Melatonin: Biological basis of its function in health and disease

MEDICAL INTELLIGENCE UNIT

PANDI-PERUMAL • CARDINALI MIU

S.R. Pandi-Perumal and Daniel P. Cardinali

Melatonin: Biological Basis of Its Function in Health and Disease

Melatonin:

Biological Basis of Its Function in Health and Disease

MEDICAL INTELLIGENCE UNIT

Melatonin: Biological Basis of Its Function in Health and Disease S.R. Pandi-Perumal, M.Sc. Comprehensive Center for Sleep Medicine Department of Pulmonary, Critical Care and Sleep Medicine Mount Sinai School of Medicine New York, New York, U.S.A.

Daniel P. Cardinali, M.D., Ph.D. Departamento de Fisiología Facultad de Medicina Universidad de Buenos Aires Buenos Aires, Argentina

LANDES BIOSCIENCE GEORGETOWN, TEXAS U.S.A.

EUREKAH.COM AUSTIN, TEXAS U.S.A.

MELATONIN:

BIOLOGICAL BASIS OF ITS FUNCTION IN HEALTH AND DISEASE Medical Intelligence Unit Eurekah.com Landes Bioscience

Copyright ©2006 Eurekah.com All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: Eurekah.com / Landes Bioscience, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081 http://www.eurekah.com http://www.landesbioscience.com ISBN: 1-58706-244-5 While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data Melatonin : biological basis of its function in health and disease / [edited by] S.R. Pandi-Perumal, Daniel P. Cardinali. p. ; cm. -- (Medical intelligence unit) Includes bibliographical references and index. ISBN 1-58706-244-5 1. Melatonin. I. Pandi-Perumal, S. R. II. Cardinali, Daniel P. III. Series: Medical intelligence unit (Unnumbered : 2003) [DNLM: 1. Melatonin--physiology. 2. Pineal Gland--physiology. 3. Pineal Gland--physiopathology. WK 102 M5165 2004] QP572.M44M437 2004 612.4'92--dc22 2004016759

This book is dedicated to our families, without whom there would be nothing.

CONTENTS Preface ............................................................................................... xvii 1. Mechanisms Underlying Seasonal Regulation of Melatonin Synthesis in Rodents ......................................................... 1 Valérie Simonneaux, Marie-Laure Garidou, Christophe Ribelayga and Paul Pévet Annual Variations of the Melatonin Pattern .......................................... 1 Nervous and Endocrine Inputs Regulating the Annual Rhythm in Melatonin Synthesis ...................................................................... 3 Molecular Mechanisms Underlying the Annual Changes in Melatonin Secretion ...................................................................... 5 2. Oxidative Stress-Mediated Damage during in Vivo Ischemia-Reperfusion Injury: Protective Effects of Melatonin .............. 12 Russel J. Reiter, Rosa M. Sainz, Dun-Xian Tan and Juan C. Mayo Melatonin and Ischemia-Reperfusion Injury ....................................... 12 Melatonin and Cardiac I/R Injury ....................................................... 13 Melatonin and Neural I/R Injury ........................................................ 16 Melatonin and I/R Injury in Other Organs ......................................... 19 3. Melatonin and the Thyroid Gland ....................................................... 26 Andrzej Lewinski Melatonin and Thyroid Growth Processes ........................................... 27 Melatonin and Thyroid Function ........................................................ 29 Oxidative Stress, the Thyroid Gland and Melatonin ........................... 29 Pineal-Thyroid Relationship in Humans ............................................. 30 Thyroid Hormone-Stimulation of Pineal Function or Growth Processes ........................................................................ 31 4. The Role of Melatonin in the Development of Scoliosis ...................... 35 Keith M. Bagnall, Talib Rajwani, Jessie Kautz, Marc Moreau, V. James Raso, James Mahood, Ariana Daniel, Christina Demianczuk, Janet Wilson and Xaioping Wang Problems with Studying Scoliosis and Melatonin ................................ 36 The Pinealectomised Chicken Model for the Study of Scoliosis ........... 38 Serum Melatonin Levels in Humans with Scoliosis ............................. 41 A Proposed Model by Which Low Levels of Serum Melatonin Can Affect Vertebral Growth and Produce Scoliosis ........................ 41 5. Effect of Melatonin on Life Span and Longevity .................................. 45 Vladimir N. Anisimov Effect of Melatonin on Longevity in Mice ........................................... 45 Effect of Melatonin on Longevity in Rats ............................................ 48 Effect of Melatonin on Longevity in Fruit Flies ................................... 51 Effect of Melatonin on Longevity in Worms ....................................... 51

Melatonin as Antioxidant .................................................................... 52 Melatonin DNA Damage and Mutagenesis ......................................... 52 Melatonin and Apoptosis .................................................................... 53 Melatonin and Immune System .......................................................... 53 Effect of Melatonin on Gene Expression ............................................. 54 6. Cardiovascular Effects of Melatonin ..................................................... 60 Ewa Sewerynek 7. Pineal Gland and Cancer—An Epigenetic Approach to the Control of Malignancy: Evaluation of the Role of Melatonin ............................ 71 Christian Bartsch and Hella Bartsch Effect of Melatonin on Tumor Growth ............................................... 72 Analysis of Melatonin and of Its Metabolite 6-Sulfatoxymelatonin in Cancer Patients ........................................................................... 76 Analysis of Melatonin in Tumor-Bearing Animals ............................... 78 In Which Way Does the Depression of Circulating Melatonin in Cancer Patients Offer a Rationale for a Substitutional Therapy? ........................................................... 78 Potential Diagnostic Relevance of Melatonin in Oncology .................. 79 Potential Significance of (Patho)Physiological Changes of Melatonin for the Aetiology of Cancer ........................................ 80 8. Expression and Signal Transduction Pathways of Melatonin Receptors in the Pituitary ..................................................................... 88 Hana Zemkova, Ales Balik and Stanko S. Stojilkovic Photoperiods, Melatonin and Reproduction ........................................ 90 Localization of Melatonin Receptors ................................................... 91 Melatonin Actions in Gonadotrophs ................................................... 92 GnRH-Induced Signaling ................................................................... 92 Melatonin Effects on GnRH Signaling ................................................ 96 Development and Receptor Expression ............................................... 99 Perspectives ....................................................................................... 100 9. The Role of Thermoregulation in the Soporific Effects of Melatonin: A New Perspective ....................................................... 106 Saul S. Gilbert, Cameron J. van den Heuvel, Drew Dawson and Kurt Lushington Melatonin ......................................................................................... 106 Historical Overview: Sleep, Body Temperature and Melatonin ......... 107 The Thermoregulatory Effect of Melatonin ....................................... 107 Relationship between Sleep and Thermoregulation: An Overview ..... 108 Exploring the Mechanism of Action of Melatonin ............................. 109

10. The Role of Melatonin in Human Aging and Age-Related Diseases ................................................................... 118 Michal Karasek The Reasons Why a Role of Melatonin in Aging Is Postulated .......... 119 Melatonin in Postmenopausal Women .............................................. 123 Melatonin and Age-Related Diseases ................................................. 123 Possible Supplementation of Melatonin in Elderly Individuals .......... 124 11. Role of Endogenous and Exogenous Melatonin in Inflammation ....... 127 Salvatore Cuzzocrea Oxygen Radical Generation in Inflammation .................................... 127 Relative Importance of Endogenous Melatonin in Acute Inflammation .................................................................. 129 Melatonin Is Effective in Experimental Inflammation ....................... 131 Inflammatory Bowel Disease ............................................................. 132 12. Heterologous Modulation of Androgen Receptor Nucleo-Cytoplasmic Shuttling by Melatonin: A Novel Mode of Regulating Androgen Sensitivity ............................ 138 Nava Zisapel Effect of Melatonin on Androgen-Induced Gene Expression ............. 139 Effects of Melatonin on AR Protein Levels ........................................ 140 Effects of Melatonin on Androgen Binding Capacity ........................ 141 Effects of Melatonin on Target DNA Binding ................................... 141 Effects of Melatonin on AR Localization ........................................... 142 Clinical Implications Melatonin’s Effects .......................................... 143 13. Extrapineal Melatonin: Location and Role in Pathological Processes ...................................... 148 Igor M. Kvetnoy, Natalia S. Sinitskaya and Tatiana V. Kvetnaia Location of Extrapineal Melatonin .................................................... 149 Extrapineal Melatonin and Pathological Processes ............................. 152 Extrapineal Melatonin and Seasonal Rhythm Disorders .................... 152 Extrapineal Melatonin and Regulation of Gastrointestinal Functions ......................................................... 153 Extrapineal Melatonin: Oncological Aspects of Biological Significance ............................................................... 154 14. Sleep and Melatonin in Diurnal Species ............................................. 162 Irina V. Zhdanova Melatonin and Circadian Regulation of Sleep ................................... 163 Melatonin and Homeostatic Regulation of Sleep ............................... 163

15. The Effect of Different Wavelengths of Light in Changing the Phase of the Melatonin Circadian Rhythm ................................... 170 Helen R. Wright and Leon C. Lack Circadian Rhythm Sleep Disorders .................................................... 171 Light Therapy ................................................................................... 173 Phase Change Studies ........................................................................ 174 Clinical Effectiveness ......................................................................... 178 Photoreceptors .................................................................................. 179 16. Clinical Utility of the Antioxidant Melatonin in the Newborn ........... 184 Eloisa Gitto, Russel J. Reiter, Aurelio Amodio and Ignacio Barberi Introduction on Oxidative Stress ....................................................... 184 Oxidative Stress and Perinatal Asphyxia ............................................ 185 Respiratory Distress Syndrome and Oxidative Stress ......................... 185 Oxidative Stress and Neonatal Sepsis ................................................. 186 Antioxidant Therapy ......................................................................... 186 Melatonin as Antioxidant .................................................................. 187 17. Diurnal 5-HT Production and Melatonin Formation ........................ 193 Jimo Borjigin and Jie Deng 18. Melatonin and Mitochondrial Respiration ......................................... 196 Yuji Okatani, Akihiko Wakatsuki and Russel J. Reiter Mitochondria and Oxygen Free Radicals ........................................... 197 Melatonin and Ischemia/Reperfusion-Induced Oxidative Damage to Mitochondria .............................................. 198 Hepatic Ischemia/Reperfusion ........................................................... 198 Fetal Ischemia and Reperfusion ......................................................... 200 Potential Links between Melatonin and Aging .................................. 201 Age-Related Changes in Peroxidation Products of Lipids, Proteins and DNA in SAM ........................................................... 202 Age-Related Changes in Hepatic Mitochondrial Function ................. 203 19. Melatonin Use As a Bone-Protecting Substance ................................. 209 Daniel P. Cardinali, Marta G. Ladizesky, Verónica Boggio, Rodolfo A. Cutrera, Ana I. Esquifino and Carlos Mautalen Mammalian Bone Is Continuously Remodeled ................................. 209 Early Studies Indicated an Effect of Melatonin on Bone .................... 209 Melatonin Acts on Both Osteoblasts and Osteoclasts in Vitro ........... 210 Low Melatonin Levels Correlate with Osteoporosis ........................... 210 Melatonin Decreases Bone Loss in Vivo ............................................ 211 Promotion of Growth Hormone (GH) Release Could Partly Explain Melatonin Effect on Bone ................................................ 212

20. Melatonin, Light and Migraine .......................................................... 214 Bruno Claustrat, Christophe Chiquet, Jocelyne Brun and Guy Chazot The Regulating System of Melatonin Secretion ................................. 215 Migraine and Light ............................................................................ 216 Melatonin and Migraine .................................................................... 216 21. Melatonin in Protection against Oxidative Damage Caused by Potential Carcinogens .................................................................... 220 Malgorzata Karbownik Oxidative Damage Caused by Potential Carcinogens— Protective Effects of Melatonin ...................................................... 221 22. Influence of Melatonin on the Health and Diseases of the Retina .................................................................. 232 Allan F. Wiechmann Sites of Retinal Melatonin Synthesis and Action ................................ 233 Putative Functions of Melatonin in the Retina .................................. 234 Potential Role of Melatonin in Photoreceptor Cell Death ................. 237 23. Melatonin Synchronizes Cell Physiology through Cytoskeletal Rearrangements.................................................................................. 243 Gloria Benítez-King, Gerardo Ramírez-Rodríguez, David García and Fernando Antón-Tay Melatonin Synchronizes Dome Formation in MDCK Cells .............. 244 Melatonin Synchronizes Microfilament Reorganization in MDCK Cells ............................................................................. 246 Characterization of the Cellular Pathway by Which Melatonin Increases Ion and Water Transport ................................................ 248 Role of Protein Kinase C in the Mechanism by Which Melatonin Induces Microfilament Reorganization and Dome Formation ....... 248 24. Melatonin in Winter Depression ........................................................ 253 Arcady A. Putilov, Galena S. Russkikh and S.R. Pandi-Perumal Winter Depression ............................................................................ 253 Day Length Measurement ................................................................. 254 Daytime MLT Levels ........................................................................ 255 Circadian Phase ................................................................................. 255 Timing of Light Treatment ............................................................... 256 Sensitivity to Light ............................................................................ 257 Multi-Component Physiological Response to Light........................... 258

25. Delayed Sleep Phase Syndrome: A Melatonin Onset Disorder ........... 263 Marcel G. Smits and S.R. Pandi-Perumal Clinical Aspects of DSPS ................................................................... 263 Epidemiology .................................................................................... 264 Comorbidity ..................................................................................... 265 Onset of DSPS .................................................................................. 265 Familial Traits ................................................................................... 265 DSPS versus Owls ............................................................................. 266 Treatment ......................................................................................... 266 Pathophysiology ................................................................................ 267 Drug Induced Delayed Sleep Phase Syndrome .................................. 267 Diagnosis .......................................................................................... 267 Biological Clock ................................................................................ 268 Practice Points ................................................................................... 270 26. Melatonin as an Antidepressant for Treatment of Delayed Sleep Phase Syndrome with Comorbid Depression ............................ 273 Leonid Kayumov, Alan Lowe, Raed Hawa and Colin M. Shapiro Index .................................................................................................. 279

EDITORS S.R. Pandi-Perumal, M.Sc. Comprehensive Center for Sleep Medicine Department of Pulmonary, Critical Care and Sleep Medicine Mount Sinai School of Medicine New York, New York, U.S.A. Email: [email protected] Chapters 24, 25

Daniel P. Cardinali, M.D., Ph.D. Departamento de Fisiolgia Facultad de Medicina Universidad de Buenos Aires Buenos Aires, Argentina Email: [email protected] Chapter 19

CONTRIBUTORS Aurelio Amodio Institute of Medical Pediatrics Neonatal Intensive Care Unit University of Messina Messina, Italy

Keith M. Bagnall University of Alberta Edmonton, Alberta, Canada Email: [email protected] Chapter 4

Chapter 16

Vladimir N. Anisimov Department of Carcinogenesis and Oncogerontology N.N. Petrov Research Institute of Oncology St. Petersburg, Russia Email: [email protected] Chapter 5

Fernando Antón-Tay Departamento de Biología de la Reproducción División de Ciencias Biológicas y de la Salud Universidad Autónoma MteropolitanaIztapalapa México, D.F., México Chapter 23

Ales Balik Institute of Physiology Academy of Sciences of the Czech Republic Prague, Czech Republic Email: [email protected] Chapter 8

Ignacio Barberi Institute of Medical Pediatrics Neonatal Intensive Care Unit University of Messina Messina, Italy Chapter 16

Christian Bartsch Institute of Physiological Chemistry University of Tübingen Tübingen, Germany Email: [email protected] Chapter 7

Hella Bartsch Institute of Physiological Chemistry University of Tübingen Tübingen, Germany Chapter 7

Bruno Claustrat Service de Radioanalyse Hôpital Neuro-Cardiologique and Insitut Fédératif de Neurosciences Lyon, Cedex, France Email: [email protected]

Gloria Benítez-King Departamento Neurofarmacologia Subdirección de Investigaciones Clínicas Instituto Nacional de Psiquiatria Ramón de la Fuente Muñiz México, D.F., México Email: [email protected]

Chapter 20

Chapter 23

Chapter 19

Verónica Boggio Departamento de Fisiología Facultad de Medicinia Universidad de Buenos Aires Buenos Aires, Argentina

Salvatore Cuzzocrea Institute of Pharmacology School of Medicine University of Messina Torre Biologica—Policlinico Universitario Via C. Valeria Messina, Italy Email: [email protected]

Chapter 19

Jimo Borjigin Department of Embryology Carnegie Institution of Washington Baltimore, Maryland, U.S.A. Email: [email protected] Chapter 17

Rodolfo A. Cutrera Departamento de Fisiología Facultad de Medicinia Universidad de Buenos Aires Buenos Aires, Argentina

Chapter 11

Ariana Daniel University of Alberta Edmonton, Alberta, Canada Chapter 4

Jocelyne Brun Service de Radioanalyse Hôpital Neuro-Cardiologique Lyon, Cedex, France Chapter 20

Drew Dawson Centre for Sleep Research University of South Australia South Australia, Australia Chapter 9

Guy Chazot Service de Neurologie Hôpital Neuro-Cardiologique Lyon, Cedex, France

Christina Demianczuk University of Alberta Edmonton, Alberta, Canada

Chapter 20

Chapter 4

Christophe Chiquet Institut Fédératif de Neurosciences INSERM, Sérvice d’Ophtalmologie Hôpital Edouard Herriot Lyon, Cedex, France

Jie Deng Department of Embryology Carnegie Institution of Washington Baltimore, Maryland, U.S.A.

Chapter 20

Chapter 17

Ana I. Esquifino Departamento de Bioquímica y Biología Molecular III Facultad de Medicina Universidad Complutense Madrid, Spain

Michal Karasek Department of Electron Microscopy Medical University of Lodz Lodz, Poland Email: [email protected] Chapter 10

Chapter 19

David García Departamento Neurofarmacologia Subdirección de Investigaciones Clínicas Instituto Nacional de Psiquiatria Ramón de la Fuente Muñiz México, D.F., México

Malgorzata Karbownik Department of Thyroidology Institute of Endocrinology Medical University of Lodz Lodz, Poland Email: [email protected] Chapter 21

Chapter 23

Marie-Laure Garidou UMR-CNRS Neurobiologie des Rythmes Strasbourg, France Chapter 1

Saul S. Gilbert Centre for Sleep Research University of South Australia South Australia, Australia Chapter 9

Jessie Kautz University of Alberta Edmonton, Alberta, Canada Chapter 4

Leonid Kayumov Department of Psychiatry University of Toronto University Health Network Toronto Western Hospital Toronto, Ontario, Canada Email: [email protected] Chapter 26

Eloisa Gitto Institute of Medical Pediatrics Neonatal Intensive Care Unit University of Messina Messina, Italy Chapter 16

Raed Hawa Department of Psychiatry University of Toronto University Health Network Toronto Western Hospital Toronto, Ontario, Canada Chapter 26

Tatiana V. Kvetnaia Department of Cell Biology and Pathology St. Petersburg Institute of Bioregulation and Gerontology of the Russian Academy of Medical Sciences St. Petersburg, Russia Chapter 13

Igor M. Kvetnoy Department of Cell Biology and Pathology St. Petersburg Institute of Bioregulation and Gerontology of the Russian Academy of Medical Sciences St. Petersburg, Russia Email: [email protected] Chapter 13

Leon C. Lack School of Psychology Flinders University Adelaide, South Australia, Australia Email: [email protected] Chapter 15

Marta G. Ladizesky Sección Osteopatías Médicas Hospital de Clínicas “José de San Martín” Facultad de Medicina Universidad de Buenos Aires Buenos Aires, Argentina Chapter 19

Carlos Mautalen Sección Osteopatías Médicas Hospital de Clínicas “José de San Martín” Facultad de Medicina Universidad de Buenos Aires Buenos Aires, Argentina Chapter 19

Juan C. Mayo Department of Cellular and Structural Biology University of Texas Health Science Center San Antonio, Texas, U.S.A. Chapter 2

Andrzej Lewinski Department of Thyroidology Institute of Endocrinology Medical University of Lodz Lodz, Poland Email: [email protected] Chapter 3

Alan Lowe Department of Psychiatry University of Toronto University Health Network Toronto Western Hospital Toronto, Ontario, Canada Chapter 26

Kurt Lushington Centre for Sleep Research School of Psychology University of South Australia South Australia, Australia Email: [email protected]

Marc Moreau University of Alberta Alberta, Canada Chapter 4

Yuji Okatani Department of Clinical Nursing Science Kochi Medical School Nankoku, Kochi, Japan Email: [email protected] Chapter 18

Paul Pévet UMR-CNRS Neurobiologie des Rythmes Strasbourg, France Email: [email protected] Chapter 1

James Mahood University of Alberta Edmonton, Alberta, Canada

Arcady A. Putilov Institute for Medical and Biological Cybernetics Siberian Branch RAMS Novosibirsk, Russia Email: [email protected]

Chapter 4

Chapter 24

Chapter 9

Talib Rajwani University of Alberta Edmonton, Alberta, Canada Chapter 4

Gerardo Ramírez-Rodríguez Departamento Neurofarmacologia Subdirección de Investigaciones Clínicas Instituto Nacional de Psiquiatria Ramón de la Fuente Muñiz México, D.F., México Chapter 23

V. James Raso University of Alberta Edmonton, Alberta, Canada Chapter 4

Russel J. Reiter Department of Cellular and Structural Biology University of Texas Health Science Center San Antonio, Texas, U.S.A. Email: [email protected]

Ewa Sewerynek Institute of Endocrinology Medical University of Lodz Lodz, Poland Email: [email protected] Chapter 6

Colin M. Shapiro Department of Psychiatry University of Toronto University Health Network Toronto Western Hospital Toronto, Ontario, Canada Email: [email protected] Chapter 26

Valérie Simmoneaux UMR-CNRS Neurobiologie des Rythmes Strasbourg, France Email: [email protected] Chapter 1

Christophe Ribelayga UMR-CNRS Neurobiologie des Rythmes Strasbourg, France

Natalia S. Sinitskaya Department of Cell Biology and Pathology St. Petersburg Institute of Bioregulation and Gerontology of the Russian Academy of Medical Sciences St. Petersburg, Russia

Chapter 1

Chapter 13

Galena S. Russkikh Institute for Medical and Biological Cybernetics Siberian Branch RAMS Novosibirsk, Russia

Marcel G. Smits Department of Neurology and Sleep-Wake Disorders Hospital “De Gelderse Vallei” Ede, The Netherlands

Chapters 2, 16, 18

Chapter 25

Chapter 24

Rosa M. Sainz Department of Cellular and Structural Biology University of Texas Health Science Center San Antonio, Texas, U.S.A. Chapter 2

Stanko S. Stojilkovic ERRB, NICHD National Institutes of Health Bethesda, Maryland, U.S.A. Email: [email protected] Chapter 8

Dun-Xian Tan Department of Cellular and Structural Biology University of Texas Health Science Center San Antonio, Texas, U.S.A.

Helen R. Wright School of Psychology Flinders University Adelaide, South Australia, Australia Email: [email protected] Chapter 15

Chapter 2

Cameron J. van den Heuvel Centre for Sleep Research University of South Australia South Australia, Australia Email: [email protected]

Hana Zemkova Institute of Physiology Academy of Sciences of the Czech Republic Prague, Czech Republic Email: [email protected]

Chapter 9

Chapter 8

Akihiko Wakatsuki Department of Obstetrics and Gynecology Kochi Medical School Nankoku, Kochi, Japan

Irina V. Zhdanova Department of Anatomy and Neurobiology Boston University School of Medicine Boston, Massachusetts, U.S.A. Email: [email protected]

Chapter 18

Chapter 14

Xaioping Wang University of Alberta Edmonton, Alberta, Canada Chapter 4

Allan F. Wiechmann Department of Cell Biology University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma, U.S.A. Email: [email protected] Chapter 22

Janet Wilson University of Alberta Edmonton, Alberta, Canada Chapter 4

Nava Zisapel Department of Neurobiochemistry The George S. Wise Faculty of Life Sciences Tel Aviv University Tel Aviv, Israel Email: [email protected] Chapter 12

PREFACE In the last two decades, our understanding of the organization of the pineal gland and the functional significance of its major secretory product, melatonin, has considerably increased. While there have been many volumes written and edited on melatonin and its clinical usage, it is unusual to see one with such interdisciplinary breadth, contributions ranging from the very basic concepts of melatonin action at a cellular level to immediate applications in clinical medicine. This book represents an integration of clinical experiences and research of the contributors. Indeed the book was written, compiled, and edited for clinical endocrinologists. Every effort has been taken to make the text as accurate and up to date as possible. As vast amount of information was processed, inaccuracies or omissions may have occurred. Readers are encouraged to contact us about such errors. Such feedback is essential to the continued development of the book. S.R. Pandi-Perumal D.P. Cardinali

Two are better than one, Because they have a good reward for their labor. For if they fall, one will lift up his companion. But, woe to him who is alone when he falls, For he has no one to help him up. -Ecclesiastes 4:9

One of the great pleasures of editing this volume was the help and encouragement we received from various sources. We acknowledge here the contributions of numerous individuals who were instrumental in the production of this volume. The editors wish to express their sincere appreciation and owe endless gratitude to all the contributors for their scholarly contributions that facilitated the development of this book. We are grateful to the authors of the chapters, many of whom worked within a tight page constraints to conform to the space limitations of the book, and the same time to infuse their creativity and knowledge into their contribution. We wish to express our appreciation for the careful reading, critique, and support of this book by Dr. Landes at Landes Bioscience. We are indebted to Dr. Landes for the helpful advice and encouragement throughout the development of this book. We owe an infinite debt and gratitude to the staff at Landes Bioscience for their patience, perseverance, and help at every stage. They have been extremely helpful in guiding us through the process of publishing this volume. They deserve recognition and special thanks. Finally, it is our hope that this book conveys some of our own gratification from the opportunity afforded to us in the rapidly growing area of Neuroendocrinology. Because the field is a dynamic one, this book is intended to be thought-provoking rather than definitive. We hope the readers will find this volume even more informative and helpful. Last but not least, the editors would like to thank their families for their unfailing and everlasting support, love, kindness and patience, and for sacrificing all the precious time during the development and production of this volume. Our families have been patient and understanding of our need to spend time on this project. To all these people goes our sincere gratitude. S.R. Pandi-Perumal D.P. Cardinali

CHAPTER 1

Mechanisms Underlying Seasonal Regulation of Melatonin Synthesis in Rodents Valérie Simonneaux, Marie-Laure Garidou, Christophe Ribelayga and Paul Pévet

Abstract

S

ynthesis and release of pineal melatonin are increased at night with a season-dependent characteristic pattern. The seasonal alterations in melatonin production constitute a key endocrine message used to time annual functions with seasons. Although the nervous pathway and cellular/molecular mechanisms involved in the daily regulation of melatonin production have been intensively investigated, those responsible for the seasonal variations in melatonin synthesis and release have just started to be studied. This review aims at defining how the melatonin pattern transduces seasonal information in various rodent species, and what could be the nervous and endocrine inputs, as well as the cellular and molecular elements underlying the seasonal alterations of the melatonin pattern.

Introduction One of the most challenging adaptive processes mammals have to face with is to measure and anticipate the drastic annual changes of their environment in order to synchronize many biological functions with seasons. Several nervous and endocrine systems are involved in these biological adjustments, among which is the periodic synthesis and release of the pineal hormone melatonin. Melatonin indeed exhibits strong synchronizing properties based on its steady and reproducible daily and annual rhythms of circulating levels. Numerous studies have described the nervous pathways controlling the daily rhythm in pineal activity and dissected the molecular and cellular events leading to the marked nocturnal increase in melatonin release. Although the synchronisation of annual functions with the seasons is the most important function of melatonin, much less is known on the mechanisms underlying the annual regulation of its synthesis. The aim of this article is to review, in rodent species, (1) the critical parameters of the melatonin profile read as seasonal information, (2) the nervous and endocrine inputs controlling the annual rhythm of melatonin production and (3) the molecular mechanisms involved in the annual variations of melatonin synthesis.

Annual Variations of the Melatonin Pattern The main feature of melatonin is the gating of its synthesis and release to the night whatever animals are diurnal or nocturnal. Because night length depends on seasons, the nocturnal peak of melatonin exhibits typical seasonal alterations, which are pivotal for the timing of annual functions, especially reproduction.1,2

Melatonin: Biological Basis of Its Function in Health and Disease, edited by S.R. Pandi-Perumal and Daniel P. Cardinali. ©2006 Eurekah.com.

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Melatonin: Biological Basis of Its Function in Health and Disease

Figure 1. Schematic representation of the photoperiodic variations of the daily melatonin profile in various rodent species. LP= long photoperiod; SP= short photoperiod.

Strikingly, however, the photoperiodic modification of melatonin peak displays strong species differences, particularly among the rodents (Fig. 1, see ref. 3 for review). In rat, lengthening of the night in short photoperiod results in a increased delay between dark onset and the nocturnal increase in melatonin production. However, the decrease in melatonin is always locked to the end of the darkness (falling just before lights-on) whatever the photoperiod.4 In Syrian hamster, the delay between dark and melatonin onset lasts about 6 hours whatever the night length but the decrease in melatonin occurs at light onset in long photoperiod and before light onset in short photoperiod.5,6 In Siberian hamster, the delay between night onset and melatonin rise augments when night extends but, as in the Syrian hamster, the morning decline in melatonin occurs at light arrival in long photoperiod and before light arrival in short photoperiod.7-9 In European hamster, melatonin synthesis occurs earlier when the night length extends, in contrast to other species, and ends with light onset in long photoperiod and before lights-on in short photoperiod,10-12 this pattern resulting in very large seasonal variations in the melatonin peak. Finally, in the subtropical diurnal rodent Arvicanthis ansorgei, there is small night length variation on site (Mali) and therefore barely detectable annual changes in the melatonin peak duration. However, when animals are kept in animal facilities, the melatonin peak shows significant photoperiodic variations similar to that observed in the Syrian hamster (Garidou et al, submitted). These examples show that in general the duration of the nocturnal peak of melatonin increases with night length but not in a linear manner and up to a limit value (supposed to correspond to a maximal decompression of the hypothalamic clock driving the melatonin rhythm13). In addition, the manner the nocturnal peak is adjusted to dusk and dawn during change in night length varies among species suggesting that the “reading” of this seasonal endocrine message depends on species. In addition to the change in the peak length, several species undergo large variation in the amplitude of the melatonin peak (Fig. 1). This is the case for the Siberian hamster where the nighttime melatonin level is twice higher in short than in long photoperiod.7-9,14-16 More importantly, the European hamster exhibits a very large increase in melatonin peak amplitude in short compared to long photoperiod. This increase is even more important when animals are kept outdoors with the peak amplitude being approximately 5 times higher in winter than in summer.10-12 Arvicanthis ansorgei, exhibits annual (in outdoors conditions) but no photoperiodic (in indoors conditions) changes of the melatonin peak amplitude (Garidou et

Mechanisms Underlying Seasonal Regulation of Melatonin Synthesis in Rodents

3

al submitted). Even in the laboratory rat, considered as a non photoperiodic species, a small increase in melatonin peak amplitude was observed in short photoperiod.17 In most studies carried out to understand how seasonal information is transmitted to the body, the annual modification of the photoperiod is taken as the best (because the most reliable) indicator of seasons. Other environmental factors however exhibit marked seasonal changes: temperature, humidity, food availability. It is not known how these factors may interfere with the seasonal regulation of melatonin synthesis, but several studies have already pointed that changes in temperature11,18 or food quality (Garidou et al submitted) alter the rate (amplitude) of melatonin synthesis. Because each species appears to display its own way of transducing seasons into alteration of the melatonin peak, it is therefore probable that the “reading” of this endocrine message differs among species. According to the species, the length, the amplitude, the internal coincidence of the melatonin peak or a combination of any parameter may be important factors for the transmission of seasonal information and synchronisation of annual functions.1

Nervous and Endocrine Inputs Regulating the Annual Rhythm in Melatonin Synthesis The nervous pathways and neurotransmitters involved in the daily regulation of melatonin synthesis are rather well known (Fig. 2; for review see ref. 3). The daily pace is given by the endogenous clock located in the suprachiasmatic nucleus which is synchronized to a 24 h-period by the day/night variation in light intensity forwarded along the retino-hypothalamic tract.19 The day/night rhythm in suprachiasmatic nucleus activity (high at daytime and low at nighttime) is transmitted to the hypothalamic paraventricular nucleus (PVN), the intermediolateral part of the spinal chord and the superior cervical ganglia whose neurons project massively to the pineal gland.20 Recently, it was found that the PVN is constantly active to stimulate pineal activity; at daytime SCN projections release GABA to inhibit PVN and therefore pineal activities; at night-time in contrast the GABA inhibition is cancelled and additionally another neurotransmitter may stimulates PVN and pineal activities.21,22 The PVN output is forwarded to the spinal chord mainly via oxytocin23,24 then to the superior cervical ganglia mainly via acetylcholine.25 The sympathetic neurons contain various neurotransmitters but the main one controlled by the SCN activity and involved in the regulation of melatonin is norephinephrine (NE).26-28 NE release in the pineal gland is restricted to the night-time and induces a marked increase in melatonin synthesis. Neuropeptide Y (NPY) is also present in the pineal sympathetic nerve terminals and has been reported to alter melatonin synthesis.29-31 In addition to the dense sympathetic innervation, other fibers originating from various structures are present in the pineal gland (Fig. 2; for reviews see refs. 3, 32). Several central structures project numerous neurotransmitters to the pineal gland: PVN (vasopressin and oxytocin), lateral hypothalamus (hypocretin), habenula nucleus (substance P), thalamic intergeniculate nucleus (NPY) and raphe nuclei (serotonin). Additionally, the neurons of pterygopalatine (vasoactive intestinal peptide, VIP), trigeminal (pituitary activating adenylate cycle peptide, PACAP; substance P, calcitonin gene-related peptide) and otic ganglia (PACAP) innervate the pineal gland. Most of these peptides have been found to regulate melatonin synthesis in in vitro conditions. Finally, a panel of other molecules (acethylcholine, GABA, glutamate, steroid hormones, etc) have been identified in the pineal gland, some of them being able to modulate the noradrenergic control of pineal activity. Whereas the acute effect of the pineal transmitters and their involvement in the daily regulation of melatonin synthesis have been investigated in detail, it is not known which transmitters drive the seasonal regulation of melatonin synthesis. Because the hypothalamic clock integrates photoperiodic information.33-36 the release of NE in the pineal gland probably exhibits seasonal variations with a larger duration of release in short photoperiod. This assumption,

4

Melatonin: Biological Basis of Its Function in Health and Disease

Figure 2. Schematic representation of the various neural and endocrine inputs of the mammalian pineal gland. The main neural pathway, which transmits light information to the pineal gland, is shown with thick arrows. In addition, numerous other neural or endocrine inputs are known to reach the pineal gland. Note that there are inter-species differences in the density and origin of the afferent pineal nerve fibers and the nature of the different pineal transmitters. 5-HT= 5-hydroxytryptamine/serotonin; ACh= acetylcholine; CGRP= calcitonin gene-related peptide; Glu= glutamate; HCRT= hypocretin; IGL= intergeniculate leaflet of the geniculate body; IML= intermediolateral part of the spinal cord; NE= norepinephrine; NPY= neuropeptide Y; OT= oxytocin; PACAP= pituitary adenylate cyclase activating peptide; PVN= paraventricular nuclei of the hypothalamus; SCG= superior cervical ganglia; SCN= suprachiasmatic nuclei of the hypothalamus; SOM= somatostatin; sP= substance P; VIP= vasoactive intestinal peptide; VP= vasopressin.

however, requires to be verified. It would be interesting to explore whether the species differences in the seasonal pattern of melatonin peak are related to similar variations in NE release. Most likely, NE is not the only factor involved in the seasonal control of melatonin synthesis. Among the other pineal transmitters, NPY displays marked seasonal variations in the European hamster.37 Interestingly, the marked increase in NPY immunoreactivity observed in winter is associated with a 2-fold increase in the activity of hydroxyindole-O-methyltransferase, the last enzyme in melatonin synthesis.38 Seasonal variations in the pineal content of VP and OT have also been described in hedgehog,39 the VPergic and OTergic innervation of the pineal gland being very low in summer and increasing in winter. Similarly, a marked seasonal variation in OT content has also been observed in the bovine pineal with a 3-fold higher value in September compared to the other months.40 Pineal SOM content displays a seasonal variation with higher values during autumn/winter.41 Noteworthy, the few peptides found to exhibit annual changes are increased in autumn/winter, when the melatonin peak is larger. It has long been debated whether the annual change in circulating level of the gonadal hormones may alter pineal metabolism. However, no clear demonstration has been brought so far. In addition, intra-pineal factors may participate in the building of the seasonal rhythm in melatonin synthesis because it has been reported that pineal responsiveness to NE varies according to the photoperiod in rat.42,43

Mechanisms Underlying Seasonal Regulation of Melatonin Synthesis in Rodents

5

Figure 3. Metabolic pathway from the essential amino acid tryptophan (TRP) to melatonin in the pineal gland. The daily variations of the enzyme coding-mRNA level and activity are schematized according to data obtained from the rat pineal gland. AAAD= aromatic amino acid decarboxylase; AA-NAT= arylalkylamine-N-acetyltransferase; HIOMT= hydroxyindole-O-methyltransferase; TPOH= tryptophan hydroxylase.

Molecular Mechanisms Underlying the Annual Changes in Melatonin Secretion Melatonin is synthesised from the essential amino-acid tryptophan (Fig. 3) which is hydroxylated by tryptophan hydroxylase (TPOH) and decarboxylated into 5-hydroxytryptophan (5-HT or serotonin). Serotonin is the starting point of several metabolic routes but the main

6

Melatonin: Biological Basis of Its Function in Health and Disease

Figure 4. Cellular and molecular events induced by the nocturnal adrenergic stimulation of the rat pineal gland. NE released at night activates two types of postsynaptic adrenergic receptors: β1-type and a1-type. Activation of the β1-type results in a dramatic accumulation of the cyclic nucleotide cAMP. Activation of the a1-type AR substantially potentiates β1-AR activation through Ca2+ mobilization and PKC activation. The marked increase in cAMP content activates PKA, which 1) phosphorylates CREB into P-CREB that switches-on the expression of different genes, especially the Aa-nat coding gene and consequently increases AA-NAT activity; 2) phosphorylates AA-NAT allowing its interaction with the chaperone protein 14-3-3; 3) protects AA-NAT from lysis by the cytosolic proteasome. AA-NAT= arylalkylamine-N-acetyltransferase; AC= adenylate cyclase; cAMP= cyclic adenosine monophosphate; cAMP response element; CREB= cAMP-response element binding protein; IP3= inositol triphosphate; NE= norepinephrine; PKA= protein kinase cAMP-dependent; PKC= protein kinase Ca2+-dependent; PLC= phospholipase Ca2+-dependent.

one includes a first step of acetylation by arylalkylamine-N-acetyl transferase (AA-NAT) followed by methylation by hydroxyindole-O-methyltransferase (HIOMT) to give melatonin. Importantly, melatonin is not stored in the pinealocytes but released in the blood stream as soon as synthesized, where its half life is approximately 20 min. This implies that any variation in melatonin synthesis is rapidly translated into similar changes of melatonin circulating levels. This peculiar dynamic is of pivotal importance for the time-giving property of melatonin.

Molecular Mechanisms Underlying the Daily Changes in Melatonin Synthesis The cellular and molecular mechanisms involved in the nocturnal noradrenergic stimulation of melatonin synthesis have long been investigated in the rat pineal gland (Fig. 4; for review see refs. 3, 44, 45, 46). Briefly, NE released during the night binds to both α- and β-adrenergic receptors. Activation of β-adrenergic receptors is a necessary step leading to the increase in cAMP and cGMP levels. Activation of α-adrenergic receptors increases intracellular levels of Ca2+ and diacylglycerol, and activates PKC activity which further amplifies the β-adrenergic-induced increase in cAMP and cGMP levels. Whereas the effect of cGMP is still unknown, the nocturnal increase in cAMP induces PKA activation, a key event leading to the synthesis of melatonin: (1) PKA induces the phosphorylation of CREB, and P-CREB binding to the CRE site of the Aa-nat gene causes a large increase in Aa-nat mRNA followed by a rapid synthesis of AA-NAT protein; (2) PKA phosphorylates AA-NAT, which in turn binds to a 14-3-3 chaperone protein to allow binding with serotonin and acetylCoA and finally conversion of serotonin into N-acetylserotonin;

Mechanisms Underlying Seasonal Regulation of Melatonin Synthesis in Rodents

7

(3) PKA-induced phosphorylation of AA-NAT protects the enzyme from proteasome proteolysis. Nocturnal PKA activation therefore results into a large (approximately 50-70 fold) AA-NAT activation by transcriptional, translational and post-translational mechanisms.47,48 By contrast, the nighttime increase in TPOH and HIOMT activity is small. Besides NE, other pineal transmitters have been reported to acutely regulate enzyme activity and melatonin synthesis: VIP and PACAP bind to VPAC1 receptors to increase cAMP levels, Aa-nat gene expression and enzyme activity and melatonin synthesis and release; VP binds to V1a receptors to potentiate the β-adrenergic increase in the cAMP-induced AA-NAT activation and melatonin synthesis; NPY binds to presynaptic Y2 receptors to reduce NE release and to postsynaptic Y1 receptors to slightly reduce the NE-induced activation of AA-NAT activity but also to increase Ca2+ level and HIOMT activity; acetylcholine binds to presynaptic mACh receptors to reduce NE release and to postsynaptic nACh receptors to induce cell depolarization with a resulting release of glutamate, from pineal microvesicles, which in turn inhibits the secretion of melatonin; GABA inhibits NE-induced melatonin synthesis via GABAA receptors and inhibits the NE release via GABAB receptors (for review see ref. 3). All these studies performed in the rat pineal gland show that NE is the main neurotransmitter triggering the nocturnal stimulation of AA-NAT activity and melatonin synthesis but other transmitters are susceptible of modulating this adrenergic stimulation through modulation of NE release or interaction with several second messenger transduction pathways. It is important to note that the mechanisms involved in the stimulation of AA-NAT activity and melatonin synthesis display important species differences. In the golden hamster, an intermediate transcription factor is necessary between CREB phosphorylation and Aa-nat gene transcription, resulting in a long delay before the onset of melatonin synthesis;49 in human, sheep and bovine pineal glands, Aa-nat gene is constitutively expressed and the protein is constantly synthesized but destroyed at daytime by proteosomal proteolysis whereas at nighttime, AA-NAT is protected from proteasome by the cAMP/PKA pathway.50,51 At the end of the night the stop in NE release (initiated by clock or light according to species and night length, see above) results in a rapid decrease in cAMP level and PKA activity, which in turn: (1) stops CREB phosphorylation and Aa-nat transcription, (2) decreases AA-NAT activity and (3) allows proteosomal proteolysis of AA-NAT protein. Additionally a β-adrenergic-induced inhibitory transcription factor (ICER: immediate cAMP early responsive protein) inhibits Aa-nat gene transcription towards the end of the night.52-54

Molecular Mechanisms Underlying the Photoperiodic and Seasonal Changes in Melatonin Synthesis All mammalian species studied so far are experiencing seasonal alteration of melatonin secretion. Although the major (and most reproductive) seasonal modification of the environmental factor is the variation in day length (photoperiod), other factors such as temperature and food availability are modified and may interfere with melatonin synthesis. In the following part, a distinction between photoperiodic (indoors) and seasonal (outdoors) changes of the environment is made. When rodents are moved from a long to a short photoperiod, the duration and, in some cases, the amplitude of the nocturnal peak of melatonin are increased. The part taken by both AA-NAT and HIOMT in these modifications has been investigated.

Changes in Duration

In all rodent species studied, rat.4,17 Siberian hamster.7-9 European hamster,11,12 Syrian hamster,5,6,49 Arvicanthis (Garidou et al, submitted), there is a tight relationship between the onset/ offset of AA-NAT activity and that of melatonin indicating that AA-NAT drives the duration of the melatonin peak. In rat,17 European hamster12 and Syrian hamster.55 the onset of AA-NAT activity is preceded by that of Aa-nat mRNA while the offset of AA-NAT activity occurs before that of Aa-nat mRNA confirming the post-traductional regulation of AA-NAT activity at the end of the night.

8

Melatonin: Biological Basis of Its Function in Health and Disease

Figure 5. Photoperiodic variations in HIOMT activity in the pineal gland of rat (A) and Siberian hamster (B). A) Photoperiodic variations in the length of the nocturnal peak of Hiomt mRNA and the mean HIOMT activity over 24h in the rat pineal gland. Rats were raised under 16L/8D, 12L/12D or 8L/16D for 8 weeks and sacrificed at different time points throughout the 24h cycle. Values are given as mean ± SEM (n = 5), *: p < 0.05 as compared to other values (data obtained from Ribelayga et al, 1999). B) In the Siberian hamster, the increase in the nocturnal melatonin peak amplitude in a short photoperiod is associated with higher HIOMT activity but lower AA-NAT activity. Schematic presentation of the daily variations in AA-NAT and HIOMT activities and melatonin content in the pineal gland of Siberian hamster raised under short photoperiod (SP) or long photoperiod LP (extrapolated from Ribelayga et al, 2000).

