increase in DNA strand breaks as analyzed by single cell gel electrophoresis [2]. Incubation with 500 mM H2O2 for 24 h causes an increase in the average image length (“comet”) of H4IIE cells from 14,01 e 0,26 mm (control) to 56,07 e 4,7 mm. To investigate antioxidative effects of flavonoids, a preincubation (1 h) was performed. Both quercetin and fisetin were found to be potent suppressors of H2O2-mediated “comet”-formation (Fig. 14.2). Other flavonoids (50 mM) showed minor (taxifolin, myricetin) or no effects (Table 14.1). Oxidative DNA damage is one of the mechanisms postulated to play an important role in mutagenesis and carcinogenesis. Therefore effects of quercetin and fisetin could be important for protection against neoplastic lesions which may undergo promotion and progression to potential malignancy.

60 55

quercetin fisetin

50 45 40 35 30 25 20 15 10 0

10

20

30

40

50

Figure 14.2. Effect of quercetin and fisetin on H2O2-mediated DNA strand breaks: H4IIE cells were pre-incubated with different concentrations of quercetin or fisetin for 1 h (control DMSO) followed by an incubation with 500 mM H2O2 for 2 h. DNA strand breaks are analyzed by single cell gel electrophoresis (comet assay). The average image length of 50 cells in mm e S. E. M. (n i 3) is shown representing the extent of H2O2-mediated DNA strand breaks (e quercetin or fisetin).

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caspase-3-activation (% of control)

500 450 400 350 300 250 200 150 100 50 0

Figure 14.3. Effects of quercetin and fisetin on caspase-3-activation: H4IIE cells were incubated either with different concentrations of quercetin or fisetin, or pre-incubated with 25 mM quercetin or fisetin for 1 h, followed by incubation with 1000 mM H2O2. After 16 h cells were lysed and incubated with a colorimetric labelled caspase-3-substrate. Results are expressed as percentage of control value e S. E. M. (n i 3).

14.2.2

Protection against H2O2-mediated Apoptosis

In H4IIE cells H2O2 causes apoptosis as confirmed by cellular “blebbing”, DNA-ladder formation (data not shown) and caspase-3-activation. Pre-incubation with quercetin and fisetin decreases the activation of this proteolytic enzyme (Fig. 14.3). This could be an important mechanism for flavonoidinduced protective cellular effects.

14.2.3

Cytotoxicity of Flavonoids

We investigated the effect of flavonoids on H4IIE cell viability (24 h) using the Neutral Red assay [3] and found great differences among the 7 selected flavonoids: While rutin and catechin up to 1000 mM showed no significant reduction in cell viability (no IC50-value determined), fisetin (IC50 62 e 14 mM) and quercetin (IC50 135 e 12 mM) were relatively toxic. Data of other flavonoids are summarized in Table 14.1.

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40

35

30

25

20

15

quercetin fisetin

10 0

100

200

300

400

500

Figure 14.4. Generation of DNA strand breaks by quercetin and fisetin: H4IIE cells were incubated with different concentrations of quercetin or fisetin for 3 h, DNA strand breaks were analyzed by single cell gel electrophoresis (comet assay). The average image length of 50 cells in mm e S. E. M. (n i 3) is shown representing the extent of flavonoid-mediated DNA strand breaks.

14.2.4

Induction of DNA Strand Breaks by Flavonoids

Incubation with quercetin and fisetin for 3 h causes DNA strand breaks (Fig. 14.4), other flavonoids (500 mM) showed minor or no effects as analyzed by single cell gel electrophoresis (Table 14.1). This result confirms previous findings by Musoda and Chipman [4], who demonstrated that on the one hand, low concentrations (10 mM) of quercetin protect against H2O2-induced cell damage, on the other hand 100 mM quercetin was able to generate DNA strand breaks in HepG2 cells.

14.2.5

Induction of Apoptosis by Flavonoids

Out of the 7 flavonoids tested, quercetin and fisetin were shown to be the most potent inducers of apoptotic DNA ladder formation [5, 6] in H4IIE cells as shown in Fig. 14.5. Induction of apoptosis by quercetin and fisetin was further confirmed by caspase-3-activation (Fig. 14.3). Myricetin and taxifolin showed less apoptosis-inducing potential leading to the conclusion that the double bond in the ring C causing the planarity of the ring system has an impact on the potential of apoptosis induction. Rutin, morin and catechin showed no apoptotic features. 342

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Pro- and Antiapoptotic Effects of Flavonoids in H4IIE-Cells

Figure 14.5. Induction of apoptosis by quercetin and fisetin: H4IIE cells were incubated with different concentrations of quercetin or fisetin for 24 h, then DNA was isolated and analyzed electrophoretically for formation of oligosomal fragmentation of DNA as a characteristic feature of apoptotic cell death (n i 3 with essentially the same results). A: quercetin (0, 50, 100, 250, 500, 1000 mM) B: fisetin (0, 50, 100, 250, 500, 1000 mM)

We conclude that there is a correlation between induction of apoptotic cell death and formation of DNA strand breaks by flavonoids. Fisetin and quercetin, the most potent inducers of apoptosis in H4IE cells, also increased the formation of DNA strand breaks as analyzed in the comet-assay. Besides morin, other flavonoids (up to 500 mM) did not lead to a significant induction of DNA strand breaks. For morin there must exist a cytotoxic mechanism involving the induction of DNA strand breaks without induction of apoptotic cell death. We tried to estimate the generation of ROS by high concentrations of flavonoids using the fluorescent probe DCF, but interference occurred due to quenching effects. The investigation of antioxidative capacity of flavonoids showed trends contrasting to the oxidative capacity of flavonoids. Quercetin, the compound with the strongest increase in DNA strand breaks inducing apoptotic cell death was also a potent inhibitor of H2O2-mediated DNA strand break formation and H2O2-induced caspase-3-activation. In a lesser extent this was also found for fisetin, taxifolin and myricetin. These apoptosis-inducing compounds showed also potential to inhibit H2O2-mediated DNA strand breaks but to a lesser extent. It appears that the antioxidative potential of a flavonoid is associated with the potential of this flavonoid to induce DNAstrand breaks and apoptosis.

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Table 14.1. Cytotoxicity, induction of apoptosis, induction of DNA strand breaks by various flavonoids and protection against H2O2-mediated DNA strand break formation. Results were obtained by neutral red accumulation assay (24 h), DNA-ladder formation (24 h) or single cell gel electrophoresis (comet-assay). Data are IC50-values (24 h), occurrence of a DNA-ladder (agarose gel electrophoresis) or image length of an average cell in mm (500 mM flavonoid for 3 h or 1 h pre-incubation with 50 mM of flavonoid followed by 2 h 500 mM H2O2), data are means e SEM (n i 3).

DMSO rutin catechin morin taxifolin myricetin

cytotoxicity

apoptotic DNA-ladder formation

Induction of DNA strand breaks

– i 1000 mM i 1000 mM 252 e 32 mM 449 e 85 mM 49 e 11 mM

– no no no i 250 mM i 500 mM

14,01 16,05 16,73 19,62 15,16 16,08

e e e e e e

0,26 1,65 0,82 2,37 0,88 0,52

DNA strand breaks after H2O2-treatment mm mm mm mm mm mm

56,07 56,67 59,19 54,73 47,67 47,8

e e e e e e

4,7 7,7 4,4 6,5 4,9 6,8

mm mm mm mm mm mm

Acknowledgement We thank S. Ohler and I. Köhler for excellent technical assistance.

References 1. Formica, J. V.; Regelson, W. (1995) Review of the biology of quercetin and related bioflavonoids. Fd Chem. Toxic. 33, 1061–1080. 2. Singh, N. P.; McCoy, M. T.; Tice, R. R.; Schneider, E. L. (1988) A simple technique for quantification of low levels of DNA damage in individual cells. Exper. Cell. Res. 175, 184–191. 3. Babich, H.; Shopsis, C.; Borenfreund, E. (1986) In vitro cytotoxicity testing of aquatic pollutants using established fish cell lines. Ecotox. Environ. Safety 11, 91–99. 4. Musonda, C. A.; Chipman, J. K. (1998) Quercetin inhibits hydrogen peroxideinduced NFkB DNA binding activity and DNA damage in HepG2 cells. Carcinogenesis 19, 1583–1589. 5. Lee, W.-R.; Shen, S.-C.; Lin, H.-Y.; Hou, W.-C.; Yang, L.-L. Chen, Y.-C. (2002) Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by a decrease of reactive oxygen species and activation of caspase-3 and Ca2 -dependent endonuclease. Biochem. Pharmacol. 63, 225–236. 6. Wang, I.-K.; Lin-Shiau, S.-Y., Lin, J.-K. (1999) Induction of apoptosis by apigenin and related flavonoids through cytochrome C release and activation of caspase-9 and caspase-3 in leukemia HL-60 cells. Europ. J. Cancer 35, 1517–1525.

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Analysis of the Hypotheses

15 Analysis of the Hypotheses on b-Carotene/ Tobacco Smoke Interactions in the A/J Mouse Lung Cancer Model Goralczyk, R., Wertz*, K., Riss, G., Bachmann, H., Buchwald, P., Hansen, T., Niehof, M., Dangers, M., and Borlak J.

15.1 Objective Analyze potential mechanisms of b-carotene (bC)/tobacco smoke interaction in a short term animal study.

15.2 Model A/J mice were fed a diet containing 120 ppm or 600 ppm bC for 6 weeks. For the last 2 weeks, the mice were exposed to main stream tobacco smoke.