Changes in Amplitude Some rodent species exhibit photoperiodic (Siberian hamster, European hamster) or seasonal (Syrian hamster, Siberian hamster, European hamster and Arvicanthis) changes in the melatonin peak amplitude, namely an increase in amplitude associated with lengthening of the night. In the rat,17 Siberian hamster7,9 (Fig. 5B), Syrian hamster,79 and Arvicanthis (Garidou et al, submitted) the amplitude of the peak of AA-NAT activity and Aa-nat mRNA is markedly reduced in short compared to long photoperiod. Strikingly, this diminution in AA-NAT activity is not associated with a decrease in melatonin peak amplitude (it may even be higher in some species) demonstrating that AA-NAT activity in the above-cited species is not the limiting factor for the rate of melatonin synthesis at night. The reason why AA-NAT activity is lowered in short photoperiod is still unknown. Several observations, however, suggest that it may result from an increase of the transcription repressor ICER in short photoperiod.42,56 Only in the European hamster, the large winter increase in melatonin peak amplitude is related to a similar rise in Aa-nat mRNA and AA-NAT activity.12

Mechanisms Underlying Seasonal Regulation of Melatonin Synthesis in Rodents

9

Although Hiomt gene expression is increased every night by NE (Fig. 3, this transcriptional activation has no short term (several hours) effect on HIOMT activity because of the long half life (>24h) of the protein.57 By contrast, we demonstrated that HIOMT activity is regulated on a long term range (several days) by the every night noradrenergic stimulation of Hiomt gene expression.57,58 Correspondingly, we reported that in short photoperiod, the nocturnal peak of Hiomt mRNA is longer and, more protein being synthesised, HIOMT activity is higher than in long photoperiod17 (Fig. 5A). The increase of HIOMT activity in short (or winter) compared to long (or summer) photoperiod is observed in the pineal gland of rat,17 European hamster38 and Siberian hamster.9 In rat, the increase in HIOMT activity is barely associated with an increase in melatonin production.17 By contrast, in Siberian hamster we have shown that the increase in HIOMT activity in short photoperiod is clearly and specifically related to the increase in melatonin amplitude, because in the same time the activity of AA-NAT is twice lower9 (Fig. 5B). In European hamster, both AA-NAT and HIOMT contribute to the large increase in melatonin peak amplitude in winter.12,38 Finally, in Arvicanthis there is 2 to 3 fold increase in melatonin peak amplitude in April compared to the other months. This increase, however, is not accompanied by a similar increase in AA-NAT and HIOMT activities, but could be related to tryptophan availability in the food because the diet is changing from green grass to seed in April (Garidou et al unpublished). In all rodent species, the photoperiodic/seasonal modification of the melatonin peak duration is clearly driven by the onset/offset of AA-NAT activity with species differences in the kinetic of this adjustment. The factors involved in the photoperiodic/seasonal modification of the melatonin peak amplitude, by contrast, appears to depend on species: HIOMT in Siberian and European hamsters, AA-NAT in European hamster, food quality (tryptophan availability) in Arvicanthis. In conclusion, the mechanisms underlying the photoperiodic/seasonal variations in melatonin synthesis in mammals have just started to be understood and need further investigation. Our studies suggest that photoperiod is not the only environmental factor involved in the seasonal regulation of melatonin synthesis. Future research will aim at identifying the part taken by other environmental cues, in particular temperature. Besides NE, numerous neurotransmitters especially neuropeptides, are known to regulate melatonin synthesis. Studies are in progress to determine whether these neurotransmitters would exert a fine seasonal tuning of melatonin synthesis. The understanding of seasonal regulation of melatonin synthesis has physio-pathological consequences since several human disorders are associated with seasonal changes.

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Melatonin: Biological Basis of Its Function in Health and Disease

8. Miguez JM, Récio J, Vivien-Roels B et al. Diurnal changes in the content of indoleamines, cathecholamines, and methoxyindoles in the pineal gland of the Djungarian hamster (Phodopus sungorus): Effect of photoperiod. J Pineal Res 1996; 21:7-14. 9. Ribelayga C, Pévet P, Simonneaux V. HIOMT drives the photoperiodic changes in the amplitude of the melatonin peak of the Siberian hamster. Am J Physiol 2000; 278:R1339-R1345. 10. Vivien-Roels B, Pévet P, Masson-Pévet M et al. Seasonal variations in the daily rhythm of pineal gland and/or circulating melatonin and 5-methoxytryptophol concentrations in the European hamster, Cricetus cricetus. Gen Comp Endocrinol 1992; 86:239-247. 11. Vivien-Roels B, Pitrosky B, Zitouni M et al. Environmental control of the seasonal variations in the daily pattern of melatonin synthesis in the European hamster, Cricetus cricetus. Gen Comp Endocrinol 1997; 106:85-94. 12. Garidou ML, Vivien-Roels B, Pévet P et al. Mechanisms regulating the marked seasonnal variations in melatonin synthesis in the European hamster pineal gland. Am J Physiol 2003; 284:R1043-R1052 13. Illnerova H, Vanecek J. Pineal N-acetyltransferase: A model to study properties of biological clocks. In: Trentini GP, De Gaetani C and Pévet P eds. Fundamentals and Clinics in Pineal Research. New-York: Raven Press, 1987:165-178. 14. Hoffmann K, Illnerova H, Vanecek J. Comparison of pineal melatonin rhythms in young adult and old Djungarian hamsters (Phodopus sungorus) under long and short photoperiods. Neurosci Lett 1985; 56:39-43. 15. Lerchl A, Schlatt S. Serotonin content and melatonin production in the pineal gland of the male Djungarian hamster (Phodopus sungorus). J Pineal Res 1992; 12:128-134. 16. Steinlechner S, Baumgartner I, Klante G et al. Melatonin synthesis in the retina and pineal gland of Djungarian hamsters at different times of the year. Neurochem Int 1995; 27:245-251. 17. Ribelayga C, Garidou Ml, Malan A et al. Photoperiodic control of the rat pineal arylalkylamine-N-acetyltransferase and hydroxyindole-O-methyltransferase gene expression and its consequence on melatonin synthesis. J Biol Rhythms 1999; 14:105-115. 18. Nir I, Hirschmann N, Sulman FG. The effect of heat on rat pineal hydroxyindole-O-methyltransferase activity. Experientia 1975; 31:867-868. 19. Klein DC, Moore RY. Pineal N-acetyltransferase and hydroxyindole-O-methyltransferase: Control by the retinohypothalamic tract and the suprachiasmatic nucleus. Brain Res 1979; 174:245-262. 20. Larsen PJ. Tracing autonomic innervation of the rat pineal gland using viral transneuronal tracing. Microsc Res Tech 1999; 46:296-304. 21. Kalsbeek A, Garidou ML, Palm IF et al. Melatonin sees the light: Blocking GABA-ergic transmission in the paraventricular nucleus induces daytime secretion of melatonin. Eur J Neurosci 2000 ; 12:3146-3154. 22. Perreau-Lenz S, Kalsbeek A, Garidou ML et al. Suprachiasmatic control of melatonin synthesis in rats : Inhibitory and stimulatory mechanisms. Eur J Neurosci 2003; 17:221-228 23. Gilbey MP, Coote JH, Fleetwood-Walker S et al. The influence of the paraventriculo-spinal pathway, and oxytocin and vasopressin on sympathetic preganglionic neurons. Brain Res 1982; 251:283-290. 24. Cechetto DF, Sapper CB. Neurochemical organization of the hypothalamic projection to the spinal cord. J Comp Neurol 1988; 272:579-604. 25. Kasa P, Dobo E, Wolff JR. Cholinergic innervation of the mouse SCG: Light and electron microscopic immunocytochemistry for choline acetyltransferase. Cell Tissue Res 1991; 265:151-158. 26. Kappers JA. The development, topographical relations and innervation of the epihysis cerebri in the albino rat. Z Zellforsch 1960; 52:163-215. 27. Axelrod J, Shein HM, Wurtman RJ. Stimulation of C14-melatonin synthesis from C14-tryptophan by noradrenaline in rat pineal in organ culture. Proc Natl Acad Sci USA 1969; 62:544-549. 28. Drijfhout WJ, van der Linde AG, De Vries JB et al. Microdialysis reveals dynamics of coupling between noradrenaline release and melatonin secretion in conscious rats. Neurosci Lett 1996; 202:185-188. 29. Zhang ET, Mikkelsen JD, Møller M. Tyrosine hydroxylase- and neuropeptide Y-immunoreactive nerve fibers in the pineal complex of untreated rats and rats following removal of the superior cervical ganglia. Cell Tissue Res 1991; 265:63-71. 30. Simonneaux V, Ouichou A, Craft C et al. Presynaptic and postsynapic effects of neuropeptide Y in the rat pineal gland. J Neurochem 1994; 62:2464-2471. 31. Simonneaux V, Rodeau JL, Calgari C et al. Neuropeptide Y increases intracellular calcium in rat pinealocytes. Eur J Pharmacol 1999; 11:725-728. 32. Simonneaux V. Neuropeptides of the mammalian pineal gland. Neuroendocrinol Lett 1995; 17:115-130.

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33. Sumova A, Travnickova Z, Peters R et al. The rat suprachiasmatic nucleus is a clock for all seasons. Proc Natl Acad Sci USA 1995; 92:7754-7758. 34. Vuillez P, Jacob N, Teclemariam-Mesbah R et al. In Syrian and European hamsters, the duration of sensitive phase to light of the suprachiasmatic nuclei depends on the photoperiod. Neurosci Lett 1996; 208:37-40. 35. Pévet P, Pitrosky B, Vuillez P et al. The suprachiasmatic nucleus: The biological clock for all seasons. In: Buijs RM, Kalsbeek A, Romijn HJ, Pennartz CMA and Mirmiran M, eds. Hypothalamic Integration of Circadian Rhythms. Progress in Brain Research, Vol 111. Amsterdam: Elsevier Science BV, 1996:369-384. 36. Hastings M. Modeling the molecular calendar. J Biol Rhythms 2001; 16:117-123. 37. Møller M, Masson-Pévet M, Pévet P. Annual variations of the NPYergic innervation of the pineal gland of the European hamster (Cricetus cricetus). A quantitative immunohistochemical study. Cell Tissue Res 1998; 291:423-431. 38. Ribelayga C, Pévet P, Simonneaux V. Possible involvement of neuropeptide Y in the seasonal control of hydroxyindole-O-methyltransferase in the pineal gland of the European hamster (Cricetus cricetus). Brain Res 1998; 801:137-142. 39. Nürnberger F, Korf HW. Oxytocin- and vasopressin-immunoreactive nerve fibers in the pineal gland of the hedgehog, Erinaceus europaeus L. Cell Tissue Res 1981; 220:87-97. 40. Badiu C, Badiu L, Coculescu M et al. Presence of oxytocinergic neuronal-like cells in the bovine pineal gland: An immunocytochemical and in situ hybridization study. J Pineal Res 2001; 31:273-280. 41. Peinado M, Viader M, Reiter RJ et al. Immunoreactive somatostatin diurnal rhythms in pineal, retina and Harderian gland: Effects of sex, season, continuous darkness and estrous cycle. J Neural Transm 1990; 81:63-72. 42. Foulkes NS, Duval G, Sassone-Corsi P. Adaptative inducibility of CREM as transcriptional memory of circadian rhythms. Nature 1996; 381:83-85. 43. Guillaumond F, Becquet D, Bosler O et al. Adrenergic inducibility of AP-1 binding in the rat pineal gland depends on prior photoperiod. J Neurochem 2002; 83:157-66. 44. Klein DC. Photoneural regulation of the mammalian pineal gland. In Everet D and Clark D, eds. Photoperiodism, melatonin and the pineal. Pitman, London: Ciba Foundation Symposium, 1985:38-56. 45. Sugden D. Melatonin biosynthesis in the mammalian pineal gland. Experientia 1989; 45:922-932. 46. Korf HW, Schomerus C, Stehle JH. The pineal organ, its hormone melatonin, and the photoneuroendocrine system. Adv Anat Embryol Cell Biol 1998; 146:1-100. 47. Ganguly S, Gastel JA, Weller JL et al. Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis. Proc Natl Acad Sci USA 2001; 98:8083-8088. 48. Ganguly S, Coon SL, Klein DC. Control of melatonin synthesis in the Mammalian pineal gland: The critical role of serotonin acetylation. Cell Tissue Res 2002; 309:127-137. 49. Garidou ML, Diaz E, Pévet P et al. Transcription factors may frame Aa-nat gene expression and melatonin synthesis in the Syrian hamster pineal gland. Endocrinology (in press). 50. Klein DC, Coon SL, Roseboom PH et al. The melatonin rhythm-generating enzyme: Molecular regulation of serotonin N-acetyltransferase in the pineal gland. Recent Prog Horm Res 1997; 52:307-358. 51. Schomerus C, Korf HW, Laedtke E et al. Selective adrenergic/cyclic AMP-dependent switch-off of proteasomal proteolysis alone switches on neural signal transduction: An example from the pineal gland. J Neurochem 2002; 75:2123-2132. 52. Klein DC, Buda MJ, Kapoor CL et al. Pineal serotonin N-acetyltransferase activity: Abrupt decrease in adenosine 3'-5'-monophosphate may be signal for “turnoff”. Science 1978; 199:309-311. 53. Gastel JA, Roseboom PH, Rinaldi PA et al. Melatonin production: Proteosomal proteolysis in serotonin N-acetyltransferase regulation. Science 1998; 279:1358-1360. 54. Maronde E, Pfeffer M, Olcese J et al. Transcription factors in neuroendocrine regulation: Rhythmic changes in PCREB and ICER levels frame melatonin synthesis. J Neurosci 1999; 19:3326-3336. 55. Garidou ML, Gauer F, Vivien-Roels B et al. Pineal arylalkylamine N-acetyltransferase gene expression is highly stimulated at night in the diurnal rodent, Arvicanthis ansorgei. Eur J Neurosci 2002; 15:1632-1640. 56. Foulkes NS, Borjigin J, Snyder SH et al. Transcriptional control of circadian hormone synthesis via the CREM feedback loop. Proc Natl Acad Sci USA 1996; 93:14140-14145. 57. Ribelayga C, Gauer F, Pévet P et al. Photoneural regulation of rat pineal hydroxyindole-Omethyltransferase (HIOMT) messenger ribonucleic acid expression: An analysis of its complex relationship with HIOMT activity. Endocrinology 1999; 140:1375-1384. 58. Ribelayga C, Pévet P, Simonneaux V. Adrenergic and peptidergic regulations of hydroxyindole-Omethyltransferase in rat pineal gland. Brain Res 1997; 777:247-250.

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Melatonin: Biological Basis of Its Function in Health and Disease

CHAPTER 2

Oxidative Stress-Mediated Damage during in Vivo Ischemia-Reperfusion Injury: Protective Effects of Melatonin Russel J. Reiter, Rosa M. Sainz, Dun-Xian Tan and Juan C. Mayo

Melatonin and Ischemia-Reperfusion Injury Summary

T

he temporary interruption of blood flow to an organ followed by reperfusion of the tissue with oxygenated blood is highly destructive to the affective cells. While this process, generally referred to as ischemia/reperfusion, can happen in any organ, when this sequence of events occurs in the heart (as a heart attack) or in the brain (as a stroke) the consequences are especially devastating, often leading to death of the individual. While the pathophysiological changes that occur during ischemia/reperfusion are highly complex, a major feature accounting for the resulting damage is the generation of destructive oxygen and nitrogen-based reactants, some of which are free radicals. These menacing agents mutilate essential molecules thereby compromising their function and leading to cellular death. This review summarizes the data which documents the high efficacy of the antioxidant melatonin in limiting tissue damage and death during ischemia/reperfusion injury. This protective action of melatonin has been documented in experimental models of ischemia/reperfusion in the brain, heart, stomach, lower gastrointestinal tract, liver, pancreas, lung and urinary bladder. Regardless of the tissue examined, melatonin has never failed to reduce the damage resulting from temporary interruption of the blood flow followed by reperfusion. Considering these findings, melatonin should be tested in humans in an attempt to mute ischemia/reperfusion damage.

Introduction

The discovery of melatonin as an antioxidant and free radical scavenger1-4 has encouraged an extensive series of reports in which melatonin has been tested as a protector against a vast array of conditions in which free radicals and/or associated reactants account for at least part of the molecular and tissue damage that occurs in these situations.5-9 Virtually without exception, melatonin has proven to be highly effective in attenuating molecular mutilation and cellular death in these conditions and some of the mechanisms whereby melatonin functions as a free radical scavenger and antioxidant have been identified.10-12 Ischemia/reperfusion (I/R) injury is a condition in which the blood supply to an organ is temporarily interrupted followed by its reperfusion with oxygenated blood. This series of events precipitates in a cascade of reactions which result in the generation of massive numbers of oxygen as well as nitrogen-based radicals and other toxic reactants (Fig. 1) which destroy the affective tissue which leads to serious impairment of function or death.13-15 An episode of I/R

Melatonin: Biological Basis of Its Function in Health and Disease, edited by S.R. Pandi-Perumal and Daniel P. Cardinali. ©2006 Eurekah.com.

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Figure 1. While the bulk of the inhaled oxygen (O2) is used in mitochondria for the generation of ATP, a small percentage (< 5%) is chemically reduced to metabolites that can be highly reactive. Some of the most reactive, and therefore damaging, are the hydroxyl radical (•OH) and the peroxynitrite anion (ONOO-). Melatonin scavenges these reactants as summarized in several recent reviews (see text). NOS= nitric oxide synthase; GPx= glutathione peroxidase; GRd= glutathione reductase; GSH= reduced glutathione; GSSG= oxidized glutathione.

in cardiac tissue is known as heart attack while in the brain it is referred to as stroke. Both these conditions are prevalent in virtually all societies and ethnic groups and, besides compromising the quality of life of the individuals in whom they occur, the associated medical costs are straining family as well as governmental health care resources. In recent years several procedures have been introduced with the intent of reducing the severity of the tissue damage and function that occurs during I/R injury. One such therapy is the administrations of antioxidants which neutralize the oxygen and nitrogen-based reactants that cause much of the tissue mutilation.16,17 The current review summarizes the use of the newly-discovered antioxidant, melatonin,8-12 in reducing tissue damage resulting from I/R. In the models that have been used for these studies, melatonin has generally been shown to be highly effective in lowering the amount of tissue damage as well as improving organ function.18,19 The success of these studies should encourage melatonin’s use in clinical situations of I/R.

Melatonin and Cardiac I/R Injury A number of recent reports have examined the ability of melatonin to curtail the severity of the damage inflicted on cardiac tissue when it is subjected to experimentally induced, transient ischemia followed by reperfusion (Fig. 2). Using the Langendorff rat heart model, Tan et al20 interrupted the blood flow in the descending coronary artery, a procedure which reduces the

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Melatonin: Biological Basis of Its Function in Health and Disease

Figure 2. Some of the mechanisms by which ischemia and reperfusion induce structural and functional damage to tissues. While free radicals contribute significantly to the molecular destruction, other processes add to the resulting damage. Melatonin, due to its direct free radical scavenging and indirect antioxidative activities, among other actions, protects against ischemia/reperfusion as summarizes in this current report.

total blood supply to the heart by an estimated 25%, for 10 min and then removed the ligature to permit reperfusion to occur. During a 10 min reperfusion period electrophysiological measurements showed the hearts were undergoing premature ventricular contractions (PVC) and/ or ventricular fibrillation (VF). Melatonin, infused during both the occlusive as well as the period of reflow, significantly attenuated both the PVC and VF. In this study, concentrations of

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melatonin in the perfusate ranged from 1-50 µM. Vitamin C also was used to compare its efficacy in protecting the heart from the aberrant contractile activity with that of melatonin. At a concentration (500 µM) that greatly exceeded that of melatonin, ascorbate was less effective in limiting the cardiac arrhythmias induced by I/R. Soon thereafter, the ability of melatonin to reduce abnormal contractions in the hypoxic/ reoxygenated rat heart were confirmed.21 Also using the isolated rat heart model, the addition of melatonin to the reperfusion medium (after a 30 min ischemic episode) was found to decrease ventricular tachycardia and VF relative to their frequencies in the hearts that were only subjected to I/R. Other physiological parameters of the heart, i.e., left ventricular pressure, were also improved as a consequence of melatonin administration. Furthermore, when 2,3-dihydroxybenzoic acid [a product that is formed when salicylate scavenges a hydroxyl radical (•OH)] was measured in the perfusate from the I/R heart vessels, melatonin was found to significantly reduce the amount of this metabolite indicating that melatonin had scavenged the highly destructive •OH. This observation, coupled with the reduced level of lipid peroxidation products in the I/R hearts that were treated with melatonin argue for the mechanism of the indole’s protective actions being related to its free radical scavenging activity. When melatonin (1 or 10 mg/kg BW) was given to rats 30 min in advance of the use of their hearts in the I/R Langendorff model, Lagneux and coworkers22 described what they referred to as “spectacular” protection of cardiac function and morphology. Given that free radicals are widely accepted as accounting for the functional alterations of the heart during hypoxia and reoxygenation (Fig. 2), the authors conjectured that the ability of melatonin to scavenge free radicals explained its marked protective actions. The morphological measurements in this study included an estimation of infarct volume which was noticeably reduced in the rats that had been given melatonin. The high efficacy of melatonin in protecting the heart from a transient interruption of blood flow followed by reoxygenation stimulated others to examine these relationships as well and, without exception, they reaffirmed the ability of melatonin to pharmacologically reduce cardiac damage and improve cardiac function under conditions of I/R. Szarszoi and coworkers23 infused melatonin either before ischemia and during cardiac reperfusion or only during the reperfusion interval and the indole, at a concentration of 10 µmol/l, improved contractile function (reduced VF and improved the arrhythmia score in both cases). Again, these authors concluded that melatonin’s beneficial actions are in accordance with its potent antioxidant activity. The first totally in vivo study in which melatonin was tested for its ability to reduce cardiac injury under conditions of I/R was reported by Lee et al.24 This group injected a single bolus of melatonin, either 0.5, 1.0 or 5.0 mg/kg BW, before temporary occlusion of the left coronary artery and, as endpoints, they evaluated the degree of ventricular tachycardia and fibrillation as well of PVC. In terms of each parameter, melatonin improved cardiac function. Additionally, melatonin reduced superoxide anion radical (O2•-) production and lowered myloperoxidase activity (an index of neutrophil infiltration) in the damaged heart tissue. Most importantly, none of the rats that received one of the two highest doses of melatonin, i.e., either 1 or 5 mg/ kg BW died (10 or 10 survived), while 8 or 9 of the rats in the nonmelatonin treated group died. The ability of melatonin to reduce polymorphonuclear leucocyte infiltration contributes to its total antioxidant protection given that these cells generate a host of oxygen-based reactants in damaged tissue.25 Although, based on the studies they performed, Szarszoi and colleagues23 speculated that physiological levels, whatever they are,26 probably would not be effective in reducing cardiac damage during I/R injury. This, however, may not be the case given that Sahna et al27,28 found that pinealectomy, which removes a major source of circulating melatonin, caused the cardiac lesions that developed after left coronary artery occlusion to be more severe compared to those in intact controls (which had normal endogenous levels of melatonin). Additionally, they reported a 63% mortality in the melatonin-deficient rats compared to only 25% death rate in the controls. Infarct volume was also greater after pinealectomy. These findings not only indicate

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Melatonin: Biological Basis of Its Function in Health and Disease

that the quantity of melatonin endogenously produced provides significant protection against damage to the heart when an I/R incident occurs, but the known reduction of melatonin in advanced age29 is likely detrimental since an important antioxidant, which is normally protective of the heart, is greatly reduced. To date melatonin has not been used as a potential treatment to reduce tissue damage in the heart of humans with cardiac I/R; however, circulating levels of melatonin as well as indices of oxidative damage have been investigated in these patients. The study was performed in subjects in the first 24 hours after admission to the coronary care unit.30 Venous blood samples were collected during the day (10:00h) and at night (03:00h) for the measurement of melatonin, products of lipid peroxidation and glutathione peroxidase activity. Compared to controls, the patients with myocardial infarction had reduced levels of nocturnal melatonin, lower glutathione peroxidase activity and generally elevated levels of products of lipid peroxidation; the latter two measures are indicative of elevated oxidative stress in the subjects with acute I/R injury. How or if the lower circulating melatonin concentrations relate to the cardiovascular episode that occurred in these subjects is unknown. It is feasible that melatonin is reduced in patients with I/ R injury because the indole is more rapidly taken into the damaged tissue where it functions as an antioxidant to resist the increased oxidative stress (which is apparent from the elevated lipid peroxidation and decreased glutathione peroxidase activity). There are animal studies which document a rapid drop in circulating melatonin values during periods of elevated utilization of O2 and heightened free radical generation. Conversely, the patients may have had reduced levels of melatonin in advance of the damaging myocardial incident which, theoretically at least, may have enhanced the likelihood of the I/R episode or led to greater damage, as suggested by the rat studies summarized above.27,28 Finally, it is also possible that the observed lower melatonin levels were unrelated to the either the onset or the progression of the I/R episode. The report by Dominguez-Rodriguez and coworkers30 is not the first one to document reduced levels of melatonin in patients with coronary artery disease. Brugger et al31 and Brugger and Herold32 also measured lower levels of circulating melatonin in patients with I/R injury while Sakotnik and colleagues33 observed reduced excretion of a major melatonin metabolite, 6-hydroxymelatonin sulfate, in the urine of such patients. Thus, compromised melatonin production or elevated melatonin uptake seems to be a common feature associated with coronary artery disease but specifically how or if it relates to these conditions remains unknown. Finally, in patients with cardiac syndrome X an attenuated rise in nocturnal serum melatonin levels relative to those in age-matched controls has also been reported.34 Clearly, what is desperately needed are more complete studies on the association of melatonin with cardiovascular diseases of all types, e.g., atherosclerosis, I/R injury, etc., to determine if in fact physiological levels of melatonin, which decrease with age, are functionally related to cardiovascular function. Additionally, the use of melatonin in the treatment of patients with I/ R injury should be considered.18 Arguments for melatonin’s use in these conditions can certainly be justified and are supported by the data summarized above.17,35

Melatonin and Neural I/R Injury As with I/R of the heart, the consequences of hypoxia/reoxygenation in the brain are devastating and often lead to permanent disability or death. Furthermore, many of the processes are the same and the involvement of free radicals as destructive agents is accepted. Several models of brain focal I/R have been used to examine the protective actions of melatonin against the resulting damage. Pinealectomy, which results in a relative melatonin deficiency, was shown to exacerbate neurological damage after both photothrombotic-induced stroke as well as after transient interruption of the blood supply to the brain via occlusion of the middle cerebral artery.36 The extent of brain injury was greater 24h after photothromobotic stroke in rats lacking their pineal gland relative to that in pineal-intact animals. Likewise, the amount of damage resulting from middle cerebral artery occlusion (MCAO) was magnified in

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pinealectomized rats. The endpoints in this study included infarct volume and the number of apoptotic cells in the brain. The same group showed that replenishing melatonin by means of its injection (2.5 mg/kg) 30 min in advance of ischemia onset and at 1 and 2 h after the reestablishment of blood flow, reversed the negative effects of pinealectomy on neurological damage.37 A relative melatonin deficiency may not be the only consequence of pinealectomy. Thus, loss of the pineal gland may alter arterial blood pressure, cerebral blood flow, arterial blood gases, hemodynamic parameters and the hematocrit, all of which could impact the degree of cerebral damage during I/R. Because of this, these were monitored in a subsequent study and found not to be substantively changed, yet melatonin (4 mg/kg) reduced brain injury and neurological disability that accompanied I/R injury in pinealectomized rats.38 In intact rats as well, providing supplemental melatonin before interrupting blood flow to the brain as well as during reperfusion attenuates the resulting brain damage. When melatonin was given before endovascular MCAO, at 11 and 19 days following the insult the infarct volume in the cortex and striatum of rats was reduced by roughly 60% and 30%, respectively.39 Furthermore, the locomotor deficits that followed I/R were significantly less severe in the melatonin-treated rats; this correlated with increased glial cell survival as a result of indole administration. Consistent with the findings of Borlongan and coworker,39 Sinha et al40 found a decrease in the volume of the ischemic lesion (estimated on diffusion weighted magnetic resonance imaging at 30 min after reperfusion) in the brain of melatonin-treated rats after MCAO. Additionally, melatonin reduced the severity of the neurological deficiencies and the level of lipid peroxidation resulting from I/R injury. Similar results were obtained recently by Kondoh and colleagues.41 Realizing that the reduction of cerebral edema is an important factor in improving the outcome after I/R injury, this group used magnetic resonance imaging (MRI) to evaluate the degree of ischemia-induced edema in rats after MCAO and they correlated these findings with neural infarct volume when the brains were collected at the termination of the study. The MRI observations on live animals revealed a marked reduction of edema as a consequence of melatonin administration; this lowering of edema was especially obvious in the cortex (Fig. 3). The volume of the resulting infarcts also positively correlated with the reduced edema. Thus, infarct volume was less in the cortex than in the striatum in the melatonin-treated rats suffering from MCAO. The degree of edema and infarct volume was highly significantly reduced as a result of melatonin administration (Fig. 3). The most active investigators in this field have been the group of Cheung et al.19 They have used various melatonin treatment schedules to attenuate neurological damage resulting from MCAO. Treatment of rats before ischemia onset with melatonin (5 or 15 mg/kg) significantly reduced infarct volume by roughly 40% at 72 hours after ischemia onset.42,43 Melatonin achieved this effect without changing the hemodynamic parameters or cerebral blood flow relative to these indices in rats with MCAO only. This group also found that a 5 mg/kg dose of melatonin given 1 h after ischemia onset during a 3 h endovascular MCAO in rats significantly reduced neural damage and the degree of protection was enhanced if additional melatonin injections were given.44 Finally, in an attempt to define, in part, the mechanisms by which melatonin protects against neural I/R injury, Pei et al45 used electron paramagnetic resonance spectroscopy to estimate nitric oxide (NO) concentrations in the brain at 15 min after MCAO. NO levels were clearly elevated at the site of the lesion in these rats; however, if the animals had been treated with melatonin (1.5, 5 or 50 mg/kg) 30 min before ischemia onset, NO levels were noticeably suppressed. Under conditions of I/R, NO is highly toxic and contributes to neurological damage. Cyclooxygenase 2 (COX-2) also plays a significant pathogenetic role in I/R injury. When melatonin was combined with meloxican, a COX-2 inhibitor, in MCAO-induced stroke in the rat, the neurological outcome was improved relative to that in rats treated only with the COX-2 inhibitor;46 indeed, meloxican by itself was ineffective in reducing neural damage suggesting that, in the combined treatment, the only protection may have been that provided by melatonin.

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Melatonin: Biological Basis of Its Function in Health and Disease

Figure 3. The top panel shows the degree of edema (white patches), as estimated by MRI on live rats, after middle cerebral artery occlusion without (a) and with (b) melatonin treatment. The middle panel is a quantification of these results. Bottom panel shows the size of the lesioned area (white patches) in the brain of the same animals at the conclusion of the study. As with edema, melatonin treatment (b) greatly reduced the size of the infarct seen in the nontreated rats (a). From reference 41.

Global ischemia models have been less frequently used to test the efficacy of melatonin in reducing I/R injury in the brain. After temporary bilateral occlusion (for 10, 20 or 30 min) of the carotid arteries in rats, melatonin given at the onset of reperfusion significantly preserved the integrity of the CA1 pyramidal neurons of the hippocampus which, in the absence of melatonin treatment, were destroyed as a consequence of I/R.47

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A frequently used animal model of global ischemia is the Mongolian gerbil (Meriones unguiculatus). Its utility for this purpose is due to an incomplete circle of Willis at the base of the brain. A single injection of melatonin (10 mg/kg) given 30 min before bilateral common carotid artery occlusion in the gerbil reduced NO production and nitric oxide synthetase (NOS) activity resulting from the hypoxia and reoxygenation.48 Using the same model of global ischemia, Cuzzocrea et al49 used multiple indices of tissue damage to evaluate the beneficial actions of melatonin in I/R injury. In this complete study, melatonin reduced NO generation, lowered lipid peroxidation, attenuated neutrophil accumulation in the hippocampus, reduced the severity of neurobehavioral effects, limited cerebral edema and CA1 neural loss, and reduced the nitrosylation of proteins after global I/R in the gerbil. When global ischemia (15 min) followed by reperfusion was induced by cardiopulmonary arrest in cats, melatonin also proved highly effective in reducing loss of neurons in the hippocampus and lowering the resulting neurological deficits.50 In this study, there was extensive loss of CA1 and CA4 pyramidal neurons 8 days following cardiopulmonary arrest along with significant neurological deficits measured on both day 1 and day 7. When melatonin was continually infused at 10 mg/kg/h for 6 h beginning 30 min after reperfusion onset, hippocampal neuronal loss and the neurological deficits were much less severe. A model of fetal rat brain global ischemia due to temporarily clamping the ovarian arteries of pregnant rats was used by Wakatsuki and coworkers51 to test the ability of melatonin to reduce neural oxidative damage. The endpoints in this study included neural thiobarbituric acid reactive substances (products of lipid peroxidation) and 8-hydroxy-2-deoxyguanosine (8-OHdG) (a damaged DNA product). A 20 min ischemic episode increased both oxidative parameters in the fetal brain; in contrast, when a 10 mg/kg dose of melatonin was given 60 min in advance of ovarian artery occlusion, both indices of oxidative damage were significantly attenuated. The results summarized herein are conclusive in documenting the protective actions of melatonin at the level of the central nervous system during I/R. Both physiological and obviously pharmacological levels of melatonin are effective in limiting neurological damage under conditions of anoxia and reoxygenation. Besides reducing the amount of tissue damage in these studies, when tested the severity of neurobehavioral deficits was also reduced by melatonin. Considering the unexpectedly high efficacy of melatonin in reducing oxidant injury, it is likely that melatonin not only directly scavenges free radicals but that it also stimulates the activities of several antioxidative enzymes which metabolize oxidants in innocuous species (Fig. 4).

Melatonin and I/R Injury in Other Organs Due to the debilitating physical and behavioral deficits that occur, tissue damage resulting from I/R is of greatest concern when it involves the heart and the brain. Other organs, however, also sometimes experience transient deprivation of oxygen due to a stoppage of blood flow for brief periods which compromises the function of these organs. The mechanisms of molecular and cellular destruction that occurs in these tissues are similar to those which develop in the heart and brain under the same conditions; as a consequence it is expected that melatonin would have protective actions against I/R in these organs as well and the published reports to date indicate this is the case. One of the first organs in which melatonin was used to stymie tissue damage following a temporary interruption of the blood flow was the liver. In this report the interval of ischemia had a duration of 40 min and this was followed by a 1 h reperfusion period.52 When the ischemic/reperfused livers were harvested there were elevated levels of products of lipid peroxidation, increased oxidized glutathione (GSSG) concentrations, and depressed activities of antioxidative enzymes (glutathione peroxidase and glutathione reductase); each of these parameters is indicative of elevated oxidative distress. Without exception, the administration of melatonin prior to ischemia induction returned each of these oxidative parameters to near control levels. Additionally, when the I/R liver were morphologically studied there was obvious

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Melatonin: Biological Basis of Its Function in Health and Disease

Figure 4. Some of the actions of melatonin that contribute to its ability to protect against ischemia/ reperfusion injury. Besides scavenging a number of oxygen and nitrogen-based reactants via nonreceptor-mediate mechanisms, melatonin also alters the activities of a number of enzymes that contribute to the ability of the indole to reduce oxidative damage. Thus, possibly via receptor-mediated processes, melatonin stimulates the activities of the following antioxidative enzymes: superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and gamma-glutamylcysteine synthase. Additionally, under some circumstances melatonin inhibits the pro-oxidative enzyme, nitric oxide synthase.

structural damage and polymorphonuclear leukocyte infiltration, both of which were prevented when elevated melatonin levels were present during the anoxic and reoxygenation periods. The stomach was the target organ studied by de la Lastra53 after its blood supply was interrupted by clamping the celiac artery. The indices of damage measured by this group included the extent of the morphologically-identified lesion, lipid breakdown products, myloperoxidase activity (an index of neutrophil infiltration) and gastric glutathione peroxidase activity. In a dose-response manner, melatonin was found to revert each of the measured changes to near normal levels. Given the known antioxidant potential of melatonin, the authors surmised that its effectiveness related to this property of the molecule.53 The most complete studies on the ability of melatonin to arrest damage to the gastrointestinal tract after I/R were provided by the group of Konturek and coworkers54-56 in a series of three reports. In addition to assessing the degree of structural and oxidative damage,

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they employed a fluorescent assay to estimate the quantity of free radicals in the blood draining the ischemic damaged area of the gut. Melatonin not only preserved the integrity of the mucosal lining but also reduced the severity of the oxidative changes which positively correlated with lowered free radical generation. While the results of the three reports indicated that melatonin’s protective actions related to its free radical scavenging activity, Konturek et al54 also found that the indole had microcirculatory actions which may have contributed to its ability to limit gastrointestinal damage after I/R. Cuzzocrea et al57 also carried out a detailed investigation in rats wherein the superior mesenteric artery and celiac trunk were clamped for 45 min to deprive the gastrointestinal tract of oxygenated blood. In the absence of melatonin there was massive mucosal damage, a rise in immunoreactive nitrotyrosine levels [an index of tyrosine nitration by the peroxynitrite anion (ONOO-)], increased positive staining for P-selectin and the intercellular adhesion molecule (ICAM) along with rises in myloperoxidase activity and malondialdehyde levels. The administration of 3 mg/kg melatonin at the end of the ischemic episode and an additional 3 mg/kg infused during the 1 h reperfusion period substantially reduced the morphological damage to the mucosa and reversed the indices of oxidative stress. Elevated levels of P-selectin and ICAM contribute to tissue damage during I/R by permitting arriving leukocytes to adhere to the endothelium; this results in a number of damaging changes including obstruction of blood flow in capillaries, augmention of edema and increased free radical generation due to the heightened myloperoxidase activity. This report shows, as some others have, that the antioxidant capabilities of melatonin are not the only actions by which the indole reduces tissue destruction during I/R injury. Recently, Jaworek and coworkers58 investigated the efficacy of melatonin in reducing I/R damage in the pancreas after clamping the inferior splenic artery of rats for 30 min followed by a 2 h reperfusion. Melatonin (10, 25 or 50 mg) was given as a bolus intraperitoneal injection 30 min prior to the interruption of the blood supply to the spleen. I/R destruction of the pancreas in this study was confirmed by the histological structural damage, the marked edematous response, the pronounced increase in plasma levels of both amylase activity and tumor necrosis factor-alpha, and elevated levels of lipid peroxidation products in the pancreas itself. An improvement in each of these indices was apparent in the rats given melatonin and the authors concluded that the beneficial effects of the indole related to both its antioxidant properties and immunomodulatory actions. As with other organs, the urinary bladder is functionally and morphologically compromised when the blood supply is discontinued. Sener and collegues59 used such a model to examine the potential protective effects of melatonin in this organ. Besides examining biochemical parameters of oxidative stress in bladder after I/R this group also tested the functional changes in the bladder musculature. After I/R, the contractile responses of bladder strips in response to carbachol were reduced. This physiological change was reversed in the urinary bladder of the melatonin-treated rats. Likewise, the biochemical indices that were altered by I/ R injury were improved as a consequence of melatonin treatment. Renal injury as a result of I/R is manifested as oxidatively damaged products in the tissue and changes in the excretion of certain molecules in the urine. Nephrotoxicity in rats was obvious when the kidneys were subjected to a 15 min period of ischemia followed by 1, 3, 6, 24, or 48 h or 1 week reperfusion. Besides the usual biochemical measures of oxidative damage, Sener et al60 also evaluated renal function by measuring serum blood urea nitrogen (BUN) and creatinine levels, both of which were increased as a result of I/R. These changes, as well as the tissue indices of molecular damage, were reversed when melatonin (10 mg/kg) was given twice, i.e., 15 min before ischemia and again prior to reperfusion. I/R injury is a natural consequence of organ transplantation. To test melatonin’s efficacy in protecting the donated organ from transient ischemia and reperfusion, Inci et al61 transplanted a lung into a group of rats, half of which were subsequently given melatonin. Two hours after transplantation, melatonin had lowered the level of oxidized lipids and myloperoxidase activity in the transplants. Additionally, the transplanted lungs of rats treated with melatonin exhibited

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better oxygenation and bronchoalveolar lavage nitrite levels in the transplants were reduced. These measures uniformly indicate melatonin is highly protective against ischemia/reperfusion injury that occurs during transplantation.

Concluding Remarks While this brief resume summarizes the efficacy of melatonin in reducing the morphological and physiological damage that occurs as a result of in vivo anoxia and reoxygenation to a variety of organs, there are an equivalent number of reports (not discussed in this review) that document the beneficial actions of melatonin on in vitro models of I/R as well. Collectively, the results unequivocally document the ability of melatonin to arrest and/or reduce the severity of damage that accompanies transient interruption of blood flow followed by reestablishment of the flow of oxygenated blood. Given that a component of the resulting damage is a consequence of free radical mutilation of critical molecules, melatonin’s antioxidant and free radical scavenging activities are assumed to be of paramount importance in limiting I/R-induced tissue damage. Melatonin, however, has a variety of additional actions, e.g., immunomodulatory effects57 as well as potential beneficial effects on mitochondrial oxidative phosphorylation,62 that probably contribute to its protective effects during I/R insults. As summarized herein, pharmacological levels of melatonin were typically given to combat the damage that occurred in the I/R models. Of course, administering higher than physiological concentrations, i.e., pharmacological levels, of antioxidants is always necessary under conditions of massive induced oxidation stress. Indeed, the reason oxidative damage occurs in these situations is that all physiological antioxidants combined are obviously incapable of coping with and preventing the destruction induced by greatly elevated levels of free radicals. In regard to this discussion, however, the reader is reminded that it is difficult to determine what constitutes a physiological/pharmacological level of melatonin.63 A final issue relates to the functional nature of the melatonin molecule. Although classically referred to as a hormone, because these were the initial actions that were described, its functions far outstrip those implied by the term hormone which are, by definitiaon, receptor mediated. In fact, melatonin’s direct scavenging actions are receptor-independent.1,6,8,11,12,64,65 For this and other reasons to refer to melatonin exclusively as a hormone seems erroneous and outdated.66

Definitions Antioxidant – a molecule that detoxifies a free radical or associated reactant by one of several means Edema – tissue fluid that escapes from blood and lymphatic vessels leading to puffiness and damage to tissues Free radical – a molecule, or portion thereof, which has an unpaired electron in its valence orbital Free radical scavenger – a molecule that neutralizes a free radical by one of several means, often by electron donation Heart attack – the temporary or permanent interruption of blood flow to a portion of the heart Hydroxyl radical – the devastating reactive product generated by the 3 electron reduction of oxygen Ischemia/reperfusion – the partial or total interruption of blood supply to an organ/tissue followed by the reopening of the vessel and reperfusion with blood Lipid peroxidation – the breakdown of polyunsaturated fatty acids and associated lipids caused by free radicals Melatonin – an endogenously produced antioxidant that has a variety of actions by which it reduces oxidative stress Oxidative stress – the accumulated molecular damage that is a consequence of free radicals and associated reactants

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Oxygen – based reactants – metabolites of oxygen that are highly reactive and damage a variety of molecules Stroke – temporary or permanent interruption of the blood supply to a portion of the brain

References 1. Tan DX, Chen LD, Poeggeler B et al. Melatonin: A potent, endogenous hydroxyl radical scavenger. Neurosci Lett 1993; 1:57-60. 2. Hardeland R, Reiter RJ, Poeggeler B et al. The significance of the metabolism of the neurohormone melatonin: Antioxidant protection and formation of bioactive substances. Neurosci Biobehav Dev 1993; 17:347-357. 3. Poeggeler B, Reiter RJ, Tan DX et al. Melatonin, hydroxyl radical-mediated oxidative damage and aging: A hypothesis. J Pineal Res 1993; 14:151-163. 4. Reiter RJ, Melchiorri D, Sewerynek E et al. A review of the evidence supporting melatonin’s role as an antioxidant. J Pineal Res 1995; 18:1-11. 5. Reiter RJ. Oxidative processes and antioxidative defense mechanisms in the aging brain. FASEB J 1995; 9:526-533. 6. Reiter RJ, Oh CS, Fujimori O. Melatonin: Its intracellular and genomic actions. Trends Endocrinol Metab 1996; 7:22-27. 7. Pappolla MA, Chyan YJ, Poeggeler B et al. An assessment of the antioxidant and antiamyloidogenic properties of melatonin: Implications for Alzheimer’s disease. J Neural Transm 2000; 107:203-231. 8. Tan DX, Manchester LC, Reiter RJ et al. Significance of melatonin in antioxidative defense system: Reactions and products. Biol Signals Recept 2000; 9:137-159. 9. Reiter RJ, Tan DX, Sainz RM et al. Melatonin: Reducing the toxicity and increasing the efficacy of drugs. J Pharm Pharmacol 2002; 54:1299-1321. 10. Reiter RJ, Tan DX, Osuna C et al. Actions of melatonin in the reduction of oxidative stress: A review. J Biomed Sci 2000; 7:444-458. 11. Tan DX, Reiter RJ, Manchester LC et al. Chemical and physical properties and potential mechanisms: Melatonin as a broad-spectrum antioxidant and free radical scavenger. Curr Topics Med Chem 2002; 2:181-197. 12. Allegra M, Reiter RJ, Tan DX et al. The chemistry of melatonin’s interaction with reactive species. J Pineal Res 2003; 34:1-10. 13. Granger DN. Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury. Am J Physiol 1988; 255:H1269-H1275. 14. Chen Y, Miles AM, Grisham MB. Pathophysiology and reactive oxygen metabolites. In: Ahmed S, ed. Oxidative Stress and Antioxidative Defense in Biology, Chapman and Hall: London, 1995: 62-95. 15. Omar B, McCord J, Downey J. Ischemia-reperfusion. In: Sies H, ed. Oxidative Stress: Oxidants and Antioxidants, Academic Press: San Diego, 1991: 493-527. 16. Kogwe K. The dawn of a new era for stroke management. Life Sci 2002; 72:575-581. 17. Reiter RJ, Tan DX, Sainz RM et al. Melatonin protects the heart against both ischemia/reperfusion injury and chemotherapeutic drugs. Cardiovasc Drugs Ther 2002; 16:5-6. 18. Reiter RJ, Tan DX. Melatonin: A novel protective action against oxidative injury of the ischemic/ reperfused heart. Cardiovasc Res 2003; 58:10-19. 19. Cheung RTF. The utility of melatonin in reducing cerebral damage resulting from ischemia and reperfusion. J Pineal Res 2003; 34:153-160. 20. Tan DX, Manchester LC, Reiter RJ et al. Ischemia/reperfusion-induced arrhythmias in the isolated rat heart: Prevention by melatonin. J Pineal Res 1998; 25:184-191. 21. Kaneko S, Okumura K, Numaguchi Y et al. Melatonin scavenges hydroxyl radical and protects isolated rat hearts from ischemic reperfusion injury. Life Sci 2000; 67:101-112. 22. Lagneux C, Joyeux M, Demenge P et al. Protective effect of melatonin against ischemia-reperfusion injury in the isolated rat heart. Life Sci 2000; 66:503-509. 23. Szarszoi O, Asemu G, Vanecek J et al. Effects of melatonin on ischemia and reperfusion injury of the rat heart. Cardiovasc Drugs Ther 2001; 15:251-257. 24. Lee YM, Chen HR, Hsiao G et al. Protective effects of melatonin on myocardial ischemia/reperfusion injury in vivo. J Pineal Res 2002; 33:72-80. 25. Cuzzocrea S, Reiter RJ. Pharmacological actions of melatonin in acute and chronic inflammation. Curr Topics Med Chem 2002; 2:153-166. 26. Reiter RJ, Tan DX. What constitutes a physiological concentration of melatonin? J Pineal Res 2003; 34:79-80.

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27. Sahna E, Olmez E, Acet A. Effects of physiological and pharmacological concentrations of melatonin on ischemia-reperfusion arrhythmias in rats: Can the incidence of sudden cardiac death be reduced? J Pineal Res 2002; 32:194-198. 28. Sahna E, Acet A, Ozer MK et al. Myocardial ischemia-reperfusion in rats: Reduction of infarct size by either supplemental physiological or pharmacological doses of melatonin. J Pineal Res 2002; 33:234-238. 29. Reiter RJ. Aging and oxygen toxicity: Relation to changes in melatonin. Age 1997; 20:201-213. 30. Dominguez-Rodriquez A, Abreu-Gonzalez P, Garcia MJ et al. Decreased nocturnal melatonin levels during acute myocardial infarction. J Pineal Res 2002; 33:248-252. 31. Brugger P, Marktl W, Herold M. Impaired nocturnal secretion of melatonin in coronary heart disease. Lancet 1995; 345:1408. 32. Brugger P, Herold M. Human melatonin and cortisol circadian rhythms in patients with coronary heart disease. Biol Rhythms Res 1998; 29:121-128. 33. Sakotnik A, Liebmann PM, Stoschitsky K et al. Decreased melatonin synthesis in patients with coronary artery disease. Eur Heart J 1999; 20:1314-1317. 34. Altun A, Yaprak M, Aktoz M et al. Impaired nocturnal synthesis of melatonin in patients with cardiac syndrome X. Neurosci Lett 2002; 327:143-145. 35. Duncker DJ, Verdouw PD. Has melatonin a future as a cardioprotective agent? Cardiovasc Drugs Ther 2001; 15:205-207. 36. Manev H, Uz T, Kharlamov A. Increased brain damage after stroke or excitotoxic seizures in melatonin-deficient rats. FASEB J 1996; 10:1546-1551. 37. Joo JY, Uz T, Manev H. Opposite effects of pinealectomy and melatonin administration on brain damage following cerebral focal ischemia in rats. Restr Neurol Neurosci 1998; 13:185-191. 38. Kilic E, Özdemir YG, Bolay H et al. Pinealectomy aggravates and melatonin administration attenuates brain damage in focal ischemia. J Cerebr Blood Flow Metab 1999; 19:511-516. 39. Borlongan CV, Yamamoto M, Takei N et al. Glial cell survival is enhanced during melatonin-induced neuroprotection against cerebral ischemia. FASEB J 2000; 14:1307-1317. 40. Sinha K, Degaonkar MN, Jagannathan NR et al. Effect of melatonin on ischemia reperfusion injury induced by middle cerebral artery occlusion in rats. Eur J Pharmacol 2001; 428:185-192. 41. Kondoh T, Uneyama H, Nishino H et al. Melatonin reduces cerebral edema formation caused by transient forebrain ischemia in rats. Life Sci 2002; 72:583-590. 42. Pei Z, Ho TH, Cheung RT. Pretreatment with melatonin reduces volume of cerebral infarction in a permanent middle cerebral artery occlusion stroke model in the rat. Neurosci Lett 2002; 318:141-144. 43. Pei Z, Pang SF, Cheung RT. Pretreatment with melatonin reduces volume of cerebral infarction in a rat middle cerebral artery occlusion stroke model. J Pineal Res 2002; 32:163-172. 44. Pei Z, Pang SF, Cheung RT. Administration of melatonin after onset of ischemia reduces the volume of cerebral infarction in a rat middle cerebral artery occlusion stroke model. Stroke 2003; 34:770-775. 45. Pei Z, Fung PC, Cheung RT. Melatonin reduces nitric oxide level during ischemia but not blood-brain-barrier breakdown during reperfusion in a rat middle cerebral artery occlusion stroke model. J Pineal Res, 2003; 34:110-118. 46. Gupta YK, Chaudhary G, Sinha K. Enhanced protection by melatonin and meloxican combination in a middle cerebral artery occlusion model of acute ischemic stroke in rat. Can J Physiol Pharmacol 2002; 80:210-217. 47. Cho S, Joh TH, Baik HH et al. Melatonin administration protects CA1 hippocampal neurons after transient forebrain ischemia in rats. Brain Res 1997; 755:335-338. 48. Guerrero JM, Reiter RJ, Ortiz GG et al. Melatonin prevents increases in neural nitric oxide and cyclic GMP production after transient brain ischemia and reperfusion in the Mongolian gerbil (Meriones unguiculatus). J Pineal Res 1997; 23:24-31. 49. Cuzzocrea S, Costantino G, Gitto E et al. Protective effects of melatonin in ischemic brain injury. J Pineal Res 2000; 29:217-227. 50. Letechipia-Vallejo G, Gonzalez-Burgos I, Cervantes M. Neuroprotective effect of melatonin in brain damage induced by global cerebral ischemia in cats. Arch Med Res 2001; 32:186-192. 51. Wakatsuki A, Okatani Y, Izumiya C et al. Melatonin protects against ischemia and reperfusioninduced oxidative lipid and DNA damage in fetal rat brain. J Pineal Res 1999; 26:147-152. 52. Sewerynek E, Reiter RJ, Melchiorri D et al. Oxidative damage to the liver induced by ischemia-reperfusion: Protection by melatonin. Hepatogastroenterology 1996; 43:898-905. 53. de la Lastra CA, Cabeza J, Motilva V et al. Melatonin protects against gastric ischemia-reperfusion in rats. J Pineal Res 1997; 23:47-52.

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54. Konturek PC, Konturek SJ, Majka J et al. Melatonin affords protection against gastric lesions induced by ischemia-reperfusion possibly due to its antioxidant and mucosal microcirculatory effects. Eur J Pharmacol 1997; 322:73-77. 55. Konturek PC, Konturek SJ, Brzozowski T et al. Gastroprotective effect of melatonin and its precursor, L-tryptophan, against stress-induced and ischemia-induced lesions is mediated by scavenging of oxygen free radicals. Scand J Gastroenterol 1997; 32:433-438. 56. Brzozowski T, Konturek PC, Konturek SJ et al. The role of melatonin and L-tryptophan in prevention of acute gastric lesions induced by stress, ethanol, ischemia and aspirin. J Pineal Res 1997; 23:79-89. 57. Cuzzocrea S, Costantino G, Mazzon E et al. Beneficial effects of melatonin in a rat model of splanchnic artery occlusion and reperfusion. J Pineal Res 2000; 28:52-63. 58. Jaworek J, Leja-Szpak A, Bonior J et al. Protective effect of melatonin and its precursor L-tryptophan on acute pancreatitis induced by caerulian overstimulation or ischemia/reperfusion. J Pineal Res 2003; 34:40-52. 59. Sener G, Sehirli AO, Paskaloglu K et al. Melatonin treatment protects against ischemia/reperfusion induced functional and biochemical changes in rat urinary bladder. J Pineal Res 2003; 34:226-230. 60. Sener G, Sehirli AO, Keyer-Uysol M et al. The protective effect of melatonin on renal ischemia-reperfusion injury in the rat. J Pineal Res 2002; 32:120-126. 61. Inci I, Inci D, Dutly A et al. Melatonin attenuates posttransplant lung ischemia-reperfusion injury. Ann Thorac Surg 2002; 73:220-223. 62. Acuña-Castroviejo D, Martin M, Macias M et al. Melatonin, mitochondria and cellular bioenergetics. J Pineal Res 2001; 30:65-74. 63. Reiter RJ, Tan DX. What constitutes a physiological concentration of melatonin? J Pineal Res 2003; 34:79-80. 64. Hardeland R, Poeggeler B, Niebergall R et al. Oxidation of melatonin by carbonate radicals and chemiluminescence emitted during pyrrole ring cleavage. J Pineal Res 2003; 34:17-25. 65. Tan DX, Hardeland R, Manchester LC et al. Mechanistic and comparative studies of melatonin and classic antioxidants in terms of their interactions with the ABTS cation radical. J Pineal Res 2003; 34:249-259. 66. Tan DX, Manchester C, Hardeland R et al. Melatonin: A hormone, a tissue factor, an autocoid, a paracoid, and an antioxidant vitamin. J Pineal Res 2003; 34:75-78.

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CHAPTER 3

Melatonin and the Thyroid Gland Andrzej Lewinski

Abstract

I

n this review, data from reports on relationships, observed between melatonin—the main pineal hormone—and the thyroid gland, are briefly summarized. The prevailing part of the survey is devoted to melatonin influence on thyroid growth processes and function. Much evidence has been accumulated, suggesting an inhibitory action of melatonin on thyroid growth and secretion; this effect has been revealed by using different experimental models: short-term and/or chronic melatonin administration in vivo to various animal species, pinealectomized animals, light restriction, which is known to increase the activity of the pineal gland, etc., as well as by employing in vitro conditions. Oxidative stress plays a crucial role in physiological and pathological processes in the thyroid gland. Accordingly, it has been documented that oxidative stress accompanies certain thyroid disturbances or diseases. Up-to-date literature, although not abundant, indicates that melatonin can protect against oxidative damage in the thyroid and in other organs. Furthermore, much data has been gathered, indicating—in experimental conditions— a mutual relationship between the pineal gland and the thyroid. The confirmation of these relations in clinical studies in humans meets numerous difficulties, resulting—among others— from the fact that—nowadays—human beings, as well as animal species, used in experimental studies, have been living far away from their natural and original habitats. It makes almost impossible to compare the results of studies on the pineal-thyroid interrelationship, obtained in particular experiments performed in different species.

Introduction Melatonin (N-acetyl-5-methoxytryptamine)—the main secretory product of the pineal gland—displays several functions in living organisms. The accumulated evidence for the relationship between the pineal and the thyroid gland derives, mainly, from studies performed in experimental animals.1 Whereas it is generally accepted that thyroxine (T4)—under physiological conditions—is exclusively produced in the thyroid gland and peripherally metabolized into a more active hormone—triiodothyronine (T3) (80% of the entire amount of T3, present in the body, is a product of T4-monodeiodination reaction), there are probably different sources of melatonin. It is already known that, beside the pineal gland, other organs, tissues or cells serve as sources of melatonin production.2,3 Among others, positive immunostaining with antibodies against melatonin has been described with respect to C cells of the thyroid gland.2,4 Unfortunately, no studies have yet been performed, which would be demonstrating the presence of melatonin in thyroid follicular cells. Thus, not only typical endocrine but, at least, paracrine (if not autocrine) regulation should be considered between melatonin and thyroid hormones.

Melatonin: Biological Basis of Its Function in Health and Disease, edited by S.R. Pandi-Perumal and Daniel P. Cardinali. ©2006 Eurekah.com.

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Melatonin and Thyroid Growth Processes As mentioned before, the inhibitory effect of melatonin on thyroid growth processes has been demonstrated in numerous studies, using different experimental models. In early experiments, pinealectomy was shown to increase thyroid weight in hypophysectomized mice.5,6 Conversely, light restriction, i.e., experimental protocol, known to activate the pineal gland, suppressed the thyroid growth in male mice.7 Consistently, melatonin, applied in mice in late-afternoon s.c. injections for 10 days, inhibited the basal and thyrotropin (TSH)-stimulated mitotic activity of thyroid follicular cells.8 Additionally, melatonin prevented the pinealectomy-induced increase of the mean mitotic activity rate in the rat thyroid gland.9 When that indoleamine was injected (s.c.) to male rats for 5 days, the hormone—in a dose of 25 µg/daily—reduced 3H-thymidine incorporation into DNA of thyroid lobes transferred into the incubation in vitro (the experiment ex vivo in vitro), whereas—in the dose of 100 µg/ daily—it revealed a stimulatory effect.10 Under the in vitro conditions, melatonin revealed an inhibitory effect on 3H-thymidine incorporation into DNA of rat thyroid lobes.11 Melatonin and another indoleamine—5-methoxytryptamine—decreased the mean nuclear volume of thyroid follicular cells in Syrian hamsters.12 In turn, melatonin and N-acetylserotonin (NAS) decreased the mitotic activity in the rat thyroid gland in vivo.13 The inhibitory effect of short photoperiod on thyroid growth processes was shown in mice7 and in Indian palm squirrels (Funambulus pennanti).14 It has been suggested that melatonin directly influences thyroid follicular cells;8,15,16 accordingly, the increase of thyroid weight after pinealectomy occurred without involvement of the pituitary, i.e., in mice subjected to hypophysectomy.5,6 On the other hand, it is worth stressing that the direction of melatonin action on growth processes depends on several experimental and, presumably, clinical conditions. It has been found that the inhibitory effect of late-afternoon melatonin injections on growth processes in rat thyroid was prevented by the indoleamine, released continuously from s.c. pellets;17 that phenomenon was named the “counter-antithyroid action” of melatonin on the growth-inhibiting response of the gland, following melatonin injections late in light period. We measured the activity of certain enzymes related to growth processes in the thyroid tissue; these are the following enzymes: thymidine kinase, thymidine phosphorylase, and adenosine kinase. Additionally, we examined the effect of indoleamines on cyclic AMP generation in rat thyroids in vitro. Thymidine kinase (TK: thymidine 5'-phosphotransferase, EC 2.7.1.21) is an enzyme responsible for catalyzing the phosphorylation of thymidine, functioning as a part of the pyrimidine salvage pathway involved in DNA synthesis and being closely correlated with 3H-thymidine incorporation and mitosis.18 Adenosine kinase (AK; EC 2.7.1.20) is an enzyme which catalyses the phosphorylation of adenosine (Ado) and deoxyadenosine (dAdo) to adenosine monophosphate (AMP) and deoxyadenosine monophosphate (dAMP), respectively. Adenosine kinase functions as a part of the purine metabolic pathway involved in DNA synthesis and is the key enzyme regulating the Ado content. Thymidine phosphorylase (dThdPase, EC 2.4.2.4) is an enzyme catalyzing the reversible phosphorolysis of thymidine, deoxyuridine, and of their analogues to the respective bases and to 2-deoxyribose-1-phosphate. This enzyme has been proved to be identical to the platelet-derived endothelial cell growth factor (PD-ECGF), which is involved in the process of angiogenesis. Our experiments revealed diverse effects of melatonin on the activity of the enzymes in question. We have shown that melatonin and NAS decreased the concentration of cyclic AMP19 and reduced the activity of TK20 in rat thyroid lobes incubated in vitro. It seems that the influence of melatonin on TK activity in the thyroids depends on the age of animals; when the employed thyroid tissue had been collected from rats much younger than those applied in the previous experiment,20 melatonin, added to the incubation medium, increased TK activity in thyroids collected from intact, sham-operated and hemithyroidectomized animals.21

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Melatonin: Biological Basis of Its Function in Health and Disease

In another study, hemithyroidectomy increased dThdPase activity in the remaining thyroid lobe. Melatonin, applied in vitro, decreased the dThdPase activity in thyroid lobes collected from intact animals, sham-operated animals, and hemithyroidectomized rats.22 The results suggest an involvement of melatonin in the regulation of thyroid growth, hypothetically—by an impairment of the process of angiogenesis. Hemithyroidectomy decreased AK activity in the remaining thyroid lobe; melatonin, used in vitro, increased AK activity in thyroid lobes, collected from intact and sham-operated rats, but it did not change AK activity in the remaining thyroid lobes after hemithyroidectomy.21 The results suggest a certain role of AK in the regulation of (patho)physiological processes in the thyroid gland after hemithyroidectomy. Karyometry is a method, used in order to assess—in an indirect way—the activity of various tissues and organs. An increased volume of cell nuclei may result either from the enhanced DNA synthesis or from the increased functional activity (e.g., the increased protein synthesis). Using this method, we have found that a short-photoperiod exposure caused a decrease in the mean volume of thyrocyte nuclei in male gerbils,23 and melatonin, administered in late-afternoon injections, decreased the mean nuclear volume of thyrocytes in male Syrian hamsters;13 under in vitro conditions, melatonin significantly decreased the mean nuclear volume and the nuclear intersection area of thyrocytes.24 Taking into consideration all the results presented above, a question has arisen about the detailed mechanism of antiproliferative melatonin action. This mechanism remains unclear but this action seems, at least in part, to be exerted directly.8,25 Such an assumption is also supported by the observations of Haldar and Shavali,17 who have succeeded in demonstrating a direct effect of melatonin on T4 release from squirrel thyroid lobes in vitro. However, the presence of specific melatonin binding sites in the thyroid has not been documented.26 The following ways of antiproliferative melatonin action are suggested: 1. inhibition of arachidonate metabolism; 2. inhibition of calcium channels; 3. protection against the damaging effects of toxic and highly reactive free radicals (e.g., hydroxyl radicals—•OH) or, the so called, reactive oxygen species (ROS).

It has been found that melatonin inhibits prostaglandin synthesis in the hypothalamus.27,28 On the other hand, prostaglandin synthesis inhibitors were shown to suppress the proliferogenic responses to certain hormones.29 Furthermore, melatonin and indomethacin—prostaglandin synthesis inhibitor—reveal a similarity with regard to their chemical structure. Melatonin was also suggested to block the voltage-dependent calcium channels.30 It was demonstrated that agents which block this type of calcium channels, like dihydropyridines, suppressed the proliferation of rat prolactinoma cells in vitro.31 The role of melatonin as a potent antioxidant will be discussed in the subsequent parts of this review. Growth processes are undoubtfully involved in the complex process of carcinogenesis. The protective effects of melatonin against cancer are a subject of an intensive research.32-34 Because of the potential role of ionizing radiation in the pathogenesis of thyroid cancer, the studies on protective effects of melatonin against radiation-induced oxidative stress and cancer of the thyroid gland seem to be of special value. However, the data on this particular issue have been rather scarce in available literature. It has been found that histoenzymological changes in rat thyroid gland, caused by an exposure to 8 Gy of γ-radiation, were partially reversed by pretreatment with melatonin.35 In another study, when using morphometric parameters, melatonin was shown to decrease the height of thyroid follicular cells and the nuclear volume of the cells from rats exposed to 8 Gy-radiation.36 The potential protective effects of melatonin against thyroid cancer will unquestionably become a subject of future studies.

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Melatonin and Thyroid Function Generally, the influence of melatonin on the thyroid secretion in photo-sensitive animals (mainly rodents) in laboratory conditions seems to be inhibitory one, however, it varies, depending on animal species, as well as on applied melatonin dose and administration protocol. Melatonin, employed in late afternoon s.c. injections, decreased circulating thyroid hormone concentrations in adult male and female Syrian hamsters (melatonin—25 µg/daily)37 and in male Wistar rats (melatonin—50 µg/daily).38 In turn, pinealectomy brought about an increase in serum T4 concentrations in male Wistar rats 10 weeks after operation.39 Under conditions of constant darkness, reduced plasma T4 concentrations were found, accompanied by lower thyroid weight in squirrels (Funambulus pennanti); conversely, an enhanced thyroid function was observed after pinealectomy.40 On the other hand, melatonin, chronically released from s.c. pellets, implanted to male Wistar rats, increased both T3 and T4 levels after 10 days and also, however to a lesser degree, after 10 weeks; this effect is called the “prothyroid” action of melatonin.38 Additionally, the joint effect of late-afternoon melatonin injections and melatonin-implants caused no changes in thyroid hormone concentrations (“counter-antithyroid action”).38 The above mentioned observations, as well as the “counter-antithyroid action” of melatonin in reference to the growth processes, suggest that this indoleamine is a “keeping-balance” molecule.

Oxidative Stress, the Thyroid Gland and Melatonin Much evidence has been accumulated, indicating that melatonin is a highly effective antioxidant and free radical scavenger.37,41 On the other hand, the role of oxidative stress in the pineal-thyroid reciprocal relationships has not been examined, so far. Melatonin, as a molecule widely distributed in the organism, should—certainly—be also present in the thyroid. Positive immunostaining with antibodies against melatonin has been observed in C cells of the thyroid gland.4 According to the current views, ROS and free radicals participate in the physiological and pathological processes in the thyroid gland. Especially, the role of hydrogen peroxide (H2O2) should be stressed; it serves as an electron acceptor and accompanies thyroid peroxidase (TPO), participating in all the steps of thyroid hormone synthesis. It is not clear, what is the source of H2O2 in the thyroid; the most convincing theory suggests that H2O2 is produced close to the apical part of the thyroid follicular cell, from superoxide anion radical (O2-•), in the reaction involving NADPH, calcium, and in the presence of NADPH oxidase. Interestingly, it has recently been documented that thyroid H2O2 is produced by divalent reduction of oxygen without O2-• generation.42 What might be the consequence of H2O2 overproduction, remains to be experimentally proved. It is well known that H2O2 plays a role in pathological processes of the thyroid. Hydrogen peroxide—when present in excessive amounts—may decrease the activity of TPO, and, subsequently, inhibit the thyroid hormone synthesis.42 Thus, it is probable that H2O2, i.e., reactive oxygen species which normally participates in Fenton reaction producing hydroxyl radical (•OH), initiates the oxidative stress (especially if H2O2 is present in high concentrations). Under such circumstances, the best defense mechanism could be the application of antioxidants, melatonin included. The are some observations explaining the role of H2O2 in pathological processes in the thyroid gland. It has recently been documented that H2O2 may participate in induction of thyroid autoimmunity. In studies with use of human thyroid cells, H2O2—present in excessive amounts—has been shown to produce immunoreactive fragments of thyroglobulin, one of the main antigens in the thyroid.43 Moreover, it has been found that, under in vitro conditions, H2O2 activates p38-MAPK (p38 mitogen-activated protein kinase) in hTSHR-CHO cells (Chinese hamster ovary cells transfected with the human TSH receptor);44 p38-MAPK is a component of the signaling pathway, activated by TSH and cAMP in thyroid cells, that plays a role in the expression of sodium-iodine symporter (NIS). This finding speaks in favour of the

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Melatonin: Biological Basis of Its Function in Health and Disease

role of ROS in NIS expression, thus, in iodine transport processes. It has been found in another study, that H2O2 induces death of pig thyrocytes in culture; H2O2, when used in the concentration below 0.5 mM, caused apoptosis.45 In contrast, after application of H2O2 in higher concentrations—cell necrosis was observed.45 Nitric oxide synthase (NOS) is an enzyme which catalyzes the formation of nitric oxide (NO•), an endogenous free radical. The expression of mRNA, specific for the three isoforms of NOS—brain (type I), endothelial (type III), and inducible (type II)—has been detected in the rat thyroid gland.46-48 It has been shown that NO• participates in the regulation of iodide uptake in the thyroid gland. It was documented that NO• suppressed the TSH-stimulated iodide uptake;49 on the other hand, NO• stimulated guanylyl cyclase (GC) activity and cyclic GMP (cGMP) production in the human and calf thyroid.47,49 The above data suggest that the inhibition of iodine uptake is probably mediated by the GC-cGMP pathway.49 It has recently been suggested that not only H2O2 but also other ROS may participate in thyroid autoimmunity. Belgian authors have shown that NO• is involved in interleukin-1α-induced cytotoxicity in polarized human thyrocytes and suggested that free radicals may promote the exposure of autoantigens to the immune system.50 Regarding the protective effects of antioxidants, it has been found that vitamin E can reduce the parameters of thyroid enlargement due to low iodine diet; this observation suggests an antigoitrogenic effect of antioxidants in iodine-deficient rats.51 The potential protective effect of antioxidants against goiter formation requires further elucidation. Hyperthyroidism is a disease in the course of which oxidative stress and peroxidation of lipids can be generated. The main role is played by the overproduction of thyroid hormones.52 Additionally, elevated levels of cytokines—as observed in hyperthyroidism—can be an additional source of free radicals. We have noticed increased Schiff bases (SB) levels—a parameter of oxidative stress—in lung, brain, and kidney homogenates in L-T4-administered animals. Melatonin in the lung, brain and kidney homogenates decreased the elevated SB concentrations in thyrotoxic animals. Additionally, melatonin decreased the basal SB concentration in the kidney, brain, and lung homogenates. We concluded that: (1) thyrotoxicosis stimulated the oxidative damage in examined organs; (2) melatonin protected against the oxidative stress, induced by L-T4 injections; and (3) melatonin reduced the basal SB concentrations in all the examined homogenates.53 The same decreasing tendency has been shown for malondialdehyde (MDA) and conjugated dienes.54,55 The meaning of all the above cited observations on the role of free radicals and/or ROS in the thyroid should be emphasized, taking into consideration that melatonin is able directly or indirectly neutralize all of these toxic species; this indoleamine influences also the activities of anti- or prooxidative enzymes, causing reduction of oxidative damage to biological molecules.37,51,56 As regards the protective effects of melatonin against oxidative stress in the thyroid gland, experimental data are, unfortunately, very scarce. It has recently been shown at our laboratory, that melatonin effectively prevented the process of ferrous (Fe2+) plus H2O2-induced lipid peroxidation in homogenates of porcine and calf thyroid (Karbownik et al, unpublished data). In conclusion, the effect of melatonin on free radicals or ROS generated in the thyroid, seems to be similar or even it is the same as that observed in other tissues and organs.

Pineal-Thyroid Relationship in Humans The clinical data on the pineal-thyroid relationship are scarce. Whereas no changes have been observed by some authors in melatonin levels, in either hypothyroidism or hyperthyroidism in human subjects,57 other investigators have found increased nocturnal melatonin concentrations in hypothyroid patients.58 Blood concentrations of melatonin were also evaluated in patients with very large nontoxic nodular goitre before and after thyreoidectomy; unexpectedly, nocturnal melatonin

Melatonin and the Thyroid Gland

31

concentrations were significantly higher after the surgery than before.59 The authors have drawn a conclusion that the goitre of a very large size can—possibly—compress the superior cervical ganglia, and—in consequence—alter indirectly the Mel synthesis. However, according to the current views, melatonin could be actively taken up by enlarged thyroid with a subsequent decrease in blood concentration of this indoleamine.

Thyroid Hormone-Stimulation of Pineal Function or Growth Processes The stimulatory effect of the thyroid hormones on the pineal gland is supported by many morphological, biochemical, and clinical findings. Peschke60-62 reported that T4 significantly increased the surface area of nuclei cross sections of rat pinealocytes in vivo; Thyroidectomy (TX) and/or methylthiouracil (MTU) treatment caused a significant decrease of the surface area in question. Also the results of our studies speak in favor of thyroid stimulation of pineal growth; thyroid hormones increased the MNV of pinealocytes in organ culture, as well as slightly increased the MMAR of pinealocytes.63 In turn, Milcou et al64 have found a significantly increased amount of DNA in rat pineals, following the administration of T4 to culture medium. A further support for our hypothesis has been provided by the results of Nir and Hirschman65 who showed that thyroid hormones enhanced melatonin concentration and induced an increase of norepinephrine-stimulated Nac-5HT content in cultured rat pineals. Consistently, in studies in vivo, treatment with T4 resulted in increased night peaks of melatonin in rats.66

Concluding Remarks On the basis of our early results, a reciprocal relationship between the pineal and the thyroid has been suggested.67,68 In agreement with this hypothesis, melatonin could act directly on thyroid follicular cells, inhibiting their proliferation. Accordingly, it is possible that plasma concentrations of thyroid hormones are direct modulators of the pineal function and growth (see Chapter 6 and above). The influence of melatonin on thyroid growth processes and thyroid hormone synthesis seems to be complex. It should be stressed once again that the evidence on the mutual relationship between the pineal gland and the thyroid is derived, almost exclusively, from studies performed in experimental animals. The confirmation of these relations in clinical studies meets numerous difficulties and pitfalls, resulting—among others—from the fact that, nowadays, human beings, as well as animal species used in experimental studies, live far away from their natural and original habitats. However, still much evidence indicates an undoubtful role of melatonin in physiological and pathological processes of the thyroid gland, providing “green light” for the future use of this indoleamine under certain clinical conditions. Taking into account the relationships between the pineal and thyroid gland, several questions still remain to be answered. These are the following: 1. 2. 3. 4. 5. 6. 7. 8.

to what extent is the relationship in question a direct one? are there any intermediate substances or factors involved in this regulation? are there any cells in the body capable of producing both thyroid hormones and melatonin? is there a local (paracrine) regulation of thyroid hormone synthesis and of thyrocyte proliferation by melatonin in the gland in question? does melatonin participate in the regulation of the peripheral metabolism of thyroid hormones (i.e., monodeiodination processes)? does melatonin regulate the activity of type II T4-5'-monodeiodinase in pinealocytes? does melatonin control the expression of certain thyroid gland-related genes, e.g., NIS, TPO, thyroglobulin, pendrin, TSH, TSH receptor (TSHR), etc.? is T3 involved in the regulation of the expression level of genes, encoding for key enzymes, which participate in melatonin synthesis (hydroxy-indole-O-methyltransferase - HIOMT; N-acetyltransferase—NAT)?

Further studies are needed to elucidate these problems.

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References 1. Lewinski A, Wajs E, Klencki M et al. Pineal-thyroid interrelationships update: 1996. In: Webb SM, Puig-Domingo M, Møller M et al, eds. Pineal Update. From Molecular Mechanisms to Clinical Implications. Westbury, New York: PJD Publ, Ltd., 1997:173-181. 2. Kvetnoy IM. Extrapineal melatonin: Location and role within diffuse neuroendocrine system. Histochem J 1999; 31:1-12. 3. Conti A, Conconi S, Hertens E et al. Evidence for melatonin synthesis in mouse and human bone marrow cells. J Pineal Res 2000; 28:193-202. 4. Raikhlin NT, Kvetnoy IM. The APUD system (diffuse endocrine system) in normal and pathological states. Physiol Gen Biol Rev 1994; 8:1-44. 5. Houssay AB, JH Pazo, Epper CE. Effects of the pineal gland upon the hair cycles in mice. J Invest Derm 1966; 47:230-234. 6. Pazo JH, Houssay AB, Davidson PA et al. On the mechanism of thyroid hypertrophy in pinealectomized rats. Acta Physiol Pharmacol Latinoam 1968; 18:332-340. 7. Lewinski A, Vaughan MK, Champney TH et al. Dark exposure inhibits the mitotic activity of thyroid follicular cells in male mice with intact pineal. Experientia 1984; 40:1284-1285. 8. Lewinski A, Sewerynek E. Melatonin inhibits the basal and TSH-stimulated mitotic activity of thyroid follicular cells in vivo and in organ culture. J Pineal Res 1986; 3:291-299. 9. Wajs E, Krotewicz M, Fryczak J et al. Melatonin suppresses the pinealectomy-induced increase of mitotic incidence in the rat thyroid gland. Med Sci Res 1989; 17:61-62. 10. Wajs E, Lewinski A, Krotewicz M et al. [3H]-thymidine incorporation into DNA of thyroid lobes incubated in vitro, following pretreatment of animals with melatonin and thyrotropin. Neuroendocrinol Lett 1992; 14:75-81. 11. Wajs E, Lewinski A. Melatonin and N-acetylserotonin—two pineal indoleamines inhibiting the proliferation of jejunal epithelium cells in rats. Med Sci Res 1988; 16:1125-1126. 12. Lewinski A, Webb SM, Sewerynek E et al. Influence of melatonin and 5-methoxytryptamine on the nuclear volume of thyroid follicular cells in the Syrian hamster (Mesocricetus auratus). Neuroendocrinol Lett 1986; 8:63-68. 13. Sewerynek E, Lewinski A, Szkudlinski M et al. The effect of melatonin and N-acetylserotonin on mitotic activity of thyroid gland and adrenal cortex in the rat. Endokrynol Pol 1988; 39:269-275. 14. Haldar C, Shavali SS, Singh S. Photoperiodic response of pineal-thyroid axis of the female Indian palm squirrel, Funambulus pennanti. J Neural Transm 1992; 90:45-52. 15. Wajs E, Lewinski A. Effects of melatonin on [3H]-thymidine incorporation into DNA of rat thyroid lobes in vitro. Biochem Biophys Res Commun 1991; 181:1187-1191. 16. Haldar C, Shavali SS. Influence of melatonin on thyroxine (T4) release from thyroid glands of female Funambulus pennanti: An in vitro study. Neuroendocrinol Lett 1992; 14:411-416. 17. Wajs E, Lewinski A. Inhibitory influence of late-afternoon melatonin injections and the counter-inhibitory effect of melatonin pellets on thyroid growth processes in male Wistar rats; Comparison with effects of other indole substances. J Pineal Res 1992; 13:158-166. 18. Zieve L, Anderson WR, Lindblad S. Course of hepatic regeneration after 80% to 90% resection of normal rat liver: Comparison with two-lobe and one-lobe hepatectomy. J Lab Clin Med 1985; 105:331-336. 19. Lewinski A, Sewerynek E, Zerek-Melen G et al. Influence of melatonin and N-acetylserotonin on the cyclic AMP concentration in the rat thyroid lobes incubated in vitro. J Pineal Res 1989; 7:55-61. 20. Lewinski A, Wajs E, Modrzejewska H et al. Inhibitory influence of melatonin on thymidine kinase activity in the rat thyroid lobes incubated in vitro. Neuroendocrinol Lett 1994; 16:221-226. 21. Gesing A, Modrzejewska H, Karbownik M et al. Thymidine kinase and adenosine kinase activities in homogenates of thyroid lobes in hemithyroidectomized rats; Effects of melatonin in vitro. Neuroendocrinol Lett 2000; 21:453-459. 22. Gesing A, Miszczak-Zaborska E, Karbownik M et al. Effects of hemithyroidectomy on thymidine phosphorylase in homogenates of rat thyroid lobes incubated in vitro in the presence of melatonin. Thyroidology Clin Exp 1999; 11:19-24. 23. Lewinski A, Vaughan MK, Champney TH et al. Inhibitory action of the pineal gland on the volume of thyroid follicular cells in male gerbils (Meriones unguiculatus). Exp Clin Endocrinol 1984; 84:239-244. 24. Klencki M, Slowinska-Klencka D, Kunert-Radek J et al. Melatonin-induced decrease of the size of thyrocytes nuclei in rat thyroids incubated in vitro. Cytobios 1994; 78:159-162. 25. Sewerynek E, Lewinski A. Melatonin inhibits mitotic activity of adrenocortical cells in vivo and in organ culture. J Pineal Res 1989; 7:1-12. 26. English J, Arendt J. Characterization of a melatonin binding site in the rat hypothalamus using 2-(125I)-iodomelatonin. Chinese J Physiol 1988; 4:236 (abstract).

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27. Cardinali D, Ritta MN, Fuentes AM et al. PGE release by medial basal hypothalamus in vitro: inhibition by melatonin at submicromolar concentrations. Eur J Pharmacol 1980; 67:151-153. 28. Pawlikowski M, Juszczak M, Karasek E et al. Melatonin inhibits prostaglandin E release from the medial basal hypothalamus of pinealectomized rats. J Pineal Res 1984; 1:317-321. 29. Pawlikowski M. Are prostaglandins involved in the mitogenic actions of hormones? Exp Clin Endocrinol 1983; 81:233-238. 30. Cardinali D, Vacas MI, Rosenstein RE. Cellular effects of melatonin: Receptors, second messengers and cell targets in brain. In: Gupta D, Attansio A, Reiter RJ, eds. The Pineal Gland and Cancer. London, Tubingen: Brain Research Promotion, 1988: 77-88. 31. Kunert-Radek J, Stepien H, Stawowy A et al. Involvement of calcium channels in control of the pituitary tumoral cell proliferation in vitro. Neuroendocrinol Lett 1989; 11:339-345. 32. Karasek M, Pawlikowski M. Pineal gland, melatonin and cancer. Neuroendocrinol Lett 1999; 20:139-144. 33. Karbownik M, Reiter RJ. Antioxidative effects of melatonin in protection against cellular damage caused by ionizing radiation. Proc Soc Exp Biol Med 2000; 225:9-22. 34. Karbownik M, Lewinski A, Reiter RJ. Anticarcinogenic actions of melatonin which involve antioxidative processes: comparison with other antioxidants. Int J Biochem Cell Biol 2001; 33:735-753. 35. Kundurovic Z, Scepovic M. Histoenzymological reactions of the thyroid gland in irradiated and previously melatonin-treated irradiated rats. Acta Med Iugosl 1989; 43:337-347. 36. Kundurovic Z, Mornjakovic Z. Morphometric characteristics of thyroid cells in irradiation stressed rats treated with pinealectomy and melatonin [In Serbo-Croatian (Roman)]. Med Arh 1992; 46:9-10. 37. Vaughan MK, Richardson BA, Petterborg LJ et al. Effects of injection and/or chronic implants of melatonin and 5-methoxytryptamine on plasma thyroid hormone in male and female Syrian hamsters. Neuroendocrinology 1984; 39:361-366. 38. Krotewicz M, Lewinski A, Wajs E. The inhibitory effect of late afternoon melatonin injections, but not of melatonin-containing subcutaneous implants, on thyroid hormone secretion in male Wistar rats. Neuroendocrinol Lett 1992; 14:405-411. 39. Krotewicz M, Lewinski A. Effects of pinealectomy and of late afternoon injections of pineal indole substances on thyroid hormone secretion in male Wistar rats. Biochem Lett 1994; 50:101-107. 40. Shavali SS, Haldar C. Effects of continuous light, continuous darkness and pinealectomy on pineal-thyroid-gonadal axis of the female Indian palm squirrel, Funambulus pennanti. J Neural Transm 1998; 105:407-413. 41. Reiter RJ, Tan D-X, Qi W et al. Pharmacology and physiology of melatonin in the reduction of oxidative stress in vivo. Biol Signals Recept 2000; 9:160-171. 42. Sugawara M, Sugawara Y, Wen K et al. Generation of oxigen free radicals in thyroid cells and inhibition of thyroid peroxidase. Exp Biol Med 2002; 227:141-146. 43. Duthoit C, Estienne V, Giraud A et al. Hydrogen peroxide-induced production of a 40 kDa immunoreactive thyroglobulin fragment in human thyroid cells: The onset of thyroid autoimmunity. Biochem J 2001; 360:557-562. 44. Pomerance M, Abdullah HB, Kamerji S et al. Thyroid-stimulating hormone and cyclic AMP activate p38 mitogen-activated protein kinase cascade. Involvement of protein kinase A, Rac1, and reactive oxygen species. J Biol Chem 2000; 275:40539-40546. 45. Riou C, Remy C, Rabilloud R et al. H2O2 induces apoptosis of pig thyrocytes in culture. J Endocrinol 1998; 156:315-322. 46. Esteves RZ, van Sande J, Dumont JE. Nitric oxide as a signal in thyroid. Mol Cell Endocrinol 1992; 90:R1-R3. 47. Millatt LJ, Jackson R, Williams BC et al. Nitric oxide stimulates cyclic GMP in human thyrocytes. J Mol Endocrinol 1993; 10:163-169. 48. Colin IM, Nava E, Toussaint D et al. Expression of nitric oxide synthase isoforms in the thyroid gland: evidence for a role of nitric oxide in vasculatur control during goiter formation. Endocrinology 1995; 136:5283-5290. 49. Bocanera LV, Krawiec L, Silberschmidt D et al. Role of cyclic 3’5’guanosine monophasphate and nitric oxide in the regulation of iodide uptake in calf thyroid cells. J Endocrinol 1997; 155:451-457. 50. Van den Hove M-F, Stenoiu MS, Croizet K et al. Nitric oxide is involved in interleukin-1α-induced cytotoxicity in polarised human thyrocytes. J Endocrinol 2002; 173:177-185. 51. Mutaku JF, Many M-C, Colin I et al. Antigoitrogenic effect of combined supplementation with dl-α-tocopherol, ascorbic acid and β-carotene and of dl-α-tocopherol alone in the rat. J Endocrinol 1998; 156:551-561. 52. Sewerynek E, Wiktorska J, Nowak D et al. Methimazole protection against oxidative stress induced by hyperthyroidism in Graves; disease. Endocrine Regul 2000; 34:83-89.

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53. Wiktorska J, Sewerynek E, Lewinski A. Effects of melatonin and of other antioxidants on the Schiff bases induced by thyrotoxicosis in rats. 12th International Thyroid Congress, Kyoto 22-27 October, 2000. Endocrine J 2000; suppl:239, abstract P-530. 54. Sewerynek E, Wiktorska J, Lewinski A. Effects of melatonin on the oxidative stress induced by thyrotoxicosis in rats. Neuroendocrinol Lett 1999; 20:157-163. 55. Wiktorska J, Sewerynek E, Lewinski A. Effects of different antioxidants on the oxidative damage induced by L-thyroxine injections in rats. 11th International Congress of Endocrinology. Sydney: 29 October to 2 November, 2000:294, abstarct P-797. 56. Tan D-X, Manchester LC, Reiter RJ et al. Significance of melatonin in antioxidative defense system: Reactions and products. Biol Signals Recept 2000; 9:137-159. 57. Soszynski P, Zgliczynski S, Pucilowska J. The circadian rhythm of melatonin in hypothyroidism and hyperthyroidism. Acta Endocrinol (Copenh) 1988; 119:240-244. 58. Rojdmark S, Berg A, Rossner S et al. Nocturnal melatonin secretion in thyroid disease and in obesity. Clin Endocrinol (Oxf) 1991; 35:61-65. 59. Karasek M, Stankiewicz A, Bandurska-Stankiewicz E et al. Melatonin concentrations in patients with large goiter before and after surgery. Neuroendocrinol Lett 2000; 21:437-439. 60. Peschke E. Morphologische, physiologische und statistische Untersuchungen an der maennlicher Wistar-Ratte zum Problem eines moeglichen funktionellen Connexus: Epiphysis cerebri-Schilddruese. Teil IV: Neurosekretorischer Hypothalamus und Epiphyse. Zool Jb Anat 1981; 105:147-176. 61. Peschke E. Morphologische, physiologische und statistische Untersuchungen an der maennlicher Wistar-Ratte zum Problem eines moeglichen funktionellen Connexus: Epiphysis cerebri-Schilddruese. Teil V: Zusammenfassung der Befunde und Diskussion. Zool Jb Anat 1981; 105:297-319. 62. Peschke E. Morphologische, physiologische und statistische Untersuchungen an der maennlicher Wistar-Ratte zum Problem eines moeglichen funktionellen Connexus: Epiphysis cerebri-Schilddruese. Teil VI: Ergebnisse der Untersuchungen und Literatur. Zool Jb Anat 1981; 105:320-340. 63. Lewinski A, Sewerynek E, Zerek-Melen G. Thyroid hormone-induced activation of rat pinealocytes in organ culture. Neurosci Lett 1986; Suppl 26:S302. 64. Milcou SM, Holban R, Tasca C et al. In vitro study of thyroxine effects on enzymatic activity and cell differentiation in the pineal gland. Rev Roum Endocrinol 1968; 5:203-207. 65. Nir I, Hirschmann N. The effect of thyroid hormones on rat pineal indoleamine metabolism in vitro. J Neural Transm 1978; 42:117-126. 66. Bondarenko LA. Effects of excess and deficiency of thyroid hormones in the body upon blood melatonin in pubertal male rats. Bull Eksp Biol Med 1991; 111:590-591. 67. Lewinski A, Webb SM, Reiter RJ. Possible mechanisms of TSH-independent thyroid growth. Med Hypothesis 1984; 14:141-160. 68. Lewinski A, Sewerynek E, Karbownik M. Melatonin from the past into the future—our own experience. In: Haldar C, Singaravel M, Maitra SK, eds. Treatise on Pineal Gland and Melatonin. Enfield, Plymouth: Science Publishers, Inc., 2002:157-175.

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CHAPTER 4

The Role of Melatonin in the Development of Scoliosis Keith M. Bagnall, Talib Rajwani, Jessie Kautz, Marc Moreau, V. James Raso, James Mahood, Ariana Daniel, Christina Demianczuk, Janet Wilson and Xaioping Wang

Abstract

S

coliosis is an abnormal lateral curvature of the spine often accompanied by vertebral rotation. The most common form of scoliosis is adolescent idiopathic scoliosis (AIS). It is believed that there are several, separate causes of scoliosis all with a common end result which hinders experimental research design. Although abnormal spinal curves have been produced using several methods in a variety of species, none of the curves created mimic those seen in AIS. Consequently, there is no current animal model that can be used to study this problem. In recent years, it has been shown that removal of the pineal gland in young chickens results in the development of scoliosis and that the curves produced have many of the characteristics seen in patients with AIS. This model is receiving much attention as it has much potential for developing an understanding of a mechanism by which scoliosis might be produced at least in some cases of AIS and also as a model for studying scoliosis in general. While serum melatonin levels are significantly reduced in all chickens following pinealectomy, not all the chickens develop scoliosis. While there is much evidence to suggest that removal of the pineal gland with subsequent reduction in serum melatonin levels is the cause of the scoliosis, there remains some suggestion that it might be an artifact of the extensive surgery or reduced levels of another product of the pineal gland that might be responsible. Unfortunately, the phenomenon does not appear to be duplicated following pinealectomy in mammals but, nevertheless, an understanding of the reasons why this is so would provide a large step forward in the understanding of scoliosis in humans. A model to explain the process by which reduced levels of melatonin might produce scoliosis includes the involvement and connection of melatonin with growth hormone and its subsequent effect on bone growth within the vertebrae. This is a very dynamic area of research and one where there is possibly immediate clinical application accompanied by the potential to revolutionize thinking about the treatment methods for scoliosis.