15.3 Results CYP1A1 and 1B1 were induced in all smoke-exposed groups, without being influenced by bC. None of the CYP enzymes analyzed were induced by bC. Rather, CYP2B9/B10, 2D10, 3A16, and 4A10 transcripts were downregulated by bC in the lung. bC/smoke exposure did not cause an altered bC metabolite pattern in lung, as compared to bC treatment alone. Both smoke and bC caused an elevated lung retinol concentration. Retinyl esters were increased by bC, while decreased by smoke. RARb was not repressed by the treatment. In general, RAR and RXR expression profiles indicate a bC/smoke interaction that leads to mutual quenching of the single treatment effect. PCNA protein was mildly more abundant in combined treatment groups, whereas the other proliferation-associated genes analyzed were not informative. Cytoprotective enzymes and the 8OHdG level in lung indicate a very mild prooxidative effect of the treatment.

* Human Nutrition and Health, Roche Vitamins Ltd., Basel, Switzerland

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15.4 Conclusion CYP induction leading to an altered bC metabolite profile, decreased RARb expression, increased proliferation, or increased oxidative stress were proposed to be responsible for an adverse bC/smoke interaction. However, none of these mechanisms were confirmed in our model. To extend this interpretation to the human situation, we are in the progress of verifying these results in human and mouse primary lung cell cultures.

16 Carob Fibre – Functional Effects on Human Colon Cell Line HT29 Stefanie Klenow*, Michael Glei, Gabriele Beyer-Sehlmeyer, Bernd Haber**, Beatrice L. Pool-Zobel

16.1 Introduction Carob fibre is a food ingredient from the mediterranean carob pod (Ceratonia siliqua L.). The product is obtained by a mild procedure during which most of the soluble carob constituents are removed by water extraction and the insoluble dietary fibre (e. g. lignin, cellulose, hemicellulose) is retained in addition to tannins and other polyphenols in the residue.

16.2 Aim To obtain first information on biological or functional properties of this novel food ingredient, we investigated its potential to modulate selected parameters of chemoprotection using the human colon cell line HT29. These were inhibition of cell proliferation, modulation of genotoxicity by the lipid oxidation product 4-hydroxy-2-nonenal (HNE) and effects on activity of glutathione S-transferase (GST). * Department of Nutritional Toxicology, Institute for Nutrition, Friedrich Schiller Universität, Dornburger Str. 25, D-07743 Jena, Germany ** Nutrinova Nutrition Specialties & Food Ingredients GmbH, Industriepark Höchst, 65926 Frankfurt/Main, Germany

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16.3 Material and Methods An aqueous extract of the carob preparation (10 g carob fibre/100 ml cell culture medium) and the supernatant of a slurry obtained after fermenting the fibre with human gut bacteria (1 g carob fibre/100 ml bacterial culture medium) were prepared. The samples were characterised for their potential to modulate cell growth and survival after 72 hours treatment by determining cellular DNA content fluorimetrically. The resulting IC50’s (inhibitory concentrations) were used as the key concentration to study the impacts on markers of chemoprotection. For this HT29 cells were first pretreated with effective concentrations of the samples for 24, 48 and 72 h and then DNA damage, induced by HNE, was determined with single cell microgelelectrophoresis assay (Comet Assay). To study the detoxification capacity, GST activity was determined after 72 h pre-incubation using 1-chlor-2,4-dinitrobenzol as substrate. To verify the influence of short-chain fatty acids alone, the cells were treated with concentrations equivalent to those present in the fermentation supernatant. Controls were cells which remained untreated, or which were treated with the solvents.

16.4 Results The aqueous extract from carob fibre significantly inhibited growth of HT29 cells at concentrations up to 0.2 % (one-way ANOVA; IC50 : 0.4 % v/v). The impact of its fermentation supernatant was apparent as well (i 2 %; IC50 : 10 % v/v) (Fig. 16.1). However, the reconstituted mixture of short chain fatty acids (acetate, propionate, butyrate) and butyrate (Tab. 16.1) on its own were less effective, yielding IC50 of 13 % and 23 %, respectively (Fig. 16.1). The supernatant of the pure human inoculum (produced without carob fibre) was also effective, but less than the carob fibre fermentation sample (IC50 : 15 %) (Fig. 16.2). Using the comet assay, DNA damage could be observed in HNE treated HT29 cells. Protection against HNE was apparent after pre-incubating a nearly confluent layer of cells for 24 h with 0.5 % to 5 % of the fermentation supernatant (Fig. 16.3). Table 16.1. Concentrations of acetate, propionate, butyrate in mM equivalent to those in 10 % of the fermentation supernatant.

fermentation supernatant 10 % (IC50)

acetate

propionate

butyrate

sort-chain fatty acids

3.11

10.9

1.55

5.75

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proliferation assay fermentation supernatant, n=4

120 110

proliferation (%)

100 90 80

mixture of short-chain fatty acids, n=3

70 60 50 40

butyrate, n=3

30 20 10 0 0

5

10

15

20

25

30

concentration (%)

Figure 16.1. Proliferation assay determined by DNA content: Fermentation supernatant (produced with carob fibre) yielding an IC50 of 10 %. The reconstituted mixture of the short-chain fatty acids and butyrate alone are less effective than the fermentation supernatant.

proliferation (%)

proliferation assay 120 110 100 90 80 70 60 50 40 30 20 10 0

fermentation supernatant, n=4

pure fermentation supernatant, n=1

0

5

10

15

20

25

30

concentration (%)

Figure 16.2. Proliferation assay determined by DNA content: Fermentation supernatant (produced with carob fibre) yielding an IC50 of 10 %, pure fermentation supernatant (produced without carob fibre) as the control is less effective (IC50 : 15 %).

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Figure 16.3. Genotoxic effects of 150 mM of 4-hydroxy-2-nonenal (HNE) in HT29 and protective effects of a pre-incubation with various concentrations of the fermentation supernatant. n 2.

Figure 16.4. GST-activity per protein content (nmol/(min q mg protein). The modulation of GST activity with half of the IC50 concentration of the aqueous extract was not significantly different from the untreated control and the solvent treated control, respectively. The IC50 concentration of the aqueous extract as well as both concentrations of the fermentation supernatant had no effect on GST-activity in HT29. n 3 for IC50, n 2 for 1⁄2 IC50.

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Pre-incubation with 10 % ( IC50) of the fermentation supernatant has distinct effects on cell viability, and protection against HNE was not observed. First results on the modulation of GST activity by half of the IC50 concentration of the aqueous extract were not significantly different (0.50 e 0.03 nmol q min-1 q mg protein-1) from the untreated control (0.41 e 0.02) (Fig. 16.4). Higher concentrations up to the IC50 also had no effect on GST-activity, and neither did the fermentation supernatant at concentrations equivalent to IC50 and one half of IC50, respectively. Studies on the effects of the supernatant of the pure human inoculum and quercetin are ongoing.

16.5 Conclusions Both aqueous extracts and fermentation samples from carob fibre significantly modulate growth of human colon tumor cells. At concentrations less than the IC50’s, protection against HNE-induced genotoxicity is observed. One of the mechanisms could be the detoxification capacity of GST enzymes. Since no significant modulation of GST activity could be observed after treatment with the fermentation supernatant so far, other mechanisms probably play a role in this context. The butyrate concentration in the fermentation supernatant (IC50 O 0.5 mM) seems to be not responsible for protective effects, since butyrate only moderately induces GST activity at 4 mM. Ongoing kinetic studies on concentration effect ranges will disclose comparative functional potencies of the different sample preparations.

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Influence of Food Constituents on Cytochrome P450 1A Activity

17 Influence of Food Constituents on Cytochrome P450 1A Activity Annette Baumgart*, Melanie Schmidt, Hans-Joachim Schmitz, and Dieter Schrenk

17.1 Introduction Plant-derived foods contain a wide variety of different classes of substances, e. g. flavonoids, which show significant effects on cytochrome P450 (CYP) isoenzymes. Constituents of grapefruit juice inhibit a variety of CYP-isoenzymes, especially CYP3A4. This may lead to an increase in the oral availability of many relevant drugs such as cyclosporin A, midazolam or felodipine, caused mainly by an altered first-pass metabolism [1]. Studies revealed that grapefruit juice contains furocoumarins which are mostly responsible for its inhibitory potency and selectivity. Furocoumarins are phytoalexines found e. g. in plants belonging to the families Umbelliferae and Rutaceae such as lime, celery, parsley and parsnip. In vitro studies have shown that single furocoumarin derivatives are relatively selective and potent inhibitors of CYP isoenzymes [2–5]. The present study was conducted to determine the effects of selected furocoumarins on CYP1A activity in vitro. To obtain more information about the specificity of action and potential involvement of the aryl hydrocarbon receptor (AhR), we tested the inhibition during cotreatment with the typical AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, we investigated the influence of selected furocoumarins on CYP1A1 mRNA expression and on a reporter gene construct.

* Department of Food Chemistry and Environmental Toxicology, University of Kaiserslautern, D-67663 Kaiserslautern, Germany

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Table 17.1. Structures of furocoumarins. R

1

R

O R

2

O

O

2

R

1

O

O

O

angelicin-type

psoralen-type

Compound

Type

R1

R2

psoralen 8-methoxypsoralen (8-MOP) bergapten isopimpinellin angelicin sphondin bergamottin (BM)

psoralen psoralen

H H

H OCH3

psoralen psoralen angelicin angelicin psoralen

H OCH3 H OCH3 H

dihydroxybergamottin (DHB)

psoralen

OCH3 OCH3 H H OCH2–CHC(CH3)–(CH2)2– CHC(CH3)–CH3 OCH2–CHC(CH3)–(CH2)2–CHOH– C(CH3)2OH

H

17.2 Materials and Methods 17.2.1

Cell Culture

Primary rat hepatocytes were prepared from male Wistar rats weighing 150–220 g. Hepatocytes cultured in DMEM including 20 % fetal bovine serum (FCS) were grown in 60 mm-diameter plates. Furocoumarins and TCDD dissolved in dimethyl sulfoxide (DMSO) were added. The cells were incubated at 37 hC for 48 hours. TCDD in a final concentration of 1 nM was used as positive control. After incubation cell homogenates were prepared as described previously [6]. The catalytic activity of CYP1A in rat hepatocytes was measured as 7-ethoxyresorufin O-deethylase (EROD) using a spectrofluorometer (PerkinElmer LS-5B).