Introduction Scoliosis is the development of abnormal lateral curvatures of the spine often accompanied by vertebral rotation. There are several ages at which there are peaks of incidence (congenital, infantile (0-3 years), juvenile (3-puberty), and adolescence (around puberty)1,2 and there are several known causes such as neurofibromatosis, poliomyelitis, cerebral palsy, and Friedrich’s ataxia. However, most cases are of unknown cause (idiopathic) and develop at the time of adolescence. Consequently, most research focuses on adolescent idiopathic scoliosis (AIS) as Melatonin: Biological Basis of Its Function in Health and Disease, edited by S.R. Pandi-Perumal and Daniel P. Cardinali. ©2006 Eurekah.com.

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this is the type that is most prevalent (80%). Scoliosis appears to affect males and females equally but more females attend the scoliosis clinic because their curves are more severe and progressive. A scoliosis curve can develop but then remain small and even become reduced on its own. In these cases there are often no problems but in cases where the curve continues to progress it has the potential to cause severe problems.1 These abnormal spinal curvatures are mainly a cosmetic deformity which develop at a time when personality development is particularly fragile but if they progress they can also cause a large ‘rib hump’ as the ribs rotate in concert with the vertebrae. This rotation can cause the ribs to impinge on the heart and lungs eventually creating cardiopulmonary compromise. As the curve gradually progresses, treatment may include ‘bracing’ but if the curve continues to progress then extensive surgery becomes necessary which can involve the implantation of long, metal rods along the spine and fusion of the vertebrae.3 In some cases at least, the development of scoliosis has all the characteristics associated with a hormonal problem but when the involvement of the hormone melatonin is considered several concepts need to be clarified.

Problems with Studying Scoliosis and Melatonin Scoliosis has been recognised literally for thousands of years and extensive research into its aetiology has been conducted particularly during the last century. This research has focused on biomechanical, biochemical, morphological, hormonal and genetic factors (for a comprehensive review see Robin2) but very few definitive facts have been uncovered. In fact, despite this extensive research, the only real knowledge that has been discovered is that a growth spurt is closely associated with the curve development5-7 and that most of the patients who attend the scoliosis clinics are female. A review of the literature shows that for every group of patients in which positive results have been found to support a theory, there is a comparable group that shows negative results. The literature is often contradictory and certainly confusing! This lack of knowledge despite the extensive research might actually be informative of itself. While many people believe that AIS has a single cause8 (which might be multifactorial) the lack of research progress might be indicative of AIS having several, completely separate, underlying causes that all result in a common end feature, namely an abnormal spinal curve. Originally, all cases of scoliosis were idiopathic (of unknown cause). Then, as patients with common problems (e.g., poliomyelitis, neurofibromitosis etc.) were identified within the pool of AIS patients, these groups were able to be culled from the idiopathic pool. As 80% of the patients still remain in the AIS pool, it seems logical to suggest that we are not yet at the stage where there is just one remaining cause still to be identified that would explain the curve formation in all these remaining patients. For example, at the present time, identification of a right-sided curve in a young male is thought to be highly indicative of a possible syrinx. If this proves to be true, then another group of patients with an identifiable cause might be able to be removed from the AIS pool. Perhaps research into scoliosis would be more productive if it was recognised that there are probably several different, separate causes that remain to be identified within the pool of AIS patients. This would mean moving away from the idea that just one underlying cause remains that would explain the abnormal curve development in all cases of idiopathic scoliosis. In this respect, reduced levels of serum melatonin might well be a cause of abnormal spinal curve development but perhaps in only a small (5%?) of cases. Such an approach is different to that previously used and significantly affects experimental design. For example, in the past if a theory was developed in which it was believed that scoliosis development was caused by, say, a reduced number of muscle spindles, then the experimental design would probably involve collecting muscle samples from a group of patients with AIS and similar controls and comparing average counts of the number of muscle spindles. However, if a lack of muscle spindles is the cause of scoliosis (which it might well be) but is only the cause in a small number of patients with AIS, then the results from the group of affected patients will be hidden and their effects significantly reduced within the normal values from the other scoliosis

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patients. The average results from the total group of AIS patients will therefore not be significantly different from those results from the normal subjects because the abnormal values from the few AIS patients who have reduced numbers of muscle spindles as the underlying cause cannot be recognised among the average values of the AIS patient group. Consequently, with an experimental design such as this, no progress is made in understanding AIS. An alternative approach is to develop a plausible theory of scoliosis development, show its ability in an appropriate animal model and then predict the clinical presentation that would be exhibited by AIS patients. While this might be considered a ‘needle in a haystack’ approach, it avoids the problems associated with studying a problem that has multiple, separate causes. It is interesting to note that current genetic studies of AIS are starting to show multiple genetic foci which could be predicted by the belief that there are several completely separate causes of scoliosis (which might, of themselves, be multifactorial but which are nevertheless separate from each other).9-11 If it is believed that multiple, different causes of scoliosis remain within the AIS pool of patients, then a comparison of average values from data collected from patients with AIS with similar data collected from normal subjects is probably meaningless. Such experimental designs should therefore be avoided and the results from such experiments viewed with considerable caution. Another problem that may affect scoliosis research and related melatonin studies, is that there could possibly be be two phases of curve development.12 When the curve first forms and is small, it is quite possible that the curve might correct itself if the cause could be identified and removed. However, if the curve is present for a length of time sufficient for morphological changes to occur (e.g., wedging of the involved vertebrae and intervertebral discs) then even if the cause was removed, the curve might remain or continue to progress because the problem is now a mechanical one involving a column of separate segments being arranged in a spiralling curve. On this basis, it is entirely possible that when a scoliosis patient appears and the curves are already established, the underlying cause might well have gone and the patient might be entirely normal-apart from the abnormal spinal curve. More specifically, if this concept is true then serum melatonin levels obtained from blood samples collected from these patients might well reveal normal levels. For success, it might be necessary to obtain samples of blood from AIS patients as their curves are forming initially, which, unfortunately, is often before the patients can be identified! Certainly, it would seem that the younger the patient and the less developed the spinal curves, the better the chance of detecting abnormal serum melatonin levels as a cause of scoliosis. Currently, scoliosis is evaluated primarily by measuring the Cobb angle13 from radiographs taken at various intervals of time when the patient visits the scoliosis clinic. However, scoliosis is a continuous process that develops in three dimensions and therefore is difficult to evaluate using static, occasional two-dimensional radiographs.14 Scoliosis research might make more progress if scoliosis was evaluated as a continuous process in three dimensions. While this aspect affects all scoliosis research, it particularly affects melatonin research because an improved evaluation system might provide clear identification of some of the symptoms manifested only by AIS patients whose melatonin levels have been affected and are the underlying cause of the scoliosis. There have been many successful attempts to produce an animal model for scoliosis (see Robin2) which have used a variety of different methods in several species. However, few, if any of these models have produced curves which bear any resemblance to that seen in patients with AIS. This could possibly be due to the unique bipedal stance of humans which affects spinal mechanics and influences spinal curvature to a great extent. Consequently, the value of these models in understanding scoliosis is limited. This is unfortunate because an appropriate animal model would allow the development of scoliosis curves to be observed from their initial stages (unlike with humans) and would allow the curve development to be controlled using methods appropriate for novel treatment strategies based on aetiology rather than symptoms.

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The Pinealectomised Chicken Model for the Study of Scoliosis

In 1959 Thillard15 discovered (serendipitously?) that removal of the pineal gland in young chickens resulted in some (~70%) developing scoliosis. At that time, pinealectomy in hamsters was a common procedure for the study of gonadal development and their association with circadian rhythms.16,17 It is interesting to note that there is no record of scoliosis development in these animals. It is also interesting to note that scoliosis development in children with pineal tumours is not regularly reported either. Therefore, even in these early stages, there is evidence that perhaps this phenomenon of scoliosis development following pinealectomy is restricted only to chickens. Nevertheless, it is now well established that pinealectomy in young chickens consistently results in the development of scoliosis that has many of the characteristics seen in patients with AIS.18-23 The types of curve that develop, the number and range of vertebrae involved, the progression pattern of the curve, and the degree of curvature are all similar to that seen in patients with AIS.19 Furthermore, the scoliosis develops quickly with some chickens developing spinal curves within 7 days and almost all the chickens that are going to develop scoliosis doing so within 3 weeks after surgery. The development of scoliosis in these chickens occurs during a period of very rapid growth. This pinealectomised chicken model holds much promise in the study of scoliosis and is the current model of choice for many people. However, the mechanism underlying the development of the scoliosis remains a mystery. Several questions still surround the production of the scoliosis in this actual model. For example, are the characteristics really the same as those seen in patients with AIS? The chicken spine has very different biomechanics to that of the human and, although the chicken has a pseudo-bipedal stance, it is possible that the very flexible intervertebral joint at T6/7 might be an important factor in the development of the spinal curves in the chicken. Evaluation of the spinal curves in the chicken also relies on two-dimensional radiographs and it is possible that if three-dimensional assessment could be made then significant differences in morphology could be identified especially in the shape changes of the actual vertebrae themselves. While the pineal gland is removed during the pinealectomy, there are several other factors that must be considered as possible causes for the spinal curve development. The pineal gland in the chicken lies just deep to the confluence of sinuses and although other approaches have been tried, the best way to gain access to the pineal gland is directly through the skull above the confluence of sinuses. Understandably, there is much bleeding during the surgery and blood pressure must drop significantly. Furthermore, opening the skull must reduce cerebrospinal fluid pressure if only for a short time. While every effort is made to reduce these effects to a minimum, their influence on curve development is unknown. Removal of the pineal gland, either by cutting or by suction, also inevitably involves at least the touching of other adjacent brain tissue. Therefore, it is possible that damage or even contact with this adjacent brain tissue might be responsible for the production of the scoliosis. However, experiments in which large areas of adjacent brain tissue have been extensively damaged with no effect on subsequent curve development suggest that it is the actual removal of the pineal gland that is the underlying cause.24,25 Certainly, none of the chickens in which ‘sham’ surgery (the same steps but without the actual removal of the pineal gland) has been performed has ever developed scoliosis. In fact, experiments have shown that only the pineal stalk needs to be cut for scoliosis to develop, not the actual removal of the gland itself.26,27 While not entirely conclusive, the results from these experiments suggest strongly that it is removal of the pineal gland that is responsible for the development of the scoliosis and not some artifact of the extensive surgery. It would be interesting to determine precisely which structure in the pineal stalk is required to be cut to produce the scoliosis although it is suspected that it would be the nerve supply. The pinealectomy model for the production of scoliosis was resurrected in the 1980s by Machida et al21,28 who reported that 100% of their chickens developed scoliosis. Subsequent experiments by others have reported much less incidence even as low as 55% (consistently).18-20,24-33 The reasons for these discrepancies in incidence are not clear. While the basic pinealectomy technique for chickens is well established and described in the literature, there

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might be subtle, unreported differences that are significant. There might be a difference in the genetic make-up of the flocks used by the different groups with one flock being more susceptible to developing scoliosis than another.34 There does not appear to be a difference if broilers (Mountain Hubbards, mature in 9 weeks-for eating) and layers (White Leghorn, mature in 21 weeks-for egg laying) are used other than the rate of scoliosis development being different.35 The more rapidly growing broilers develop scoliosis more quickly than the layers but the eventual scoliosis is the same in both groups. Nor does there appear to be a difference if males or females are used.35 There is a reduced incidence if older chickens (3 weeks and 8 weeks after hatching) are used but this seems to be associated with a reduction in growth rate accompanying increased age. In contrast, this age factor does not apply to the first week after hatching. Experiments have shown that the number of days between hatching and surgery are within the first week. The effects of pinealectomy on other avian species have not apparently been studied. This difference between research groups in incidence of scoliosis development following pinealectomy in young chickens is disconcerting but perhaps holds an important key for finding the solution to this phenomenon. Pinealectomy in young rats and hamsters does not appear to produce scoliosis although there have been reports that pinealectomy in bipedal rats does eventually lead to scoliosis.32 However, the rat spine is so flexible that a reliable method for determining the degree of any scoliosis is difficult to achieve and makes such results questionable. Even if it is accepted that removal (or at least ‘disconnection’ by cutting the stalk) of the pineal gland in young chickens results in the development of scoliosis,26 then the underlying mechanism remains a mystery. The main product (or at least the one most studied) of the pineal gland is melatonin and so most research in this area has focused on the effects of reduced levels of melatonin as a possible solution to the problem. Melatonin production is responsive to light cycles and so has a circadian rhythm with serum levels being high in the dark and low in the light.17,36 Consequently, removal of the pineal gland would be expected to reduce serum melatonin levels unless there is another source. It has been reported that both the retina and cells in the lining of the gut tube produce melatonin.37 Measurement of serum melatonin levels in pinealectomised chickens has shown that they remain low for at least 4 weeks following pinealectomy which is long after the development of any scoliosis. Even if there is another potential source of melatonin in the chicken, its production does not appear to have any consequence on serum melatonin levels during the time of curve development. Two points of concern regarding melatonin research and the development of scoliosis must be mentioned at this time. 1 ml of blood is required to generate sufficient serum for evaluation using competitive- binding radioimmunassay techniques for determining serum melatonin levels. This amount of blood cannot be withdrawn from a chicken until 3 weeks after hatching without there being a high probability that the chicken will die. Three weeks after hatching a chicken has sufficient blood to adequately withstand withdrawal of such an amount. Consequently, all measurements are expressed as average values from a group of chickens since longitudinal assessment of serum melatonin levels within a single chicken is not currently possible. Methods by which smaller amounts of serum can be used are being explored and, with their successful development, it will be possible to follow the serum melatonin levels within a single chicken. The competitive-binding radioimmunassay technique is also not sufficiently sensitive to be able to state accurately that serum melatonin levels are at the zero level. While the error of measuring serum melatonin levels at 50pgs/ml have been calculated to be + 5 pgs/ml, the error is much larger at zero levels because of the nature of the technique. Therefore, it is only possible to state that serum melatonin levels are ‘close to zero’ or ‘significantly reduced’ and not that they are actually zero. Following pinealectomy in young chickens, average serum melatonin levels drop to being close to zero. Machida et al21 found that 100% of their chickens developed scoliosis and made the logical assumption that these reduced levels were responsible for the development of the

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scoliosis. However, subsequent work, particularly by others,15,20,24-27,29-33 has shown that not all the chickens develop scoliosis and yet the average serum melatonin levels have been significantly reduced to close to zero. By looking at the serum melatonin values from individual chickens in a series of experiments and correlating them with the development or absence of scoliosis development in that particular chicken, it appears that there might be a threshold level of serum melatonin below which scoliosis might develop. The serum melatonin levels of chickens that developed scoliosis are not randomly scattered along the continuum of serum melatonin values and are clearly below a specific level. In contrast, there are many chickens who did not develop scoliosis whose serum melatonin levels are much higher than this proposed threshold level. However, it must be clearly stated that there is also a large group of chickens in whom individual serum melatonin levels were below this threshold value and did not develop scoliosis. Perhaps the only statement that can be made with certainty, up to this point, is that reduced levels of serum melatonin are associated with the development of scoliosis in young chickens due to the fact that no chickens with normal levels of serum melatonin have been shown to develop scoliosis. The traditional methods of clarifying whether or not the absence of a particular hormone is responsible for a particular effect have both been tried in this model but with very contrasting results. Machida et al21 gave melatonin therapy to their pinealectomised chickens and showed that such treatment eliminated the production of scoliosis and even reduced the scoliosis if scoliosis was allowed to develop for a time before treatment was initiated. However, Machida et al21 gave a dose approximately 10X the calculated physiological dose, they gave the treatment every other day despite melatonin being related to a circadian rhythm, and they gave the injection in the middle of the light cycle when average serum melatonin levels are at their lowest. In contrast, Bagnall et al31 gave a predetermined physiological dose each day to the chickens in the middle of the dark cycle but were unable to show any effects on the developing scoliosis at all. Machida et al23,31,38 also transplanted the removed pineal gland to the musculature of the chest wall in the chicken and were also able to prevent the development of any scoliosis. Again in contrast, Bagnall et al31 transplanted 10 pineal glands to the chest wall and yet were unable to prevent the development of the scoliosis. It is difficult to imagine how a transplanted of a pineal gland to the body wall musculature can function immediately after transplantation. This immediate functioning would be necessary for the prevention of scoliosis which occurs within the first week after surgery in chickens.40 Furthermore, previous experiments have shown that simply cutting the stalk of the pineal gland is sufficient to produce the scoliosis.26 Removal of the actual pineal gland is not necessary and such experiments, where the pineal gland has been left in place after the stalk has been cut, perhaps represent the ideal ‘transplant’ study because they have had minimal interference and yet scoliosis still developed. These contradictory results are very confusing. It is as if two entirely separate models of pinealectomised chicken for the production of scoliosis are being developed. Conversely, if it could be shown that another product of the pineal gland was responsible for the production of the scoliosis and that, in some way, this product was being replaced in the successful therapy experiments then these differing results could be reconciled. Currently, there remains some doubt as to whether or not reduced levels of serum melatonin are responsible for the development of scoliosis in young chickens. Methods by which serum melatonin levels can be reduced without surgery are therefore being examined. If another method to reduce serum melatonin levels could be established then it would be possible to prove that reduced levels of serum melatonin are responsible for the production of the scoliosis. If another method copuld be found, it might also be possible to control serum melatonin levels more precisely and explore this phenomenon more carefully. As melatonin is only produced in the dark, it has been postulated that growing chickens in 24h light would prevent the production of melatonin and so reduce serum melatonin levels to zero. Nette et al (submitted) grew chickens in 24h light levels of 1200 Lux and found that 15% of the normal chickens developed scoliosis and the incidence of scoliosis in the pinealectomised chickens increased

The Role of Melatonin in the Development of Scoliosis

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from 50% to 80% when compared to control groups of chickens. These results supported the suggestion that there is a threshold level of serum melatonin for the development of scoliosis and that the increased light reduced levels sufficiently to increase the incidence of scoliosis. It is particularly exciting that some of the normal chickens developed scoliosis without any extensive surgery. Maintaining the chickens in such an environment proved difficult and they have been unable to repeat their results in subsequent experiments involving slightly different environmental conditions. Apparently, the chickens would provide their own ‘darkness’ by huddling together and burying their heads under their wings to achieve a dark environment. Nevertheless, this method seems to be effective and supports the idea that reduced levels of serum melatonin are responsible for the development of scoliosis. Another method that is being explored involves feeding the chickens a tryptophan-free or tryptophan-reduced diet.42 Tryptophan is an essential amino acid and is involved in the first stage of melatonin production apparently in all animals. It has been postulated that if tryptophan is removed from the diet, then serum melatonin levels will be reduced. Unfortunately, however, tryptophan is also necessary in the production of many other substances and tissues in the body, especially cell membranes. Therefore, the use of a tryptophan-free diet can be extremely difficult in terms of survival for the animals and so a tryptophan-reduced diet has often been used in experiments. It remains to be seen whether or not a tryptophan-free or tryptophan-reduced diet will be effective in chickens for the production of scoliosis. If either is, then controlling the amount of tryptophan might be an ideal way to manipulate serum melatonin levels. Further investigation may also lead to determination of the specific times of the circadian cycle when the amounts can be altered to affect serum melatonin levels. Such an approach would have enormous benefits in the study of scoliosis. It must be noted that a tryptophan-reduced diet given to trout has resulted in scoliosis development but whether or not this can be achieved in chickens remains to be seen.

Serum Melatonin Levels in Humans with Scoliosis An obvious extension of the pinealectomised chicken model for the study of scoliosis production in young chickens is the examination of serum melatonin levels in patients with AIS. This has already been performed by Hilebrand et al43 and Bagnall et al.29 Neither of these studies found any significant differences in average serum melatonin levels between patients with AIS and normal, age- and maturity-matched controls. However, both these studies can be criticised on at least two grounds as already discussed earlier: reduced levels of serum melatonin might only be the cause of AIS in a small percentage of patients; serum melatonin deficiency might have already been corrected in the selected patients used in both groups (~16 years) due to age. In another study, Machida et al44 collected serum melatonin values from hospitalised patients with AIS at regular intervals over 24 h and found that those patients who exhibited a progressive curve demonstrated significantly lower levels of serum melatonin compared to patients with non-progressive curves. If these experiments can be replicated and the results confirmed, this would be an enormous step forward in the treatment of scoliosis because at the present time there is no known marker to differentiate these two significant groups of patients. Unconfirmed reports have also detailed results of treating scoliosis patients with melatonin and having success in reducing the curves significantly.

A Proposed Model by Which Low Levels of Serum Melatonin Can Affect Vertebral Growth and Produce Scoliosis With so few of the pieces of the puzzle available and unclear, the model to describe the development of scoliosis following pinealectomy in chickens is difficult to visualise. With this in mind, it is proposed that pinealectomy in young chickens results in reduced levels of serum melatonin. Melatonin receptors are ubiquitous with two types of receptor in the cell membrane and one in the cytosol. In the cell membrane, the attachment of the melatonin

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affects the shape of the G-proteins which then bind to and activate adenylate cyclase. Alternatively, melatonin can bind intracellularly to cytosolic calmodulin which may then affect calcium signalling by interacting with target enzymes such as adenylate cyclase and phosphodiesterase as well as structural proteins.45-47 Calmodulin is a calcium-binding receptor protein which regulates cellular calcium through transport across the cell membrane. Kindsfater et al48 produced results which suggested that increased calmodulin levels in platelets were associated with the progression of curves in patients with AIS. Initially this was a very attractive proposition because it is difficult to see how scoliosis could affect calmodulin levels in platelets. However, further studies have shown that the calmodulin levels usually decrease in patients undergoing brace treatment or spinal fusion and so it is difficult to separate these changes from cause and effect principles. It is unfortunate that such principles always haunt scoliosis research because patients are only examined and available after curve development has occurred. There does appear to be an association between serum melatonin levels and growth hormone levels although the precise connection mechanism is unclear and vague at best.21,23 The response of the body to growth hormone is dependent on age and actual dose and varies both within and between species. The situation is further complicated because there is a temptation to assign the reverse effects to a reduced level of growth hormone to those experienced with an increased dosage. Nevertheless, a review of the literature suggests that in the chicken, melatonin acts as a serotonin receptor antagonist so that low levels of melatonin results in low levels of serotonin. Serotonin stimulates somatostatin release which, in turn, inhibits the release of growth hormone. Therefore, low levels of serotonin results in high levels of growth hormone. In summary for the chicken, the literature would suggest that low levels of melatonin would result in high levels of growth hormone. In contrast in the rat, serotonin appears to inhibit somatostatin release which, in turn, would inhibit growth hormone release. Consequently, in the rat, low levels of melatonin would be predicted to result in low levels of growth hormone. Studies of the effect of pinealectomy on serum melatonin and growth hormone levels in chickens and rats have not yet been performed but are currently underway. If these predictions from the literature are shown to be true, then it might explain why a pinealectomy in chickens produces scoliosis whereas the same procedure in rats does not. In the human, the picture is even more confusing. Certainly, some studies have shown that growth hormone levels are higher in patients with AIS49-51 and that patients with AIS are taller than controls at least in the early stages of puberty. While two studies were unable to show significant differences in serum melatonin levels between patients with AIS and controls, another found significantly lower levels of serum melatonin in AIS patients. This would fit well with Machida et al44 who found that patients with AIS whose curves were progressive had lower levels of serum melatonin than controls but perhaps all that can be stated at this stage is that low levels of melatonin may result in increased levels of growth hormone which leads to scoliosis. The results from the combined growth hormone and melatonin studies are awaited with interest. Even if the interaction of abnormal serum melatonin and growth hormone levels is the primary cause of scoliosis development in some patients, the model must include a mechanism that translates this difference into a means by which abnormal spinal curves can develop. As both growth hormone and melatonin can affect bone growth, it is proposed that these abnormal levels act directly on vertebral growth. In evolutionary terms a vertebra is a composite of several bones each with its own growth pattern. It is envisaged that these abnormal serum levels of both growth hormone and melatonin both act on the vertebra in such a way that abnormal vertebral growth develops, especially between the anterior and posterior components. This abnormal vertebral growth then leads to abnormal spinal curve development and the initiation of scoliosis.

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References 1. Keim H. Scoliosis. Clinical Symposia, Ciba 1979; 31. 2. Robin G. The aetiology of idiopathic scoliosis: a review of a century of work. 1990;Boca Raton, Florida. CRC Press. 3. Krismer M, Bauer R, Sterzinger W. Scoliosis correction by Cotrel-Dubousset instrumentation. Spine 1992; 17:S263-269. 4. Nordwall A, Willner S. A study of skeletal age and weight in girls with idiopathic scoliosis. Clin Orth Rel Res 1975; 110:6-10. 5. Willner S, Nilsson K, Kastrup K et al. Growth hormone and soamtomedin A in girls with idiopathic scoliosis. Acta Ortho Scand 1976; 65:547-552. 7. Hagglund G, Karlberg J, Willner S. Growth in girls with adolescent idiopathic scoliosis. Spine 1992; 17:108-111. 8. Bagnall K, Goldberg C, Burwell G. Adolescent Idiopathic Scoliosis: is the cause neuromuscular? In: Research into Spinal Deformities 2. (Ed. IAF Stokes) Series Study in Health Technology and Informatics. 1999; 59:91-93. IOS Press, Oxford. 9. Miller N. Genetics of familial idiopathic scoliosis. Clin Orth Rel Res 2002; 401:60-64. 10. Miller N, Schwab D, Sponseller P et al Characterization of idiopathic scoliosis in a clinically well-defined population. Clin Orth Rel Res 2001; 392:349-357. 11. Wise C, Barnes R, Gillum J et al. Localisation of susceptibility to familial idiopathic scoliosis. Spine 2000; 25;2372-2380. 12. Lonstein J. Adolescent idiopathic scoliosis. Lancet 1994; 344:1407-1412. 13. Cobb J. The problem of the primary curve. J Bone Jt Surg 1960; 42A:1413-1425. 14. Bagnall K, Thomas B, Moreau M et al. A new tool by which to visualise adolescent idiopathic scoliosis as a continuous process. In: Research into Spinal Deformities 2. (Ed. IAF Stokes) Series Study in Health Technology and Informatics. 1998; 59:65-68. IOS Press, Oxford. 15. Thillard M. Deformations de la colonne vertebrale consequtives a l’epiphysectomie shez le poussin. Extrait des comptes Rendus de l’Assoc des Anat 1959; 46:22-26. 16. Volrath L. The pineal organ. New York, Springer Verlag. 1981. 17. Reiter R. Pineal melatonin: Cell biology of its synthesis and of its physiological interreactions. Endocrine reviews 1991; 12:151-180. 18. Wang X, Moreau M, Raso J et al. Changes in serum melatonin levels in response to pinealectomy in the chicken and its correlation with development of scoliosis. Spine 1998; 23(22):2377-81. 19. Wang X, Jiang H, Raso J et al. Characterisation of the scoliosis that develops following pinealectomy in the chicken and comparison with the scoliosis seen in adolescent idiopathic scoliosis in humans. Spine 1997; 22(22):2626-2635. 20. Kanemura T, Kawakami N, Deguchi M et al. Natural course of experimental scoliosis in pinealectomised chickens. Spine 1997; 22(14):1563-1567. 21. Machida M, Dubousset J, Imamura Y et al. An experimental study in chickens for the pathogenesis of idiopathic scoliosis. Spine 1983; 18:1609-1615. 22. Machida M, Dubousset J, Imamura Y et al. Role of melatonin deficiency in the development of scoliosis in pinealectomised chickens. J Bone Jt Surg (Brit) 1995; 77:134-138. 23. Machida M, Dubousset J, Imamura Y et al. Melatonin: A possible role in the pathogenesis of adolescent idiopathic scoliosis. Spine 1996; 21:1147-1152. 24. Bagnall K, Raso J, Moreau M et al. The development of scoliosis following pinealectomy in young chickens is not the result of an artifact of the surgical procedure. International Research Society for Spinal Deformities. 2002; 3-9. 25. Bagnall K, Raso J, Moreau M et al. The development of scoliosis following pinealectomy in young chickens is not the result of an artifact of the surgical procedure. Microsc Res Tech 2001; 1:53(1)81-6. 26. Beuerlein M, Wilson J, Moreau M et al The critical stage of pinealectomy surgery following which scoliosis is produced in young chickens. Spine 2001; 1:26(3)237-40. 27. Bagnall K, Wang X, Raso J et al The pinealectomised chicken model for the study of Adolescent Idiopathic Scoliosis 2001; Internet publication for ISBE. 28. Dubousset J, Queneau P, Thillard M. Experimental scoliosis induced by pineal and diencephalic lesions in young chickens. Orth Trans 1983; 7:7. 29. Bagnall K, Beuerlein M, Johnson P et al The effects of pineal transplantation on the production of scoliosis in pinealectomised chickens. Spine 2001; 1:26(9)1022-7. 30. Bagnall K, Deyell M, Wang X et al. Pinealectomy and scoliosis: The day of surgery is critical to the development of scoliosis following pinealectomy in young chickens. J Bone Jt Surg (Amer) 2000; 82A/8 1197-1198.

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31. Bagnall K, Raso J, Moreau M et al The effects of melatonin therapy on the development of scoliosis following pinealectomy in the chicken. J Bone Jt Surg (American) 1999; 81-A:191-199. 32. O’Kelly C, Wang X, Raso J et al The production of scoliosis following pinealectomy in young chickens, rats and hamsters. Spine 1999; 24:35-43. 33. Coillard C, Rivard C. Vertebral deformities and scoliosis. Eur Spine J 1996; 5:91-100. 34. Riggins R, Abbott U, Ashmore R et al. Scoliosis in chickens. J Bone Jt Surg 1977; 59:1020-1026. 35. Beuerlein M. Scoliosis in pinealectomised chickens. 1999 MSc thesis. University of Alberta. 36. Bagnall K, Raso J, Hill D et al. Diurnal and nocturnal serum melatonin levels in girls with adolescent idiopathic scoliosis. Spine 1996; 21(17):1974-1978. 37. Weichmann A. Melatonin: Parallels in pineal gland and retina. Exp Eye Res 1986; 42:507-527. 38. Machida M, Dubousset J, Imamura Y et al. Pathogenesis of idiopathic scoliosis: SEPs in chickens with experimentally-induced scoliosis and in patients with idiopathic scoliosis. J Ped Orth 1994; 14:329-335. 39. Machida M, Miyashita Y, Murai I et al. Role of serotonin for scoliotic deformity in pinealectomised chickens. Spine 1997; 22:1297-1301. 40. Wu W, Scott D, Reiter R. Transplantation of the mammalian pineal gland: studies of survival, revascularisatiojn, reinnervation, and recovery of function. Exp Neurol 1993; 122:88-99. 41. Nette F, Bagnall K, Daniel A et al. The effects of intense 24h light on scoliosis development in young chickens. Submitted. 42. Zimmermann R, McDougle C, Schumaker M et al. Effects of acute tryptophan depletion on nocturnal melatonin secretion in humans. J Clin Endocrin Met 1993; 76:1160-1164. 43. Hilebrand A, Blackmore L, Loder R et al. The role of melatonin in the pathogenesis of adolescent scoliosis. Spine 1996; 21:1140-1146. 1996 44. Machida M. Cause of idiopathic scoliosis. Spine 1999; 24:2576-2587. 45. Haeseleer F, Imanishi Y, Sokal I et al. Calcium-boinding proteins: intracellular sensors from the calmodulin superfamily. Bichem Biophys Res Commun 2002; 290:615-623. 46. Von Gall C, Stehl J, Weaver D. Mammalian melatonin receptors. Cell Tissue Res 2002; 309:151-162. 47. Thomas L, Purvis C, Drew J et al. Melatonin receptors in human fetal brain. J Pin Res 2002; 33:218-224. 48. Kindsfater K, Lowe T, Lawellin D et al. Levels of platelet calmodulin for the prediction of progression and severity of adolescent idiopathic scoliosis. J Bone Jt Surg 1994; 76:1186-1192. 49. Shohat M, Shohat T, Nitzam M et al. Growth and ethnicity in scoliosis. Acta Orth Scand. 1988; 59:310-313. 50. Ahl T, Albertsson-Kikland K. Twenty-four-hour growth hormone profiles in pubertal girls with idiopathic scoliosis. Spine; 1988:13:139-142. 51. Wilner S. A study of growth in girls with idiopathic structural scoliosis. Clin Orth 1974; 101:129-135.

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CHAPTER 5

Effect of Melatonin on Life Span and Longevity Vladimir N. Anisimov

Introduction

D

uring the past decade, a number of reports, sometimes contradictory, appeared concerning the role of the pineal gland in aging.1-4 Melatonin is the main pineal hormone synthesized from tryptophan, predominantly at night time.5 Melatonin is critical for the regulation of circadian and seasonal changes in various aspects of physiology and neuroendocrine function.5,6 As age advances, the nocturnal production of melatonin decreases in animals of various species, including in humans.7 The performance of a pinealectomy on rats produced a reduced life span8,9 whereas the syngeneic transplantation of a pineal gland from young donors into the thymus of old mice or in situ into pinealectomized old mice prolonged the life span of the recipients.10,11 In this chapter the results of studies on the effect of administration of melatonin to mice, rats, fruit flies, or worms are reviewed.

Effect of Melatonin on Longevity in Mice

Pierpaoli and Maestroni12 were the first in demonstration of life extension induced by melatonin. In 1985 they started a daily administration of melatonin with drinking water (10 mg/ l) into 10 male C57BL/6J mice aged 575 days. Ten control mice received a 0.01% solution of ethanol as a drinking water. Melatonin was given from 18.00 hrs to 8.30 hrs. In 5 months after start of the experiment, control mice became bold, less active and had decreased body weight. Treatment with melatonin prevent body weight loss. Mean life span of mice under the influence of melatonin increased by 20%. In 1991 Pierpaoli et al10 reported the results of 3 new experiments with melatonin. In all of them melatonin was given with drinking water (10 mg/l) during night time. 15 female C3H/ He mice were given melatonin starting at the age 12 months. 14 mice of the same strain served as a control. The treatment failed to increase a longevity of C3H/He mice, and increased the incidence of spontaneous tumors (lympho- and reticulosarcomas and ovarian tumors). It is worthy to note that female c3H/He mice characterized by a high incidence of spontaneous mammary carcinoma,13 however authors did not reported any data on mammary tumors in his paper. Analysis of presented survival curves has shown that the mice exposed to melatonin lived 2 months shorter that controls. In the 2nd series of the experiment, melatonin was given at day time or at night time to female NZB mice, characterized by high incidence of autoimmune hemolytic anemia, nephrosclerosis and systemic reticulo-cell tumors. Each group included 10 mice and melatonin was given from the age of 4 months. The administration of the hormone during the day time failed influence survival, and all mice of this group were died before the age of 20 months (in the control group—before the age of 19 months). The night exposure to

Melatonin: Biological Basis of Its Function in Health and Disease, edited by S.R. Pandi-Perumal and Daniel P. Cardinali. ©2006 Eurekah.com.

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melatonin reduced the mortality of NZB mice—at the age of 20 months 4 of 10 mice were alive, and 2 of them survived the age of 22 months. The last mouse was died 4 months after the death of the last mice in the control group. In the 3rd series, the mean life span of 20 control male C57BL/6J mice was 743 ± 84 days whereas the mean life span of 15 males treated with melatonin from the age of 19 months was 871 ± 118 days. Treatment with melatonin failed to modify body weight as compared to the controls in any series. In 1994 Pierpaoli and Regelson14 presented new results. Melatonin (10 mg/l) was given with drinking water during night hours (18.00 to 8.30) to female BALB/c mice aged 15 months. Mean life span of 26 control mice was 715 days whereas of 12 mice exposed to melatonin – 843 days (+ 18%). Maximum life span was 27.2 months in the control and 29.4 months in the treated group. There was no any differences in the body weight between groups. In another experiment melatonin (10 mg/l) was given with drinking water at night to male BLAB/c mice starting at the age 18 months.15 Animals were sacrificied by small groups in 4, 7 and 8 months after the start of the experiment. In 8 months after start the weight of thymus, adrenal glands and testicles of mice treated with melatonin was significantly differ from age-matched controls but was similar to more young control mice. The similar changes were observed in the number of peripheral blood leucocytes, the level of zinc, testosterone and thyroid hormones. Authors believe that the cyclic administration of melatonin has positive influence on animals maintaining the young status of endocrine and thymicolymphoid organs. Lenz et al16 injected melatonin into female NZB/W mice in a single dose 100 mkg/mouse (2 - 3,5 mg/kg) daily during 9 months in the morning hours (between 8 and 10 a.m.) or in the evening (between 5 and 7 p.m.), starting at the age of 8 months. There were 15 mice in each group. It was shown that morning injections of melatonin significantly increased the survival of mice whereas the evening injection failed influence the survival. Twenty percent of control mice survived 34 weeks whereas 65% of mice exposed to the morning injections of melatonin survived this age. Unfortunately the observation was finished before a natural death of all animals and no complete autopsy has been performed. Mocchegiani et al17 administered melatonin (10 mg/l) with drinking water to 50 male Balb/c mice starting at the age of 18 months. Fifty other mice were given water with supplementation of zinc sulphate (22 mg/l) and 50 mice served as the control group. The treatment with melatonin and zinc significantly shifted to right survival curves and increased maximal life span of mice by 2 and 3 month, respectively. Unfortunately, no numerical data on mean and maximum life span was presented. Both compounds failed influence food consumption or body weight dynamics. Conti and Maestroni18 have studied effect of melatonin on longevity of female NOD mice prone to high incidence of insulin-dependent diabetes. One of mouse groups (n= 25) was neonatally pinealectomized, the 2nd group (n= 30) was given subcutaneous injections of melatonin in a single dose of 4 mg/kg at 16.30 hours daily 5 times a week from the age 4 weeks until the age of 38 weeks. Mice of the 3rd group were injected with bovine serum (PBS) as a control to the group 2. In the 4th group mice were given melatonin (10 mg/l) with drinking water during night time 5 times a week from the age of 4 weeks until the 38th weeks of life. Fifth group included 29 intact mice. Survival of pinealectimized mice was significantly reduced due to autoimmune diabet and at the age of 32 weeks only 8% of mice in this group survived. In the control group 65.6% of mice died before the 50th week of the age. Long-term injections with melatonin slow down the diabetes development and mortality. Only 10% of mice in this group were died before the age of 50 weeks. Administration of melatonin with drinking water was less effective than injections—to the end of the period of observation survived 58.8% of melatonin-treated mice whereas in the control group 34.5%. Thus, pinealectomy accelerated diabetes development and reduced life span of mice whereas treatment with melatonin slow down the disease development and increased longevity.18 Oxenkrug et al19 have studied effect of long-term administration of melatonin and N-acetylserotonin (NAS) in male and female C3H mice. Melatonin being given with drinking

Effect of Melatonin on Life Span and Longevity

47

water during night hours at the daily dose 2.5 mg/kg starting at the age 1 month increased life span in male mice about 20% versus control animals but did not affect the life span of female mice. The ability of melatonin to alter disease incidence and longevity was studied in adult male C57BL/6 mice.20 Mice were fed died supplemented with melatonin (11 ppm) starting at 18 months of age. Melatonin failed influence dynamics of the body weight and food consumption as well as mortality of mice. Fifty percent mortality was at the age 26.5 months in the control group and at the 26.7 months – in melatonin-treated. Survival curves were not presented. A part of animals was sacrificied at the age of 24 months (cohort 1) or at the age of 50% mortality (cohort 2). The 3rd cohort included mice died before the age of 2 years. There were 20 control and 20 melatonin-treated mice in the cohort 1, 7 and 13 mice, correspondingly, in the cohort 2 and 28 and 30 mice, correspondingly, in the cohort 3. Authors claimed that diet supplementation with melatonin initiated during middle age did not appear to affect age-associated lesions patterns, lesion burden or longevity in male C57BL/6 mice. Data presented in the paper show that incidence of lymphomas was similar in the control and melatonin-treated mice in the cohort 3 (21.1 and 23.3%, respectively), however in the cohort 2 the incidence of lymphomas was 28.6% in controls and 77.9% in melatonin-treated animals. In our experiments21 50 female CBA mice from the age of 6 months until their natural deaths were given melatonin with their drinking water (20 mg/l) for 5 consecutive days every month. Fifty intact mice served as controls. The results of this study show that the consumption of melatonin did not significantly influence food consumption, but it did increase the body weight of older mice; it did not influence physical strength or the presence fatigue; it decreased locomotor activity and body temperature; inhibited free radical processes in serum, brain, and liver; it slowed down the age-related switching-off of estrous function. The survival rate dynamics were similar in both groups up to the age of 22 months. Afterward, a pronounced decrease in mortality rate was observed under the effect of melatonin. Under the influence of melatonin the number of mice that reached the age of 24 months increased 5.4-fold in comparison with the controls. The mean life span of mice treated with melatonin was slightly increased compared with controls (+ 5.4%; p < 0.05). The life span in the last 10% of the mice increased by the duration of melatonin treatment (by 2 months). The maximum life span expanded by almost 4 months under the effect of melatonin. At the same time the treatment with melatonin was followed by a 20% increase in malignant tumor incidence in comparison with that of the control group. Five cases of lymphomas and 5 cases of lung adenocarcinomas were observed in the group treated with melatonin, whereas no cases of similar tumors were found in the control group. It is worth noting that the mean life span of fatal tumor-bearing mice in the group treated with melatonin was increased by 2.3 months as compared with that of the control group. In new set of experiments conducted in our laboratory melatonin (2 mg/l or 20 mg/l) was given with drinking water (20 mg/l) for 5 consecutive days every month to female Swiss-derived SHR mice starting at the age of 3 months.22 It was 50 mice in each group. Fifty other mice served as controls. The last mouse in the control group died at the age of 772 days. In the groups treated with melatonin at the doses 2 and 20 mg/l 6% and 12% of mice survived this age, correspondingly. The maximum life span was 881 and 890 days in melatonin-treated groups, respectively. The mean life span of mice treated with melatonin was not increased compared to controls. Life span in the last 10% of survivors was increased with melatonin treatment (by 55 days at the dose of 2 mg/l and 85 days at the dose of 20 mg/l). Treatment with melatonin at the dose of 2 mg/l was followed by a 1.9 fold decrease in total, and a 2.2- fold decrease in malignant tumor incidence as compared to the controls. Incidence of mammary carcinomas with the lower dose of melatonin decreased 4.3-fold. The mean latent period of mammary carcinomas was significantly increased (by 2 months) in mice treated with 2 mg/l melatonin as compared to controls. There was no effect of treatment with the higher dose of melatonin (20 mg/l) on the total incidence of tumors or on the incidence of tumors at any site.

48

Melatonin: Biological Basis of Its Function in Health and Disease

The effect of melatonin was studied in our laboratory in senescence accelerated mouse model (SAM).23 Female SAMP-1 and SAMR-1 mice were given melatonin with their drinking water (20 mg/l) for 5 consecutive days every month until natural death. There was no any significant effect of melatonin on life span or spontaneous tumor incidence in senescence accelerated SAMP-1 mice and senescence resistant strain SAMR-1. In Table 1 we presented available data on survival and tumor incidence in mice exposed to long-term treatment with melatonin. Melatonin did not induce malignancies in male C57BL/ 6 mice when administered at 10 mg/l (1.5-2.0 mg/kg) in the night drinking water from 19 months.10,12 Lipman et al20 observed lymphomas in 77.9% of male C57BL/6 mice that received melatonin with food (11 ppm or 68 mkg/kg) from the age of 18 months and survived to a 50% mortality (26.5 months). In controls only 28.6% of mice developed lymphomas. Leukemia was detected in 70-98% of C57BL/6 mice and 78% of CC57Br mice (both males and females) treated subcutaneously with melatonin at a dose of 2.5 mg/mouse (~ 80 mg/kg) twice a week for of 2.5-5 months.24,25 Thus, being administered in significantly higher dose (80 mg/ kg) melatonin induced lymphomas and leukemias in C57BL/6 mice. At low dose (1.5-2.0 mg/ kg) it did not induce them. In CBA mice, melatonin given in night drinking water in an interrupted (course) regimen at a relatively low dose (3-3.5 mg/kg) induced lymphomas and lung carcinomas.21 In female SHR mice, melatonin given approximately in the same dose (20mg/l; 2.7-3.3 mg/kg) failed increase the total incidence of tumors or tumors of any localization. Strain differences in susceptibility to chemical carcinogens is well known.26 There are strong critical comments on the anti-aging effects of melatonin in mice. These comments mainly related to the observation that murine strains used in some studies do not synthesize melatonin as a result of a genetic defect (BALB/c, NZB, and C57BL/6).29 Later it was shown that the pineal gland did produce melatonin in the above-mentioned mouse strains with genetic defects, but the production night peak was very short, so its presence was difficult to detect.18 It is worthy to note that the major signal transduction cascades in the pineal gland did not differ between melatonin-proficient C3H mice and melatonin-deficient C57BL mice.30

Effect of Melatonin on Longevity in Rats In the experiment with male CD rats melatonin was given with drinking water (4 mg/l) during the whole day starting at 11-13 months age during 16 months.31 Additional groups of rats received with drinking water melatonin antagonist M-(1,4-dinitrophenyl)-5-methoxytryptamin (ML-23) at the dose of 0.4 mg/l or combination of melatonin and ML-23) in the same doses. Observation was stopped when the age of rats was 26-29 months. In the control group to this age survived 7 from 16 rats (44%), whereas in the group exposed to melatonin alone – 13 from 15 rats (87%). Surprisingly, in the group exposed to ML-23 survived 6 from 10 mice; and in the group treated with combination of melatonin and ML-23 – survived 8 from 10 rats. Body weight was similar in all groups. Five from 7 rats of the control group revealed a pneumonia at autopsy whereas in the group treated with melatonin there were no cases of pneumonia. Authors believe that melatonin antagonist ML-23 induces chronic deprivation of melatonin receptors followed by their hypersensitivity to the melatonin. It is worthy of note that in this experiment was small number of animals per group and the experiment was finished before natural death of all animals. Meredith et al32 studied effect of lifelong supplementation with melatonin on reproductive senescence. Holtzman rats were divided into three treatments on day 10 after pupping. Treatment 1 pups had access to water, whereas treatment 2 and 3 pups had access to water containing 10 mg/l melatonin only at night (treatment 2) or continuously (treatment 3). There were fewer (p < 0.001) abnormal-length estrous cycles from 180 to 380 days of age in the treatment 2 as compared with the treatments 1 or 3. There was no effect of treatment on number of primordial follicles. The authors concluded that nighttime, but not continuous treatment with melatonin, delayed reproductive senescence without any effect on number of primordial follicles.

Female

Male

Male & female Male & female Male & female Male

Male

Male

Female

Male Female Female

Female

Balb/c

Balb/c

C57BL/6

C57BL/6J

C57BL/6

C57BL/6

CBA

C3H

C3H/He

C3H/Jax

CC57Br

C57BL/6

Sex

Strain

16/39

20/20 20/20 14/15

1:20/20 2:7/13 3:38/30 50/50

20/15

10/10

26/57

29/57

25/45

50/50

26/12

Nos. of Mice C/M

3 wk

1 8 12

6

18

19

19

1.5

1.5

1.5

18

15

Age at Start of Treatment, mo

ND

ND

22 mo

22 mo

22 mo

ND

ND

2.5 mg/kg/day in night drinking water 10 mg/l in night drinking water 25-50 mkg/mouse/d with drinking water

12 mo

23 mo 27 mo ND

1: 24 mo; 2: 50% survival 3: died < 2 y 20 mg/l in night drinking water ND

11 ppm (68 mkg/kg) with lab chow ad libitum

10 mg/l in night drinking water

2.5 mg/mouse s.c. twice a wk x 5 mo 2.5 mg/mouse s.c. twice a wk x 2.5 mo 2.5 mg/mouse s.c. twice a wk x 2.5 mo 10 mg/l in night drinking water

10 mg/l in night drinking water

10 mg/l in night drinking water

Treatment with Melatonin

Age at the End of Observation

No data

+ 20% No effect No effect

+5%

No effect

+17%

+ 20%

-12%

- 20.6%

Shift to right of the survival curve -13%

+18%

Anisimov et al, 200121 Oxenkrug et al, 200119 Pierpaoli et al, 199110 Subramanian and Kothari, 199127

Romanenko, 198324 Romanenko, 198524 Romanenko, 198525 Pierpaoli and Maestroni, 198712 Pierpaoli et al, 199110 Lipman et al, 199820

Pierpaoli and Regelson, 199414 Mocchegiani et al, 199817

References

Table continued on next page

Decreases

Increases

No data

1: No effect; 2: Increases; 3: No effect Increases

No data

No data

Increases

Increases

Increases

No data

No data

Effects of Melatonin on: Mean Tumor Life Span Incidence

Table 1. Summary of experiments on the effect of melatonin on life span and spontaneous tumor incidence in mice

Effect of Melatonin on Life Span and Longevity 49

Female

Female

Female

Female

Female

Female

Female

Female

HER-2 / neu

NOD

NOD

NZB

NZB/W

SAMP-1

SAMR-1

SHR

50/50/50

10/12

20/20

15/15/15

10/10

29/17

25/30

30/27/22

Nos. of Mice C/M

3

3

3

8

4

1

1

2

Age at Start of Treatment, mo

2 or 20 mg/l in night drinking water

20 mg/l in night drinking water

20 mg/l in night drinking water

2-3.5 mg/kg s.c.daily at 8-10 hrs (M1) or at 17-19 hrs (M2) x 9 mo

10 mg/l in night drinking water, 5 times a wk, 4-38 wk 10 mg/l in night drinking water

2.5 mg/kg/day in night drinking water 5 d/w monthly or constantly 4 mg/kg s.c. at 4:30 PM, 5 times a wk, 4-38 wk

Treatment with Melatonin

ND

ND

ND

34 wk

20 mo

50 wk

50 wk

ND

Age at the End of Observation

No effect; M1, M2: +3 mo. MLS

-11%

Survivors: C:0; M: 40% Survivors: C: 20%; M1: 60%; M2: 60% No effect

C:32% survivors M:90% survivors + 17%

M1: No effect M2: -13%

M1: ↓ 1,9-fold M2: No effect

No effect

No effect

No data

No data

No data

No data

M1: No effect; M2: Decreases

Effects of Melatonin on: Mean Tumor Life Span Incidence

Rosenfeld, 200223 Rosenfeld, 200223 Anisimov et al, 2003

Conti and Maestroni, 199818 Pierpaoli et al, 199110 Lenz et al, 199516

Conti and Maestroni, 199818

Anisimov et al, 200228

References

Note: C= control group; M= melatonin-treated group; MLS= maximum life span; ND= animals were survived until natural death; NOD= non-obese diabetic; SAMP-1= senescence accelerated mouse-prone; SAMR-1= senescence accelerated mouse-resistant.

Sex

Strain

Table 1. Continued

50 Melatonin: Biological Basis of Its Function in Health and Disease

Effect of Melatonin on Life Span and Longevity

51

Effect of Melatonin on Longevity in Fruit Flies Melatonin synthesizes and arylalkylamine N-acetyltransferase, a key enzyme in melatonin biosynthesis, were identified in Drosophila melanogaster.33 We have studied effect of melatonin on longevity in D. melanogaster strain HEM.34 Melatonin was added to the nutrition medium (100 µg/ml) during 2-3rd age of larvas. Exposure to melatonin was followed by a decrease in the level of conjugated hydroperoxides and ketodienes in females, and failed to influence the activity of catalase in males, but increased it in females by 24% (p < 0.02) and failed to influence of Cu,Zn-superoxide dismutase (SOD) activity both in males and females. It was shown that melatonin did not influence life span of this strain of fruit flies. The life span of D. melanogaster wild strain Canton-S was studied under effect of melatonin at a concentration of 0.08%.35 The hormone was introduced into a nutrient medium only at the stage of development. Five experiments with melatonin have shown a variations in the effect of melatonin on life span: the mean life span in males varied from—10.0% to +18.5%, whereas in females—from +2.3% to 12.1%. An inverse correlation was observed between the change in life span after melatonin supplementation and the value of life span in the corresponding control group. For a relative low life span in a population from which the control and experimental group were formed, the geroprotector effect of melatonin was the most distinct; for a relatively high life span, the effect of melatonin was either not detected or appeared as a toxic reduction in life span. Recently effect of melatonin on life span was studied in D. melanogaster Oregon wild strain.36 Melatonin, added daily to the nutrition medium at a concentration of 100 µg/ml during the all experiment, increased significantly the life span of flies. The maximum life span was 61.2 days in controls and 81.5 days in melatonin fed group (+33.2%). Relative to the controls, the percentage in the melatonin fed flies was 19.3% in the onset of 90% mortality and 13.5% in median life span. Authors have shown also that melatonin treatment increased the resistance of fruit flies to superoxide mediated toxicity of paraquat and to termal stress. Thus, if melatonin was added to food throughout the life span of life it increases the longevity of fruit flies.

Effect of Melatonin on Longevity in Worms

Bakaev et al37 have studied effect of various doses of melatonin on life span of nematode Caenorhabditis elegans. Three to five-day-old adult nematodes (Bristol, N2) were kept 4 hours in melatonin-free nutrient medium with E. coli and then transfered into stanard vessels with melatonin. The temperature was +21ºC and animals were kept in constant darkness. The mean life span of C. elegans in control group was 23.7 ± 1.8 days and maximum life span was 32 days. At the concentrations 10-4 and 10-5 melatonin failed influence the life span of worms. However at concentration from 10-6 to 10-10 the hormone significantly decreased mean life span (by 31 to 55.7%, p < 0.05). It was shown that melatonin-synthesizing enzyme activities and melatonin level has a circadian rhythm in planarians.38 Melatonin supplementation at a dose of 10 mg/l) of nutrient media per day, followed by incubation for 23 hours in darkness, increased the mean clonal life span of an aerobic single-cell organism, planaria Paramecium tertaurelia in days by percentages ranging from 20.8 to 24.2%. 39 Maximum clonal life span was also increased in melatonin-supplemented cells, from 14.8% to 24.0% over controls. It is worthy to note that the increase in the concentration of melatonin in the nutrient medium was follow by decrease in life span of planaria. Mean clonal life span in fissions was not significantly increased in melatonin-supplemented cells, with values ranging from 6.0% to 15.5% over controls. Authors suggest that geroprotector effect of melatonin in worms depend on its capacity to scavenge free radicals in cells.

52

Melatonin: Biological Basis of Its Function in Health and Disease

Melatonin as Antioxidant According to the free radical theory of aging, various oxidative reactions occuring in the organism (mainly in mitochondria) generate free radicals as byproduct which cause multiple lesions in macromolecules (nucleic acids, proteins and lipids), leading to their damage and aging. The recent evidence suggest that key mechanisms of both aging and cancer are linked via endogenous stress-induced DNA damage caused by reactive oxygen species (ROS).40-42 Since 1993 when melatonin was firstly discovered to be a free radical scavenger 43 there were published many papers confirming the ability of melatoni n to protect DNA from free radical damage.44 There are evidence that melatonin in vitro directly scavenges ·OH, H2O2, singlet oxygen (↑O2-), and inhibits lipid peroxidation. Melatonin stimulates a number of antioxidative enzymes including SOD, glutathione peroxidase, glutathione reductase, and catalase. It was shown that melatonin enhances intracellular glutathione levels by stimulating the rate-limiting enzyme in its synthesis, γ-glutamylcysteine synthase, inhibits the proxidative enzymes nitric oxide synthase and lipoxygenase.43-48 There is evidence that melatonin stabilizes cellular membranes, thereby probably helping them resist oxidative damage. Melatonin has been shown to increase the efficiency of the electron transport chain and, as a consequence, to reduce electron leakage and the generation of free radicals.44 It was shown that melatonin reduced the formation of 8-hydroxy-2-deoxyguanosine, a damaged DNA products, 60 to 70 times more effectively that did some classic antioxidants (ascorbate and α-tocopherol).47 Thus, melatonin acts as a direct scavenger of free radicals with the ability to detoxify both reactive oxygen and reactive nitrogen species and indirectly increasing activity of antioxidative defense systems.44 However, melatonin does not necessarily act as an intracellular antioxidant, in some conditions it can be prooxidant.49-52

Melatonin DNA Damage and Mutagenesis There is evidence of an age-related accumulation of spontaneous mutations in somatic and germ cells.53 Accumulation with age of some spontaneous mutations or mutations evoked by endogenous mutagens can induce genome instability and, hence, increase the sensitivity to carcinogens and/or tumor promoters. It has been shown that clonally expanded mtDNA mutations accumulate with age in normal human tissues.54 The finding that deleted mtDNA accumulated in human muscle tissue as well as evidence for partially duplicated mtDNA in aged human tissues55 allow to suggest the important role of clonal expansion of mutant mtDNA in the age-related increase of systemic oxidative stress in the whole organism.56 A significant trend toward increasing p53 mutations frequency with advancing age was found in some normal and malignant tissue.57 Simpson58 suggests that the aging human body accumulates enough mutations to account for multistep carcinogenesis by selection of preexisting mutations. Melatonin has been found to inhibit X-ray induced mutagenesis in mouse and human lymphocytes in vitro,59,60 to reduce cis-platinum-induced genetic damage in the bone marrow of mice,61 to decrease hepatic DNA adduct formation caused by safrole in rats62 and to protect rat hepatocytes from chromium(VI)-induced DNA single-strand breaks in vitro63 We studied the effect of melatonin on the induction of chromosome aberrations and sperm head anomalies in mice treated with cyclophosphamide, 1,2-dimethylhydrazine (DMH) and N-nitrosomethylurea (NMU) and found that melatonin inhibited greatly the mutagenicity of these carcinogens.64 Since melatonin can protect cells directly as an antioxidant and indirectly through receptor-mediated activation of antioxidative enzymes we applied two different in vitro test systems: the Ames test and the Single Cell Gel Electrophoresis assay (SCGE assay).65 Melatonin alone turned out neither toxic nor mutagenic in the Ames test and revealed clastogenic activity at the highest concentration tested (100 µM) in the SCGE assay.65

Effect of Melatonin on Life Span and Longevity

53

As oxidative mutagens we used DMH, bleomycin (with S9-mix) and mitomycin C (without S9-mix) which are believed to generate oxygen radical species.66 Additionally, we tested eight other intercalating and alkylating agents both direct-acting and requiring metabolic activation. Melatonin inhibited the mutations induced by promutagens 7,12-dimethylbenz(a) anthracene (DMBA), benzo(a)pyrene (BP), 2-aminofluorene, DMH and bleomycin in all the strains used. The mutagenicity of 4-nitroquinoline-N-oxide, 2,4,7-trinitro-9-fluorenone, 9-aminoacridine, NMU and sodium azide remained unaffected by melatonin. It is to be noticed that melatonin was effective as an antimutagen only at extremely high doses (0.25 - 2 µM/plate).65 As melatonin display its protective effect towards promutagens only we speculate that it can exert its activity on the metabolic activation process, perhaps by inhibiting the cytochrome P-450-dependant mono-oxygenase enzyme system of S9-mix. Melatonin modulate the clastogenicity of DMBA, BP and cyclophosphamide. Compared to the data obtained in the Ames test, melatonin inhibited the clastogenicity of the chemicals at lower concentrations (0.1-1 nM). In combination with mitomycin C a significant dose-related exacerbating effect of melatonin has been observed in both tests.65 The available data shows that melatonin may play an important role in defending cells from DNA damage induced not only by oxidative mutagens but also by different alkylating agents.

Melatonin and Apoptosis In the series of studies it was shown that melatonin inhibits apoptosis in cells of the brain, induced by ROS,45 amilod β-peptide, related to Alzheimer disease,67 kainate, neuromediators and neurotixins, but not by N-methyl-D-aspartate, 68,69 staurosporine or neurotoxin ethylcholinazyridine.70 It was suggested that protective effect of melatonin on neurocyte apoptosis depends on a model used and do not mediated by caspase-dependent programmed cell death, but it include prevention of glycosylation derivative-induced necrosis.70 In some cases melatonin can enhance the damage of neurones in primary culture.70 Melatonin supplementation suppresses NO-induced apoptosis via induction of Bcl-2 expression in immortalized pineal PGT-β cells.71 Similar pathway mediates inhibitory effect of melatonin on apoptosis induced by ischemic neuronal injury.72 It was shown that low doses of melatonin (10-7-10-9 M) inhibits apoptosis in both the intact thumus and dexamethasone-treated cultured thymocytes. This effect of melatonin was mediated by its inhibitory influence on proliferation of thymocytes.73 Long-term (during 8 months) admnistration of melatonin in a daily dose of 40-50 µg/mouse prevents thymic involution in very old animals. This effect was mediated by inhibitory effect of melatonin on dexamethazone-induced apoptosis in thymocytes and splenocytes.74 Administration of melatonin to mice with drinking water (15 mg/l) during 40 days also attenuated apoptotic thymocyte death caused by free radicals.75 Administration of melatonin with night drinking water (20 mg/l) to rats exposed to DMH failed influence an apoptotic index in normal colon mucosa but significantly (by 1.8 times) inhibits it in colon tumors.76 Treatment with aflatoxin B1 leads to direct or indirect caspase-3 activation and consequently to apoptosis in rat liver. Melatonin treatment of rats enhances hepatic antioxidant/detoxification system which consequently reduces the apoptotic rate and necrobiotic changes in the liver.77

Melatonin and Immune System Immunopharmacological activity of melatonin has been demonstrated in various experimental models. Treatment with melatonin increases production of antibodies to sheep erythrocytes and immune response to primary immunization with T-dependent antigen.78 There are evidence of an involvement of melatonin in complex relationships between nervous and endocrine system.18,78 There are melatonin membrane receptors on T-helpers (Th). Activation of melatonin receptors leads to the increase of release of some type Th1 cytokines, including γ-interferon, interleukin-1 and opioid cytokines related to interleukin-4 and dinorphine.78

54

Melatonin: Biological Basis of Its Function in Health and Disease

Melatonin stimulates production of interleukins-1, -6 and -12 in human monocytes. These mediators can prevent stress-induced immunodepression defending mice from virus- and bacteria-induced lethal diseases. Important chain in mechanism of influence of melatonin on hemopoiesis includes the effect of melatonin-induced opioids on κ-opioid receptors at stromal macrophages of bone marrow.78

Effect of Melatonin on Gene Expression The available data on the genomic effect of melatonin is rather scarce. In cytogenetic study, it was shown that pinealectomy was followed by decrease in ribosomal gene activity in rats.79 Melatonin decreases the level of mRNA for the rate-limiting enzyme in porphyrin synthesis, 5-aminolevulinate synthase, in Harderian glands of Syrian hamsters.80 Melatonin decreased levels of mRNAfor histone H4 and prevent age-related decrease mRNA for Bcl-2, but not for p53 in thymocytes of mice81,82,and increases the mRNA for some antioxidant enzymes (Mn-SOD, Cu,Zn-SOD) in Harderian gland of Syrian hamster48 Melatonin caused a marked increase in relative mRNA levels for Mn-SOD, Cu,Zn-SOD and glutathione peroxidase in rat cerebral cortex.83 Using the GT1-7 cell line, an in vitro model of gonadotropin-releasing hormone (GnRH)-secreting neurons of hypothalamus, Roy and Belsham84 have shown that melatonin induced protein kinase C activity by 1.65-fold over basal level and activated c-fos and junB mRNA expression. Melatonin (1 nM) significantly downregulates GnRH Mrna.85 Male C57 mice were injected in the morning hours with melatonin (5mg/kg) during 10 days and the level of gene expression in splenocytes and peritoneal exudate cells (PEC) was analyzed with the reverse transcription-polymerase chain reaction.86 It was observed that melatonin up-regulated the level of gene expression of transforming growth factor-β, macrophage-colony stimulating factor (M-CSF), tumor necrosis factor-α (TNF-α) and stem cell factor in PEC and the level of gene expression of interleukin-1β, M-CSF, TNF-α, interferon-γ and stem cell factor in splenocytes. Melatonin reduced transcription of genes correlated to T lymphocyte activation (HLA-DBR, thymosin-β 10) and to lymphokine activated killer (LAK) activity (thymosin-β, tumor rejection antigen - TRA 1), nRap 2 in human lymphocyte culture.81 Transcription of genes correlated to T lymphocye activation and to lymphokine activated killer (LAK) activity in human lymphocyte culture.81 Administration of dietary melatonin to 26-month-old mice for 6 weeks resulted in reduction of basal level of cytokine mRNA levels (interleukin-6 and TNF-α) to values found in 5-month-old mice.87 Supplementation of 1 nM melatonin into cultural media inhibited cell proliferation of breast cancer cells MCF-7 coincident with a significant increase in the expression of p53 as well as p21WAF1 proteins.82 In transgenic HER-2/neu mice treatment with melatonin inhibited mammary tumor development and down regulated the expression of HER-2/neu oncogene in mammary tumors.88 To identify molecular events regulated by melatonin, gene expression profiles were studied in hearts melatonin-treated CBA mice in comparison to the control using cDNA gene expression arrays (15,247 cDNA clone set, NIA, USA).89 The dose and schedule of the treatment were similar to these in the long-term study.21 It was shown that primary effectors for melatonin are the genes controlling the cell cycle, cell/organism defense, protein expression and transport. Melatonin also increased the expression of some mitochondrial genes (16S, cytochrome c oxidases 1 and 3 (COX1 and COX3), and NADH dehydrogenase 1(ND1), which agrees with its ability to inhibit free radical processes (see section 7). The using differential display RT-PCR, it was shown that the cytochrome b gene is also a putative target for melatonin in brown adipocytes of Syberian hamster.90 A significant effect of melatonin on expression of some oncogenesis-related genes was detected.89 While expression of myeloblastosis oncogene-like 1 (Mybl1) was down-regulated by melatonin (exceeded 2-fold confidence level), melatonin up-regulated an expression of RAS p21 protein activator 1, Enigma homolog 2 and myeloid/lymphoid or mixed-lineage leukemia (MLLT3) gene. Of a great interest is an effect of melatonin onto a large number of genes related to calcium exchange, such as cullins, Kcnn4 and Dcamkl1, calmodulin, calbindin,

Effect of Melatonin on Life Span and Longevity

55

Kcnn2 and Kcnn4. Whereas the expression of cullin-1 in the mouse heart is down-regulated, that of cullin-5 is highly up-regulated, and expression of cullins-2 and -3 is not altered significantly. The cullin family, comprising at least six members, is involved in ubiquinone-mediated protein degradation required for cell-cycle progress through the G1 and S phases. Nevertheless, cullin-1, but not other members of the cullin family, is generally thought to be implicated in SCFs (Skp1-cullin-F-box protein ligase complexes), that control the ubiquinone-dependent degradation of G1 cyclins and inhibitors of cyclin-dependent kinases, thus playing an important role in cell proliferation and differentiation.91,92 Like the effects of other proteins of this family, the effect of cullin-5 is mediated by a Skp1/F-box-independent mechanism. It is believed that melatonin may influence tumor growth by interfering with calcium binding and blocking the formation of the MAPs/calmodulin and tubulin/calmodulin complexes to prevent cytoskeletal degradation93 Four serine/threonine kinases (Pctk3, FUSED, TOPK and Stk11) with expression increased by both peptides can be found in the same functional category (cell signaling/communication).89 At least one of these, Stk11 kinase with an unclear function, have anticarcinogenic effects and mutations in it lead to the Peutz-Jeghers syndrome, associated with high risk of tumor development in multiple localizations.94 Thus, these data present a direct evidence for the various effect of melatonin on expression of different genes in vivo.

Conclusion In general, the analysis of available data on the effect of melatonin on longevity support the hypothesis on antiaging effect of melatonin. At the same time, a critical review of real results has shown that the majority of studies are invalid from the point of view of the current guidelines for long-term testing of chemicals for carcinogenic safety95 and, to some extent, from the point of view of the correctness of the gerontological experiment.96 Often in reviewed experiments with rodents, melatonin was given to a small number of animals (10-20); the treatments start when the animals are old; the observations stop at some voluntary time, but not at the natural death of the last survivor; an autopsy and a correct pathomorphological examination sometimes are not performed; the body weight gain and food consumption of the animals are not monitored; and so on. We believe that the study of long-term effects of melatonin at a variety of doses in different strains and species (e.g., in rats) will be useful for making a conclusion about it safety. There are data on suppressive effect of melatonin on development of spontaneous and induced by chemical agents and ionizing radiation mammary carcinogenesis in mice and rats,97 on colon carcinogenesis induced by DMH in rats,98 spontaneous endometrial adenocarcinomas in BDII/Han rats,99 on induced by N-nitrosodiethylamine premalignant foci in rats liver,100 on DMBA-induced carcinogenesis of the uterine cervix and vagina in mice,101 It is very important that melatonin administration at middle age decreased visceral fat, plasma insulin and insulin-like growth factor-I levels in rats.102 Since visceral fat is associated with increased insulin resistance, diabetes, and cardiovascular disease, these results suggests that appropriate administration of melatonin may potentially provide prevention of some age-associated pathology. Thus, melatonin has two faces—it is both a potent geroprotector, anticarcinogen and inhibitor of tumor growth in vivo and in vitro and in some models may induce tumors and promote tumor growth. There is no contradictions between data on the carcinogenic and anticarcinogenic potential of melatonin. Some antioxidants, including natural ones have both geroprotector and tumorigenic potential and could be potent anticarcinogens as well.103 The results of treatment of patients with advanced cancer with melatonin104 and of administration of melatonin to perimenopausal women are promising.105

Acknowledgments This study was supported by grant 02-04-07573 from the Russian Foundation for Basic Research and by the grant # 02-SC-NIH-1047 from Duke University, NC, U.S.A.

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30. Stehle JH, von Gall C, Korf HW. Organization of the circadian system in melatonin-proficient C3H and melatonin-deficient C57BL mice: A comparative investigation. Cell Tissue Res 2002; 309:173-182. 31. Oakin-Bendahan S, Anis Y, Nir I et al. Effects of long-term administration of melatonin and a putative antagonist on the ageing rat. Neuro Report 1995; 6:785-788. 32. Meredith S, Jackson K, Dudenhoeffer G et al. Long-term supplementation with melatonin delays reproductive senescence in female rats, without an effect on number of primordial follicles. Exp Gerontol 2000; 35:343-352. 33. Hintermann E, Jeno P, Meyer UA. Isolation of an arylalkylamine N-acetyltransferase from Drosophila melanogaster. FEBS Lett 1995; 375:148-150. 34. Anisimov VN, Mylnikov SV, Oparina TI et al. Khavinson VKh Effect of melatonin and pineal peptide preparation epithalamin on life span and free radical oxidation in Drosophila melanogaster. Mech. Ageing Dev 1997; 97:81-91. 35. Izmaylov DM, Obukhova LK. Geroprotector effectiveness of melatonin: Investigation of life span of Drosophila melanogaster. Mech Ageing Dev 1999; 106:233-240. 36. Bonilla E, Medina-Leendertz S, Diaz S. Extension of life span and stress resistance of Drosophila melanogaster by long-term supplementation with melatonin. Exp Gerontol 2002; 37:629-638. 37. Bakaev VV, Efremov AV, Anisimov VN. An attempt to slow aging in C. elegans. 8. Melatonin reduces life span of C. elegans. The Worm Breeder Gazette 1997; 15(1):36. 38. Itoh MT, Shinozawa T, Sumi Y. Circadian rhythms of melatonin-synthesizing enzyme activities and melatonin levels in planarians. Brain Res 1999; 830:165-173. 39. Thomas JN, Smith-Sonneborn J. Supplemental melatonin increases clonal lifespan in the protozoan Paramecium tetraurelia. J Pineal Res 1997; 23:123-130. 40. Harman DH Free-radical theory of aging: Increasing the functional life span. Ann NY Acad Sci 1994; 717:257-266. 41. Hamilton ML, Van Remmen H, Drake JA et al. Does oxidative damage to DNA increase with age? Proc Natl Acad Sci USA 2001; 98:10469-10474. 42. Skulachev VP. The programmed death phenomena, aging, and the Samurai law of biology. Exp Gerontol 2001; 36:995-1024. 43. Tan DX, Chen LD, Poeggeler B et al. Melatonin: A potent, endogenous hydroxyl radical scavanger. Endocrine J 1993; 1:57-60. 44. Reiter RJ, Tan DX, Allegra M. Melatonin; reducing molecular pathology and dysfunction due to free radicals and associated reactants. Neuroendocrinol Lett 2002; 23 Suppl 1:3-8. 45. Pozo D, Reiter RJ, Calvo JR. Physiological concentrations of melatonin inhibit nitric oxide synthase in rat cerebellum. Life Sci 1994; 55:455-460. 46. Pieri C, Marra M, Moroni F et al. Melatonin: A peroxyl radical scavenger more effective than vitamin E. Life Sci 1994; 55:271-276. 47. Qi W, Reiter RJ, Tan DX et al. Increaesd level of oxidatively damaged DNA induced by chromium (III) and H2O2: Protection by melatonin and related molecules. J Pineal Res 2001; 29:54-61. 48. Antolin I, Rodriguez C, Sainz RM et al. Neurohormone melatonin prevents cell damage: Effect on gene expression for antioxidant enzymes. FASEB J 1996; 10:882-890. 49. Pieri C, Recchioni R, Moroni F et al. Melatonin regulates the respiratory burst of human neutrophils and their depolarization. J Pineal Res 1998; 24:43-49. 50. Recchioni R, Marcheselli F, Moroni F et al. Melatonin increases the intensity of repsiratory burst and prevents L-selectin shedding in human neutrophils in vitro. Biochim Biophys Res Communic 1998; 252:20-24. 51. Osseni RA, Rat P, Bogdan A et al. Evidence of prooxidant and antioxidant action of melatonin on human liver cell line HepG2. Life Sci 2000; 68:387-399. 52. Wolfler A, Caluba HC, Abuja PM et al. Proxoidant activity of melatonin promotes fas-induced cell death in human leukemic Jurkat cells. FEBS Lett 2001; 502:127-131. 53. Vijg J. Somatic mutations and aging: A reevaluation. Mutat Res 2000; 447:117-135. 54. Coller HA, Bodyak ND, Khrapko K. Frequent intracellular clonal expansions of somatic mtDNA mutations. Ann NY Acad Sci 2002; 959:434-447. 55. Bodyak ND, Nekhaeva E, Wei JY et al. Quantitation and sequencing of somatic deleted mtDNA in single cells: Evidence for partially duplicated mtDNA in aged human tissues. Human Mol Genetics 2001; 10:17-24. 56. de Grey AD. The reductive hotspot hypothesis: Un update. Arch Biochem Biophys 2000; 373:295-301. 57. Liang SB, Ohtsuki Y, Furihata M et al. Sun-expoure and aging-dependent p53 protein accumulation results in growth advantage for tumour cells in carcinogenesis of nonmelanocytic skin cancer. Virchows Arch 1999; 434:193-199.

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58. Simpson AJG. A natural somatic mutation frequency and human carcinogenesis. Adv Cancer Res 1997; 71:209-240. 59. Vijayalaxmi, Reiter RJ, Meltz ML. Melatonin protects human blood lymphocytes from radiation-induced chromosome damage. Mutation Res 1995; 346:23-31. 60. Vijalaxmi, Meltz ML, Reiter RJ et al. Melatonin and protection from genetic damage in blood and bone marrow: Whole-body irradiation studies in mice. J Pineal Res 1999; 27:221-225. 61. Koratkar R, Vasudha A, Ramesh G et al. Effect of melatonin on cisplatinum induced genetic damage to the bone marrow cells of mice. Med Sci Res 1992; 20:179-180. 62. Tan DX, Reiter RJ, Chen LD et al. Both physiological and pharmacological levels of melatonin reduce DNA adduct formation induced by the carcinogen safrole. Carcinogenesis 1994; 15:215-218. 63. Susa N, Ueno S, Furukawa Y et al. Potent protective effect of melatonin on chromium (VI)-induced DNA strand breaks, cytotoxicity and lipid peroxidation in primary cultures of rat hepatocytes. Toxicol Appl Pharmacol 1997; 144:377-384. 64. Musatov SA, Rosenfeld SV, Togo EF et al. The influence of melatonin on mutagenicity and antitumor action of cytostatic drugs in mice. Vopr Onkol 1997; 43:623-627. 65. Musatov SA, Anisimov VN, Andre V et al. Modulatory effects of melatonin on genotoxic response of reference mutagens in the Ames test and the COMET assay. Mutat Res 1998; 417:75-84. 66. Mahmutoglu I, Kappus H. Redox cycling of bleomycin-Fe(III) by an NADP-dependent enzyme, and DNA damage in isolated rat liver nuclei. Biochem Pharmacol 1987; 36:3667-3671. 67. Shen YX, Xu SY, Wei W et al. Melatonin blocks rat hippocampal neuronal apoptosis induced by amyliod beta-peptide 25-35. J Pineal Res 2002; 32:163-167. 68. Iacovitti L, Stull ND, Hohnston K. Melatonin rescues dopamine neurones from cell death in tissue culture models of oxidative stress. Brain Res 1997; 768:317-326. 69. Skaper SD, Floreani M, Ceccon M et al. Excitotoxicity, oxidative stress, and the neuroprotective potential of melatonin. Ann NY Acad Sci 1999; 890:107-118. 70. Harms C, Lautenschlager M, Bergk A et al. Melatonin is protective innecrotic but not in caspase-dependent, free radical-independent apoptotic neuronal cell death in primary neuronal cultures. FASEB J 2000; 14:1814-1824. 71. Yoo YM, Yim SV, Kim SS et al. Melatonin suppresses NO-induced apoptosis via induction of Bcl-2 expression in PGT-beta immortalized pineal cells. J Pineal Res 2002; 33:146-150. 72. Sun FY, Lin X, Mao LZ et al. Neuroprotection by melatonin against ischemic neuronal injury associated with modulation of DNA damage and repair in the rat following a transient cerebral ischemia. J Pineal Res 2002; 33:48-56. 73. Sainz RM, Mayo JC, Kotler M et al. Melatonin decreases mRNA for histone H4 in thymus of young rats. Life Sci 1998; 63:1109-1117. 74. Provinciali M, Di Stefano G, Bulian D et al. Effect of melatonin and pineal grafting on thymocyte apoptosis in aging mice. Mech Ageing Dev 1996; 90:1-19. 75. Tian YM, Li PP, Jiang XF et al. Rejuvenation of degenerative thymus by oral melatonin administration and the antagonistic action of melatonin against hydroxyl radical-induced apoptosis of cultured thymocytes in mice. J Pineal Res 2001; 31:214-221. 76. Anisimov VN, Popovich IG, Shtylik AV et al. Melatonin and colon carcinogenesis.III. Effect of melatonin on proliferative activity and apoptosis in colon mucosa and colon tumors induced by 1,2-dimethylhydrazine in rats. Exp Toxicol Pathol 2000; 52:71-76. 77. Meki AR, Abdel-Ghaffar SK, El-Gibaly I. Alatoxin B1 induces apoptosis in rat liver: Protective effect of melatonin. Neuroendocrinol Lett 2001; 22:417-426. 78. Maestroni GJM. The immunotherapeutic potential of melatonin. Expert Opin Invest Drugs 2001; 10:467-476. 79. Payao SL, de Carvalho CV, da Silva ER et al. Pinealectomy-associated decrease in ribosomal gene activity. Biogerontology 2001; 2:105-108. 80. Menendez-Pelaez A, Rodriguez C, Dominguez D. 5-Aminolevulinate synthase mRNA levels in the Harderian gland of Syrian hamsters: Correlation with porphyrin concentrations and regulation by androgens and melatonin. Mol Cell Endocrinol 1991; 80:177-182. 81. Capelli E, Campo I, Panelli S et al. Evaluation of gene expression in human lymphocytes activated in the presence of melatonin. Int Immunopharmacol 2002; 2:885-892. 82. Mediavilla MD, Cos S, Sanchez Barcelo EJ. Melatonin increases p53 and p21WAF1 expression in MCF-7 human breast cancer cells in vitro. Life Sci 1999; 65:415-420. 83. Kotler M, Rodriguez C, Sainz RM et al. Melatonin increases gene expression for antioxidant enzymes in rat brain cortex. J Pineal Res 1998; 24:83-89. 84. Roy D, Belsham DD. Melatonin receptor activation regulates GnRH gene expression and secretion in GT1-7 GNRH neurons. Signal transduction mechanisms. J Biol Chem 2002; 277:251-258.

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85. Roy D, Angelini NI, Fujieda H et al. Cyclical regulation of GnRH gene expression in GT1-7 GnRH secreting neurons by melatonin. Endocrinology 2001; 142:4711-4720. 86. Liu F, Ng TB, Fung MC. Pineal indoles stimulate the gene expression of immunomodulating cytokines. J Neural Transm 2001; 108:397-405. 87. Sharman KG, Sharman EH, Yang E et al. Dietary melatonin selectively reverses age-related changes in cortical cytokine mRNA levels, and their responses to an inflammatory stimulus. Neurobiol Aging 2002; 23:633-638. 88. Baturin DA, Alimova IA, Anisimov VN et al. The effect of light regimen and melatonin on the development of spontaneous mammary tumors in HER-2/neu transgenic mice is related to a down regulation of HER-2/neu gene expression. Neuroendocrin Lett 2001; 22:439-445. 89. Anisimov SV, Boheler KR, Anisimov VN. Microarray technology in studying the effect of melatonin on gene expression in the mouse heart. Dokl Biol Sci 2002; 383:90-95. 90. Prunet-Marcassus B, Ambid K, Viguerie-Bascands N et al. Evidence for a direct effect of melatonin on mitochondrial genome expression of Sberian hamster brown adipocytes. J Pineal Res 2001; 30:108-115. 91. Michel JJ, Xiong Y. Human CUL-1, but not other cullin family members, selectively interacts with SKP1 to form a complex with SKP2 and cyclin A. Cell Growth Differ 1998; 9:435-449. 92. Deshaies RJ SCF and Cullin/Ring H2-based ubiquitin ligases. Annu Rev Cell Dev Biol 1999; 15:435-467. 93. Blask DE, Sauer LA, Dauchy RT. Melatonin as a chronobiotic/anticancer agent: Cellular, biochemical, and molecular mechanisms of action and their implications for circadian-based cancer therapy. Curr Top Med Chem 2002; 2:113-132. 94. Hemminki A. The molecular basis and clinical aspects of Peutz-Jeghers syndrome. Cell Mol Life Sci 1999; 55:735-750. 95. Gart JJ, Krewski D, Lee PN et al. Statistical Methods in Cancer Research. Vol. III - The Design and Analysis of Long-Term Animal Experiments. Lyon: IARC Scientific Publication 79 IARC, 1986. 96. Warner HR, Ingram D, Miller RA et al. Program for testing biological interventions to promote healthy aging. Mech Ageing Dev 2000; 155:199-208. 97. Cos S, Sanchez-Barcelo EJ Melatonin and mammary pathological growth. Front Neuroendocrin 2000; 17:133-170. 98. Anisimov VN, Popovich IG, Zabezhinski MA Melatonin and colon carcinogenesis: I. Inhibitory effects of melatonin on development of intestinal tumors induced by 1,2-dimethylhydrazine in rats. Carcinogenesis 1997; 18:1549-1553. 99. Deerberg F, Bartsch C, Pohlmeyer G et al. Effect of melatonin and physiological epiphysectomy on the developmet of spontaneous endometrial carcinoma in BDII/HAN rats. Cancer Biother Radiopharmacol 1997; 12:420. 100. Imaida K, Hagiwara A, Yoshino H et al. Inhibitory effects of low doses of melatonin on induction of preneoplastic liver lesions in a medium-term liver bioassay in F344 rats: Relation to the influence of electromagnetic near field exposure. Cancer Lett 2000; 155:105-114. 101. Anisimov VN, Zabezhinski MA, Popovich IG et al. Inhibitory effect of melatonin on 7,12-dimethylbenz[a]anthracene-induced carcinogenesis of the uterine cervix and vagina in mice and mutagenesis in vitro. Cancer Lett 2000; 156:199-205. 102. Wolden Hanson T, Mitton DR, McCants RL et al. Daily melatonin administration to middle-aged male rats suppresses body weight, intraabdominal adiposity, and plasma leptin and insulin independent of foof intake and total body fat. Endocrinology 2000; 141:487-497. 103. Anisimov VN Life span extension and cancer risk: Myths and reality. Exp Geront 2001; 36:1101-1136. 104. Lissoni P. Is there a role for melatonin in supportive care? Support Care Cancer 2002; 10:110-116. 105. Bellipanni G, Bianchi P, Pierpaoli W et al. Effects of melatonin in perimenopausal and menopausal women: A randomized and placebo controlled study. Exp Gerontol 2001; 36:297-310.

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CHAPTER 6

Cardiovascular Effects of Melatonin Ewa Sewerynek

Abstract

I

n the course of aging, the incidence of both acute and chronic heart diseases, systematically increases. Concentrations of some hormones decrease in the course of aging, e.g., melatonin concentrations in serum and urinary levels of its main metabolite, 6-sulphatoxymelatonin, are lower in older, when compared to values observed in younger population. The evidence obtained during the last 10 years suggests that melatonin exerts certain effects upon the cardiovascular system. The presence of vascular melatoninergic receptors (binding sites) has been demonstrated; these receptors are functionally associated with either vasoconstrictor or vasodilatory effects of melatonin. Melatonin clearly indicates a certain contribution in general cardioprotection of the rat heart, following myocardial ischemia-reperfusion and adriamycin-induced cardiotoxicity. It has been shown that patients with coronary heart disease have a low melatonin production rate, especially those with higher risk of cardiac infarction and/or of sudden death. There are clinical data, reporting alterations of melatonin concentrations in serum in coronary heart disease. The suprachiasmatic nucleus and, possibly, the melatoninergic system may also modulate cardiovascular rhythmicity. Other problems, related to age, include hypercholesterolemia and hypertension. People with high levels of LDL-cholesterol have low levels of melatonin. It has been shown that melatonin suppresses the formation of cholesterol, reduces LDL accumulation in serum and modifies fatty acid composition of rat plasma and liver lipids. People with hypertension demonstrate lower melatonin levels vs. those with normal blood pressure. The administration of the hormone in question declines blood pressure to normal range. This chapter summarizes the actual knowledge of the relationships between the cardiovascular system and melatonin.

It is well-known that serum melatonin concentrations and urinary levels of its main metabolite, 6-sulphatoxymelatonin, decrease in the course of ageing.1 In elderly subjects, the incidence of heart diseases, both acute and chronic, systematically increases. The evidence from the last 10 years indicates that melatonin influences the cardiovascular system.2 Similarly to other organs and systems, the cardiovascular system exhibits diurnal and seasonal rhythms, including the heart rate, cardiac output, and blood pressure.3 It has been shown that: 1. heart rate variability is the lowest in winter,4 2. the incidence of cardiac arrest is the highest in winter,5 3. blood pressure values are higher in winter than in summer.6

Melatonin: Biological Basis of Its Function in Health and Disease, edited by S.R. Pandi-Perumal and Daniel P. Cardinali. ©2006 Eurekah.com.