17.2.2

Reporter Gene Assay

A sequence of the 5l-flanking region of the CYP1A1 gene containing two xenobiotic responsive elements (XREs) was amplified with specific primers from genomic rat DNA. The PCR product was cloned into a firefly luciferase reporter vector (pGL3-Promotor, Promega, Heidelberg) as described previously [7]. 352

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H4IIE rat hepatoma cells were grown in DMEM supplemented with 20 % FCS and 1 % penicillin/streptomycin. The cells were plated in 60 mmdiameter plates. After 24 hours the cells were transiently cotransfected with the CYP1A1 reporter gene construct and the control plasmid pRL-SV40 (Promega, Heidelberg, Germany), expressing the renilla luciferase gene, using the calcium phosphate coprecipitation method as described previously [8]. After transfection cells were incubated with the furocoumarin in a final concentration of 100 mM for 48 hours. TCDD and DMSO were used as control. Reporter gene assays were performed using the Dual luciferaseTM system (Promega, Heidelberg, Germany). Cell homogenates were analysed luminometrically (Lumat LB 9507, Berthold, Wildberg, Germany) according to the instructions of the manufacturer. After background correction (activities in untreated cells) relative reporter gene activities were determined by dividing the firefly luciferase activity (reporter gene) by the renilla luciferase activity (control gene).

17.3 Results and Discussion Investigations in primary rat hepatocytes and H4IIE rat hepatoma cells of the aryl hydrocarbon receptor (AhR)-mediated activity of CYP1A show an inhibitory potency of selected furocoumarins in co-incubations with the classic agonist TCDD. The IC50 values are in the mM range. Semiquantitative RT-PCR analysis shows, however, induction of CYP1A1 mRNA by several furocoumarins like 8-MOP and angelicin, but in reporter gene experiments no induction of the luciferase activity was determined at the investigated concentrations. These findings demonstrate that certain food constituents can both inhibit and induce CYP1A1. Further experiments are necessary to determine the mechanisms of these effects and the relevance for the metabolism of drugs and endogenous compounds.

Table 17.2. IC50 values and confidence intervals of inhibition of EROD activity in coincubations of furocoumarins with TCDD in rat hepatocytes. Furocumarin

IC50 value [mM]

CI (95 %)

8-MOP angelicin isopimpinellin bergamottin

9.34 1.68 5.19 1.80

– 0.65 1.24 0.32

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Acknowledgements The authors thank Dipl.-Chem. Ragna Hussong for kindly providing the reporter gene plasmids. This study was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany.

References 1. Schmiedlin-Ren, P.; Edwards, D. J.; Fitzsimmons, M. E., He, K.; Lown, K. S., Woster, P. M.; Rahman, A.; Thummel, K. E.; Fisher, J. M.; Hollenberg, P. F.; Watkins, P. B. (1997) Mechanisms of enhanced oral availability of CYP3A4 substrates by grapefruit constituents. Decreased enterocyte CYP3A4 concentration and mechanism-based inactivation by furanocoumarins. Drug Metab.Dispos. 25 (11), 1228–1233. 2. Guo, L.-Q.; Fukuda, K.; Ohta, T.; Yamazoe, Y. (2000) Role of furanocoumarin derivatives on grapefruit juice-mediated inhibition of human CYP3A4 activity. Drug Metab. Dispos. 28 (7), 766–771. 3. Gwang, J. H. (1996) Induction of rat hepatic cytochrome P4501A and P4502B by the methoxsalen. Cancer Lett. 109 (1-2), 115–20. 4. Tassaneeyakul, W.; Guo, L.-Q.; Fukuda, K.; Ohta, T.; Yamazoe, Y. (2000) Inhibition selectivity of grapefruit juice components on human cytochromes P450. Arch. Biochem. Biophys. 378, 356–363. 5. Zhang, W.; Kilicarslan, T.; Tyndale, R. F.; Sellers, E. M. (2001) Evaluation of methoxsalen, tranylcypromine, and tryptamine as specific and selective CYP2A6 inhibitors in vitro. Drug Metab. Dispos. 29 (6), 897–902. 6. Schmitz, H. J.; Hagenmaier, A.; Hagenmaier, H. P.; Bock, K. W.; Schrenk, D. (1995) Potency of mixtures of polychlorinated biphenyls as inducers of dioxin receptorregulated CYP1A activity in rat hepatocytes and H4IIE cells. Toxicol. 99, 47–54. 7. Hussong, R. (1999) Charakterisierung von Agonisten des Dioxinrezeptors aus dem Tryptophanmetabolismus der intestinalen Mikroflora. Diplomarbeit, University of Kaiserslautern. 8. Kauffmann, H. M.; Schrenk, D. (1998) Sequence analysis and functional characterization of the 5’-flanking region of the rat ‘multidrug resistance protein 2’ (mrp2) gene. Biochem. Biophys. Res. Commun. 245, 325–331.

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Red Grape Products and Ethanol Modulate Coagulation

18 Red Grape Products and Ethanol Modulate Coagulation and Fibrinolysis in Healthy Male Volunteers Achim Bub*, Bernhard Watzl, Gerhard Rechkemmer

Moderate consumption of red wine may lower the risk of cardiovascular disease by reducing coagulation. The role of polyphenols from wine and other red grape products on hemostasis is not well examined. We therefore studied the effects of red wine (RW), dealcoholized red wine (DRW), red grape juice (RGJ), or a 12 % ethanol solution on coagulation and fibrinolysis in healthy young men. In a randomized cross over design, 24 volunteers (31 e 7 years) consumed either 500 ml of each beverage for 2 weeks with a washout period of 1 week between each intervention. Total polyphenol content of the grape products were 4.7 mM (RW), 4.2 mM (DRW), and 5.1 mM (RGJ), respectively. Blood was drawn weekly after an overnight fast. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and platelet activator inhibitor (PAI) were determined. aPTT did not change during any intervention period. We found a significant increase in PT after ethanol consumption (p I 0.05) and a reduction in fibrinogen after ethanol (p 0.084) and red wine (p 0.086) consumption, respectively. PAI significantly increased after ethanol (p I 0.05) and red wine (p I 0.05) consumption, respectively. Red wine and ethanol decrease fibrinolysis (PAI) and coagulation by acting on the extrinsic pathway (PT), while the intrinsic pathway was uneffected (aPTT) in this study. We conclude, that modulation of hemostasis by red wine is rather due to the ethanol and not the polyphenol content.

* Federal Research Centre for Nutrition, Institute of Nutritional Physiology, Haid- und Neu-Str. 9, Karlsruhe, 76131 Germany

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19 Long-Term Treatment with a Prebiotic Modulates the Gut-Associated Immune System of Azoxymethane-Treated F344 Rats M. Roller*, G. Caderni**, G. Rechkemmer*, B. Watzl*

Several studies have shown that probiotics stimulate the immune system in animals and humans. For prebiotics however, no data are available on their modulatory effects on the systemic and the gut-associated lymphoid tissue (GALT). In this study, we investigated the effects of probiotics (PRO), a prebiotic (PRE), and a combination of both (synbiotic, SYN) on the GALT of rats treated orally with the carcinogen azoxymethane (AOM). Male F344 rats received for 32 wks a high fat diet (40 %) as the basic diet supplemented with PRO (B. lactis BB12 and L. rhamnosus GG, each 5 q 108 cfu/g diet), PRE (inulin, RAFTILOSE Synergy1, 10 % of the diet), or SYN. Control animals were not treated with AOM. Immune cell suspensions were prepared from spleens, mesenteric lymph nodes (MLN) and Peyer’s patches (PP) and the following immune parameters were measured: Lytic activity of natural killer (NK) cells, phagocytic activity, lymphocyte proliferation and cytokine secretion capacity (IL-10, IFN-g). The AOM-treatment suppressed lytic activity of NK cells prepared from spleens and PP. In contrast, PRE supplementation enhanced NK cell activity (PP), IL-10 secretion (PP, MLN), IFN-g secretion (PP) as well as lymphocyte proliferation (MLN). SYN also increased NK cell activity (PP), IL-10 secretion (PP), while lymphocyte proliferation (spleen, PP) was decreased. PRO, PRE, and SYN each inhibited the monocyte phagocytic activity in the spleen, respectively. The immunomodulatory effects of PRO and PRE supplements observed in rats not treated with AOM (controls) differed from those observed in the carcinogen-treated rats. In conclusion, PRE and SYN supplementation of AOM-treated rats on a high-fat diet modulated immune functions primarily affecting the GALT (PP, MLN).

Acknowledgement Supported by the European Union (SYNCAN-QLKI-1999-00346).

* Federal Research Centre for Nutrition, Institute of Nutritional Physiology, 76131 Karlsruhe, Germany ** Department of Pharmacology, University of Florence, Italy

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b-Carotene Inhibits Growth of Human Colon Carcinoma Cells (HT 29)

20 b-Carotene Inhibits Growth of Human Colon Carcinoma Cells (HT 29) in vitro by Induction of Apoptosis Karlis Briviba*, Kerstin Schnäbele, Elke Schwertle, Gerhard Rechkemmer

20.1 Introduction Epidemiological studies suggest that b-carotene is able to modulate the risk of cancer. A number of in vitro studies reported that b-carotene inhibits growth of cancer cells, however, so far little is known about the molecular mechanisms of the antiproliferative effect of b-carotene. The effects of two b-carotene preparations, (i) b-carotene dissolved in tetrahydrofuran (the final concentration of tetrahydrofuran in the cell culture medium was 0.5 %) and (ii) b-carotene incorporated in a water dispersible bead form, on cultured human colon carcinoma cells HT29 was investigated [1].