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There is some information about some seasonal variations in the incidence of cardiac events, e.g.,: 1. seasonal variations of acute myocardial infarction show winter peaks and summer drops,7 2. cardiac mortality rate has its peak in July and during the cold season, from December to February,8 3. cardiac output of rats has demonstrated low values in spring and summer but high values in autumn and winter.9

The results of epidemiological studies show that cardiological events occur most often between 6 a.m. and 12 a.m., with the highest risk of heart disease at 9 a.m.10 Additionally, it has been observed that the incidence of cardiac arrests is the highest at 8 a.m. to 11 a.m. and from 4 p.m. to 7 p.m.5,11 Daily variations in the incidence of cardiac events have also been observed. Coca12 demonstrated that blood pressure falls during the night. Nicolau et al8 suggested that cardiac mortality rate has its peaks early in the morning, coinciding with the peaks in systolic and diastolic blood pressure. It has been known that acute heart attack has daily, seasonal and, perhaps, ultradian rhythm; on the other side, cardiological events, such as angina pectoris and sudden death, indicate circadian rhythms.13 Also, a number of known cardiovascular risk factors, such as hormones, metabolic parameters, lifestyle, blood pressure, fibrinogenesis, and fibrinolytic activity demonstrate periodical oscillations. The suprachiasmatic nucleus and, possibly, the melatoninergic system can modulate the cardiovascular rhythm. During the night, when the level of melatonin is the highest, the heart rate decreases, the cardiac output is higher, the blood pressure drops, the level of cholesterol declines and the activity of calcium pump increases. The data, concerning the chronobiological considerations of time-dependent incidents of cardiovascular diseases, compared to circadian and seasonal variations in melatonin concentrations, are not very well documented. Data obtained in animals indicate that the cardiovascular response to melatonin may be mediated, at least in part, by reducing noradrenergic activity.14,15 Also in men, melatonin administration may exert suppressive effects on the sympathetic tone.16 The fact of seasonal variations in blood pressure of patients on chronic beta-adrenergic receptor blockers3 and that the circadian rhythm of the heart rate was maintained in patients after heart transplantation,17 indicate that seasonal and daily variations in the sympathetic tone may not be the only controlling factors, thus suggesting an involvement of some other mechanisms. The presence of vascular melatoninergic receptors (binding sites) has been demonstrated, together with their functional associations with vasoconstrictor or vasodilatory effects of melatonin. The receptors for melatonin have been detected in walls of cerebral and caudal arteries of rats,18,19 in myoblasts, and coronary arteries of chick20 as well as in walls of cerebral arteries of subhuman primates.21 The expression of the MT1 receptor in human coronary arteries, derived from healthy heart donors, has been described.22 It has been suggested that MT2 melatonin receptors, expressed in vascular smooth muscles, mediate vasodilation, in contrast to vascular MT1 receptors mediating vasoconstriction.23 Direct actions of melatonin on blood vessels have also been reported.24,25 A decrease in nocturnal serum melatonin levels has been observed in patients with clinically non-characterised coronary artery disease. Also decreased nocturnal melatonin levels were observed during acute myocardial infarction.26 Urinary 6-sulphatoxymelatonin excretion was significantly lower in patients with unstable angina, compared to healthy subjects or patients with stable angina 27 (Fig. 1). Additionally, they observed that the concentrations of 6-sulphatoxymelatonin correlated negatively with the age in healthy subjects, but not in coronary patients. Brugger et al28 have shown that serum melatonin concentrations at night were more than five times lower in patients with coronary heart disease than in those in controls. The authors have suggested that melatonin reduces sympathetic activity, which is higher during the day. This effect is important for the body to relax at night. In the morning, an opposite effect can be observed—melatonin concentrations decrease and, automatically, the sympathetic

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Figure 1. Urinary 6-sulphatoxymelatonin excretion (µg in urine collected from 18:00 to 06:00 hr) in coronary patients. Shown are the means ± S.D., of aged-matched healthy subjects (n=24), stable angina patients (n=32) and unstable angina patients (n=27). Asterisks designate significant differences as compared with controls (P=0.000054) and stable angina patients (P=0.000092). The presenting data are from paper modified from Girotti et al. Low urinary 6-sulphatoxymelatonin levels in patients with coronary artery disease. J Pineal Res 2000; 29:138-142.

activity is regained. The results of Harris et al29 indicate that melatonin is unlikely to drive the previously observed presleep increase in cardiac parasympathetic activity. It has been observed that patients with coronary heart disease have a low melatonin production rate, which correlates with the stage of the disease, e.g., deeper decreases are observed in patients with higher risk of cardiac infarction and/or sudden death. Several studies suggest that some immunological factors can play an important role in the pathogenesis of coronary diseases, for example, the reactive C protein or cytokines. By activation of cytokine receptors in the endothelium of cerebral vessels, increased serum cytokine levels augment the synthesis of hypothalamic corticotropin-releasing hormone (CRH) and suppress the activity of the pituitary-adrenal axis.30,31 The data indicate that an increased circulating CRH levels suppress melatonin secretion32 or 6-sulphatoxymelatonin excretion with urine in humans.27 In addition, the possible use of β-adrenoceptor blockers, which reduce melatonin synthesis, may be an important factor responsible for low melatonin levels in patients with coronary disease. Stoschitzky et al33 have shown that beta-blockers decrease melatonin release via a specific inhibition of beta1-receptors. Nathan et al34 have demonstrated a dose-dependent relationship between β1-receptor blockade and the suppression of nocturnal plasma melatonin in humans. On the other hand, Girotti et al27 did not observe any significant difference in the levels of 6-sulphatoxymelatonin excretion in patients, either treated or not treated with β-adrenoceptor blockers. Lower nocturnal melatonin levels may be the cause of sleep disturbances which are well-known side effects of beta-adrenergic antagonists. Several studies indicate that sleep disorders occur more frequently in coronary patients than in non-coronary or normal subjects. Since low melatonin levels can be associated with sleep disturbances, at least, in elderly patients, low melatonin secretion, reported in coronary patients, could play a causal role in this respect. The other problems related to age are hypercholesterolemia and hypertension. It has been shown that chronic melatonin administration decreases serum total cholesterol levels.35,36 Hoyos et al37 have shown that melatonin diminishes total cholesterol and LDL-cholesterol levels, while increasing high-density lipoprotein (HDL)-cholesterol in diet-induced hypercholesterolemia in rats (Fig. 2). The results of that study confirm that melatonin participates in the

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Figure 2. Levels of serum total cholesterol and LDL-cholesterol in rats fed with the regular diet (Control), regular diet plus melatonin (melatonin), cholesterol-rich diet (Cholesterol) and cholesterol rich diet plus melatonin (Chol + Mel). Each values is the mean ± S.E. of 12 rats. The presenting data are from paper modified from Hoyos et al. Serum cholesterol and lipid peroxidation are decreased by melatonin in diet-induced hypercholesterolemic rats. J Pineal Res 2000; 28:150-155.

regulation of cholesterol metabolism and in the prevention of oxidative damage to membranes. Pita et al38 have shown that oral melatonin administration modifies fatty acid composition of rat plasma and liver lipids in rats fed with high-cholesterol diet for 3 months. In this long-term experiment, the analysis of lipid fractions revealed that only cholesterol ester fraction was affected by melatonin. Additionally, they found that melatonin reduced arterial fatty infiltration, induced by cholesterol feeding. The authors suggest that these effects may, at least in part, be related to antioxidative properties of melatonin. Although, a possible modulation of the activity of some hepatic enzymes can be suggested (e.g., delta-9desaturase, lecithin-cholesterol acyltransferase). Also, other authors have shown that melatonin can inhibit LDL oxidation.39-41 Furthermore, Seegar et al42 have demonstrated that, although melatonin itself appears to have little anti-atherogenic activity during LDL oxidation, melatonin precursors and breakdown products inhibit LDL oxidation, as compared to vitamin E. In contrast, Abyja et al43 have reported that melatonin cannot prevent LDL lipid peroxidation. Wakatsuki et al44 found that melatonin treatment reduced LDL susceptibility to oxidative modification in normolipidemic post-menopausal women. Thus, the oxidised form of LDL-cholesterol (ox-LDL) plays a principal role in the development of atherosclerosis. The findings of Okatani et al45 suggest that ox-LDL potentiates the vascular tension in human umbilical artery, probably by suppressing the endothelial synthesis of nitric oxide (NO). In that experiment, melatonin significantly suppressed the vasospastic effect of ox-LDL, possibly because it generally scavenges that hydroxyl radical induced by this lipid fraction. The administration of melatonin reduces blood pressure in normal,15 pinealectomized,46 and spontaneously hypertensive rats,47 whereas hypertension is induced by pinealectomy in rats.48 Laflamme et al49 have suggested that melatonin may act as the main antihypertensive agent by stimulating the central inhibitory adrenergic pathways, thereby diminishing the basal tone of the peripheral sympathetic nervous system. The hypotensive action of melatonin appears to be, at least partly, associated with the inhibition of basal sympathoadrenal tone and, finally, it could be mediated by blocking the postsynaptic α1-adrenergic receptor-induced inositol phosphate formation. On the other hand, a group from Canada50 have concluded that the hypotensive effect of melatonin in rats is not mediated either by melatonin receptors or α-adrenoceptors. Rather, the antioxidative effect of melatonin may be important in hypertensive rats, which either demonstrate a lower content of endogenous antioxidants or a greater sensitivity to free radicals of the vascular tissue.

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It has been shown that ageing and gonadal steroids influence the expression of vascular melatonin receptors in animals.51,52 Cagnacci et al53 examined the effect of melatonin on the vascular reactivity in postmenopausal women, either on or without hormone replacement therapy (HRT). They have found that the circulatory response to melatonin is preserved in postmenopausal women on HRT but not in untreated postmenopausal women. In their subsequent paper, Cagnacci et al54 found that melatonin increased NO levels only in HRT-treated but not in unreplaced postmenopausal women. These results indicate that melatonin may amplify the reported estrogen capacity to increase a nitric oxide synthase (NOS). The authors have suggested that, because a normal night-time decline of blood pressure protects women from cardiovascular accidents,55 it may be the case that estradiol capability to maintain the circulatory response to melatonin represents one of the mechanisms mediating the reduction of the cardiovascular risk in postmenopausal women. Doolen et al56 attempted at determining whether oestrogen modulates the function of vascular melatonin receptors. They have found that estradiol appears to enhance MT2 melatonin receptor function in the thermoregulatory caudal artery of female rat, resulting in an increased vasodilatation in response to melatonin. In that experimental model, as mention above, MT1 receptors mediated melatonin-induced vasoconstriction, while MT2 receptors mediated melatonin-induced vasodilatation.57 Weekley45 found that melatonin relaxed the smooth muscles, lining the rat aorta. The vascular endothelium may contribute to the regulation of vascular smooth muscle tone by producing such vasoconstrictors as endothelin-159 and thromboxane,60 as well as vasodilators, such as prostacyclin61 and NO.62 Nitric oxide was originally identified as the principal endothelium-derived vascular relaxation factor. Okatani et al63 demonstrated that a pre-treatment with L-NG-monomethyl arginine, a NOS inhibitor, suppressed the potentiating effect of hydrogen peroxide (H2O2) on the vascular tension in umbilical artery segments, suggesting that H2O2 may exert its vasospastic effect by inhibiting NOS in the endothelium. Melatonin modulates NOS activity and, thereby, influences NO production.64-65 Cuzzocrea et al66 demonstrate that melatonin treatment in a model of splanchnic artery occlusion shock exerts a protective effect due to inhibition of the expression of adhesion molecule and peroxynitrite-related pathways and subsequent reduction of neutrophil-mediated cellular injury. The results of the study of Wakatsuki et al67 indicate that H2O2 may impair NO synthesis in the endothelium of human umbilical arteries. Melatonin significantly suppresses the H2O2-induced inhibition effect of NO production, most likely through its ability to scavenge hydroxyl radicals. Ca+2 plays an important role in physiology of the heart. Melatonin may participate in the regulation of myocardial Ca+2 homeostasis. It has been shown that this indoloamine enhances the activity of the membrane calcium pump and regulates calmodulin.68-70 This indoloamine regulates intracellular calcium levels by preventing calcium overloading. The results of Mei et al71 suggest a specific melatonin receptor-mediated action on the calcium channel of the chick myocyte. The melatonin-induced increase in high-voltage calcium current may enhance myocyte contractility and cardiac output. The results of many publications suggest an impending decrease in circulating melatonin concentrations at different stages of the coronary disease. The antioxidative property of melatonin has been demonstrated during the last 10 years of studies.72-78 The results of epidemiological studies have demonstrated a lower incidence of coronary artery disease and mortality rate in persons who consume larger quantities of antioxidants, like vitamin E, beta-carotene, and vitamin C in their diet.79 The antioxidants, including melatonin, can play a beneficial role in reducing the incidence of coronary events. Tan et al80 observed that melatonin protected against arrhythmia induced by ischemia-reperfusion in isolated rat hearts. Sahna et al81,82 suggested that physiological melatonin concentrations are important to reduce the ischemia-reperfusion arrhythmias, myocyte damage and mortality, while pharmacological concentrations of this hormone did not increase its beneficial effect. The cardioprotective activity of this indoloamine may be mediated by its antioxidative property and its capacity for neutrophil inhibition in myocardial ischemia-reperfusion.83 Melatonin reduced the damage induced by chemical hypoxia and reoxygenation in rat cardiomyocytes. 84 Melatonin alone or in

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Figure 3. Lipoperoxide levels in rat heart, expressed as nmol (MDA and 4-HDA) per g of tissue. Bars represent mean values ± S.E.M., n= 6 animals per group. *** P2500 lux) sufficient to suppress endogenous melatonin increased core temperature.21,30 The findings from these studies resulted in the hypothesis that, in addition to its effect on the circadian system, melatonin may affect sleep-wake behavior via direct thermoregulatory mechanisms. Although this hypothesis represented a significant shift in the conceptualization of melatonin’s mechanism of action, its seminal influence can be seen in the results from an animal study over 20 years earlier (e.g., ref. 31). From a broader perspective, however, it is also important to consider that the development of this hypothesis was influenced by the investigation into the relationship between sleep and temperature. A brief description of this research is outlined in the section below.

Relationship between Sleep and Thermoregulation: An Overview A large body of indirect data supports a link between thermoregulation and sleep. It is well documented, for example, that sleep onset is presaged by an increase in peripheral temperature and a concomitant decrease in core body temperature.32-35 There is also a considerable amount of experimental evidence demonstrating a close temporal relationship between the rhythms of core body temperature and sleep propensity with the major sleep period coinciding with the time of the core body temperature minimum.36-40 More direct support for this relationship between sleep and thermoregulation has come from studies showing that various treatments that increase core body temperature also delay sleep onset. Examples of such experimental treatments include: bright light,41 capsaicin administration,42 and melatonin-suppressive agents, such as the beta-adrenergic antagonist atenolol and anti-inflammatory drugs.43-45 In contrast, treatments that decrease core body temperature typically increase sleep propensity such as hand thermal biofeedback,46 ethanol47 and temazepam.48,49 Additionally, several studies reported a reduction in sleep quality following whole body heating sufficient to increase core body

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temperature.50 Support for the thermoregulatory model of sleep onset has also come from clinical research demonstrating that impaired heat loss capacity is associated with a prolonged latency to sleep onset in patients with vasospastic syndrome,51 while raised core body temperature is characteristic of poor sleepers.7 In the next section, we detail recent work leading to suggestions that the rate of peripheral heat loss at the feet, hand and face is perhaps the strongest predictor of sleep propensity.52

The Effects of Melatonin on Both Sleep and Temperature The inherent relationship between sleep and temperature, taken together with the capacity of melatonin to affect both sleep and thermoregulation, resulted in the suggestion that melatonin may influence sleep through its effect on temperature.53,54 To examine this hypothesis, a number of research groups investigated the effects of oral melatonin on temperature and sleep. Across a range of doses (1-10 mg) most research groups reported that melatonin reduced core body (and oral) temperature and increased sleep propensity or sleepiness.55-61 This work was extended in later studies with the effects of melatonin on temperature being explored in more detail. It was demonstrated, for example, that the rate of decline in core temperature was a better predictor of sleep onset latency than just the magnitude of core temperature decline.48,62 Later, peripheral and proximal skin temperatures were included in addition to core body temperature in studies of both daytime and evening melatonin administration.52,63 From these results, an index of peripheral heat loss (the distal-proximal gradient) was found to be a better predictor of sleep onset than the rate of core temperature decline.52,64 As such, it was concluded that the peripheral thermoregulatory action of melatonin may be functionally involved in the regulation of sleep. At around the same time, the effects of melatonin were being examined in subject groups such as the aged and insomniacs.6,65,66 The results obtained from the aged and insomniac studies were particularly informative and revealed that, in individuals where the soporific effects of melatonin were attenuated, the thermoregulatory effects were also attenuated. As such, these studies provided additional support for the suggestion that melatonin may exert its soporific effect directly via its effects on body temperature. Figure 1 illustrates the relationship between peripheral heat loss and sleepiness following daytime exogenous melatonin administration. However, the apparent variability in the soporific and thermoregulatory effects of exogenous melatonin, combined with the possibility that melatonin may act through peripheral rather than only central mechanisms, resulted in a realization that much of the basic melatonin research had not yet been performed. With the benefit of hindsight it is perhaps surprising that, following the purification of melatonin, research moved straight onto the administration of exogenous melatonin without first attempting to investigate its physiological role, long term safety, or mechanism of action. This being said, it is clear that recent research focusing on the mechanism of action of melatonin has steadily increased.

Exploring the Mechanism of Action of Melatonin Melatonin Receptors Traditionally, endocrinologists have considered that hormones and many pharmacological agents exert their influence through action on specific receptors. In line with this perception, researchers initially sought to identify target sites where melatonin may act.67-69 By so doing, it was hoped that melatonin’s physiological mechanisms of action would be elucidated. The radioligand 2-iodomelatonin was initially extensively used to localize binding sites in both the brain and peripheral tissues.70 In general these binding sites were found to be high affinity, with binding constant (Kd) in the low picomolar range, and selective for structural analogues of melatonin. An early investigation of melatonin receptor molecular actions was carried out by White and colleagues.71 Using amphibian dermal melanosome preparations, this group found that a regulatory protein, with homology to a mammalian protein, mediated melatonin’s binding.

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Figure 1. A sequence of thermographic images taken at 0, 10, 20, 30, 45 and 60 minutes after ingestion of a rapid-release melatonin capsule (3 mg) at 1400 hrs. The six panels show the dynamic changes in upper-body skin temperatures following ingestion. The subject was a healthy 27-year old male, lying supine in bed covered to the waist by a sheet with room temperature controlled at 25 degrees Celsius. After skin temperatures were stable, thermographs were taken with a thermal imaging camera (Meditherm, Queensland, Australia) placed directly above the bed. Each thermograph covers a range of temperatures from 22.0-35.5 degrees Celsius, as indicated on the scale at the bottom of the figure; black represents temperatures at or below 22.0 degrees and white temperatures at or above 35 degrees Celsius. It can be seen in the left and right hands, and to a lesser extent the face and neck, that administration of melatonin elicited an increase in peripheral skin temperature. Analyses of images using WinTES thermal evaluation software (Compix Ltd, Tualatin, OR), revealed that the average temperatures recorded over both hands were 30.2, 31.1, 31.0, 30.9, 31.9 and 32.2 degrees Celsius at 0, 10, 20, 30, 45 and 60 minutes after ingestion, respectively. The data from these images supports previous findings that daytime melatonin administration significantly elevates skin temperatures at distal sites to allow reductions in core temperature.

Mammalian cells use this related protein to mediate the action of hormones that inhibit adenylate cyclase through a cell surface receptor. An early autoradiographic study localized putative melatonin receptors predominantly in the median eminence (implicated in seasonal/reproductive regulation) and the SCN.72 Soon after, two distinct melatonin binding sites were identified; so called melatonin 1 (MEL1) from chicken brain,73 and melatonin 2 (MEL2) from hamster tissues.73,74 Since then, melatonin binding sites have been observed in the median eminence/arcuate nucleus, pars tuberalis, suprachiasmatic nucleus, pineal gland, anterior pituitary and preoptic area in the fetal brain (e.g., refs 75, 76). In decreasing order of abundance in

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humans, melatonin receptors have been detected by in-situ hybridization in the cerebellum, occipital cortex, parietal cortex, temporal cortex, thalamus, frontal cortex and hippocampus.77 Despite the identification of these binding sites, it is not known whether melatonin has a functional role in all of these areas. Initially, there was some contention as to whether there were multiple distinct types of melatonin receptors.78 However, when genes coding for melatonin receptors were discovered in mammalian tissue, the cloning of these receptors revealed that all melatonin receptors belong to the superfamily of G-protein coupled receptors.79,80 The discovery of melatonin receptors in the SCN indicated that endogenous melatonin was able to feedback on the circadian pacemaker and was a modulator of circadian time.75 This finding complimented the laboratory work in humans and resulted in a strong cohesive model for the role of melatonin in the circadian system. However, unlike the SCN, very few melatonin receptors were found within the PoAH.80 Such an observation indicated that a central action of melatonin on the thermoregulatory system was unlikely. Melatonin receptors were, however, not confined to the brain and in 1990, melatonin binding sites were located in the smooth muscle of the rat caudal artery, an area known to be involved in thermoregulation.81 Such a finding supported the suggestion that melatonin may exert its thermoregulatory effect through peripheral heat loss.52 Moreover, the possibility that these peripheral receptors may have a functional role in thermoregulation was highlighted by subsequent research demonstrating that melatonin was able to enhance both noradrenergic,82 and electrically-evoked,83 constriction of isolated rat tail arteries. Indirect evidence indicating a similar peripheral action of melatonin in humans was documented by Cagnacci and colleagues who reported a significant decrease in circulating noradrenaline levels in humans following the daytime administration of exogenous melatonin (1 mg).84 It should be noted, of course, that, as rats are nocturnal, melatonin acts in a contrary manner to humans and other diurnal animals (i.e., it is alerting and hyperthermic). Interestingly, however, new research has shown that melatonin is able to cause both vasodilatation and vasoconstriction in the same organism (e.g., ref. 85). It appears that activation of MEL1 receptors cause vasoconstriction on blood vessels while activation of MEL2 receptors cause vasodilatation. In humans, MEL2 receptors predominate in the peripheral vasculature while MEL1 receptors are found in the cerebral arteries.86 These findings are therefore consistent with the earlier work of Reppert who suggested that each receptor subtype might mediate a different action of melatonin in the body.79 It is interesting to note that although the rat caudal artery contains both functional MEL1 receptors and MEL2 receptors, the density of MEL2 receptors is much less.85 As such, it could be argued that type of melatonin receptor that is promoted through evolution is consistent with the organism’s diurnal or nocturnal status. It is therefore not surprising that MEL2 receptors typically predominate in the peripheral vasculature of diurnal species while MEL1 receptors predominate in the peripheral vasculature of nocturnal species. Despite the discovery of specific melatonin receptors, it has also been suggested that some actions of melatonin may be independent of these receptors. Due to melatonin’s lipophilicity, it has been suggested that it can enter all cells in the body and hence act directly on the cell nucleus.87 Alternatively, it has been suggested that melatonin may interact with other endogenous compounds or their receptors. In particular, it was suggested that melatonin may exert its soporific effect through an action on central benzodiazepine receptors,88 or through the modulation of endogenous GABA itself.89-91

A Central GABA Pathway? A possible action on GABA was first demonstrated in in vitro animal studies. Specifically, melatonin was able to enhance muscimol binding in vitro in rat brain tissue,92 as well as inhibiting diazepam binding in the rat brain.93 In both these studies, the action of melatonin was attributed to an action at the GABAA-benzodiazepine receptor complex. In the case of in vivo human research, indirect evidence supporting a role of GABA in the action of melatonin was provided by Dijk and colleagues.94 A 5mg oral dose of melatonin was given to eight healthy

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men immediately prior to a 4-hour daytime sleep episode (1300–1700h). Spectral analysis was subsequently performed on the EEG data and melatonin was found to suppress low frequency EEG activity and to enhance spindle activity. As these changes in the EEG were similar to those typically seen following benzodiazepine administration, it was suggested that melatonin’s soporific effects, like the benzodiazepines, may arise through occupation of the GABAA-benzodiazepine receptor complex.94 On this basis, it is possible that the thermoregulatory effects of melatonin, like its soporific effects, may also be mediated through action at GABA-receptors. To investigate this theory, the thermoregulatory and soporific effects of a 3mg melatonin dose were examined by Nave and colleagues with and without the additional administration of 10mg of flumazenil (administered orally), a central benzodiazepine antagonist.95 Although the flumazenil did not block either the hypothermic or soporific effects of melatonin, it is important to note that flumazenil has a very low bioavailability when administered orally.96 As flumazenil levels were not directly measured by Nave and colleagues,95 it is possible that flumazenil had no significant effect on melatonin because it simply was not present at a sufficient concentration. Therefore, it is not possible to discount a central action of melatonin through the GABA system from these results.

A Shift in Focus from Central to Peripheral Effects For the majority of the 1990s, interest in melatonin had largely been concerned with the central effects of melatonin. Physiologists examined the relationship between melatonin, core body temperature and the sleep/wake cycle.22,23,53,57,61 Similarly, neurobiologists examined the possible mechanism of action of melatonin through centrally located receptors.76,77,79,80,97 As such, it was thought that melatonin acted on central brain areas, which in turn, initiated melatonin’s systemic effects. Not surprisingly, such an approach is consistent with that used for sedative/hypnotic agents such as benzodiazepines. However, unlike benzodiazepines, the efficacy of melatonin has not been consistent, and, therefore, would seemingly discount a central action.6,55 Indeed, as the focus of melatonin’s physiological effects shifted from core temperature to peripheral temperature in the last years of the 20th Century,49,52,63,64 to understand the mechanism of action of melatonin the research focus must also shift to the peripheral vasculature. As described above, melatonin has functional binding sites in the peripheral vasculature.81,98 It is also clear that, in humans, melatonin initiates vasodilatation of peripheral blood vessels leading directly to heat loss.64 However, to fully understand melatonin’s mechanism of action, the neural relationship between thermosensitive cells and somnogenic brain areas must first be understood.

The Role of the PoAH and Thermosensitive Neurons in Sleep Regulation: The Basis for Melatonin’s Effect on Sleep Neuroanatomical research revealed that sleep could be initiated following physical warming of the PoAH,99,100 and also following chemical stimulation of the same area.101 Such a finding implicated activation of the PoAH in the normal initiation of sleep. Consistent with such an hypothesis, groups of warm-sensitive neurons in the PoAH were found to increase their firing rate at sleep onset and decrease their firing just before arousal in animals,102 an observation confirmed by immunocytochemistry.103 This finding was important as the activation of these PoAH thermosensitive neurons also affected the discharge rate of neurons in other brain areas known to regulate sleep and wakefulness.104,105 Such areas include the posterior hypothalamus, basal forebrain, and the dorsal raphe nuclei. When taken together, these findings provided evidence to support a role of temperature in sleep regulation. However, the most compelling evidence for such a role came from the finding that heating of peripheral skin resulted in an increase in the firing of warm-sensitive neurons in the PoAH and other brain areas known to be involved in sleep regulation.102,103 Not only did this indicate that a neural pathway existed between peripheral skin and somnogenic brain areas, but it provided a specific neurophysiological mechanism through which an increase in peripheral skin

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temperature may be able to initiate sleep. More specifically, a neural pathway was described which could explain why an increase in peripheral heat loss following melatonin administration could result in an increase in sleepiness. Importantly, such a theory is consistent with experimental work in humans that have documented a significant relationship between increased heat loss and increased sleep propensity following exogenous melatonin administration.63,64

Conclusions If we momentarily put the chronobiotic effects of melatonin aside, when the above neuroanatomical findings are taken together with the peripheral effects of melatonin on temperature, it is clear that to think of melatonin as a soporific or hypnotic agent is misleading. Rather, it is perhaps more accurate to conceptualize exogenous melatonin as a vasodilator agent, and that it is the stimulation of peripheral thermosensitive neural pathways feeding onto somnogenic brain areas, which underlies melatonin’s increase in sleep propensity. When exogenous melatonin is viewed in this way, much of the conflicting and inconsistent findings become clearer. If melatonin does not affect sleep directly, then this explains why exogenous melatonin is not consistently efficacious. For example, any factor that attenuates peripheral heat loss would also attenuate the soporific effects of exogenous melatonin. The fact that vascular conductance decreases with age106 may explain the age-related variability observed in melatonin’s soporific effects, as it is typically less effective in older individuals than young adults. The fact that exogenous melatonin administration in the evening has no additional effect on sleep is also consistent with such a hypothesis. That is, as the peripheral vasculature becomes maximally dilated in the evening, administration of exogenous melatonin would not additionally dilate the peripheral blood vessels and thus, sleep would be unaffected. If we presume that endogenous melatonin, like exogenous melatonin, affects sleep indirectly though an effect on thermoregulation, it is reasonable to then ask what is the purpose of such an action; especially as it is well known that a major role of endogenous melatonin is to modulate the circadian system. It seems, therefore, that to think of melatonin only as a vasodilator would be an oversimplification. Given the direct action of melatonin on the thermoregulatory system, it would be parsimonious to consider that the thermoregulatory effects of melatonin may also play a role in its effect on the circadian system. If this is the case, it is possible that the thermoregulatory effect of melatonin may augment the central action of melatonin on the SCN. Nevertheless, there is currently little or no experimental evidence to support this suggestion in humans. An additional possibility is that melatonin’s circadian modulation of sleep propensity may “gate” our daily exposure to synchronizing environmental light and dark periods. That is, by determining when we are likely to be sleeping and when we are awake through direct thermoregulatory effects, the production of melatonin thereby influences our exposure to time cues. In summary, we have presented the hypothesis that exogenous melatonin has no primary soporific properties but acts directly at the peripheral vasculature to enhance heat loss. In doing so, thermosensitive neurons in the periphery are activated that, in turn, provide feedback information to somnogenic centers in the brain to promote sleepiness. Finally, we have proposed that endogenous melatonin may act peripherally on the thermoregulatory system to augment central circadian time keeping. This may occur as a result of the peripheral action of melatonin on the thermoregulatory system, acting as an anchor for the circadian system by influencing when we are sleeping (and impervious to environmental light) or conversely when we are awake (and likely to receive exposure to light). This theory is schematically represented in Figure 2. Despite recent advances in the understanding of melatonin’s molecular targets, much basic research is still yet to be performed. Exogenous melatonin holds considerable promise in the treatment of sleep disorders, if not directly, then as a research tool with which the thermoregulatory system can be probed in more depth. This, in turn, will allow the development of better treatments for insomnia, particularly those associated with decreased peripheral heat loss capacity or an elevated nocturnal core temperature.

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Figure 2. Schematic illustration of the interaction between melatonin, sleep and the thermoregulatory and circadian systems.

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51. Pache M, Krauchi K, Cajochen C et al. Cold feet and prolonged sleep-onset latency in vasospastic syndrome. Lancet 2001; 358(9276):125-6. 52. Krauchi K, Cajochen C, Werth E et al. Warm feet promote the rapid onset of sleep. Nature 1999; 401(6748):36-7. 53. Badia P, Myers B, Murphy PJ. Melatonin and thermoregulation. In: Reiter R, Yu HS, eds. Melatonin: Biosynthesis, Physiological Effects and Clinical Applications. Boca Raton: CRC Press; 1992: 349-364. 54. Dawson D, Encel N. Melatonin and sleep in humans. J Pineal Res 1993; 15(1):1-12. 55. Cagnacci A, Soldani R, Yen SS. Hypothermic effect of melatonin and nocturnal core body temperature decline are reduced in aged women. J Appl Physiol 1995; 78(1):314-7. 56. Dawson D, Gibbon S, Singh P. The hypothermic effect of melatonin on core body temperature: is more better? J Pineal Res 1996; 20(4):192-7. 57. Reid K, Van den Heuvel C, Dawson D. Day-time melatonin administration: effects on core temperature and sleep onset latency. J Sleep Res 1996; 5(3):150-4. 58. Deacon S, English J, Arendt J. Acute phase-shifting effects of melatonin associated with suppression of core body temperature in humans. Neurosci Lett 1994; 178(1):32-4. 59. Zhdanova IV, Wurtman RJ, Lynch HJ et al. Sleep-inducing effects of low doses of melatonin ingested in the evening. Clin Pharmacol Ther 1995; 57(5):552-8. 60. Nave R, Peled R, Lavie P. Melatonin improves evening napping. Eur J Pharmacol 1995; 275(2):213-6. 61. Dollins AB, Zhdanova IV, Wurtman RJ et al. Effect of inducing nocturnal serum melatonin concentrations in daytime on sleep, mood, body temperature, and performance. Proc Natl Acad Sci USA 1994; 91(5):1824-8. 62. Campbell SS, Broughton RJ. Rapid decline in body temperature before sleep: fluffing the physiological pillow? Chronobiol Int 1994; 11(2):126-31. 63. Krauchi K, Cajochen C, Wirz-Justice A. A relationship between heat loss and sleepiness: effects of postural change and melatonin administration. J Appl Physiol 1997; 83(1):134-9. 64. Krauchi K, Cajochen C, Werth E et al. Functional link between distal vasodilation and sleep-onset latency? Am J Physiol Regul Integr Comp Physiol 2000; 278(3):R741-8. 65. Hughes RJ, Sack RL, Lewy AJ. The role of melatonin and circadian phase in age-related sleepmaintenance insomnia: assessment in a clinical trial of melatonin replacement. Sleep 1998; 21(1):52-68. 66. Mishima K, Okawa M, Satoh K et al. Different manifestations of circadian rhythms in senile dementia of Alzheimer’s type and multi-infarct dementia. Neurobiol Aging 1997; 18(1):105-9. 67. Cohen M, Roselle D, Chabner B et al. Evidence for a cytoplasmic melatonin receptor. Nature 1978; 274(5674):894-5. 68. Cardinali DP, Vacas MI, Boyer EE. Specific binding of melatonin in bovine brain. Endocrinology 1979; 105(2):437-41. 69. Niles LP, Wong YW, Mishra RK et al. Melatonin receptors in brain. Eur J Pharmacol 1979; 55(2):219-20. 70. Acuna-Castroviejo D, Pablos MI, Menendez-Pelaez A et al. Melatonin receptors in purified cell nuclei of liver. Res Commun Chem Pathol Pharmacol 1993; 82(2):253-6. 71. White BH, Sekura RD, Rollag MD. Pertussis toxin blocks melatonin-induced pigment aggregation in Xenopus dermal melanophores. J Comp Physiol [B] 1987; 157(2):153-9. 72. Vanecek J, Pavlik A, Illnerova H. Hypothalamic melatonin receptor sites revealed by autoradiography. Brain Res 1987; 435(1-2):359-62. 73. Dubocovich ML. Pharmacology and function of melatonin receptors. Faseb J 1988; 2(12):2765-73. 74. Weaver DR, Namboodiri MA, Reppert SM. Iodinated melatonin mimics melatonin action and reveals discrete binding sites in fetal brain. FEBS Lett 1988; 228(1):123-7. 75. Reppert SM, Weaver DR, Rivkees SA et al. Putative melatonin receptors in a human biological clock. Science 1988; 242(4875):78-81. 76. Krause DN, Dubocovich ML. Regulatory sites in the melatonin system of mammals. Trends Neurosci 1990; 13(11):464-70. 77. Mazzucchelli C, Pannacci M, Nonno R et al. The melatonin receptor in the human brain: cloning experiments and distribution studies. Brain Res Mol Brain Res 1996; 39(1-2):117-26. 78. Morgan PJ, Barrett P, Howell HE et al. Melatonin receptors: localization, molecular pharmacology and physiological significance. Neurochem Int 1994; 24(2):101-46. 79. Reppert SM, Godson C, Mahle CD et al. Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor. Proc Natl Acad Sci USA 1995; 92(19):8734-8.

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80. Reppert SM, Weaver DR, Ebisawa T. Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses. Neuron 1994; 13(5):1177-85. 81. Viswanathan M, Laitinen JT, Saavedra JM. Expression of melatonin receptors in arteries involved in thermoregulation. Proc Natl Acad Sci USA 1990; 87(16):6200-3. 82. Krause DN, Barrios VE, Duckles SP. Melatonin receptors mediate potentiation of contractile responses to adrenergic nerve stimulation in rat caudal artery. Eur J Pharmacol 1995; 276(3):207-13. 83. Ting KN, Dunn WR, Davies DJ et al. Studies on the vasoconstrictor action of melatonin and putative melatonin receptor ligands in the tail artery of juvenile Wistar rats. Br J Pharmacol 1997; 122(7):1299-306. 84. Cagnacci A, Arangino S, Angiolucci M et al. Influences of melatonin administration on the circulation of women. Am J Physiol 1998; 274(2 Pt 2):R335-8. 85. Masana MI, Doolen S, Ersahin C et al. MT(2) melatonin receptors are present and functional in rat caudal artery. J Pharmacol Exp Ther 2002; 302(3):1295-302. 86. Savaskan E, Olivieri G, Brydon L et al. Cerebrovascular melatonin MT1-receptor alterations in patients with Alzheimer’s disease. Neurosci Lett 2001; 308(1):9-12. 87. Menendez-Pelaez A, Reiter RJ. Distribution of melatonin in mammalian tissues: the relative importance of nuclear versus cytosolic localization. J Pineal Res 1993; 15(2):59-69. 88. Golombek DA, Escolar E, Burin LJ et al. Time-dependent melatonin analgesia in mice: inhibition by opiate or benzodiazepine antagonism. Eur J Pharmacol 1991; 194(1):25-30. 89. McIntyre IM, Norman TR, Burrows GD et al. Alterations to plasma melatonin and cortisol after evening alprazolam administration in humans. Chronobiol Int 1993; 10(3):205-13. 90. Golombek DA, Martini M, Cardinali DP. Melatonin as an anxiolytic in rats: time dependence and interaction with the central GABAergic system. Eur J Pharmacol 1993; 237(2-3):231-6. 91. Golombek DA, Pevet P, Cardinali DP. Melatonin effects on behavior: possible mediation by the central GABAergic system. Neurosci Biobehav Rev 1996; 20(3):403-12. 92. Coloma FM, Niles LP. In vitro effects of melatonin on [3H]muscimol binding in rat brain. Prog Neuropsychopharmacol Biol Psychiatry 1984; 8(4-6):669-72. 93. Marangos PJ, Patel J, Hirata F et al. Inhibition of diazepam binding by tryptophan derivatives including melatonin and its brain metabolite N-acetyl-5-methoxy kynurenamine. Life Sci 1981; 29(3):259-67. 94. Dijk DJ, Roth C, Landolt HP et al. Melatonin effect on daytime sleep in men: suppression of EEG low frequency activity and enhancement of spindle frequency activity. Neurosci Lett 1995; 201(1):13-6. 95. Nave R, Herer P, Haimov I, Shlitner A et al. Hypnotic and hypothermic effects of melatonin on daytime sleep in humans: Lack of antagonism by flumazenil. Neurosci Lett 1996; 214(2-3):123-6. 96. Whitwam JG, Amrein R. Pharmacology of flumazenil. Acta Anaesthesiol Scand Suppl 1995; 108:3-14. 97. Vanecek J. Mechanism of melatonin action. Physiol Res 1991; 40(1):11-24. 98. Viswanathan M, Scalbert E, Delagrange P et al. Melatonin receptors mediate contraction of a rat cerebral artery. Neuroreport 1997; 8(18):3847-9. 99. McGinty D, Szymusiak R. Keeping cool: A hypothesis about the mechanisms and functions of slow- wave sleep. Trends Neurosci 1990; 13(12):480-7. 100. Gong H, Szymusiak R, King J et al. Sleep-related c-Fos protein expression in the preoptic hypothalamus: effects of ambient warming. Am J Physiol Regul Integr Comp Physiol 2000; 279(6):R2079-88. 101. Ramesh V, Kumar VM, John J et al. Medial preoptic alpha-2 adrenoceptors in the regulation of sleep- wakefulness. Physiol Behav 1995; 57(1):171-5. 102. Alam MN, McGinty D, Szymusiak R. Preoptic/anterior hypothalamic neurons: thermosensitivity in wakefulness and non rapid eye movement sleep. Brain Res 1996; 718(1-2):76-82. 103. Sherin JE, Shiromani PJ, McCarley RW et al. Activation of ventrolateral preoptic neurons during sleep. Science 1996; 271(5246):216-9. 104. Szymusiak R. Magnocellular nuclei of the basal forebrain: substrates of sleep and arousal regulation. Sleep 1995; 18(6):478-500. 105. Guzman-Marin R, Alam MN, Szymusiak R et al. Discharge modulation of rat dorsal raphe neurons during sleep and waking: Effects of preoptic/basal forebrain warming. Brain Res 2000; 875(1-2):23-34. 106. Kenney WL, Morgan AL, Farquhar WB et al. Decreased active vasodilator sensitivity in aged skin. Am J Physiol 1997; 272(4 Pt 2):H1609-14.

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CHAPTER 10

The Role of Melatonin in Human Aging and Age-Related Diseases Michal Karasek

Abstract

M

any theories relating the pineal gland and its secretory product melatonin to aging have been put forward. However, the role of this agent in the aging process is still not clear. Although aging process is multifactoral, and no single element seems to be of basic importance for several reasons it seems reasonable to postulate a role of melatonin in this process. Melatonin levels fall gradually over the life-span. Reduced concentrations of melatonin may result in lowered sleep efficacy very often associated with advancing age. Diminished melatonin secretion in advanced age may be related to deterioration of many circadian rhythms. Melatonin deficiency is related to suppressed immunocompetence which plays a role in the acceleration of aging. Finally, melatonin is a potent free radical scavenger, and free radicals cause accumulating with age damage to vital cellular constituents which has significance not only for aging per se but also for many age-related diseases. The data on the possible importance of melatonin in human aging and age-related diseases, and background for its supplementation in advanced age are briefly presented in this survey.

Introduction Population aging was one of the most distinctive demographic events of the twentieth century.1 The world population increased from 1 billion at the beginning of 19th century to 6 billion at the start of 21st century, and is predicted to expand further reaching over 9 billion in 2050. Moreover, the worldwide prolongation of the mean life expectancy, partially due to developments in modern medicine, as well as the drastic reduction of fertility rate result, in the most parts of the world, in rapid increase of the size of the elderly population (over the age 60), both in numbers and as a proportion of the whole. In 1950 there were 205 million persons over 60 (less than 5% of the world population), this number increased to 606 million (about 10% of the world population) in 2000, and is expected to be tripled in the next 50 years (2 billion – about 20% of the world population).1-3 The growth rate of the older population (1.9%) is significantly higher that of total population (1.2%), and is predicted that by 2030 there will be further increase (older population – 2.8%, total population – 0.8%). Moreover, there is a constant increase in the age group of over 80. The number of persons aged 80 or more amounted to 69 million in 2000, and is expected to increase to 379 million in 2050. The same is true for the people who live beyond the age of 100. Although the proportion of such people is very small (it is estimated that currently only 180,000 centenarians live throughout the world) by 2050 they are projected to increase about 18 times (to 3.2 million).1,2 We should remember, however, that aging has to be seen as a lifelong process, not as a state which emerges acutely at the age of 60.4 The global population aging raises many social and

Melatonin: Biological Basis of Its Function in Health and Disease, edited by S.R. Pandi-Perumal and Daniel P. Cardinali. ©2006 Eurekah.com.

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economical problems because of increasing number of potential beneficiaries of health and pension funds, mainly those aged 65 and over, are supported by a relatively smaller number of potential contributors, i.e., those in the economically active ages of 15-64.2 Increase in number of people in advanced age results also increase in number of people suffering from age-related diseases, such as atherosclerosis, neurodegenerative diseases (Alzheimer’s and Parkinson’s disease), and neoplastic disease. Aging per se as well as age-related diseases lead to less or more advanced disability, and prevention and reduction of disability should be the basis of a sensible strategy for aging population. Therefore, the geriatric approach to rational health care for older people should be seriously considered.4 Any therapeutic agent improving quality of life of elderly is urgently needed. A role for melatonin as such compound was recently suggested. Although melatonin, the indoleamine secreted by the pineal gland, has been discovered over 40 years ago, for many years its role was studied mostly in experimental animals. After introducing the methods of melatonin measurements in body fluids more and more clinical studies were performed. However, melatonin received great attention just in the last decade following the hypothesis suggesting its role in the aging process. Currently melatonin is available in many countries, including e.g., USA, Argentina, and Poland, as food supplement or OTC drug, and often advertised as “rejuvenating” agent. Therefore, medical doctors should be aware of some basic principles regarding melatonin role in human aging, and its possible therapeutic use. The aim of this survey is to present data on the role of melatonin in aging and age-related diseases, and to discuss background for its therapeutic use in the advanced age.

The Reasons Why a Role of Melatonin in Aging Is Postulated First attempts relating melatonin to aging have been made in late 80s and early 90s on the basis of experimental animal studies.5-9 Moreover, the hypothesis has been put forward that “aging is secondary to pineal failure”.5,6 The most subsequent data came also from the animal experiments which utilized rather a small number of animals and the animals typically were not barrier maintained. Although the data on the relationships between melatonin and aging in humans are rather scarce, the following reasons allowed to postulate a role for melatonin in the process of aging.10-14 1. Melatonin participates in many vital life processes, and its secretion falls gradually over the life-span. 2. Melatonin acts as endogenous sleep-inducing agent, and its reduced concentrations may result in lowered sleep efficacy very often associated with advancing age. 3. Diminished melatonin secretion in advanced age may be related to deterioration of many circadian rhythms, as a consequence of a reduced function of suprachiasmatic nucleus. 4. Melatonin exhibits immunoenhancing properties, and suppressed immunocompetence has been implicated in the acceleration of aging processes. 5. Melatonin is a potent free radical scavenger, and the proposed link between oxidative stress and aging itself as well as age-related diseases (such as Alzheimer’s and Parkinson’s diseases, neoplastic disease, senile cataract) suggest a role for melatonin in these processes.

Melatonin Circadian Rhythm during Life-Span Melatonin has a well-defined circadian rhythm with low values during the daytime and 10-15 fold increase at night.15,16 This rhythm is generated by the circadian pacemaker (oscillator, biological clock) situated in the suprachiasmatic nucleus (SCN) of the hypothalamus, and synchronized to 24 hours primarily by the light-dark cycle acting via the SCN.15 Melatonin is present in all living organisms from plants, through animal kingdom to humans, and from unicellular algae to man shows this characteristic circadian rhythm. The rhythm of melatonin secretion in humans develops around the 6th month of life and reaches the highest levels between 4th and 7th year of age. There is a drop in melatonin concentrations around maturation, and thereafter its levels diminish gradually.12,15,16 As a consequence, in advanced age many individuals do not exhibit a day-night differences in melatonin secretion (Fig. 1). The

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Figure 1. A) Circadian profiles of serum melatonin concentrations at various age; grey area – darkness. B) Maximum nocturnal serum melatonin levels at various age.

amplitude of nocturnal melatonin secretion is believed to be genetically determined and shows great differences among individuals.17 Thus, some individuals produce significantly less melatonin during lifetime than others, which may have significance in terms of aging.

Melatonin and Sleep Disorders in Advanced Age Sleep promoting effects of melatonin have been well known since first experiments in early 70s, and is probably a consequence of increasing sleep propensity and of synchronizing effect on the circadian clock.18 Melatonin secretion during aging negatively correlates with sleep disturbances. Melatonin concentrations significantly decrease in advanced age, whereas the

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increased frequency of sleep disorders occurs in the elderly.19 It should be stressed that 40- 70% of the elderly population suffers from chronic sleep disturbances (only 20% do not report any sleep disturbance at all), and sleep disturbances compromise the subjective and objective general physical health of the elderly, and is associated with mental health problems including poor life satisfaction or quality of life as well as poor cognitive, psychological, and social functioning (for review see ref. 20). Although there is not clear cut evidence that impaired melatonin secretion is the main cause of sleep disturbances in advanced age, there are some data showing that melatonin or 6-hydroxymelatonin sulphate concentrations are lower in elderly insomniacs than in old individuals without sleep disorders. However, a recent study has not shown a correlation between melatonin concentrations and insomnia in aged subjects.21 It is probable that diminished melatonin secretion in the elderly is caused by insufficient environmental illumination.22 Youngstedt et al23 on the basis of 6-sulphatoxymelatonin measurements demonstrated significantly greater circadian dispersion (defined as the mean variations of 6-SMT acrophase from the median age-specific acrophase) as well as greater circadian malsynchronization (defined as absolute number of hours between 6-SMT acrophase and the middle of sleep period) in the old (60-79 years old) vs young (20-40 years old) individuals. The authors suggest that circadian malsynchronization might be a common and significant cause of disturbed sleep in elderly subjects. It has been demonstrated in several reports that administration of melatonin has beneficial effects in aged subjects suffering from insomnia. It should be noted, however, that although majority of data show that melatonin improve sleep parameters in elderly, in some studies sleep was unaffected by melatonin (for review see refs. 14, 18, 24-26).

Melatonin and Circadian Rhythm Deterioration in Advanced Age Circadian rhythmicity in many important physiological functions plays an important role in the maintenance of proper function of the body as a whole. However, advanced age is characterized by deterioration of many circadian rhythms (e.g., sleep/wake cycle, the core body temperature, performance, alertness, and secretion of many hormones, including melatonin), leading to disorganization of the temporal structure of the organism’s rhythmic physiology and behavior. Aging is often associated with earlier timing of endogenous circadian rhythmicity and reduced amplitude of many rhythms.14 It is known that the older people have a difficulty in adapting to shift work schedules and to quick time zone changes during transmeridian travel.27,28 The accumulated evidence indicate that melatonin treatment may improve adaptation to jet-lag and shift work.26 Changes in the neurons of the SCN seem to pay a crucial role in age-related deterioration in circadian clock function. As an effect of age-related changes in the SCN there is the desynchronization of overt rhythms that accompany aging due to a loss of control of these functions by the SCN.14,29 Interestingly, plasma melatonin is considered as one of the three markers (beside core body temperature and plasma cortisol) frequently used to estimate the phase of the human circadian pacemaker.30 It has been demonstrated that application of a variety of potent modulators of the circadian timing system, like bright light, melatonin, manipulation of body heat and physical activity caused improvement in sleep-wake rhythm of healthy and demented elderly people (for review see ref. 20). According to Armstrong and Redman hypothesis the stability of the circadian system correlates with the amplitude of melatonin secretion.31 The loss of melatonin in advanced age leads to disturbances in the circadian pacemaker, which causes internal temporal desynchronization which induces a variety of chronopathologies and leads to generalized deterioration of health. The authors suggest that melatonin has beneficial effects in terms of aging because of its association with circadian timing system.

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On the other hand, Rodenbeck and Hajak presented hypothesis that the diminished melatonin secretion in insomniacs seems to represent the result of very slowly developing neuroendocrine dysregulation caused by disturbed sleep.32 This hypothesis is based on the observations that the application of corticotropin releasing hormone (CRH) significantly reduce melatonin concentrations, and a decreased negative feedback inhibition in the hypothalamic-pituitary-adrenal axis with subsequently chronically increased CRH levels, in addition to the physiological decrease due to aging, may further reduce nocturnal melatonin secretion in subjects with chronic primary insomnia.32

Melatonin and Suppressed Immunocompetence in Advanced Age

Early studies by Maestroni et al7 and Pierpaoli et al8 have shown that giving melatonin in the drinking water at night increases life-span and maintains the mice in a more youthful state. These initial reports claimed that melatonin most likely prolonged survival and the youthful character of the animals because of the immunoenhancing actions. There are, indeed, numerous experimental data presented mostly by groups of Maestroni (for review see refs. 33-35) and Guerrero (for review see ref. 36) showing that melatonin exerts immunoenhancing action, both in animals and in humans. On the other hand, suppressed immunocompetence has been implicated in the acceleration of aging processes resulting in increased susceptibility to diseases.37-39 Recently, Pierpaoli and Lesnikov suggested that the pineal, due to its influence on the function of the neuroendocrine system monitors and regulates “self control” and the ability of the immune system to recognize and react against any endo- or exogenic factor.40 They suggest aging is a result of deterioration of this central role of the pineal gland. Although melatonin seems to be an important factor responsible for this function, other signals generated by the organ may also play a role.

Significance of Melatonin Secretion Decline for Reduced Antioxidant Protection in Advanced Age Free radicals, the highly reactive molecules that have an unpaired electron, continuously produced in cells as byproducts of oxidative phosphorylation and fatty acid oxidation, are destructive to intracellular and extracellular molecules.41,42 Free radical theory of aging formulated by Harman states that the deterioration of function in the elderly is in part related to the damage to subcellular constituents (DNA, RNA, proteins, carbohydrates, unsaturated lipids, etc.), cells and organs sustained as a consequence of their persistent bombardment by free radicals.43,44 This damage, with accumulates with age, has significance not only for aging per se but also for many age-related diseases, such as atherosclerosis, Alzheimer’s disease, Parkinson’s disease, senile cataract, and neoplastic disease.10,11,41,42 The contribution of free radicals to the development of degenerative and age-related diseases has been more or less clearly demonstrated.45 Although the question whether the balance between the amount of free radical formation and the activity of the antioxidative defense might influence the aging process in general seems to be still open, according to Biesalski, such balance is not only involved in but even triggers the process of aging of cells and tissues.45 The most recent theory suggesting a role for the pineal gland and melatonin in aging refers to the fact that melatonin is a very effective antioxidant and potent free radical scavenger.10,11,46,47 It scavengers both hydroxyl radicals and peroxyl radicals, although it is more efficient direct scavenger of the highly toxic hydroxyl radicals. Additionally, melatonin stimulates a number of antioxidative enzymes, e.g., glutathione peroxidase and glutathione reductase.47 It should be stressed that melatonin is the only antioxidant known to decrease substantially after middle age. This decrease strictly correlates with decrease in total antioxidant capacity of human serum with age.48

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Melatonin in Postmenopausal Women Although melatonin levels have been investigated in various groups of elderly persons the melatonin concentrations have been rarely studied in postmenopausal women. Decreased melatonin concentrations were demonstrated in postmenopausal women in comparison with premenopausal individuals.49 Age-related decline in nocturnal melatonin secretion in postmenopausal women was also demonstrated by Okatani et al50 Moreover, it has been shown that melatonin administration caused reduction in both diastolic and systolic blood pressure as well as in norepinephrine levels and decrease in blood flow in the internal carotid artery in postmenopausal women on hormone replacement therapy (HRT) but not in untreated postmenopausal individuals.51 These data show that the circulatory response to melatonin is conserved only in postmenopausal women on HRT and are in agreement with observations by Cagnacci group that biological response to melatonin is modulated by estrogens, and presumably also by progestagens.52,53 Moreover, reduced body temperature response to melatonin was also demonstrated in aged women.54

Melatonin and Age-Related Diseases Because of its antioxidant activity a role for melatonin in many age-related diseases has been suggested. This especially concerns neurodegenerative diseases (such as Alzheimer’s and Parkinson’s diseases) because of high vulnerability of the central nervous system to oxidative attack. Melatonin concentrations decrease in some, but not all, patients suffering from Alzheimer’s disease, the most common cause of progressive cognitive decline in the aged population.55,56 Although a statistically significant circadian rhythm of plasma melatonin concentrations was found by the population mean cosinor method in elderly subjects as well as in individuals with senile dementia, the rhythm amplitude was significantly lower in old (especially demented) subjects in comparison with young individuals.57 The experimental findings indicate that melatonin may act in a variety of ways to reduce neuronal loss in Alzheimer’s disease by altering the process of generation and action of amyloid-β, considered to be a factor causing cell death in this disease.47,58 Recent reports demonstrated that melatonin treatment seems to constitute a selection therapy to improve sleep, to ameliorate sundowning, and to slow evolution of cognitive impairment in Alzheimer’s patients.59,60 There are also experimental data that suggest a role of melatonin in another neurodegenerative disorder, Parkinson’s disease which is characterized by the progressive deterioration of dopamine-containing neurons in the pars compacta of the substantia nigra in the brain stem due to the oxidation of dopamine.61,62 There is evidence that melatonin may reduce dopamine auto-oxidation under experimental conditions.63 Using animal models which are a surrogate for Parkinson’s disease in humans it has been demonstrated that melatonin was able to overcome increased lipid peroxidation that occurred in the striatum, hippocampus, and midbrain after 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine injection, and to reduce the cytotoxicity 6-hydroxydopamine.64-66 Neoplastic disease is another example of age-related disease. Numerous experimental studies have shown the oncostatic action of melatonin [for review see refs. 67-69]. Moreover there are some clinical data suggesting that administration of melatonin (alone or in combination with inteleukin-2) is able to favorably influence the course of advanced malignant disease in humans (for review see refs. 70-72). Since endogenous melatonin concentrations fall markedly in advanced age, the implications of these findings is that the loss of this antioxidant may contribute to the incidence or severity of some age-associated diseases.

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Possible Supplementation of Melatonin in Elderly Individuals The generally accepted indications for therapeutic use of melatonin include sleep disorders, and circadian clock disturbances (e.g., jet-lag, phase-shifting of the circadian clock in blind people).73 However, considering a decrease in melatonin concentrations with age, keeping in mind its antioxidant action and beneficial effects on sleep as well as evidence of helpful effects in age-related diseases, recommendations of melatonin supplementation in advanced age should be considered.73 Melatonin administration may improve temporal organization in advanced age. It has been shown in clinical trials that melatonin administration may be beneficial in elderly, especially in terms of the quality of life improvement.74-75 Moreover, it should be stressed that melatonin treatment seems to be safe because of its remarkable low toxicity and absence of any significant side effects.76,78-80

Concluding Remarks Although aging process is multifactoral, and no single element seems to be of basic importance, on the basis of experimental and clinical data it seems reasonable to postulate a role for melatonin in this process. The age-related decline in melatonin secretion may have various consequences including sleep inefficiency, circadian rhythm dysregulation, depressed immune function, reduced antioxidant protection, and possibly others. However, the precise role of melatonin in the aging process remains to be determined, and melatonin presently can not be univocally recognized as a substance delaying aging. We should be aware that with increasing life expectancy (46.5 years in 1950-55, 66 years in 2000-05, 76 years projected in 2045-50) as well as increasing the median age of the world population, i.e., the age that divides the population into two equal halves (26 years in 1950, 27 years in 2000, 36 years projected in 2050), men and women will live one third of their life with some form of hormone deficiency.1-3 Therefore, this aspect of aging should also be considered. Supplementation of melatonin in the advanced age might be rational, as is widely used hormonal replacement therapy in women or suggested supplementation with dehydroepiandrosterone.

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17. Bergiannaki JD, Soldatos CR, Paparrigopoulos TJ et al. Low and high melatonin excretors among healthy individuals. J Pineal Res 1995; 18:159-164. 18. Cardinali DP, Brusco LI, Perez Lloret S et al. Melatonin in sleep disorders and jet lag. Neuroendocrinol Lett 2002; 23(suppl. 1):9-13. 19. Miles LE, Dement WC. Sleep and aging. Sleep 1980; 3:119-220. 20. Van Someren EJW. Circadian and sleep disturbances in the elderly. Exp Gerontol 2000; 35:1229-1237. 21. Baskett JJ, Wood PC, Broad JB et al. Melatonin in older people with age-related sleep maintenance problems: A comparison with age-matched normal sleepers. Sleep 2001; 24:418-424. 22. Mishima K, Okawa M, Shimizu T et al. Diminished melatonin secretion in the elderly caused by insufficient environmental illumination. J Clin Endocrinol Metab 2001; 86:129-134. 23. Youngstedt SD, Kripke DF, Elliot JA et al. Circadian abnormalities in older adults. J Pineal Res 2001; 31:264-272. 24. Zisapel N. The use of melatonin for the treatment of insomnia. Biol Signals Recept 1999; 8:84-89. 25. Monti JM, Cardinali DP. A critical assessment of the melatonin effects on sleep in humans. Biol Signals Recept 2000; 9:328-339. 26. Skene D, Lockley SW, Arendt J. Use of melatonin in the treatment of phase shift and sleep disorders. Adv Exp Med Biol 1999; 467:79-84. 27. Akerstedt T, Torsvall L. Age, sleep and adjustment to shiftwork. In: Koella WP, ed. Sleep. Basel: Karger, 1981:190-195. 28. Smolensky MH, Lee E, Mott D et al. A health profile of American flight attendants (FA). J Hum Ergol (Tokyo) 1982; 11(suppl):103-119. 29. Aujard F, Herzog ED, Block GD. Circadian rhythms in firing rate of individual suprachiasmatic nucleus neurons form adult to middle-aged mice. Neuroscience 2001; 106:255-261. 30. Klerman EB, Gershengorn HB, Duffy JF et al. Comparisons of the variability of three markers of the human circadian pacemaker. J Biol Rhytms 2002; 17:181-193. 31. Armstrong SM, Redman JR. Melatonin: A chronobiotic with anti-aging properties. Med Hypotheses 1991; 34:300-309. 32. Rodenbeck A, Hajak G. Neuroendocrine dysregulation in primary insomnia. Rev Neurol (Paris) 2001; 157:5S57-5S61. 33. Maestroni GJM. The neuroendocrine role of melatonin. J Pineal Res 1993; 14:1-10. 34. Maestroni GJM. MLT and the immune-hematopoietic system. Adv Exp Biol Med 1999; 460:395-405. 35. Maestroni GJM. The immunotherapeutic potential of melatonin. Exp Opin Invest Drugs 2001; 10:467-476. 36. Gurrero JM, Garcia-Maurino S, Pozo D et al. Mechanisms involved in the immunomodulatory effects of melatonin on the human immune system. In: Bartsch C, Bartsch H, Blask DE et al, eds. The Pineal Gland and Cancer. Berlin: Springer, 2001:408-416. 37. Ginaldi K, De Martinis M, D’Ostilio A et al. The immune system in the elderly: I. Specific humoral immunity. Immunol Res 1999; 20:101-108. 38. Ginaldi K, De Martinis M, D’Ostilio A et al. The immune system in the elderly: II. Specific cellular immunity. Immunol Res 1999; 20:109-115. 39. Ginaldi K, De Martinis M, D’Ostilio A et al. The immune system in the elderly: III. Innate immunity. Immunol Res 1999; 20:117-126. 40. Pierpaoli W, Lesnikov V. Theoretical considerations on the nature of the pineal ‘aging clock’. Gerontology 1997; 43:20-25. 41. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and degenerative diseases of aging. Proc Nat Acad Sci USA 1993; 90:7915-7922. 42. Beckman KB, Ames BN. Oxidative decay of DNA. J Biol Chem 1997; 272:19633-19636. 43. Harman D. Ageing: A theory based on free radical and radiation chemistry. J Gerontol 1956; 11:298-300. 44. Harman D. Free radical theory of aging. Mutat Res 1992; 275:257-266. 45. Biesalski HK. Free radical theory of aging. Curr Opin Clin Nutr Metab Care 2002; 5:5-10. 46. Reiter RJ, Poeggeler B, Tan DX et al. Antioxidant capacity of melatonin: A novel action not requiring a receptor. Neuroendocrinol Lett 1993; 15:103-116. 47. Reiter RJ. Oxidative damage in the central nervous system: Protection by melatonin. Progr Neurobiol 1998; 56:359-384. 48. Benot S, Goberna R, Reiter RJ et al. Physiological levels of melatonin contribute to the antioxidant capacity of human serum. J Pineal Res 1999; 27:59-64. 49. Vakkuri O, Kivela A, Leppaluoto J et al. Decrease in melatonin precedes follicle-stimulating hormone increase during perimenopase. Eur J Endocrinol 1996; 135:188-192.

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50. Okatani Y, Morioka N, Wakatsuki A. Changes in nocturnal melatonin secretion in perimenopausal women: Correlation with endogenous estrogen concentrations. J Pineal Res 2000; 28:111-118. 51. Cagnacci A, Arangino S, Angiolucci M et al. Different circulatory response to melatonin in postmenopasal women without and with hormone replacement therapy. J Pineal Res 2000; 29:152-158. 52. Cagancci A, Soladani R, Yen SSC. Melatonin enhances cortisol levels in aged women: Reversible by estrogens. J Pineal Res 1997; 22:81-85. 53. Cagnacci A, Soldani R, Laughlin G et al. Modification of circadian body temperature rhythm during the luteal menstual phase. Pole of melatonin. J Appl Physiol 1996; 80:25-29. 54. Cagnacci A, Soldani R, Yem SSC. Hypothermic effect of melatonin and nocturnal core body temperature are reduced in aged women. J Appl Physiol 1995; 78:314-317. 55. Morita Y, Uchida K, Okamoto N. Melatonin rhythm of Alzheimer patients. Front Horm Res 1996, 21:180-185. 56. Mishima K, Tozawa T, Satoh K et al. Melatonin secretion rhythm disorders in patients with senile dementia of Alzheimer’s type with disturbed sleep-waking. Biol Psychiatry 1999; 15:417-421. 57. Ferrari E, Arcaini A, Gornati R et al. Pineal and pituitary-adrenocortical function in physiological aging and in senile dementia. Exp Gerontol 2000; 35:1239-1250. 58. Pappolla MA, Chyan YJ, Poeggeler B et al. An assessment of the antioxidant and the antiamyloidogenic properties of melatonin: Implications for Alzheimer’s disease. J Neural Transm 2000; 107:203-231. 59. Brusco LI, Marquez M, Cardinali DP. Melatonin treatment stabilizes chronobiologic and cognitive symptoms in Alzheimer’s disease. Neuroendocrinol Lett 1998; 19:111-115. 60. Cardinali DP, Brusco LI, Liberczuk C et al. The use of melatonin in Anzheimer’s disease. Neuroendocrinol Lett 2002; 23(suppl. 1):20-23. 61. Fearnley JM, Less AJ. Aging and Parkinson’s disease: Substantia nigra regional selectivity. Brain 1991; 114:2283-2301. 62. Fahn S, Cohen G. The oxidant stress hypothesis in Parkinson’s disease: Evidence supporting it. Ann Neurobiol 1991; 32:804-812. 63. Miller JW, Selhub J, Joseph JA. Oxidative damage caused by free radicals produced during catocholamine autooxidation: Protective effects of O-methylation and melatonin. Free Radical Biol Med 1996; 21:241-249. 64. Acuna-Castroviejo D, Coto-Montes A, Monti MG et al. Melatonin is protective against MPTP-induced striatal and hyppocampal lesions. Life Sci 1997; 60:PL23-29. 65. Mayo JC, Sainz RM, Uria H et al. Melatonin prevents apoptosis induced by 6-hydroxydopamine in neuronal cells: Implications for Parkinson’s disease. J Pineal Res 1998; 24:179-192. 66. Mayo JC, Sainz RM, Uria H et al. Inhibition of cell proliferation: A mechanism likely to mediate prevention of neuronal death by melatonin. J Pineal Res 1998; 25:12-18. 67. Blask DE. Melatonin in oncology. In: Hu HS, Reiter RJ eds. Melatonin – Biosynthesis, Physiological Effects and Perspectives. Boca Raton: CRC Press; 1993:447-475. 68. Karasek M, Pawlikowski M. Pineal gland, melatonin and cancer. Neuroendocrinol Lett 1999; 20:139-144. 69. Pawlikowski M, Winczyk K, Karasek M. Oncostatic action of melatonin: Facts and question marks. Neuroendocrinol Lett 2002; 23(suppl. 1):24-29. 70. Lissoni P. Efficacy of melatonin in the immunotherapy of cancer using interleukin-2. In: Bartsch C, Bartsch H, Blask DE et al, eds. The Pineal Gland and Cancer. Berlin: Springer, 2001:465-475. 71. Hrushesky WJM. Melatonin cancer therapy. In: Bartsch C, Bartsch H, Blask DE et al, eds. The Pineal Gland and Cancer. Berlin: Springer, 2001:476-508. 72. Bartsch C, Bartsch H, Karasek M. Melatonin in clinical oncology. Neuroendocrinol Lett 2002; 23(suppl.1):30-38. 73. Karasek M, Reiter RJ, Cardinali DP et al. Future of melatonin as a therapeutic agent. Neuroendocrinol Lett 2002; 23(suppl. 1):118-121. 74. Siegrist C, Benedetti C, Orlando A et al. Lack of changes in serum prolactin, FSH, TSH, and estradiol after melatonin treatment in doses that improve sleep and reduce benzodiazepine consumption in sleep-disturbed, middle aged, and elderly patients. J Pineal Res 2001; 30:34-42. 75. Karasek M, Kuzdak K, Cywinski J et al. Effects of melatonin administration in advanced breast cancer patients – preliminary study. Neuroendocrinol Lett 1998; 19:15-19. 76. Pawlikowski M, Kolomecka M, Wojtczak A et al. Effects of six months melatonin treatment on sleep quality and serum concentrations of estradiol, cortisol, dehydroepiandrosterone sulfate, and somatomedin C in elderly women. Neuroendocrinol Lett 2002; 23(suppl. 1):17-19. 77. Avery D, Lenz M, Landis C. Guidelines for prescribing melatonin. Ann Med 1998; 30:122-130. 78. Seabra MLV, Bignotto M, Pinto LR et al. Randomized, double-blind clinical trial, controlled with placebo, of the toxicology of chronic melatonin treatment. J Pineal Res 2000; 29:193-200.

CHAPTER 11

Role of Endogenous and Exogenous Melatonin in Inflammation Salvatore Cuzzocrea

Abstract

A

vast number of experimental and clinical studies implicates oxygen-derived free radicals (especially, superoxide and hydroxyl radical) and high energy oxidants (such as peroxynitrite) as mediators of acute and chronic inflammation. The purpose of this review is to describe the role of endogenous and exogenous melatonin in inflammation. Reactive oxygen species can modulate a wide range of toxic oxidative reactions. These include initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3phosphate dehydrogenase, inhibition of membrane sodium/ potassium ATP-ase activity, inactivation of membrane sodium channels, and other oxidative modifications of proteins. Reactive oxygen species (e.g., superoxide, peroxynitrite, hydroxyl radical and hydrogen peroxide) are all potential reactants capable of initiating DNA single strand breakage, with subsequent activation of the nuclear enzyme poly (ADP ribose) synthetase (PARS), leading to eventual severe energy depletion of the cells, and necrotic-type cell death. All these toxicities are likely to play a role in the pathophysiology of inflammation. Melatonin has been shown to posses in vitro an important antioxidant capacity as well as to inhibits the activation of poly (ADP ribose) synthetase. Therefore a large number of experimental studies have demonstrated that melatonin may exert an important anti-inflammatory action.

Introduction Oxygen Radical Generation in Inflammation Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants and/ or a depletion of antioxidants. Oxidative stress is thought to play an important role in the pathogenesis of a number of inflammatory diseases, not only through direct injurious effects, but by involvement in the molecular mechanisms that control inflammation. The free radical nitric oxide (NO) is synthesized from the guanidino group of L-arginine by a family of enzymes termed NO synthases (NOS). Three isoforms have been described and cloned: endothelial cell NOS (ecNOS, or type 3), brain NOS (bNOS, nNOS, or type 1), and inducible macrophage type NOS (iNOS, or type 2). The cytotoxic effects of NO (in high local concentrations) involve the inhibition of key mitochondrial Fe-S enzymes, including NADH:ubiquinone oxidoreductase, NADH:succinate oxidoreductase, and aconitase. 1 cGMP-independent activation by NO of other enzymes, such as cyclooxygenase, has also been described. This action may be related to the reaction of NO with the iron-heme center at the active site of the enzyme.2 Administration of NOS inhibitors reduces blood flow to most

Melatonin: Biological Basis of Its Function in Health and Disease, edited by S.R. Pandi-Perumal and Daniel Cardinali. ©2006 Eurekah.com.

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organs. Many inflammatory conditions are associated with production of comparatively large amounts of NO, produced by iNOS, with consequent cytotoxic effects. iNOS, first identified in macrophages, can be expressed in essentially any cell type. Although constitutive expression of iNOS has been localized to the kidney, the intestine, and the bronchial epithelia, iNOS is expressed typically in response to immunological stimuli and produces nanomoles, rather than picomoles, of NO. Once produced in high local concentrations, NO may act as a cytostatic and cytotoxic molecule for fungal, bacterial, helminthic, and protozoal organisms as well as tumor cells. Induction of iNOS can be inhibited by numerous agents, including glucocorticoids, thrombin, macrophage deactivation factor, tumor growth factor beta, platelet-derived growth factor, IL-4, IL-8, IL-10, and IL-13. Induction of iNOS may have either toxic or protective effects. Factors that appear to dictate the consequences of iNOS expression include the type of insult, the tissue type, the level and duration of iNOS expression, and probably the redox status of the tissue. Much attention has focused on the toxicity of iNOS. For example, induction of iNOS in endothelial cells produces endothelial injury.3 Induction of iNOS has been shown to inhibit cellular respiration in macrophages and vascular smooth muscle cells; these processes can lead to cell dysfunction and cell death. The notion that acute and chronic inflammation is associated with overproduction of NO is hardly novel: enhanced formation of NO by the measurement of evaluation of iNOS expression was demonstrated in lung from rats subjected to experimental pleurisy or in the joint of arthritic rats.4 Therefore NO formation was also demonstrated in serum and synovial fluid samples from patients with rheumatoid arthritis (RA) and osteoarthritis (OA).5 Using iNOS inhibitors or iNOS knockout mice it has been experimentally demonstrated that NO participated as a pro-inflammatory signalling in the activation of the inflammatory cascades characterized by increased cytokine production, leukocyte adhesion molecule expression, and neutrophil infiltration into tissues during acute or chronic inflammation.6-8 As indicated above, sources of NO production during inflammation include both ecNOS and iNOS. Oxygen radicals are produced in abundance during inflammatory process. Sources of superoxide include xanthine oxidase and NADPH oxidase, as well as various metabolic and signalling pathways. Simultaneous generation of nitric oxide and superoxide favors the production of a toxic reaction product, peroxynitrite anion.9 It is important to point out that, under certain conditions, NOS can produce both precursors of peroxynitrite (NO and superoxide). Such conditions cannot be found under normal circumstances, but can occur during L-arginine depletion. Low levels of arginine might be expected following resuscitation with crystalline solutions.10 Under low cellular arginine concentrations, NOS produces both NO and superoxide, and the resulting generation of peroxynitrite can contribute to cytotoxicity. This mechanism has been confirmed in neuronal cultures, as well as in macrophages that express iNOS.11 Small amounts of peroxynitrite are produced under basal physiological conditions, since, most cells are exposed to low levels of NO due to constitutive NO production, and also superoxide from mitochondria and other cellular sources are always produced.12 It is probable that the endogenous antioxidant systems are sufficient to neutralize such low-level peroxynitrite production, which is, therefore, not cytotoxic. It may be important to note that, although peroxynitrite is generally considered as a cytotoxic molecule, peroxynitrite in low concentrations, in the presence of intact antioxidant systems, has been proposed to mediate physiological effects. For instance, a low concentration of peroxynitrite has been shown to inhibit neutrophil adhesion.13 Under these conditions, peroxynitrite is likely to form NO adducts with glucose, thiols, and other species14 which, in turn, can act as NO donors, activating guanylyl cyclase.14 Currently, little information is available regarding these “physiological” roles of peroxynitrite, while the evidence for the roles of peroxynitrite in pathophysiological conditions is expanding. Although there are a number of experimental difficulties related to delineation of the actual role of peroxynitrite in acute and chronic inflammatory conditions, theoretical considerations strongly favor the production of peroxynitrite when NO and superoxide are produced simultaneously, because the reaction of these two species is nearly diffusion-controlled. In fact, the reaction of superoxide with NO is the only reaction that outcompetes the reaction

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of superoxide with superoxide dismutase.9 Although chemical considerations favor the production of peroxynitrite, the actual demonstration of the presence or production of peroxynitrite in pathophysiological conditions is far from straightforward. The finding that peroxynitrite is produced during inflammation is not surprising, in light of the previous evidence for the overproduction of oxygenderived free radicals. The formation of nitrotyrosine staining as an indication of “increased nitrosative stress,” and peroxynitrite formation has recently been demonstrated in the lung of rats subjected to pleurisy and in the joint of rats subjected to arthritis.15 Thus, multiple lines of evidence strongly suggest that peroxynitrite is produced in inflammation. Specific peroxynitrite scavengers that could help further delineating the role of peroxynitrite in inflammation. Therefore, the evidence implicating the role of peroxynitrite in a given pathophysiological condition can only be indirect. However, recently Salvemini et al, using a specific class of peroxynitrite decomposition caytalyst have demonstrated that peroxynitrite play a role in acute inflammation.16 Therefore, it is likely that additional interactions of oxygen- and nitrogen-derived free radicals also contribute to the inflammatory cell injury. Peroxynitrite induces the oxidation of sulfhydryl groups and thioethers and the nitration and hydroxylation of aromatic compounds, such as tyrosine, tryptophan, and guanine. These reactions, when occurring during the reaction of peroxynitrite with various enzymes of the cell, can markedly suppress the catalytic activity of these enzymes. For instance, peroxynitrite has been shown to inhibit manganese superoxide dismutase, tyrosine hydroxylase, membrane sodium/potassium ATP ase, membrane sodium channels mitochondrial and cytosolic aconitase, and a number of critical enzymes in the mitochondrial respiratory chain, as well as NOS.9 While peroxynitrite inactivates many enzymes, the catalytic activity of some enzymes is actually enhanced by peroxynitrite a primary example being cyclooxygenase. In addition to the interactions of peroxynitrite with proteins, an important interaction of peroxynitrite occurs with nucleic acids. Two main types of reactions have been described: DNA base modifications and DNA single strand breakage. In addition to direct cytotoxic effects, an indirect pathway of peroxynitrite-induced cellular injury has also been proposed. The generation of peroxynitrite, either intracellularly or extracellularly, has been shown to trigger DNA single strand breakage and activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS). The PARS pathawy activation generally lead to cell death via the necrotic pathway. This is the pathway affected by pharmacological inhibitors of PARS.17 On the other hand, peroxynitrite can also lead to cell death via the apoptotic pathway. PARS, however, does not play a role in this latter process, since inhibition of PARS does not appear to prevent peroxynitrite-induced apoptosis.18 Recent investigations tested the effects of pharmacological inhibitors of PARS in rodent model of arthritis. In a mice model of potassium peroxochromate-induced arthritis, the mice treated with the PARS inhibitor nicotinamide showed a significant reduction of acute and chronic inflammation.19 A number of recent observations suggest that PARS activation plays a role in the oxidant injury in various forms of inflammation. In fact PARS activation was found in the lung of rats subjected to pleurisy and in the joint of arthritic rats. The blockade of neutrophil recruitment associated with PARS inhibition, coupled with a direct cytoprotective effect of PARS inhibition against oxidant injury20 and may explain the antiinflammatory effects seen with inhibition of PARS. Furthermore, recent studies have demonstrated the PARS-/- mice developed less arthritis when compared with PARS+/+ mice.21 Based on the these studies, it has been propose that inhibition of PARS represents a novel strategy for anti-inflammatory therapy under conditions of oxidant stress.

Relative Importance of Endogenous Melatonin in Acute Inflammation We have recently suggest that endogenous melatonin plays a crucial role as a protective factor against the carregeenan-induced development of acute inflammation.22 Although there are data that depletion of endogenous antioxidant mechanisms can increase mortality in various forms of shock23,24 and can exacerbate ischemia/reperfusion injury in some25 but not other26

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experimental models. What, then, is the mechanism by which endogenous melatonin reduced acute inflammation?

Effect of in Vivo Depletion of Endogenous Melatonin Synthesis on Nitrotyrosine Formation in Carrageenan-Induced Acute Inflammation Reactive oxidants such as hydrogen peroxide, superoxide and hydroxyl radical contributes to tissue damage in inflammation.27-30 Pharmacological inhibitors of NOS have been shown to reduce the development of carrageenan-induced inflammation and support a role of NO in this model of inflammation.27-29,31 More recent studies have shown the formation of peroxynitrite in carrageenan-induced inflammation.27-29,32 Using nitrotyrosine immunohistochemistry, we have confirmed the production of peroxynitrite in the lung of rats subjected to carrageenan-induced pleurisy.22 Moreover, we have observed that in the animals depleted of melatonin, a much more pronounced nitrotyrosine staining was present, suggesting the presence of more, biologically active peroxynitrite in the alveolar macrophage and in the airway epithelial cells. The more pronounced nitrotyrosine staining was not due to increased production of NO, as demonstrated by the measurement of lung iNOS activity.22

Endogenous Melatonin Protects against Pleural Macrophages Dysfunction It is well known that in acute inflammatory process, in which vascular permeability increases and leukocyte migration occurs, there is an involvement of several mediators including neutrophil-derived free radicals, such as hydrogen peroxide, superoxide and hydroxyl radical.27,28 It is proposed that reactive oxygen species, including oxygen radicals, and nonradicals that are either oxidising agents and/or are easily converted into radicals, such as HOCl, ozone, peroxynitrite, single oxygen and H2O2 can cause structural alteration in DNA33 with consequent cellular dysfunction.34 In ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy, we have recently reported the production of NO, superoxide and peroxynitrite, concomitant with inhibition of suppression of mitochondrial respiration, DNA single strand breakage, NAD depletion, and ATP depletion.35 Using pharmacological inhibitors and scavengers, it appears that the most important cytotoxic species under these conditions is peroxynitrite and not NO or superoxide per se. This conclusion is based on the simultaneous protective effects of NOS inhibitors27-29 and a cell-permeable superoxide dismutase scavenger compound36 against the suppression of mitochondrial respiration, and by the protective effects of various peroxynitrite scavengers.27,29 Although a variety of endogenous antioxidant systems in the cell are actively involved during the inflammatory process, it is remarkable that depletion of melatonin alone exerted a marked potentiating effect of peroxynitrite-induced cytotoxicity. These findings are in agreement with recent suggestions that endogenous antioxidant systems play an important role against the oxidant-induced injury and, specifically, against the peroxynitrite-induced injury.36,37 Several data support this hypothesis: (1) the enhancement of the appearance of DNA strand breaks (2) the demonstration of a further decrease in the conversion of MTT to formazan (3) the partially enhanced reduction of the intracellular levels of NAD+. A variety of additive or synergistic cytotoxic processes triggered by peroxynitrite may contribute to acute and delayed cytotoxicity and depletion of melatonin may interfere also with these pathways.

Role of Melatonin on NO, Oxyradicals and Peroxynitrite Formation in Carrageenan-Induced Acute Inflammation

Melatonin is a known effective scavenger of the hydroxyl radical and the peroxyl radical38,39 and it may stimulate some important antioxidative enzymes such as superoxide dismutase, glutathione peroxidase and glutathione reductase.38 Melatonin also acts as a peroxynitrite scavenger and protects cultured cells against peroxynitrite-induced injury.40 Thus, theoretically, the mechanism of the observed inflammatory alterations in the melatonin depleted animals may be related to peroxynitrite, oxyradicals, NO, or the combination of these.

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In vitro studies in macrophages and other cell types have established that endogenous antioxidants (such as glutathione) only protect against very large amount of NO, but not against lower levels of NO production,41,42 such as the ones which are relevant to the ex vivo or in vivo conditions in our experiments. In our experiments it is conceivable that a more pronounced inhibition of mitochondrial respiration by oxygen-derived free radicals and oxidants can lead to a dysfunctional electron transfer, with more superoxide production from the mitochondria. This effect would also lead to an enhancement of peroxynitrite production, with subsequent increased cytotoxicity. It is noteworthy in this context, that the production of superoxide, not the production of NO, is the rate-limiting factor in peroxynitrite formation during endotoxemia.41 Furthermore, hydrogen peroxide prolongs the half-life of peroxynitrite.43 In addition, recent reports have shown that nitrotyrosine formation may results also from reaction between nitrite and myeloperoxidase.44 Thus, it is possible that the cytotoxic effects observed in response to carrageenan represent the sum of a complex interaction between various oxygen- and nitrogen-derived radicals and oxidants. In conclusion, endogenous melatonin plays an important role against the acute inflammation.

Melatonin Is Effective in Experimental Inflammation Zymosan and Carrageenan Induced Acute Inflammation Recent studies have clearly demonstrated the role of ROS and PARS activation in various forms of local or systemic inflammation induced by the prototypical inflammatory stimuli zymosan and carrageenan. In these experimental condition have been used to test the anti-inflammatory activity of various agent such us new NSAIDs,45 anti-oxidants,46 PARS inhibitors and other new moleculre including melatonin. In this regard in carrageenan-induced paw edema it has been demonstrated that melatonin reduced paw swelling and inhibited the infiltration of neutrophils into the inflamed paw.47 Furthermore, in a model of acute local inflammation (carrageenan-induced pleurisy) melatonin (given at 25 and 50 mg/kg) inhibits the inflammatory response (pleural exudate formation, mononuclear cell infiltration, histological injury).47,48 Similar to the pharmacological effect of melatonin in local inflammation, it has been also demonstrated that melatonin was able to reduced the zymosan-induced inflammation and multiple organ failure.47 Melatonin also reduced the formation of nitrotyrosine in the inflamed tissues.47 Using nitrotyrosine as a marker for the presence of ONOO- has been challenged by the demonstration that other reactions can also induce tyrosine nitration; e.g., the reaction of nitrite with hypochlorous acid and the reaction of myeloperoxidase with hydrogen peroxide can lead to the formation of nitrotyrosine.49 Thus, increased nitrotyrosine staining is considered, as an indicator of “increased nitrosative stress” rather than a specific marker of the generation of peroxynitrite.49 We have found that nitrotyrosine is indeed present in lung sections taken after carrageenan injection or in lungs from zymosan-treated rats and that melatonin reduced the staining in these tissues. Therefore, macrophages harvested from pleural cavity from carrageenan-treated rats or from peritoneal cavity from zymosan-treated rats generated substantial amounts of ONOO- and this was significantly reduced by melatonin treatment. ROS can also cause DNA single-strand damage which is the obligatory trigger for PARS activation50,51 resulting in the depletion of its substrate NAD+ in vitro and a reduction in the rate of glycolysis. Since NAD+ functions as a cofactor in glycolysis and the tricarboxylic acid cycle, NAD+ depletion leads to a rapid fall in intracellular ATP and, ultimately, cell injury.20 Furthermore, substantial evidence exists to support the fact that PARS activation is important in inflammation.51 Melatonin reduced PARS activation and attenuated the reduction of NAD+. The overall effect of melatonin was a significant protection of cellular viability. In light of the role of PARS in inflammation, it is possible that PARS inhibition by melatonin accounts for its anti-inflammatory response.

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In addition to the reduction of ROS production and PARS activation, melatonin also reduced the development of oedema, neutrophil accumulation and lipid peroxidation and had an overall protective effect on the degree of organ injury as assessed by histological examination.52 A possible mechanism by which melatonin attenuates PMNs infiltration is by down-regulating adhesion molecules ICAM-1 and P-selectin.53 ROS also plays a role in the regulation of cytokine release since melatonin inhibited the release of the pro-inflammatory cytokines, TNFα and IL-1β in acute inflammation and post ischemia-reperfusion injury.53,54 This results is substantiated by a recent report which demonstrates that ROS increase TNFα release from macrophages.55 Local and systemic inflammation is associated with the induction of the expression of iNOS and COX-2 that in turn release large amounts of pro-inflammatory NOx and PGs.55-57 Recent studies have demonstrated that melatonin inhibits NO production,58 and reduces the expression of iNOS in the lung after carrageenan-induced pleurisy.47 This reduction in iNOS is to be related to the known inhibitory effect of melatonin on the activation of the NFκB,59-61 since this transcription factor is involved in the process of iNOS expression.1,62 In addition, in vitro studies have demonstrated that melatonin reduces 6-keto-prostaglandin-Fα production in cultured J774 and RAW.264.7 macrophages activated by lipopolysaccharide.61 Suppression of cyclooxygenase-2 expression by melatonin has been also demonstrated in carrageenan-induced pleurisy.63 The results of our study using the carrageenan or zymosan -induced inflammation cleary indicate that melatonin is anti-inflammatory agent.

Inflammatory Bowel Disease It is well established that inflammatory bowel disease is associated with the production of oxygen-derived free radicals and oxidants.64-67 Increased NO production from the inducible NO synthase has also been proposed to be responsible for various experimental models of inflammatory bowel disease,68-75 and ulcerative colitis in humans, where inducible NO synthase activity and elevated levels of luminal nitrite have been detected in rectal dialysates and in biopsy specimens.76-79 During inflammatory bowel disease, the simultaneous production of superoxide and NO is likely to produce peroxynitrite and to promote oxidative reactions. Biochemical evidence for the formation of peroxynitrite has been provided in an experimental model of ileitis in guinea pigs by immunohistochernical staining of nitrotyrosine in epithelial cells.80 Similarly, in human samples of active Crohn’s lesions, massive nitrotyrosine staining has been reported.81 The role of peroxynitrite in the pathogenesis of is further supported by the fact that intracolonic administration of exogenous peroxynitrite produces a severe mucosal damage in rats.82 Nitrotyrosine formation was found mostly localised on epithelial cells and in the area of infiltrated inflammatory cells in the colon from DNBS or TNBS-treated rats83-86 and in active Crohn’s lesions in humans.87 Recently it has been demonstrated that melatonin treatment significantly reduced the nitrotyrosine staining as well as the formation of tissue malondialdehyde.47 Furthermore, melatonin-treated rats are more resistant to DNBS induced lethal disease with a significant resolution of the macroscopic and histological signs of the inflammatory process. In addition melatonin treatment significantly reduced also poly (ADP-ribose) synthetase immunofluorescence.47 Recent studies have demonstrated that TNF-α and IL-1β play a role in the pathogenesis of experimental colitis has been obtained in animal models in which blocking of the action of these cytokines has been shown to delay the onset of experimental colitis, suppress inflammation, and ameliorate colon destruction that corresponds to the anti-inflammatory response.88,89 ROS have been shown to release cytokines such as TNFα although the precise mechanisms need to be elucidated.55 Consistent with this, the increase in TNF-α and IL-1β in the colon from DNBS-treated rats were significantly reduced by melatonin treatment may well contribute to the overall benefical effect observed. Recent studies have also point out an important role for neutrophils in the development and full manifestation of gastrointestinal inflammation, as they represent a major source of free radicals in the inflamed colonic mucosa.90,91 Melatonin administration exert a remarkable

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Figure 1. Proposed scheme of some of the delayed inflammatory pathways involving nitric oxide (NO), hydroxyl radical (OH) and peroxynitrite (ONOO-) in inflammation and potential sites of melatonin’s anti-inflammatory actions. Inflammation triggers the expression of the inducible NO synthase (iNOS), at least in part, via activation of nuclear factor κB (NF-κB). NO, in turn, combines with superoxide to yield ONOO-. OH. (produced from superoxide via the iron-catalyzed Haber-Weiss reaction) and ONOO- or peroxynitrous acid (ONOOH) induce cellular injury. Part of the injury is related to the development of DNA single strand breakage, with subsequent activation of poly (ADP-ribose) synthase, leading to cellular dysfunction. NO can directly increase the catalytic activity of the inducible isoform of cyclooxygenase (COX-2), leading to enhanced production of pro-inflammatory prostaglandin metabolites. In this system, melatonin’s anti-inflammatory effects may include 1) inhibition of the activation of NF-kB and prevention of the expression of iNOS, 2) direct inhibition of the catalytic activity of NOS; 3) OH scavenging, 4) ONOO- scavenging, 5) prevention of adhesion molecules expression and 6) specific effects related to activation of melatonin receptors.

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recovery of the mucosal morphology associated with a reduction in oxidative and nitrosative damage after DNBS administration and that in melatonin-treated rats, infiltration of polymorphonuclear neutrophils was significant reduced in tissue. Furthermore, ICAM-1 and P-selectin were expressed in endothelial and epithelial cells, and neutrophils in the distal colon in DNBS-treated rats. Thus, in contrast melatonin-treated rats had a significant reduction of ICAM-1 and P-selection expression leading to a reduction of neutrophil infiltration.

Conclusion Understanding the signal transduction mechanisms used by free radicals to modify the course of disease will undoubtedly elucidate important molecular targets for future pharmacological intervention. One question that remains to be answered is the mechanism by which melatonin protects against the inflammatory injury? There are a number of sites where melatonin may interfere with the inflammatory process (Fig. 1): (1) melatonin inhibits NO production, and reduces the expression of iNOS;63,65 (2) melatonin influences the activation of the transcription factor NF-βB59-61 and (3) melatonin reduces the expression of iNOS at the transcriptional level.58,60,92,93 These findings are consistent with a proposed novel mechanism for melatonin’s anti-inflammatory effect. Recently, prostaglandin (PG) levels in the exudate and cyclooxygenase-2 expression from carrageenan-treated rats were found to be completely inhibited by melatonin.63 This inhibitory response is likely related to a regulatory effect on gene expression as suggested by Gilad et al.60 Finally, we have also found that melatonin attenuates the formation of ONOO- and the increase in poly (ADP-ribose) synthase activity. In addition, melatonin inhibits the formation of P-selectin and ICAM-1 which in turn may contribute to the reduced recruitment of PMNs. We conclude that the observed anti-inflammatory effects of melatonin may be dependent upon a combination of the following pharmacological properties of this agent: (1) Melatonin secondarily scavenges and inactivates O2-. which reduced the formation of ONOO-. This, in turn, prevents the activation of in poly (ADP-ribose) synthase and the associated tissue injury. (2) At the same time, melatonin lowers the synthesis of NO thereby also reducing ONOO- formation. (3) In addition to O2-. melatonin also scavenges other radical oxygen species including. OH. (4) Melatonin additionally scavenges ONOO-. (5). Finally, melatonin reduces the recruitment of polymorphonucleates into the inflammatory site. This effect of melatonin is very likely secondary to the reduction endothelial oxidant injury and, hence, a preservation of endothelial barrier function. These results support the view that the over-production of reactive oxygen or nitrogen species contributes to the acute inflammatory response and we propose that small molecules, such as melatonin which permeate biological membranes and function as intracellular radical scavengers, may be useful in the therapy of conditions associated with local or systemic inflammation.