20.2 Results The treatment of cells with b-carotene up to 30 mM for 72 h led to a significant increase in the cellular b-carotene concentration and formation of retinol. b-Carotene showed only low cytotoxicity for confluent cells tested up to 30 mM, but at dietary relevant concentrations for the intestinal tract (10, 30 mM) b-carotene was strongly cytotoxic for growing cells and induced apoptosis in HT29 cells as assessed by the Annexin-V assay (the maximal effect was observed 15 h after treatment with b-carotene). Exposure of cells to retinol at concentrations causing cellular retinol levels similar to those observed by b-carotene treatment, had no antiproliferative or cytotoxic effect. Further, b-carotene did not affect the activation of the extracellular signal-regulated kinases (ERK1 and ERK2) that are essential for cellular growth.

* Federal Research Center for Nutrition, Institute of Nutritional Physiology, Haid- und Neu-Str. 9, 76131 Karlsruhe, Germany

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20.3 Conclusion b-Carotene can inhibit growth of human colon carcinoma cells in vitro by induction of apoptosis in proliferating cells.

Acknowledgement Supported by the Deutsche Krebshilfe.

Reference 1. Briviba, K, Schnäbele, K, Schwertle, E, Blockhaus, M, and Rechkemmer, G. (2001) Biol. Chem. 382, 1663–1668.

21 The Influence of Short-Chain Fatty Acids on Intracellular pH and Calcium-Concentration in the HT-29 Colon Carcinoma Cell Line Kerstin Rebscher*, Stephan W. Barth*, Gerhard Rechkemmer*

Intracellular calcium-concentration [Ca2 ]i and intracellular pH (pHi) are important parameters for the regulation of various physiological processes, including apoptosis, cell proliferation or hormone secretion. The short-chain fatty acids (SCFA) acetate, butyrate, propionate or their SCFA-mixtures, embling typical bacterial fermentation patterns of polysaccharides or oligosaccharides in the human colon, were studied. By investigating the effects of SCFA-induced [Ca2 ]i and pHi changes in the colon carcinoma cell line HT-29, underlying regulatory mechanisms of signal transduction were characterized. Both, [Ca2 ]i and pHi are measured individually or simultaneously using ratiometric fluorescence microscopy and the fluorescence dyes Fura-2 and

* Federal Research Center for Nutrition, Institute of Nutritional Physiology, 76139 Karlsruhe, Germany

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BCECF respectively. Co-incubating cells with both dyes allows to directly study regulatory mechanisms of [Ca2 ]i and pHi homeostasis and the interaction between these two important factors. Perfusion of fluorescence loaded cells with SCFA dissolved with Ca2 -containing Ringer solution lead to a significantly increase of [Ca2 ]i of 40–50 nmol/L up to 70 nmol/L during SCFAperfusion. This effect could neither be observed in Ca2 -free nor EGTA-containing Ringer solution, indicating the influx of extracellular-Ca2 through Ca2 -channels in the plasma membrane. SCFA-perfusion simultaneously leads to intense intracellular acidification (DpH –0.2 pH units), which then activates Na /H exchangers, presumably the housekeeping NHE1 subtype, to induce pHi recovery. Removing extracellular SCFA leads to transient [Ca2 ]i and pHi recovery, the latter was blocked by inhibition of the Na /H exchanger using ethylisopropylamiloride (EIPA), respectively. An intense SCFA-induced relationship between increasing [Ca2 ]i and intracellular acidification rate was shown, improving the knowledge of the participation of microbial fermentation of dietary fibre and prebiotic carbohydrates on metabolic and cellular processes during colon carcinogenesis.

22 Effect of Short Chain Fatty Acids on Cytotoxicity, Proliferation, and Apoptosis in Human Colon Carcinoma Cell Lines Silvia Roser*, Heike Lang, Gerhard Rechkemmer

22.1 Introduction The short-chain fatty acids (SCFA) acetate, propionate, and butyrate are the main products of bacterial fermentation in the colon with potential health benefits such as colon cancer prevention. One aim of functional food development is therefore to rise the SCFA-concentration in the colon, especially the concentration of butyrate, by adding e. g. prebiotic substances as inulin to the food. In this study, the potential colon cancer preventive effects with regard to cytotoxicity, cell proliferation and apoptosis of acetate, propionate, and butyrate were analyzed in three colon cancer cell lines with various differentiation status, the HT29- HT29 clone 19A- and T84-cells.

* Federal Research Center for Nutrition, Institute of Nutritional Physiology, Haid- und Neu-Str. 9, 76131 Karlsruhe, Germany

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22.2 Methods Proliferation (7 days) and cytotoxicity (24 h incubation time) were measured using WST-1, a tetrazolium salt which is cleaved by cellular enzymes of living, metabolically active cells to a water-soluble formazan dye. Flow cytometry was used to measure the rate of apoptotic cells by the Annexin V assay and by analysing the activation of caspase-3, a key enzyme of the apoptotic cascade.

22.3 Results Acetate, propionate and butyrate inhibited cell-proliferation dose-dependently. Butyrate showed significant effects in HT29-cells in the micromolar concentrations range. SCFA were cytotoxic at milimolar concentrations, e. g. butyrate i 5 mM in HT29-cells. All three SCFA were able to induce apoptosis in all of the three cell lines. Apoptosis was induced by the activation of caspase-3. Butyrate had the strongest effects of all SCFA in all test systems and in all cell lines used, followed by propionate and acetate. Differences between the various cell lines could be observed, but there was no correlation between the grade of differentiation of the cell lines and the intensity of the effects observed after incubation with SCFA.

22.4 Conclusions Inhibition of cell proliferation, cytotoxic, and apoptosis inducing effect of the three main bacterial fermentation products in the colon, acetate, propionate, and butyrate, was shown in three cell lines with various grades of differentiation. Former studies mainly focused on butyrate as a marker for potential preventive effects of SCFA. This study shows that all three SCFA can play a chemopreventive role with regard to colon cancer. Current studies with SCFA-mixes show that the total SCFA-concentration is more important than the absolute amount of one of the SCFA in the mix.

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23 Investigation of the Antihormonal Potential of Xanthohumol, the Major Prenylated Flavonoid of Humulus Lupulus Anette Höll*, Sabine Guth, Gerhard Eisenbrand

23.1 Summary Xanthohumol, a major prenylated chalcone in hop, was investigated for antiandrogenic activity using a transiently transfected mammalian reporter gene based system (COS-AR-Luc) and yeast cells stably transfected with the human androgen receptor gene. Test systems were validated using the physiological androgen DHT and the established antiandrogen linuron. DHT induced reporter gene activity in a dose dependent manner in both test systems. This androgen response was inhibited by linuron down to 60 % residual activity. Xanthohumol showed comparable antiandrogenic activity to linuron reducing DHT induced reporter gene activity in both test systems.

23.2 Introduction Hop has been used since ages in beer brewing due to its characteristic flavor and taste. Recent studies suggest that xanthohumol, a major prenylated chalcone in hop, might be useful for prevention of osteoporosis, arteriosclerosis and cancer. Xanthohumol is reported to modulate critical steps of procarcinogen activation, for example by inhibition of human CYP isoenzymes [1, 2] or by induction of quinone reductase in cells [3]. Furthermore, xanthohumol has been shown to inhibit growth of cancer cell lines [4]. In addition, marked antioxidative properties of xanthohumol have also been described [5]. Some prenylated flavonoids, such as 8-prenyl-naringenine or licochalcone A, have been described to display estrogenic properties [6, 7]. The structurally related xanthohumol has not been found to exhibit estrogenic, androgenic or progestogenic properties [6]. However, antiestrogenic activity has been ascribed to xanthohumol, as evidenced by a significant reduction of estrogen-induced alkaline phosphatase activity by xanthohumol [8].

* Division of Food Chemistry and Environmental Toxicology, University of Kaiserslautern, Erwin-Schroedinger-Str. 52, D-67663 Kaiserslautern, Germany

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OMe

OH OH

O

xanthohumol

Cl

O Me

N OMe

OH

N H

Cl

O

linuron

DHT

Figure 23.1. Structures of DHT, xanthohumol and linuron.

In the present study xanthohumol was investigated for antiandrogenic activity. A transient transactivation assay in mammalian COS-7 cells and a transactivation assay in stably transfected yeast cells were utilized.

23.3 Materials and Methods 23.3.1

Chemicals

Dihydrotestosterone (DHT) and linuron were purchased from Sigma-Aldrich. Xanthohumol was obtained from Prof. Hans Becker, Saarbrücken, Germany.

23.3.2

COS-AR-Luc Transactivation System

Fetal calf serum (FCS), RPMI 1860 medium, DMEM medium without phenol red and glutamine, penicillin-streptomycin and glutamine solution were purchased from Gibco/Life Technologies. CDFCS (Charcoal dextran FCS) was prepared by the following procedure: 50 ml of a sterile solution containing 0.5 % Norrit A and 0.05 % Dextran in 0.14 M NaCl were diluted in 500 ml FCS. After 45 min incubation at 50 hC the solution was centrifuged twice at 800 g for 20 min and 4hC. The supernatant (CDFCS) was used. x x x x x x

FCS-medium: RPMI 1860 medium supplemented with 10 % FCS. CDFCS-medium: DMEM medium supplemented with 10 % CDFCS. Luciferase Assay Kit (Promega). b-galactosidase Assay Kit (Clontech). Luciferase and b-galactosidase activity were measured according to the instructions of the suppliers using a luminometer. Luminometer: Lumistar BMG Technologies.

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Table 23.1. Sequence of operations of the COS-AR-Luc assay. Day 1

Day 2

Day 3

Day 4

Electroporation with pSG5-hAR, pSVb, pMAMneoLuc

Replace medium, incubate with substance for 24 h

Replace incubation medium by CDFCS- medium and incubate for another 24 h

Wash with PBS, cell preparation, luciferase and b-galactosidase activity assay

COS-7 cells were transiently transfected using electroporation with a human androgen receptor expression vector (pSG5-hAR, Dr. A. Cato, Karlsruhe, Germany), a luciferase reporter gene vector under the control of MMTV promotor (pMAMneoLuc, Clontech) and a control vector (pSVb, Promega), constitutively expressing b-galactosidase. After 24 h, cells were incubated in CDFCS-medium (containing 0.1 % DMSO) either with xanthohumol (1 nM – 10 mM) in coincubation with 100 nM DHT or with DHT (0.1 nM – 1 mM) as positive control. Medium served as negative control. After another 24 h, medium was replaced by CDFCS-medium. 48 h after incubation, cells were washed twice and lysed with lysis buffer. The resulting suspension was centrifuged. Luciferase and b-galactosidase activity were measured using aliquots of the supernatant. Hormone dependent luciferase activity was corrected against b-galactosidase activity (Tab. 23.1).

23.3.3

Yeast-AR Transactivation System

Chlorophenolred-b-D-galactopyranoside (CPRG) was purchased from Roche Diagnostics, Mannheim. Growth and test medium was prepared according to the instructions of Sohoni & Sumpter [9]. x x

Yeast cells were obtained from Prof. G. Vollmer, Dresden, Germany. Microplate reader: Lambda spectral 340 (MWG Biotech).

Yeast cells, stably cotransfected with the human androgen receptor gene and lacZ (b-galactosidase enzyme) reporter gene under the control of androgen responsive element, were grown in growth medium for 24 h. Cells (extinction 1 at 620 nm in 0.5 ml) were incubated in 96-well plates with xanthohumol (1 nM – 100 mM) in the presence of 50 nM DHT or with DHT (1 mM – 0.01 nM) as positive control. Test medium containing 0.5 ml CPRG solution (10 mg/ml) served as negative control. After incubation, absorbance was measured at 540 nm and optical density at 620 nm in a 96-well plate reader. Hormone dependent b-galactosidase activity was corrected against optical density (Tab. 23.2).

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Table 23.2. Sequence of operation of the yeast transactivation assay. Day 1

Day 2

Day 4

Cell growth

Incubation over 48 h

Determination of absorbance at 540 nm and 620 nm

23.4 Results In both test systems, DHT induced reporter gene expression in a dose dependent manner. Maximal response was achieved by incubating cells with 1 mM DHT. EC50 values obtained from the respective sigmoidal dose-response curves were within a similar range: 12 nM in the mammalian COS-AR-Luc system, 8 nM in the yeast system (see Fig. 23.2 A and B). To test for antiandrogenic activity, DHT concentrations selected to induce androgen stimulated reporter gene response were 100 nM in the COS-AR-Luc system and 50 nM in the yeast system, respectively (see Fig. 23.2 A and B). These concentrations were within the upper concentration range of the androgen induced dose response curve, slightly exceeding the EC50 values but not yet reaching the upper plateau, in order to afford highest sensitivity for detecting antiandrogenic effects. Test systems were validated using linuron, a phenylurea herbicide found earlier to display antiandrogenic properties [10]. In both test systems, linuron was able to inhibit DHT induced reporter gene activity dose dependently. In COS-AR-Luc cells, DHT-induced luciferase activity was reduced down to 60 % residual activity by linuron (1–10 mM). In yeast cells a similar extent of reduction, down to about 60 %, was served at 50–100 mM linuron. Likewise, xanthohumol was found to inhibit DHT induced reporter gene activity dose dependently. In the mammalian transactivation system (COSAR-Luc), maximal reduction down to 60 % was achieved by 1–10 mM xanthohumol. In stably transfected yeast cells, maximal inhibition was achieved at 50–100 mM xanthohumol. Although the same degree of maximal inhibition was observed in both systems, considerably higher concentrations of antagonists were necessary in the yeast systems.

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A

DHT sigmoidal curve fit DHT 100 nM + linuron DHT 100 nM + xanthohumol DHT 100 nM

200 180

relative luciferase activity [%]

160 140 120 100

EC50

80 60 40 20

1 10 10 0 10 00 10 00 0

1 1 10 10 0 10 0 10 0 00 0 1 10 10 0 10 00 10 00 0

0,

0,

01

0

concentration [nM] B

relative §-galactosidase activity [%]

200

DHT sigmoidal curve fit DHT 50 nM + linuron DHT 50 nM + Xanthohumol DHT 50 nM

180 160 140 120 100

EC50

80 60 40 20

0, 1 0, 5 1 5 10 50 10 100 00 1 10 10 10 0 0 50 0 10 00 0 50 00 10 000 00 1 00 10 10 10 0 0 50 0 10 00 0 50 00 10 000 00 00

0

concentration [nM] Figure 23.2 A, B. Transiently transfected mammalian transactivation system (COS-ARLuc): DHT induced dose dependently luciferase expression with an EC50 of 12 nM. Linuron and xanthohumol (1–10 mM) reduce DHT induced reporter gene expression to about the same extent. Stably transfected yeast system: A comparably sigmoidal dose response curve is obtained with DHT, with an EC50 of 8 nM. Significant reduction of reporter gene response by xanthohumol is observed at higher concentrations (50–100 mM) as compared to the mammalian system (1 mM).

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23.5 Discussion Xanthohumol was found to exhibit antiandrogenic properties, as measured in a mammalian and in a yeast based transactivation system. Maximal inhibitory effects were achieved similar to those observed for the established antiandrogen linuron. In yeast, considerably higher concentrations of antagonists were necessary, compared to the mammalian test system, to obtain the same inhibitory effect. Therefore, for detection of antiandrogenic effects, the COS-AR-Luc system appears to be more sensitive than the yeast system, although EC50 values of response to the agonist DHT are almost identical. The xanthohumol content in beer has been reported to be in a range of about 2 to 700 ppb [11]. Daily consumption of 0.5 l beer might thus result in a daily intake of 1 to 350 mg, equivalent to about 0.2–6 mg/kg body weight for a person with 60 kg body weight. This intake would be expected to result in plasma concentrations several orders of magnitude lower than those inducing significant antiandrogenic effects in the mammalian test system. However, hop extracts or hop preparations have come into focus as potential food supplements. The xanthohumol content in genuine hop was reported to be about 0.2 % to 1 % [11], but recently products have been described containing hop enriched with up to 10 % xanthohumol [12]. Furthermore a dietary supplement has been reported to have xanthohumol content of about 320 ppm [13]. In view of the substantial antiandrogenic activity of xanthohumol found in the present study as well as the antiestrogenic activity ascribed before, potential endocrine side effects resulting from such significantly increased xanthohumol intake need to be further evaluated.

Acknowledgements This study was supported by the BWPLUS-Program – “Projekt Lebensgrundlage Umwelt und ihre Sicherung”, grant BWB 99002. We thank Dr. A. Cato (Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, 66041 Karlsruhe, Germany) for providing the androgen receptor expression vector pSG5-hAR, Prof. Sumpter (Brunel University, Uxbridge, Middlesex, 66041 UK) and Prof. Vollmer (Fachrichtung Biologie, Institut für Zoologie, Dresden, Germany) for providing transfected yeast cells. The gift of xanthohumol from Prof. Becker (Pharmakognosie und Analytische Phytochemie FR 8.7, Universität des Saarlandes, Saarbrücken, Germany) is also gratefully acknowledged. Special thanks are due to Dr. M. Keme´ny for a most valuable discussion.

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References

References 1. Miranda, C. L.; Yang, Y. H.; Henderson, M. C.; Stevens, J. F.; Santana-Rios, G.; Deinzer, M. L.; Buhler, D. R. (2000) Prenylflavonoids from hops inhibit the metabolic activation of the carcinogenic heterocyclic amine 2-amino-3-methylimidazo[4,5-F]quinoline mediated by cDNA-expressed human CYP 1A2. Drug Metab. Dispos. 28, 1297–1302. 2. Henderson, M. C.; Miranda, C. L.; Stevens, J. F.; Deinzer, M. L.; Buhler, D. R. (2000) In vitro inhibition of human P450 enzymes by prenylated flavonoids from hops, Humulus lupulus. Xenobiotica 30, 235–251. 3. Miranda, C. L.; Aponso, G. L.; Stevens, J. F.; Deinzer, M. L.; Buhler, D. R. (2000) Prenylated chalcones and flavanones as inducers of quinone reductase in mouse Hepa 1c1cz cells. Cancer Lett. 149, 21–29. 4. Miranda, C. L.; Stevens, J. F.; Helmrich, A.; Henderson, M. C.; Rodriguez, R. J.; Yang, Y. H.; Deinzer, M. L.; Barnes, D. W.; Buhler, D. R. (1999) Antiproliferative and Cytotoxic Effects of Prenylated Flavonoids from Hops (Humulus lupulus) in Human Cancer Cell Lines. Food Chem. Tox. 37, 271–285. 5. Miranda, C. L.; Stevens, J. F.; Ivanov, V.; McCall, M.; Frei, B.; Deinzer, M. L.; Buhler, D. R. (2000) Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. J. Agric. Food Chem. 48, 3876–3884. 6. Milligan, S. R.; Pocock, V.; van de Kauter, V.; Stevens, J. F.; Deinzer, M. L.; Rong, H.; de Keukeleire, D. (2000) The endocrine activities of 8-prenylnaringenin and related hop (Humulus lupulus L.) flavonoids. J. Clin. Endocrinol. Metab. 85, 4912–4915. 7. Rafi, M. M.; Rosen, R. T.; Vassil, A.; Ho, C.; Zhang, H.; Ghai, G.; Lembert, G.; Dipaola, R. S. (2000) Modulation of bcl-2 and Cytotoxicity by Licochalcone-A, a Novel Estrogenic Flavonoid. Anticancer Research 20, 2653–2658. 8. Gerhauser, C.; Alt, A.; Heiss, E.; Gamal-Eldeen, A.; Klimo, K.; Knauft, J.; Neumann, I.; Scherf, H.-R.; Frank, N.; Bartsch, H.; Becker, H. (2002) Cancer Chemopreventive Activity of Xanthohumol, a Natural Product Derived from Hop. Mol. Cancer Ther. 1, 959–969. 9. Sohoni, P.; Sumpter, J. P. (1998) Some environmental oestrogens are also antiandrogens. J. Endocrinol. 158, 327–339. 10. Lambright, C.; Ostby, J.; Bobseine, K.; Wilson, V.; Hotchkiss, A. K.; Mann, P. C.; Gray, L. E. (2000) Cellular and Molecular Mechanisms of Action of Linuron: An Antiandrogenic Herbicide that Produces Reproductive Malformations in Male Rats. Toxicol. Sci. 56, 389–399. 11. Stevens, J. F.; Taylor, A. W.; Deinzer, M. L. (1999) Quantitative analysis of xanthohumol and related prenylflavonoids in hops and beer by liquid chromatographytandem mass spectrometry. J. Chromatography A 832, 97–107. 12. Biendl, M.; Eggers, R.; Czerwonatis, N.; Mitter, W. (2001) Studies of the production of a xanthohumol-enriched hops product. Cerveza y Malta 38 (150), 25–29. 13. Coldham, N. G.; Sauer, M. J. (2001) Identification, quantification and biological activity of phytoestrogens in a dietary supplement for breast enhancement. Food Chem. Tox. 39, 1211–1224.

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24 Anthocyanidins Potently Interfere with Signalling Cascades Regulating Cell Proliferation Doris Marko*, Monika Keme´ny, Michael Habermeyer, Edda Bernardy, Susanne Meiers, Ulrike Weyand

Abstract The aglycons of the most abundant anthocyanins in food, cyanidin and delphinidin, were found to inhibit the growth of human tumor cells in vitro, comparable in their potency to the green tea constituent epigallocatechin3-gallate. Malvidin, bearing methoxy substituents in 3l- and 5l-position, exhibited less but also substantial growth inhibitory properties. However, the compounds differing in their substitution pattern at the B-ring, appear to affect different signalling cascades within the cell, relevant for the regulation of cell growth. The hydroxy substituted compounds cyanidin and delphinidin represent potent inhibitors of the epidermal growth factor receptor, leading to a shut-off of the subsequent mitogen-activated protein kinase pathway. In contrast, malvidin, lacking EGFR-inhibitory properties, potently inhibits the activity of cAMP-specific phosphodiesterases, thus affecting cAMP-homeostasis. The results show that depending on the substitution pattern at the B-ring, anthocyanidins affect different cellular signalling cascades, crucial for cell proliferation.

24.1 Introduction Anthocyanins are widely spread in food of plant origin. Depending on pH and presence of chelating metal ions anthocyanins are intensely coloured in blue, violet or red, contributing substantially to the colouring of a multitude of food, like berries, grapes or cherries. Glycosides of the aglycons cyanidin (cy) and delphinidin (del) represent the most abundant anthocyanins in plants (Table 24.1).

* Department of Chemistry, Division of Food Chemistry and Environmental Toxicology, University of Kaiserslautern, Erwin-Schroedinger-Str. 52, 67663 Kaiserslautern, Germany, E-mail: [email protected]

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Table 24.1. Structure of the most abundant anthocyanidins in food. R1 R2 + O

HO A

B R3

C OR

OH

Anthocyanidin

R

R1

R2

R3

Cyanidin Delphinidin Malvidin

H H H

OH OH OCH3

OH OH OH

H OH OCH3

In grapes and grape products also substantial amounts of malvidin (mv) glycosides are found [1]. Anthocyanins have been associated with a panel of potentially positive therapeutic effects, including the treatment of diabetic retinopathy and various microcircular diseases as well as antiinflammatory and chemoprotective properties [2]. However, despite the relatively high possible intake in humans, information on potential cellular effects of these compounds is limited. Anthocyanins have repeatedly been reported to possess antioxidative properties in vitro [3, 4]. However, little is known about the effect of anthocyanins on human tumor cells. We could recently show that the aglycons of the most abundant anthocyanins in food, cy and del, inhibit the growth of human tumor cells in vitro in the micromolar range, whereas mv is less active [5]. Furthermore, we investigated the influence of anthocyanins on potential cellular targets. Especially we focussed on central signalling cascades crucial for the regulation of cell growth, like the EGFR-MAP kinase and the cAMP pathway.

24.2 Materials and Methods 24.2.1

Tyrosine Kinase Activity of the EGFR

The EGF-receptor was isolated from A431 cells and purified by affinity chromatography using wheat germ agarose (Amersham Bioscience, Freiburg, Germany). The tyrosine kinase activity was determined as described previously [5].

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Elk-1 Reporter Gene Assay

Effects of test compounds on the MAP kinase activity of A431 cells were determined using the PathDetect reporter gene system (Stratagene, La Jolla, USA) as described previously [5].

24.2.3

PDE Assay

Cyclic AMP-hydrolysing phosphodiesterase (PDE4) was isolated from solid tumor tissue of the human large cell lung carcinoma LXFL529, grown as a xenograft subcutaneously onto nude mice. The tumor tissue was homogenised in a buffer containing 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 4 mM benzamidine hydrochloride, 0.5 mM trypsin inhibitor from soy beans, 0.1 mM phenylmethylsulfonylfluoride, 1 mM b-mercaptoethanol, 0.1 mM N-a-p-tosyl-L-lysine chloromethyl ketone, 1 mM pepstatin and 1 mM leupeptin. After centrifugation (100,000 g, 60 min), the supernatant (cytosol) was carefully removed and subjected to anion exchange chromatography on a Q-Sepharose column (Amersham Bioscience, Freiburg, Germany) using a NaCl gradient. Fractions of 5 mL were collected and assayed for cAMP-hydrolysing activity. PDE4 containing samples were identified by the selective inhibition of cAMP-hydrolysis in the presence of rolipram (10 mM). PDE4-containing samples were concentrated by ultrafiltration under nitrogen atmosphere. Subsequently, gel filtration on a Superdex HiLoad column (Amersham Bioscience, Freiburg, Germany) was used for further separation of PDE4 isoenzymes. PDE4 containing samples were incubated in the presence or absence of test compounds at 37 hC with a mixture of cAMP and [3H]-cAMP in a buffer containing 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, and 1 mM AMP, resulting in a final cAMP concentration in the assay of 1 mM. Reaction was stopped at a maximal cAMP turnover of 20 % by adding ZnSO4. [3H]-5l-AMP was precipitated by addition of Ba(OH)2 and centrifugation at 10,000 g for 5 min. Nonhydrolysed [3H]-cAMP was determined by liquid scintillation counting of the supernatant. PDE activity of each sample was determined in triplicate. The whole experiment was performed three times.

24.3 Results and Discussion The aglycons of the most abundant anthocyanins in food, cyanidin and delphinidin were found to inhibit tumor cell growth in vitro in the low micromolar range (Table 24.2) [5]. Both compounds represent highly potent inhibitors of the tyrosine kinase activity of the EGFR (Fig. 24.1). The presence of the hydroxy substituents at the B-ring appears to play an important role for the 370

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interaction with the EGF receptor. Malvidin, bearing two methoxy substituents at the B-ring, was found to be inactive up 100 mM. However, also malvidin exhibited substantial growth inhibitory properties (Table 24.2), although less potent compared to cyanidin and delphinidin. These results indicate that especially in the case of malvidin other cellular targets contribute to the growth inhibitory properties. The MAP kinase pathway represents one of the major signalling cascades regulating cell proliferation. Effective inhibition of the upstream located EGF-receptor results in a shut-off of the subsequent kinase cascade, Table 24.2. Growth inhibition of anthocyanidins in the sulforhodamine B assaya [5]. Substance

Cyanidin Delphinidin Malvidin

Cell line LXFL529L IC50 [mM]

A431 IC50 [mM]

73 e 4 33 e 3 i 100

42 e 1 18 e 2 61 e 7

a

Cells were incubated for 3 days with the respective compound. Growth inhibition was calculated as survival of treated cells over control cells (treated with the vehicle 0.1 % DMSO) q 100 [T/C %]. IC50 values were calculated by linear regression (at least three points). The values given are the mean IC50 e SD of at least three independent experiments, each done in quadruplicate.

Figure 24.1. Inhibition of the tyrosine kinase activity of the EGFR. Phosphorylation of the tyrosine residues of a peptide poly (Glu/Tyr) was determined by ELISA using an antiphosphotyrosine antibody linked to peroxidase. The data presented are the mean e SD of three independent experiments, each performed in quadruplicate [5].

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leading to the inhibition of cell growth [5]. In intact cells anthocyanins were found to influence the signalling cascades downstream of the EGFR. This was measured as phosphorylation of the transcription factor Elk-1 in a reporter gene assay. A431 cells were transiently transfected with a luciferase reporter gene construct, its expression depending on the phosphorylation of a GAL4-Elk-1 fusion protein by components of the MAP kinase pathway (Fig. 24.2). Cy and del inhibit the activation of the GAL4-Elk-1 fusion protein in the concentration range where growth inhibition is observed. Thus, the anthocyanidins cy and del are potent inhibitors of the EGFR, triggering downstream signalling cascades (Fig. 24.3). Interestingly, malvidin, lacking EGFR-inhibitory properties, also affects Elk-1 phosphorylation, comparable in the extent to the potent EGFR inhibitor cyanidin. These results underline the hypothesis that malvidin probably targets signalling elements upstream of the MAP kinase, but different from the EGFR. The MAP kinase cascade is connected with several other signalling pathways in a complex network pattern. One important regulative factor is the deactivating phosphorylation of the serine/threonine kinase Raf-1 by the protein kinase A (PKA), a key element of the cAMP-pathway (Fig.

Figure 24.2. Scheme of the reporter gene assay for Elk-1 phosphorylation (Stratagene, La Jolla, USA). A431 human vulva carcinoma cells were transiently cotransfected with a plasmid encoding a fusion protein consisting of the DNA binding domain (dbd) of GAL4 and Elk-1 as well as a plasmid containing the upstream activating domain (UAS) of GAL4 and the luciferase gene as a reporter. MAPK, mitogen activated protein kinase; EGF, epidermal growth factor; GAL4, trans-acting transcriptional activator from yeast; Elk-1, transcription factor [5].

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24.4). A central element in the cAMP homeostasis represent the superfamily of cAMP-hydrolysing phosphodiesterases (PDE). We showed previously that the cAMP-specific PDE isoenzyme family PDE4 represents the highest cAMP-hydrolysing activity in many human tumor cells [6]. Selective inhibition of PDE4 in these tumor cells results in growth inhibition, arrest in the G1-phase of the cell cycle and induction of apoptosis [7]. Compared to the results obtained for the inhibition of EGFR activity, a completely different pattern of activity was observed investigating the effect of anthocyanins on PDE4 activity. Malvidin potently inhibits the cAMP hydrolysis of PDE4 isolated from solid human tumor tissue (IC50 32.7 e 3.2 mM). In contrast, cy and del even enhanced PDE4 activity at low substance concentrations (Fig. 24.5). The effective inhibition of PDE4 activity by malvidin might explain the effect of the compound on Elk-1 phosphorylation without EGFR-inhibitory properties. These results show that depending on the structure anthocyanins affect different cellular targets crucial for the regulation of cell proliferation. The results further indicate that anthocyanins exhibit a complex pattern of cellular activities which are still not fully understood. Further studies on structureactivity relationships and the potential interaction with additional yet

Figure 24.3. Inhibition of luciferase expression as a measure for the inhibition of Elk-1 phosphorylation. After transfection cells were cultivated for 24 h. Thereafter, incubation with the test compounds was started 30 min prior to stimulation with 100 ng/ml EGF and continued for 4.5 h. Luciferase activity was determined in three independent experiments. Data are presented as mean e SD [5].

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Figure 24.4. Simplified scheme of the crosstalk between the MAPK cascade and the cAMP-pathway. PTK, Protein tyrosine kinase; Ras, GTP-binding protein; Raf-1, Serin-Threonine-Kinase; MAPKK, mitogen-activated protein kinase kinase; MAPK, mitogen-activated protein kinase; Elk-1, transcription faktor; SRE, serum-responsive element; L, ligand; Rez, ceceptor; G, G-Protein; AC, adenylat cyclase; ATP, adenosine triphosphat; cAMP, 3l,5l-cyclic adenosine monophosphat; R, regulatory subunit of the protein kinase A (PKA); C, catalytic subunit.

unknown cellular targets are mandatory to fully understand the cellular effects of these compounds as an element for risk/benefit evaluation.

References 1. Mazza, G. (1995) Anthocyanins in grapes and grape products. Critical Reviews in Food Science and Nutrition 35 (4), 341–371.

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References

Figure 24.5. Effect of anthocyanidins on the cAMP-hydrolysing activity of PDE4.The data presented are the mean e SD of three independent experiments, each performed in triplicate.

2. Wang, H.; Cao, G.; Prior, R. L. (1997) Oxygen radical absorbing capacity of anthocyanins. J. Agric. Food Chem. 45, 304–309. 3. Pool-Zobel, B. L.; Bub, A.; Schroder, N.; Rechkemmer, G. (1999) Anthocyanins are potent antioxidants in model systems but do not reduce endogenous oxidative DNA damage in human colon cells. Eur. J. Nutr. 38, 227–34. 4. Tsuda, T.; Shiga, K.; Kawakishi, S.; Osawa, T. (1996) Inhibition of lipid peroxidation and the active oxygen radical scavenging effect of anthocyanin pigments isolated from Phaseolus vulgaris L. Biochem. Pharmacol. 52 (7), 1033–1039. 5. Meiers, S.; Keme´ny, M.; Weyand, U.; Gastpar, R.; von Angerer, E.; Marko, D. (2001) The anthocyanidins cyanidin and delphinidin are potent inhibitors of the epidermal growth factor receptor. J. Agric. Food Chem. 49 (2), 958–962. 6. Marko, D.; Pahlke, G.; Merz, K. H.; Eisenbrand, G. (2000) Cyclic 3l,5l-nucleotide phosphodiesterases: potential targets for anticancer therapy. Chem. Res. Toxicol. 13 (10), 944–948. 7. Marko D.; Romanakis K.; Zankl H.; Fürstenberger G.; Steinbauer B.; Eisenbrand G. (1998) Induction of apoptosis by an inhibitor of cAMP-specific PDE in malignant murine carcinoma cells overexpressing PDE activity in comparison to their nonmalignant counterparts. Cell Biochem. Biophys. 28, 75–101.

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VII Participants and Contributors of the Symposium

Prof. Dr. H.-J. Altmann Bundesinstitut für Risikobewertung Thielallee 88–92 14195 Berlin Prof. Dr. Aalt Bast University Maastricht Faculty of Medicine Dept. of Pharmacology and Toxicology P. O. Box 616 NL-6200 MD Maastricht Niederlande Dr. Matthias Baum Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern, Geb. 52/322 Erwin-Schrödinger-Str. 67663 Kaiserslautern Annette Baumgart Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern Erwin-Schrödinger-Str. 67663 Kaiserslautern Dr. Jochen Bausch Roche Vitamins Ltd CH-4070 Basel Schweiz

Prof. Dr. Hans Konrad Biesalski Institut für Biologische Chemie und Ernährungswissenschaft Universität Hohenheim (140) Fruwirthstraße 12 70593 Stuttgart Dr. Catherine Boyle Chemical Safety & Toxicology Division Food Standards Agency Room 653C, Skipton house 80 London Road London SE1 6XZ UK K. Briviba Institut für Ernährungsphysiologie Haid- und Neu-Str. 9 76131 Karlsruhe Dr. Achim Bub Institut für Ernährungsphysiologie Bundesforschungsanstalt für Ernährung Haid- und Neu-Straße 9 Fabio Dal Bello Institut für Lebensmitteltechnologie Universität Hohenheim Garberstr. 28 70599 Stuttgart

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Participants and Contributors of the Symposium

Prof. Dr. Manfred Edelhäuser Ministerium für Ernährung und Ländlichen Raum Baden-Württemberg Kernerplatz 10 70182 Stuttgart Prof. Dr. Gerhard Eisenbrand Lebensmittelchemie und Umwelttoxikologie Fachbereich Chemie Universität Kaiserslautern, Geb. 52/322 Erwin-Schrödinger-Str. 67663 Kaiserslautern Prof. Dr. Peter Stefan Elias Bertha-von-Suttner-Straße 3A 76139 Karlsruhe Dr. Eric Fabian BASF Aktiengesellschaft Postfach 120 67114 Limburgerhof Grit Festtag Institut für Ernährungswissenschaften Lehrstuhl Ernährungstoxikologie Universität Jena Dornburger Str. 25 07743 Jena Dr. Christine Gärtner Cognis Deutschland GmbH Postfach 13 01 64 40551 Düsseldorf Prof. Dr. Hans Günter Gassen Institut für Biochemie Technische Universität Darmstadt Petersenstr. 22 64287 Darmstadt

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Prof. H. P. Gelbke ZHT-Z 470 Toxikologie BASF AG 67056 Ludwigshafen Eva Gietl Institut für Ernährungswissenschaften Lehrstuhl für Ernährungstoxikologie Universität Jena Dornburger Str. 25 07743 Jena Dr. Sibylle Grub Roche Vitamins Ltd. CH-4070 Basel Schweiz Prof. Dr. Werner Grunow Bundesinstitut für Risikobewertung Thielallee 88–92 14195 Berlin Dr. Sabine Guth Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern, Geb. 52/322 Erwin-Schrödinger-Str. 67663 Kaiserslautern Natja Haag Institut für Ernährungswissenschaften Lehrstuhl für Ernährungstoxikologie Dornburgerstr. 25 07743 Jena Saskia Habben Institut für Lebensmittelchemie Technische Universität Braunschweig Schleinitzstr. 20 38106 Braunschweig

VII

Participants and Contributors of the Symposium

Dr. Bernd Haber Nutrinova GmbH Industriepark Hoechst 65926 Frankfurt Michael Habermeyer Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern, Geb. 52/322 Erwin-Schrödinger-Str. 67663 Kaiserslautern Prof. Dr. Walter P. Hammes Institut für Lebensmitteltechnologie der Universität Hohenheim Garbenstraße 25 70599 Stuttgart Christian Hertel Institut für Ernährungstechnologie Universität Hohenheim Garbenstraße 25 70599 Stuttgart Frankie Hippe Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern, Geb. 52/322 Erwin-Schrödinger-Str. 67663 Kaiserslautern Anette Höll Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern, Geb. 52/322 Erwin-Schrödinger-Str. 67663 Kaiserslautern Dr. Jürgen Hollmann Bundesanstalt für Getreide-, Kartoffel- und Fettforschung Schützenberg 12 32756 Detmold

Dr. Hans-Ulrich Humpf Institut für Lebensmittelchemie Westfälische Wilhelms-Universität Münster Corrensstr. 45 48149 Münster Willi Hunziker Roche Vitamins Ltd Dep. VFH Grenzacher Str. 124 Ch-4070 Basel Schweiz Sandra Jakobs Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern, Geb. 52/322 Erwin-Schrödinger-Str. 67663 Kaiserslautern Prof. Dr. Regine Kahl Institut für Toxikologie Geb. 22.21, Ebene 02 Heinrich-Heine-Universität Düsseldorf Universitätsstr. 1 40225 Düsseldorf Hennicke Kamp Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern, Geb. 52/322 Erwin-Schrödinger-Str. 67663 Kaiserslautern Andreas Kampkötter Institut für Toxikologie Universität Düsseldorf Universitätsstr. 1 40225 Düsseldorf

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Participants and Contributors of the Symposium

Dominique Kavvadias Lehrstuhl für Lebensmittelchemie Universität Würzburg Am Hubland 97074 Würzburg Dr. Monika Keme´ny Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern, Geb. 52/322 Erwin-Schrödinger-Str. 67663 Kaiserslautern Prof. Dr. Dr. hc. Mult. Fritz Kemper Westfälische Wilhelms-Universität Umweltprobenbank für HumanOrganproben mit Datenbank Domagkstraße 11 48129 Münster Stefanie Klenow Institut für Ernährungswissenschaften Lehrstuhl für Ernährungstoxikologie Universität Jena Dornburger Str. 25 07743 Jena Prof. Dr. Josef Köhrle Institut für Experimentelle Endokrinologie Charite´, Humboldt-Universität zu Berlin Schumannstr. 20/21 10098 Berlin Prof. Dr. Berthold v. Koletzko Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen Kinderspital Abt. Stoffwechsel und Ernährung Ludwig-Maximilian-Universität München Lindwurmstraße 4 80337 München

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Dr. Gunhild Kozianowski Südzucker AG Wormser Str. 11 67283 Obrigheim Dr. Klaus Kraemer BASF Aktiengesellschaft Scientific Affairs Human Nutririon MEM/FN – D 205 67056 Ludwigshafen Linda Lea Safety and Environmental Assurance Centre Unilever, Sharnbrook Bedford MK 44 ILQ UK Dr. Stefan Lebrun Institut für Arbeitsphysiologie Ardeystr. 67 44139 Dortmund Leane Lehmann Institut für Lebensmittelchemie Universität Karlsruhe Kaiserstr. 12 76128 Karlsruhe Prof. Dr. Eckhard Löser Schwelmer Str. 221 58285 Gevelsberg Dr. Doris Marko Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern, Geb. 52/322 Erwin-Schrödinger-Str. 67663 Kaiserslautern

VII

Participants and Contributors of the Symposium

Prof. Dr. Manfred Metzler Institut für Lebensmittelchemie und Toxikologie Universität Karlsruhe Postfach 6980 76128 Karlsruhe Christiane Meyer Verbraucherservice Bayern im KDFB e. V. Jesuitenstr. 4 85049 Ingolstadt Dr. Christoph Müller Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern, Geb. 52/322 Erwin-Schrödinger-Str. 67663 Kaiserslautern Dr. Rudi Müller Global Technology Centre Unilever Bestfoods Knorrstr. 1 74074 Heilbronn Dr. Bernd Mussler Roche Vitamins Ltd CH-4070 Basel Prof. Dr. Karl J. Netter An den Brunnenröhren 14 35037 Marburg Petra Niering Institut für Toxikologie Universität Düsseldorf Universitätsstr. 1 Dr. Ute Obermüller-Jevic Scientific Marketing MEM/FN – D 205 BASF AG 67056 Ludwigshafen

Dr. John O’Brien Danone 15 Avenue Galille´e F-92350 Le Plessis Robinson Frankreich Dr. Margreet Olthof Wageningen Centre for Food Sciences Human Nutrition & Epidemiology P. O. Box 8129 NL-6700 EV Wageningen Niederlande Dr. Christoph Persin VK Mühlen AG Trettaustr. 32–34 21107 Hamburg Prof. Dr. Friedlieb Pfannkuch Roche Vitamins Ltd. CH-4070 Basel Schweiz Erika Pfeiffer Institut für Lebensmittelchemie Universität Karlsruhe Kaiserstr. 12 76128 Karlsruhe Prof. Dr. Raymond H. H. Pieters Immunotoxicology Research Institute of Toxicology Utrecht University Postbus 80176 NL-3508 TD Utrecht Niederlande Dr. Annette Pöting Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin Thielallee 88–92 14195 Berlin

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Participants and Contributors of the Symposium

Prof. Dr. Beatrice L. Pool-Zobel Friedrich-Schiller-Universität Jena Institut für Ernährungswissenschaften Lehrstuhl für Ernährungstoxikologie Dornburger Str. 25 07743 Jena

Dr. Maria Saarela Research Manager VTT Biotechnology Tietotie 2, Espoo P. O. Box 1500 FIN-02044 VTT Finnland

K. Rebscher Institut für Ernährungsphysiologie Bundesforschungsanstalt für Ernährung Haid- und Neu-Straße 9 76131 Karlsruhe

Dr. Sandra Schäfer Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern, Geb. 52/322 Erwin-Schrödinger-Str. 67663 Kaiserslautern

Prof. Dr. Gerhard Rechkemmer Institut für Ernährungsphysiologie Bundesforschungsanstalt für Ernährung Haid- und Neu-Straße 9 76131 Karlsruhe Dr. Elke Richling Lehrstuhl für Lebensmittelchemie Universität Würzburg Am Hubland 97074 Würzburg Dr. Gerald Reinders Haarmann & Reimer GmbH Mühlenfeldstr. 1 37603 Holzminden M. Roller Institut für Ernährungsphysiologie Bundesforschungsanstalt für Ernährung Haid- und Neu-Straße 9 76131 Karlsruhe S. Roser Institut für Ernährungsphysiologie Bundesforschungsanstalt für Ernährung Haid- und Neu-Straße 9 76131 Karlsruhe 382

Dr. Josef Schlatter Bundesamt für Gesundheit Sektion Lebensmitteltoxikologie Stauffacherstr. 101 CH- 8004 Zürich Schweiz Prof. Dr. Barbara O. Schneeman University of California, Davis Department of Nutrition One Shields Avenue Davis, CA 95616 USA Prof. Dr. Peter Schreier Lehrstuhl für Lebensmittelchemie Universität Würzburg Am Hubland 97074 Würzburg Prof. Dr. Dr. Dieter Schrenk Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern Gebäude 52/409 B Erwin-Schrödinger-Straße 67663 Kaiserslautern

VII

Participants and Contributors of the Symposium

Dr. Stefan Schulte BASF AG Abt. Für Produktsicherheit GUP, Z – 470 67056 Ludwigshafen Dr. Gerrit I. A. Speijers RIVM-Rijksinstitut voor Volksgezondheid en Milieu Centrum voor Stoffen en Risicobeoordeling (CSR) Antonie van Leeuwenhoeklaan 9 NL-3720 BA Bilthoven Niederlande Dr. Franz Timmermann Grünau Illertissen GmbH Postfach 10 63 89251 Illertissen Dr. Cornelia Ulrich Fred Hutchinson Cancer Research Center Cancer Prevention Research Program 1100 Fairview Ave N, MP-900 Seattle, WA 98109-1024 Dr. Karin H. van het Hof Unilever Bestfoods Europe P. O. Box 160 NL-3000 AD Rotterdam Niederlande Jan van Loo RT – TS Services Aamdorenstraat 1 B-3300 Tiemen Belgien

Dr. Sandra Vatter Lebensmittelchemie und Umwelttoxikologie Universität Kaiserslautern, Geb. 52/322 Erwin-Schrödinger-Str. 67663 Kaiserslautern Prof. Dr. Burkhard Viell Bundesministerium für Verbraucherschutz, Ernährung und Landwirtschaft Rochusstraße 1 53123 Bonn Prof. Dr. Rudi F. Vogel Lehrstuhl für Technische Mikrobiologie Technische Universität München Weihenstephaner Steig 16 85350 Freising Prof. Dr. A. G. J. Voragen Food Science Department Wageningen Agricultural University Sparrenbos 37 NL-6705 BB Wageningen Niederlande Dr. Wim Wätjen Institut für Toxikologie Universität Düsseldorf Universitätsstr. 1 40225 Düsseldorf Prof. Dr. Ron Walker 44 Ash Hill Road, Ash, Aldershot, Hants. GU12 6AB UK

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Participants and Contributors of the Symposium

Prof. Dr. Xiang-Dong Wang Associate Professor of Nutrition and Medicine Director of Molecular Carcinogenesis Section HNRCA at Tufts University 711 Washington Street Boston, MA 02111 USA Prof. Dr. Shaw Watanabe, M. D., D. M. S. Department of Applied Bioscience Tokyo University of Agriculture 1-1-1, Sakuragaoka, Setagaya, Tokyo 156-8502 Japan Prof. Dr. Wei Yimin Institute of Food Science & Technology, CAAS P.O. Box 5109 100094 Beijing PR China

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Karin Wertz Roche Vitamins Ltd. Buildg. 93, Room 8.10 CH-4070 Basel Schweiz Prof. Dr. Johannes Westendorf Abteilung Toxikologie Universitätsklinikum Hamburg Vogt-Kölln-Str. 30 22527 Hamburg Dr. Erich Wolz Roche Vitamins Ltd. CH-7040 Basel Schweiz Dr. Jürgen Zarn Swiss Federal Office of Public Health Stauffacher Str. 101 CH-8004 Zürich Schweiz