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35. Cuzzocrea S, Zingarelli B, Caputi AP. Peroxynitrite-mediated DNA strand breakage activates poly (ADP-ribose) synthetase and causes cellular energy depletion in carrageenan-induced pleurisy. Immunology 1998; 93:96-101. 36. Cuzzocrea S, Zingarelli B, Costantino G et al. Beneficial effects of Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimetic, in carrageenan-pleurisy. Free Radic Biol Med 1999; 26: 25-33. 37. Karoui H, Hogg N, Frejaville C et al. Characterization of sulfur-centered radical intermediates formed during the oxidation of thiols and sulfite by peroxynitrite. ESR-spin trapping and oxygen uptake studies. J Biol Chem 1996; 271:6000-6009. 38. Reiter RJ. The role of the neurohormone melatonin as a buffer against macromolecular oxidative damage. Neurochem Int 1995; 27:453-460. 39. Pieri C, Marra M, Moroni F et al. Melatonin: A peroxyl radical scavenger more effective than vitamin E. Life Sci 1994; 55:271-277. 40. Gilad E, Cuzzocrea S, Zingarelli B et al. Melatonin is a scavenger of peroxinitrite. Life Sci 1997; 60:PL 169-174. 41. Walker MW, Kinter MT, Roberts RJ et al. Nitric oxide-induced cytotoxicity: Involvement of cellular resistance to oxidative stress and the role of glutathione in protection. Ped Res 1995; 37:41-49. 42. Petit JF, Nicaise M, Lepoivre M et al. Protection by gluthatione against the antiproliferative effects of nitric oxide. Dependence on kinetics of no release. Biochem Pharmacol 1996; 52:205-212. 43. Alvarez B, Denicola A, Radi R. Reaction between peroxynitrite and hydrogen peroxide: Formation of oxygen and slowing of peroxynitrite decomposition. Chem Res Toxicol 1995; 8:859-864. 44. Eiserich JP, Hristova M, Cross CE et al. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 1998; 391:393-397. 45. Cuzzocrea S, Zingarelli B, Costantino G et al. Protective effect of melatonin in a nonseptic shock model induced by zymosan in the rat. J Pineal Res 1998; 25:24-33. 46. Cuzzocrea S, Riley DP, Caputi AP et al. Antioxidant therapy: A new pharmacological approach in shock, inflammation and ischemia-reperfusion injury. Pharm Rev 2001; 53:135-159. 47. Cuzzocrea S, Zingarelli B, Gilard E et al. Protective effect of melatonin in carrageenan-induced models of local inflammation. J Pineal Res 1997; 23:106-116. 48. Dugo L, Serraino I, Fulia F et al. Effect of melatonin on cellular energy depletion mediated by peroxynitrite and poly (ADP-ribose) synthetase activation in an acute model of inflammation. J Pineal Res 2001; 31:76-84. 49. Eiserich JP, Patel RP, O’Donnell VB. Pathophysiology of nitric oxide and related species: Free radical reactions and modification of biomolecules. Mol. Aspects Med 1998; 19:221-357. 50. Salgo MG, Bermudez E, Squadrito G et al. DNA damage and oxidation of thiols peroxynitrite causes in rat thymocytes. Arch Biochem Biophys 1995; 322:500-505. 51. Szabo C. Role of poly(ADP-ribose)synthetase in inflammation. Eur J Pharmacol 1998; 350:1-19. 52. Cuzzocrea S, Reiter RJ. Pharmacological action of melatonin in shock, inflammation and ischemia/ reperfusion injury. Eur J Pharmacol 2001; 426:1-10. 53. Cuzzocrea S, Costantino G, Mazzon E et al. Beneficial effects of melatonin in a rat model of splanchnic artery occlusion and reperfusion. J Pineal Res 2000; 28:52-63. 54. Cuzzocrea S, Mazzon E, Serraino I et al. Melatonin reduces dinitrobenzene sulfonic acid-induced colitis. J Pineal Res 2001; 30:1-12. 55. Volk T, Gerst J, Faust-Belbe G et al. Monocyte stimulation by reactive oxygen species: Role of superoxide and intracellular Ca2+. Inflamm Res 1999; 48:544-549. 56. Salvemini D. Nitric Oxide regulation of eicosanoid production. Nitric Oxide, Basic Research and Clinical Applications. Gryglewski RJ, Minuz P, eds. IOS Press, 2001:59-76. 57. Tomlinson A, Appleton I, Moore AR et al. Cyclo-oxygenase and nitric oxide synthase isoforms in rat carrageenin-induced pleurisy. Br J Pharmacol 1994; 113:693-698. 58. Pozo D, Reiter RJ, Calvo JR et al. Physiological concentrations of melatonin inhibit nitric oxide synthase in rat cerebellum. Life Sci 1994; 55:PL455-460. 59. Mohan N, Sadeghi K, Reiter RJ et al. The neurohormone melatonin inhibits cytokine, mitogen and ionizing radiation induced NF-kappa B. Biochem Mol Biol Int 1995; 37:1063-1070. 60. Gilad E, Wong HR, Zingarelli B et al. Melatonin inhibits expression of the inducible isoform of nitric oxide synthase in murine macrophages: Role of inhibition of NFkappaB activation. FASEB J 1998; 12:685-693. 61. Lezovalch F, Sparapani M, Behl C. N-acetil-serotonin (normelatonin) and melatonin in protect neurons against oxidative challenges and suppresses the activity of the trascription factor NF?B. J Pineal Res 1998; 24:168-178. 62. Salzman AL, Denenberg AG, Ueta I et al. Characterization of the induction and activity of the human nitric oxide synthase in a transformed intestinal epithelial cell line. Am J Physiol 1996; 270:G565-573.

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63. Cuzzocrea S, Costantino G, Mazzon E et al. Regulation of prostaglandin production in carrageenan-induced pleurisy by melatonin. J Pineal Res 1999; 27:9-14. 64. Allgayer H. Clinical relevance of oxygen radicals in inflammatory bowel disease- facts and fashin. Klin Wochenschr 1991; 69:1001-1003. 65. Babbs CF. Oxygen radicals in ulcerative colitis. Free Radic Biol Med 1992; 13:169-181. 66. Keshavarzian A, Haydek J, Zabihi R et al. Agents capable of eliminating reactive oxygen species. Catalase, WR-2721, or Cu(II)2(3,5-DIPS)4 decrease experimental colitis. Dig Dis Sci 1992; 37:1866-1873. 67. Chamulitrat W, Spitzer JJ. Generation of nitro and superoxide radicals anions from 2,4,6trinitrobenzenesulfonic acid by rats gastrointestinal cells. Biochim Biophys Acta 1997; 1336:73-82. 68. Yamada T, Sartor RB, Marshall S et al. Mucosal injury and inflammation in a model of chronic granulomatous colitis in rats. Gastroenterology 1993; 106:759-771. 69. Miller MJS, Sadowska-Krowicka H, Chotinaruemol S et al. Amelioration of chronic ileitis by nitric oxide inhibition. J Pharmacol Exp Ther 1993; 264:11-16. 70. Aiko S, Grisham MB. Spontaneous intestinal inflammation and nitric oxide metabolism in HLA-B27 transgenic rats. Gastroenterology 1995;109:142-50. 71. Ribbons KA, Zhang XJ, Thompson JH et al. Potential role of nitric oxide in a model of chronic colitis in rhesus macaques. Gastroenterology 1995; 108:705-11. 72. Rachmilewitz D, Karmeli F, Okon E. Sulfhydryl blocker-induced rat colonic inflammation is ameliorated by inhibition of nitric oxide synthase. Gastroenterology 1995; 109:98-106. 73. Hogaboam CM, Jacobson K, Collins SM et al. The selective beneficial effects of nitric oxide inhibition in experimental colitis. Am J Physiol 1995; 268:G673-84. 74. Mourelle M, Vilaseca J, Guarner F. Toxic dilatation of colon in a rat model of colitis is linked to an inducible form of nitric oxide synthase. Am J Physiol 1996; 33:G425-G430. 75. 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CHAPTER 12

Heterologous Modulation of Androgen Receptor Nucleo-Cytoplasmic Shuttling by Melatonin: A Novel Mode of Regulating Androgen Sensitivity Nava Zisapel

Abstract

M

elatonin, the hormone secreted nocturnally from the pineal gland, is an androgen protagonist in vivo. Its effects are mostly demonstrable under conditions of low circulating androgen levels (e.g., during pubertal development, under conditions of chemical or surgical castration or androgen ablation therapy). The prostate gland is an androgen dependent organ that responds to melatonin in vivo. Benign and cancer prostate cells provide suitable systems for exploring androgen-melatonin interactions in vitro. This review summarizes recent findings on the effects of melatonin on androgen receptors (AR) level and function in two lines of human prostate cancer cells- LNCaP cells expressing an innate AR and PC3 cells stably transfected with the AR (PC3AR). In both cell lines melatonin at physiological concentrations, attenuated androgen-induced gene expression suggesting anti androgenic activity. On the other hand, melatonin did not suppress, and even upregulated, AR protein levels and did not activate the AR in terms of androgen and DNA binding capacities. Immunocytochemical and subcellular fractionation studies that melatonin caused nuclear exclusion of AR in the cells, an effect that may explain the attenuation of AR activity. The pathway eliciting this nuclear exclusion involves a melatonin-induced increase in cGMP that acts to enhance calcium entry into the cells and subsequently protein kinase C activation. These studies identify a novel mode of hormonal interference in AR nuclear effects through modulation of AR nucleo-cytoplasmic shuttling. This interference may be utilized to attenuate the sensitivity of target cells to androgens, which may be of special importance in androgen related diseases such as prostate cancer and Kennedy’s disease.

Introduction The androgen receptor (AR) is a member of the steroid/thyroid hormone receptor gene superfamily and like other steroid receptors, it functions as a ligand dependent transcription factor.1 Unliganded AR is found in the nucleus in some target tissues and in the cytosol and nucleus in others.2, 3 Upon ligand binding, the AR binds to hormone response elements in the promoter region of inducible androgen-dependent genes thus controlling their transcription.1 The androgen-dependent gene products mediate the androgen-dependent development, differentiation and maintenance of male reproductive function, support sexually dimorphic functions of non-reproductive functions and enhance prostate cancer growth.4 Besides the induction Melatonin: Biological Basis of Its Function in Health and Disease, edited by S.R. Pandi-Perumal and Daniel P. Cardinali. ©2006 Eurekah.com.

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of AR transcriptional activity, androgens induce post-translational modifications leading to increased AR stability and therefore AR protein levels in cells.5 The pineal hormone melatonin is involved in the control of reproductive physiology in seasonal breeders and sexually immature rodents and perhaps humans.6,7 Accumulating evidence indicate that the hormone may directly regulate prostate cell growth in vivo under conditions of low circulating androgens (e.g., during pubertal development or in castrated rats) but is less effective under conditions of adult androgen levels.8-11 We have previously found that primary cultures established from human benign prostatic hyperplasia tissue, and the androgen-sensitive prostatic tumor -LNCaP cells express functional melatonin receptors.12,13 Expression of mt1 melatonin receptor protein was demonstrated in LNCaP cells, but not in PC-3 cells, in vitro.14,15 In the human benign prostate epithelial cells and prostate cancer LNCaP cells melatonin, at physiological concentrations, transiently inhibited3H-thymidine incorporation, DNA content and viability.12,13 This inhibition was not evident in the androgen-insensitive prostatic carcinoma PC3 cells.16 Furthermore, in nude mice, melatonin inhibited the growth of LNCaP tumors, without affecting the growth of PC-3 xenografts. Melatonin induced significant decreases in the expression of PCNA, cyclin A, and PSA in LNCaP tumors. Expression of mt1 receptor protein was demonstrated in LNCaP cells, but not in PC-3 cells in vivo as well.15 Prostate carcinoma PC3 cells are androgen insensitive. However, when stably transfected with a wild-type AR-expressing vector (PC3-AR) their growth is suppressed by picomolar concentrations of androgens.17 Melatonin reversed the growth inhibition effected by picomolar concentrations of androgen (dihydrotestosterone, DHT) in the PC3-AR cells but not the growth suppression effected by higher (nanomolar) DHT concentrations suggesting that the effect of melatonin can be abrogated by excess androgen.17 These data demonstrate differential interaction of melatonin with AR negative and positive prostate cells. We hypothesized that melatonin interfered with the AR cascade and investigated whether melatonin might suppress AR mediated gene expression. Having found that it did, we asked whether this was due to AR down regulation, inhibition of AR androgen or DNA binding or effects on AR nuclear localization. This review summarizes our recent findings concerning the effects of melatonin on the AR cascade and androgen responses in prostate cancer cells and how such influence is achieved. The potential significance of these findings in terms of the regulation of androgen sensitivity in health and disease is discussed.

Effect of Melatonin on Androgen-Induced Gene Expression The androgen-induced induction of reporter gene activity was used as an index of transcriptional activity of the AR (Fig. 1). In LNCaP cells transfected with an androgen reporter plasmid, melatonin (100 nM) did not affect basal reporter gene activity. Treatment with the synthetic non-metabolizable androgen R1881 (10 nM) resulted in about twenty-fold induction of the androgen-regulated reporter gene activity. Melatonin attenuated the androgen induced transcriptional activity.18 In PC3 cells co-transfected with wild-type AR and androgen reporter plasmidsR1881 treatment (10 nM, 24 h) resulted in 7 fold induction of the reporter gene activity (Fig. 1). Melatonin (100 nM ) had no effect on reporter gene activity in the absence of androgens but markedly inhibited CAT activity induced by 10 nM R1881.19 Hence, melatonin inhibits gene expression mediated by the wild-type AR (in PC3AR cells) as well as the innate mutant AR (in the LNCaP cells). Apparently, excess androgen negated the effects of melatonin as evidenced from the observation that the attenuation by melatonin of reporter gene activity was more prominent at lower concentrations of androgen.18,19 This suggests that the two agents have contradicting effects on an unidentified factor in the AR cascade, leading to diminution of melatonin response at high androgen concentrations.

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Figure 1. Effects of androgen and melatonin on androgen-dependent reporter gene activity in LNCaP and PC3AR cells. Cells were transfected with an androgen-dependent reporter plasmid containing two androgen responsive elements driven CAT. The cells were then incubated for 24 hr in the absence or presence of androgen (R1881 10 nM), melatonin (100 nM) and their combinations. CAT activity was then assessed. Results are means ± SD of 6 determinations and are expressed as % of the control value in the absence of steroids. Values sharing a common letter do not differ significantly (Student-Newmann-Keul’s test).

Effects of Melatonin on AR Protein Levels In LNCaP cells, melatonin (0.1-100 nM) increased immunoreactive AR the cells in the absence as well as in the presence of DHT in a concentration dependent manner. Immunoreactive AR increased also in cells treated with DHT (1n M) but was further enhanced in the presence of melatonin, suggesting non-additive effects. 18 Cycloheximide prevents AR up-regulation by melatonin, suggesting effect on de novo synthesis of the AR protein (unpublished). Similarly, in the PC3-AR cells DHT (1 n M) increased the immunostained AR protein band significantly. A significant increase in immunostaining intensity of the 110 kDa AR band was also observed in cells treated (48 h) with melatonin alone or in combination with DHT.19 The androgen-induced increase in AR in the cells is compatible with stabilization of the androgen-AR complex previously demonstrated in other systems.5 However, the effect of melatonin is not due to AR stabilization as there is no difference in AR stability in cells treated with melatonin and control cells (unpublished data). Melatonin receptors are coupled to heterotrimeric G-proteins.20-22 Coprecipitation experiments in HEK 293 cells showed that melatonin receptors of the MT1 class couple to G(i2), G(i3) and G(q/11) proteins.23 In PC3 cells Pertussis toxin treatment ablated the enhancement by melatonin of cGMP and inhibition of cAMP.16 Cholera toxin treatment prevented the modulation by melatonin of cAMP and cGMP but its effects were dependent on cell density.16 These observations are consistent with the involvement of a heterotrimeric G protein of the Gi/Go class in melatonin responses. The effects of CTX and PTX treatment on the melatonin- and androgen mediated accumulation of AR in LNCaP cells are shown in Figure 2. CTX treatment (16 h) reduced AR levels in the cells and attenuated the up-regulation of AR by melatonin (100 nM; 24 h) both in the absence and presence of androgen. In contrast, PTX treatment (16 h) which causes sustained activation of Gi type G proteins, enhanced AR levels in the cells as does melatonin and further enhancement was evident in the presence of melatonin (100 nM; 24 h) both in the absence and presence of DHT. These data are compatible with involvement of multiple

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Figure 2. Effects of cholera (CTX) and pertussis (PTX) toxins on AR up-regulation by melatonin in LNCaP cells. Cells were grown without steroids and then treated with CTX, PTX or vehicle for 16 h. The cells were then incubated without (control) or with melatonin (100 nM), in the absence or presence of DHT (1 nM) for 24 h and the amount of immunoreactive AR in whole cell proteins was then analyzed by Western blots as described.18 Mean and SD values of densitow7etric analyses of Western blots (expressed in % of the value in control cells without toxin treatment) obtained from three repetitive experiments are presented. Bars sharing a common letter do not differ significantly (Student-Newmann-Keul’s test).

heterotrimeric G proteins in the melatonin-mediated up regulation of AR protein in the prostate cells.

Effects of Melatonin on Androgen Binding Capacity In LNCaP cells treated with melatonin (100 nM), DHT (1 nM) or their combination, the androgen binding capacity was not significantly changed compared the control cells treated with vehicle.18 Because steroid binding affinity of the AR was not reduced, and even slightly enhanced with melatonin, DHT and their combinations, the differences in Kd are unlikely to cause attenuation of transcriptional activity in the hormone treated cells. Similarly, in PC3AR cells treated with melatonin for 48 h AR binding was not reduced, and even slightly increased, in parallel to the up-regulation of the AR protein, indicating that pretreatment with melatonin did not impair androgen binding capacity of the AR.19 The possibility that melatonin directly inhibits AR activity by competing for androgen binding was also assessed. Melatonin did not compete for AR androgen binding sites in the LNCaP as well as PC3-AR cells even at very high concentrations, thus excluding direct or indirect effects of melatonin on androgen binding to the AR.

Effects of Melatonin on Target DNA Binding The interaction of the AR-androgen complex with target DNA was studied in vitro in PC3-AR and LNCaP cells using an electrophoretic mobility shift assay, which measures the migration velocity of the receptor-DNA probe complex in a non denaturing polyacrylamide gel. The capacity to form AR- DNA complex was not inhibited and even somewhat enhanced in the melatonin-treated PC3AR as well as LNCaP cells. When normalized per protein content of the sample, enhancement was estimated at ca 30%, for both cell types-consistent with the increase in AR levels in melatonin treated cells.17 Hence, the attenuated androgen dependent gene expression in melatonin treated cells is not the result of inactivation of the AR with respect to androgen or target DNA sequences.

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Figure 3. Immunocytochemical localization of AR in human prostate PC3AR cells. Cells were grown without steroids and then treated with vehicle (A,B), 100 nM melatonin (C,D), 1 nM DHT (G,H), and their combination (E,F) for 48 h. The cells were fixed and immunostained for AR (A,C,E,G) and cell nuclei stained with Hoechst (B,D,F,H). The cells were photographed in a microscope (100X enlargement) equipped with fluorescence attachment to detect AR localization (A,C,E,G) and UV light to demonstrate location of cell nuclei (B,D,F,H). (Bar=10µ.)

Effects of Melatonin on AR Localization The effect of melatonin (100 n M) on the localization of the AR in PC3AR cells is shown in Figure 3. Most of the AR staining in the PC3AR cells appeared to be associated with the cell nuclei even in the absence of androgen, as also found previously in other cells2 and this was also the case with LNCaP cells.18,19 However, in cells treated with melatonin (48 h) the AR appeared in the cytoplasm, in the absence as well as presence of androgen (Fig. 3). This effect could be detected shortly (within 1 h) after the addition of melatonin to the culture media and could not be due to increase in AR content of the cells. Moreover, the nuclear export inhibitor

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Leptomycin B completely blocked the melatonin-mediated nuclear exclusion of the AR indicating that melatonin facilitates nuclear export of the AR.19 To corroborate these findings by an independent method, the intracellular distribution of the AR was investigated by subcellular fractionation followed by Western blot analyses of the AR protein. In agreement with literature data24 most (80%) of the immunostained AR in control cells was found in the cytosol fraction, probably due to receptor redistribution during cell disruption. In melatonin treated cells, the amount of AR associated with the nucleus was greatly reduced despite an overall increase in total amount of AR in the cells (namely, cytosol+nucleus). The nuclear exclusion of the AR by melatonin that has been observed in both prostate cell types, are most probably not derived from a reduced capacity of the AR-androgen complex to bind to its DNA target site. Because as mentioned above, no reduction in binding of the AR-androgen complex to the androgen response element is seen in both transfected wild-type (in PC3-AR) or innate mutant AR (in LNCaP). The AR nuclear exclusion by melatonin may in fact be the primary cause of the attenuated androgen dependent gene expression observed in the prostate cells because the nuclear localization is mandatory for the induction of gene expression. The intracellular signaling pathways mediating the nuclear exclusion of the androgen receptor (AR) by melatonin were evaluated in PC3 cells stably transfected with the AR.25 The melatonin-induced nuclear exclusion of the AR by melatonin (100 nM, 3 h) was blocked by the LY 83583 (inhibitor of guanylyl cyclases). 8-bromo cGMP (cell-permeable cGMP analog), mimicked melatonin’s effect and so did ionomycin (calcium ionophore) and PMA (activator of protein kinase C -PKC) and their effects were blocked by GF-109203X (selective PKC inhibitor). BAPTA (intracellular calcium chelator) blocked the effects of melatonin and 8 bromo cGMP but not of PMA. Inhibition or activation of the protein kinase A pathway did not affect basal or melatonin-mediated AR localization.25 A simplified mechanism was proposed, on the basis of these findings, to explain AR nuclear exclusion by melatonin (Fig. 4). We propose that melatonin elicits an increase in cGMP that triggers an increase in intracellular Ca2+, leading to PKC activation. Active PKC promotes cellular changes within the prostate cell resulting in nuclear exclusion of the androgen receptor. The possibility for dual action of melatonin on Ca2+ (via a Gi type G protein) and phosphoinositide metabolism (via a Gq type G protein) warrants further investigation.

Clinical Implications Melatonin’s Effects The modulation by melatonin of the AR nucleo-cytoplasmic shuttling may have important implications in disorders in which the nuclear localization of the AR is part of the pathogenic process (e.g., prostate cancer and Kennedy’s disease).26 Androgens, via the androgen receptor (AR) promote growth of prostate cancer. The mainstay of treatment of this disease is hormonal therapy causing castrate-like situation. However, after a while, the tumor regrows in an androgen refractory manner. Almost all patients with metastatic prostate cancer will eventually escape the control of first-line endocrine therapy and relapse. Once the first line treatments have proven ineffective there are really no possible second line treatments.27,28 It should be noted that in androgen-independent prostate carcinoma cells in vivo expression of AR is present and often amplified compared to androgen dependent prostate carcinoma despite their being refractory to androgen ablation therapy.29 The escape from androgen ablation therapy may partially be due to selection of cancer cells containing mutant AR forms that have high affinity and low selectivity to androgens.30 Hence, the AR may potentially be activated by endogenous hormones other than DHT. In addition, ligand independent mechanisms may contribute to prostate cancer relapse. The mechanism of ligand- independent AR processes and the involvement of growth factors in such stimulation are still not completely understood. However, Culig et al31 showed that in AR transfected DU145 cells, insulin growth factor I (IGF-I), as well as interleukin-6 (IL-6), stimulated an androgen responsive reporter

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Figure 4. Hypothetical scheme by which melatonin may effect AR nuclear exclusion. Stimulation of the melatonin receptor elicits an increase in cGMP that triggers an increase in intracellular Ca2+, leading to PKC activation. Active PKC promotes cellular changes within the prostate cell resulting in nuclear exclusion of the androgen receptor. Androgen promote nuclear localization of the AR thus negating melatonin’s effect. A putative activation by melatonin of phospholipase C (PLC) is shown, which may synergistically act with Ca2+ (through generation of diacylglycerol) to elicit PKC activation. A solid arrow represents activation proven by experiments. An interrupted arrow represents a hypothetical activation step. A T shape represents inhibition.

gene to a similar degree as synthetic androgens. Furthermore, this effect was inhibited by an antiandrogen. Recently phosphorylation and activation of the AR by HER2/neu a member EGF receptor family were demonstrated. This phosphorylation was mediated by the MAP kinase.32 Another possible mechanism for ligand- independent activation of the AR may be via cross talk between the AR and the protein kinase A (PKA) pathways. It was shown that PKA activators such as forskolin and cyclic AMP analogs can induce AR activation and secretion of PSA in the prostate cancer cells in the absence of androgens.33,34 As mentioned above, in most androgen target tissues, the AR is localized within the nucleus regardless of the absence or presence of androgens. In the presence of androgen, an AR-androgen complex is formed leading to binding of the AR to androgen-response elements in the promoter region of inducible genes thus controlling their transcription.1 Mutations at the DNA binding domain of the AR, leading to nuclear exclusion of the receptor result in loss of androgen sensitivity.35 The modulation by melatonin of androgen localization in the prostate cancer cells reduces the accessibility of the AR to the DNA thereby modulating its genomic activities regardless of whether the AR is activated by non-androgen or through a ligand independent pathway. Very much in line with these conclusions are the results of a recent study showing that melatonin treatment attenuated EGF-stimulated increases in inoculated LNCaP cell proliferation and cyclin D1 levels in nude mice. Melatonin had no effect on the proliferation or growth of DU 145 cells, and of PC-3 cells that do not express the AR indicating synergistic action of melatonin and castration in inhibiting the growth of androgen-sensitive LNCaP tumor.36

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A recent clinical study has shown that melatonin restores the sensitivity of metastatic prostate cancer patients to triptolein treatment.37 Triptolein treatment results in lowering androgen concentrations in the patients’ blood to castrate levels. Our findings that melatonin promotes AR nuclear exclusion provides a mechanistic explanation for the beneficial effects of melatonin on prostate cancer patients who relapse on androgen ablation therapy. As discussed above, under conditions of low androgen, the ability of melatonin to inhibit AR activity would presumably be at its peak. Nocturnal melatonin production decreases concurrently with age and is diminished in prostate carcinoma patients.38 The endogenous production of melatonin may thus be insufficient to interfere with AR gene expression in the prostate cancer patients. Proteins containing polyglutamine (polyGln) tracts, including the androgen receptor (AR), have recently gained much scientific awareness because of their involvement in a number of inherited diseases. Shorter than normal polyGln tracts (encoded by CAG repeats) in the AR increase susceptibility to prostate cancer whereas expanded tracts have been identified as a pathogenic mutation in spinal and bulbar muscular atrophy (SBMA; Kennedy’s disease) and androgen insensitivity in male patients carrying the mutation. The polyGln stretch is part of the N terminal domain of the AR that is involved in transactivation and transrepression reactions.39 The long polyGln mutant AR supposedly adopts an altered configuration, which may lead to further resistance to proteasomal degradation or abnormal cleavage leading to aggregate formation and neuronal death. This process may be exaggerated in the presence of androgens that stabilize the AR. Therefore, agents that may regulate AR concentration and intracellular localization may have important effects on the aggregability of long polyGln stretch AR and possibly on the development and progression of the disorder. The results of melatonin treatment will very much depend on the circumstances. The AR is autoregulated by androgen, which reduces AR messenger RNA (mRNA) in vivo and increases AR protein stability. The net effects of these paradoxical activities leads to variable, tissue specific steady state levels of the AR protein and mRNA in various cells. The effects of melatonin on the AR cascade can also be divided into 3 categories based on their resemblance to the effects of the cognate AR ligand. Some of the effects of melatonin are androgen-like (i.e., up-regulation of AR, enhanced androgen binding affinity and down regulation of AR-mRNA), and some negated those of androgen (i.e., AR nuclear accumulation, androgen-dependent down-regulation of AR-mRNA and reporter gene transactivation). This broad array of effects of melatonin may all result of the primary effect of melatonin on AR nucleo-cytoplasmic shuttling. However, because AR regulation is a complex process, the overall response generated may be vary under different hormonal circumstances.

References 1. Zhou ZX, Wong CI, Sar M et al. The androgen receptor: an overview. Recent Prog Horm Res 1994; 49:249-74. 2. Husmann DA, Wilson CM, McPhaul MJ et al. Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. Endocrinology 1990; 126:2359-68. 3. Sar M, Lubahn DB, French FS et al. Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology 1990; 127:3180-6. 4. Chang C, Saltzman A, Yeh S et al. Androgen receptor: an overview. Crit Rev Eukaryot Gene Expr 1995; 5:97-125. 5. Kemppainen JA, Lane MV, Sar M et al. Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones. J Biol Chem 1992; 267:968-74. 6. Reiter RJ. Pineal melatonin: Cell biology of its synthesis and of its physiological interactions. Endocr Rev 1991;12:151-80. 7. Waldhauser F, Ehrhart B, Forster E. Clinical aspects of the melatonin action: impact of development, aging, and puberty, involvement of melatonin in psychiatric disease and importance of neuroimmunoendocrine interactions. Experientia 1993; 49:671-81.

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8. Laudon M, Yaron Z, Zisapel N. N-(2,4-dinitrophenyl)-5-methoxytryptamine, a novel melatonin antagonist: effects on sexual maturation of the male and female rat and on oestrous cycles of the female rat [corrected]. J Endocrinol 1988; 116:43-53. 9. Yamada K. Effects of melatonin on reproductive and accessory reproductive organs in male rats. Chem Pharm Bull (Tokyo) 1992; 40:1066-8. 10. Debeljuk L, Feder VM, Paulucci OA. Effects of melatonin on changes induced by castration and testosterone in sexual structures of male rats. Endocrinology 1970; 87:1358-60 11. Gilad E, Laudon M, Matzkin H et al. Evidence for a local action of melatonin on the rat prostate. J Urol 1998; 159:1069-73. 12. Gilad E, Laudon M, Matzkin H et al. Functional melatonin receptors in human prostate epithelial cells. Endocrinology 1996; 137:1412-7. 13. Lupowitz Z, Zisapel N. Hormonal interactions in human prostate tumor LNCaP cells. J Steroid Biochem Mol Biol 1999; 68:83-8. 14. Xi SC, Tam PC, Brown GM et al. Potential involvement of mt1 receptor and attenuated sex steroid- induced calcium influx in the direct anti-proliferative action of melatonin on androgen-responsive LNCaP human prostate cancer cells. J Pineal Res 2000; 29:172-83. 15. Xi SC, Siu SW, Fong SW et al. Inhibition of androgen-sensitive LNCaP prostate cancer growth in vivo by melatonin: association of antiproliferative action of the pineal hormone with mt1 receptor protein expression. Prostate 2001; 46:52-61. 16. Gilad E, Laufer M, Matzkin H et al. Melatonin receptors in PC3 human prostate tumor cells. J Pineal Res 1999; 26:211-20. 17. Rimler A, Lupowitz Z, Zisapel N. Differential regulation by melatonin of cell growth and androgen receptor binding to the androgen response element in prostate cancer cells. Neuroendocrinol Lett 2002; 23Suppl1:45-9. 18. Rimler A, Culig Z, Levy-Rimler G et al. Melatonin elicits nuclear exclusion of the human androgen receptor and attenuates its activity. Prostate 2001; 49:145-54. 19. Rimler A, Culig Z, Lupowitz Z et al. Nuclear exclusion of the androgen receptor by melatonin. J Steroid Biochem Mol Biol 2002; 81:77-84. 20. Reppert SM, Weaver DR, Godson C. Melatonin receptors step into the light: cloning and classification of subtypes. Trends Pharmacol Sci 1996; 17:100-2. 21. Bubis M, Zisapel N. Involvement of cGMP in cellular melatonin responses. Biol Cell 1999; 91:45-9. 22. Jockers R, Petit L, Lacroix I et al. Novel isoforms of Mel1c melatonin receptors modulating intracellular cyclic guanosine 3',5'-monophosphate levels. Mol Endocrinol 1997; 11:1070-81. 23. Brydon L, Roka F, Petit L et al. Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins. Mol Endocrinol. 1999; 13:2025-38. 24. Guiochon-Mantel A, Delabre K, Lescop P et al. The Ernst Schering Poster Award. Intracellular traffic of steroid hormone receptors. J Steroid Biochem Mol Biol 1996; 56:3-9. 25. Lupowitz Z, Rimler A, Zisapel N. Evaluation of signal transduction pathways mediating the nuclear exclusion of the androgen receptor by melatonin. Cell Mol Life Sci 2001; 58:2129-35. 26. Ross CA. Intranuclear neuronal inclusions: A common pathogenic mechanism for glutamine-repeat neurodegenerative diseases? Neuron 1997; 19:1147-50. 27. Mahler C, Denis LJ. Hormone refractory disease. Semin Surg Oncol 1995; 11:77-83. 28. Scher HI, Steineck G, Kelly WK. Hormone-refractory (D3) prostate cancer: refining the concept. Urology 1995; 46:142-8. 29. Koivisto P, Kononen J, Palmberg C et al. Androgen receptor gene amplification: a possible molecular mechanism for androgen deprivation therapy failure in prostate cancer. Cancer Res 1997; 57:314-9. 30. Culig Z, Stober J, Gast A et al. Activation of two mutant androgen receptors from human prostatic carcinoma by adrenal androgens and metabolic derivatives of testosterone. Cancer Detect Prev 1996; 20:68-75. 31. Culig Z, Hobisch A, Cronauer MV et al. Androgen receptor activation in prostatic tumor cell lines by insulin- like growth factor-I, keratinocyte growth factor, and epidermal growth factor. Cancer Res 1994; 54:5474-8. 32. Gioeli D, Ficarro SB, Kwiek JJ et al. Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites. 33. Sadar MD. Androgen-independent induction of prostate-specific antigen gene expression via cross-talk between the androgen receptor and protein kinase A signal transduction pathways. J Biol Chem 1999; 274:7777-83. 34. Blok LJ, de Ruiter PE, Brinkmann AO. Forskolin-induced dephosphorylation of the androgen receptor impairs ligand binding. Biochemistry 1998; 37:3850-7.

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35. Nazareth LV, Stenoien DL, Bingman WE 3rd, et al. A C619Y mutation in the human androgen receptor causes inactivation and mislocalization of the receptor with concomitant sequestration of SRC-1 (steroid receptor coactivator 1). Mol Endocrinol 1999; 13:2065-75. 36. Siu SW, Lau KW, Tam PC et al. Melatonin and prostate cancer cell proliferation: interplay with castration, epidermal growth factor, and androgen sensitivity. Prostate 2002; 52:106-22. 37. Lissoni P, Cazzaniga M, Tancini G et al. Reversal of clinical resistance to LHRH analogue in metastatic prostate cancer by the pineal hormone melatonin: efficacy of LHRH analogue plus melatonin in patients progressing on LHRH analogue alone. Eur Urol 1997; 31:178-81. 38. Bartsch C, Bartsch H. Melatonin in cancer patients and in tumor-bearing animals. Adv Exp Med Biol 1999; 467:247-64. 39. Becker M, Martin E, Schneikert J et al. Cytoplasmic localization and the choice of ligand determine aggregate formation by androgen receptor with amplified polyglutamine stretch. J Cell Biol 2000; 149:255-62.

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CHAPTER 13

Extrapineal Melatonin: Location and Role in Pathological Processes Igor M. Kvetnoy, Natalia S. Sinitskaya and Tatiana V. Kvetnaia

Introduction

M

elatonin (5-methoxy-N-acetyltryptamine) is a major hormone produced by the pineal gland. Presence of melatonin in the pineal gland was first reported in 1958 by Lerner et al.75 Since this discovery it was clearly demonstrated that melatonin plays crucial role in the regulation daily and seasonal rhythms, pigment metabolism, immune response, reproductive function and other vitally important physiological processes in all photoperiodic species.50 Melatonin synthesis depends on the duration of the day and the intensity of the light, reflecting annual photoperiodic changes. Being an endocrine messenger, melatonin quantitatively transfers a photic signal to others tissues, expressing its own receptors, deliver thus timing information to the organism.102 Indoleamine metabolic pathways in pineal gland were investigated in detail. The precursor of melatonin is tryptophan, which is metabolized into 5-hydroxy-tryptophan by the action of tryptophan-hydroxylase (TROH). Serotonin (5-HT) then derives from tryptophan by the action of aromatic amino acid decarboxylase (AAAD), Serotonin is converted into N-acetylserotonin by the arylalkylamine-N-acetyltransferase (NAT) enzyme and then into melatonin by the action of hydroxyindole-O-methyltransferase (HIOMT). It has been established that key enzymes in this pathway are NAT and HIOMT.1 Maximum of melatonin synthesis is observed at the night. Nighttime production of melatonin in pineal gland is mainly regulated by the central circadian clock, situated in the hypothalamic suprachiasmatic nucleus (SCN)12 via norepinephrine release from pineal sympathetic nerve endings. Norepinephrine as well as other pineal transmitters (such as neuropeptide Y, vasopressin, oxytocin, somatostatin, substance P, etc.) carry out transcriptional control of activity main enzymes in melatonin synthesis. It was proposed that NAT connects melatonin synthesis with photoperiodic variation in duration and that the level of HIOMT activity may tune the seasonal magnitude of melatonin production in pineal gland.46,106 However, pinealectomy does not abolishes the animal’s circadian rhythm in rest-activity though facilitates the re-synchronization of the animal to a new photoperiod.128,129 In the animal models it was shown that after pinealectomy, melatonin levels at night become greatly attenuated while daytime levels of melatonin in blood remain unaffected, indicating existence of extrapineal sources of melatonin in the organism.119 Indeed, the pineal gland is not an exclusive organ where melatonin is synthesized. Extrapineal melatonin is widespread in the organism in humans and animals: melatonin-producing cells are found in the gastrointestinal tract, airway epithelium, pancreas, adrenal glands, thyroid gland, thymus, urogenital tract, placenta and other organs. Moreover, there has been demonstrated the immunoreactivity of

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melatonin in the non-endocrine cells, such as mast cells, natural killer lymphocytes, eosinophilic leukocytes, platelets, some endothelial cells and others.53 Therefore, melatonin demonstrates a wide distribution in the organism. Its role as intercellular neuroendocrine regulator and coordinator of many complex and interrelated biological processes has not yet been elucidated. Therefore the investigation of extrapineal melatonin is of great importance to gain a better understanding of its functions and role in organism as a whole. The chapter is devoted to considering of biological role of extrapineal melatonin and its participation in pathological processes.

Location of Extrapineal Melatonin The pineal gland is undoubtedly not the exclusive site of melatonin production. During the last two decades melatonin synthesis was found in many various organs, tissues and cells: in retina, in the gastrointestinal tract, in the liver, kidneys, adrenals, in lymphocytes, in mast cells, natural killer cells, eosinophilic leukocytes, platelets, thymocytes, some endothelial cells, in placenta and endometrium.23,45,95,97,100 The melatonin content in organism and its concentration in blood are accounted for not only by the pineal gland secretion, but also by extrapineal sources of synthesis, changes in the volume of extracellular fluid, hormone binding with blood proteins, metabolism and excretion rates depending on different outer and inner regulatory factors. Functionally, cells producing extrapineal melatonin are certain to be part and parcel of the diffuse neuroendocrine system. The main concept for the diffuse neuroendocrine system was the APUD-concept, firstly reported by Pearse in 1968-1969. This author undertook an extensive series of experiments aimed to distinguish endocrine cells in different organs, to identify endocrine cell-generated products and to obtain a thorough cytochemical and ultrastructural analysis of these cells.90 Pearse suggested that a specialized, highly organized cell system should exist in the organism, whose main feature was the ability of component cells to produce peptide hormones and biogenic amines. Different types of cells widely dispersed throughout the organism have a common ability of absorbing monoamine precursors (5-hydroxytryptophan and L-dihydroxyphenylalanine) and decarboxylating them, thus producing biogenic amines. That ability accounts for the term APUD, an abbreviation of “Amine Precursor Uptake and Decarboxylation” used by Pearse to designate this cell series.91 The APUD series includes over 60 types of cells located in gut, pancreas, urogenital tract, airway epithelium, pineal gland, thyroid gland, adrenals, adenohypophysis and hypothalamus, carotid body, skin, sympathetic ganglia, thymus, placenta and other organs.4,68,72,86,97 Meanwhile the appearance of radioimmunological methods and rapid development of immunohistochemistry resulted in establishing a completely unexpected phenomenon, i.e., the same biogenic amines and peptide hormones were identified in neurons and endocrine cells. Among APUD cells, cells which produce serotonin, melatonin, catecholamines, histamine, endorphins, endothelin, matrilysin, natriuretic peptide, vasoactive intestinal peptide, neuropeptide Y, vasopressin, oxytocin, somatostatin, endothelin, insulin, substance P and others, can be found.55,85 Within the whole diffuse neuroendocrine system, two compartments can be distinguished for melatonin-producing cells, viz central and peripheral. The central compartment includes the melatonin-producing cells, which are associated with the pineal gland and the visual system (retina, Harderian gland and possibly others) whose secretion rhythm complies with the rhythmic pattern of environmental light and dark. The peripheral compartment seems to account for all cells located outside the above areas, and its function probably does not depend on the degree of illumination. It includes melatonin-producing cells of the diffuse neuroendocrine system, mainly – gastrointestinal enterochromaffin cells. Below we shall consider both compartments of melatonin synthesis. The presence and rhythmical production melatonin in pineal gland have been well investigated. As soon as highly sensitive techniques of analysis and identification became available,

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melatonin and its precursors as well as catalytic enzymes began to be found in extrapineal tissues, primarily those anatomically connected with the visual system-retina and Harderian gland. Melatonin localization in retina was found immunocytochemically.23,122 The fact that pinealectomy did not result in any alterations of retinal melatonin level, allowed to consider proved this hormone synthesis in retina as independent on epiphysis.51,128,103 Furthermore, the presence of key enzymes of melatonin biosynthesis-NAT and HIOMT were shown in retinal tissue, as well as melatonin synthesis from labeled precursors (tryptophan and serotonin) was demonstrated.6,34,35,51,56,83,84,93 There are presented the evidences of melatonin synthesis in the layer of photoreceptor cells, that seems to be more likely in cytoplasm of these cells.21,122,125,126 Light is a crucial factor for melatonin biosynthesis in retina, as well as in pineal gland.132 Thus, it is interesting to note that light influences only one of two enzymes participating in melatonin synthesis, namely, the NAT.83,131 Activity of another enzyme (HIOMT) does not depend on light action.83 Available data permit to consider that regulation of physiological processes in complex “retina-retinal pigment epithelium,” submitting to light regime, is an essential function of retinal melatonin.131 It enables to assume that in retina melatonin carries out a transductive function of coordinator in receiving, primary processing and transmiting visual and nervous information. Harderian gland (an especial type of intra-orbital lacrimal glands) is one of the sources of extrapineal melatonin synthesis.54,80 Evidently, melatonin synthesis in Harderian gland occurs as well as in pineal gland; at least, one of two key enzymes of melatonin biosynthesis, HIOMT, was found in Harderian gland.7 Melatonin synthesis in Harderian gland of birds and mammals has been shown to comply with a circadian rhythm, typical for pineal gland, but independent on it.103,118 Moreover, a compensatory increase of melatonin content occurs in Harderian gland of rats some weeks after pinealectomy.104 The physiological role of melatonin in Harderian gland is presently not completely understood. In terms of the peripheral compartment of melatonin-producing cells, as it was mentioned above, these cells are widely distributed in the organism, predominantly in the digestive tract. Taking into account the fact that gut enterochromaffin cells (EC cells) are the main serotonin depot in the organism13,44 we were the first to identify melatonin production for these cells. Three steps were followed in melatonin identification for EC cells. Initially it had to be found out whether melatonin was present in gut mucosa-in the same wall layer which houses EC cells. Then melatonin location in EC cells had to be identified by immunohistochemical method and, finally we wanted to see: could the hormone be stored or synthesized in EC cells. Using classical biological tests, the presence of melatonin in gut mucosa was confirmed.98 When purified extracts of human appendix’s mucosa (which are especially rich in EC cells) were applied onto frog skin, and the sterile extract was injected into the lymphatic sac, the skin colour was observed to become lighter, which is a characteristic of melatonin impact. Experimental studies of extracts prepared separately from appendixes with simple, phlegmonose or gangrenous inflammation (the mean number of argentaffin EC cells in their mucosa depends on the form of inflammation) showed that the frog skin bleaching rate was related to the EC cell content of the mucosa.99 Correlation between the frog skin bleaching rate which is melatonin-specific and the number of EC cells seemed to be an indirect confirmation of melatonin being present in EC cells. Chromatographic analysis of the test extracts using as indicators synthetic melatonin and its main precursors, showed the presence in gut mucosa of 5-hydroxytryptophan, 5-HT, 5-methoxytryptamine (mexamine) and melatoni.n95 The fact that gut extracts contained the immediate precursors of melatonin which are generated in the chain tryptophan > serotonin > melatonin also supported the suggestion of melatonin being synthesized in gut EC cells.99 The immunohistochemical study, using specific antibodies showed the presence of immunopositive cells to melatonin and its precursors throughout the gastro-intestinal tract in both humans and experimental animals (dogs, rabbits, rats and mice).99,96

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Thus, the integrated application of methods of biological testing, thin-layer chromatography, histochemical stain and immunohistochemical analysis, enabled the first demonstration of the possibility, in principle, of melatonin synthesis in gut EC cells. Soon these results were confirmed by Bubenik, who by using an immunohistochemical method detected melatonin in practically all parts of the rat gastro-intestinal tract.28 It was emphasized that melatonin distribution corresponded to localization of serotonin-producing argentaffin EC cells. The fact that key melatonin synthesizing enzyme HIOMT was localized in gut94 confirmed the occurrence of its synthesis rather than just passive accumulation. Mathematical analysis showed that the total number of EC cells throughout the gut would be significantly larger than the number of melatonin producing cells of the pineal gland.72 Recently it was shown that the avian and the mammalian gastrointestinal tract contain at least 400x more melatonin than the pineal gland.54 These data, and the fact that EC cell account for 95% of all endogenous serotonin, being the principal precursor of melatonin, allow us to consider gut EC cells as the main source of melatonin in humans and animals.47,120,121 By employing reverse transcription-polymerase chain reaction methodology, occurrence of the two key enzymes in melatonin synthesis (NAT and HIOMT) was established in wide variety of tissues, i.e., gut, testis, spinal cord, raphe nuclei, stomach fundus and striatum.113 NAT and HIOMT activities were also shown in bone marrow.115 Collectively, these data indicate extrapineal melatonin synthesis in several organs. The functional morphology of EC cells has been studied.82 EC cells as well as other melatonin-producing cells can serve as a classic example of APUD cells in which biogenic amines (5-HT and melatonin) and peptide hormones (substance P, motilin and enkephalins) co-exist.110 Interestingly, co-localization of melatonin and calcitonin in thyroid C-cells; of melatonin and histamine in mast cells; of melatonin, somatostatin and beta-endorphins in natural killer cells; and of melatonin and prostaglandin F2 in thymic reticulo-epithelial cells was also found.67,64 This fact testifies in favor that the “non-endocrine” cells produce peptide hormones and biologically active amines in different tissues as a part of a universal system of response, control and protection of the organism. The hormonal substances of APUD cells may act as paracrine signal molecules for the local co-ordination of intracellular, inter-tissue and inter-organ relationships.65 Accumulated data do not fit the traditional concept of hierarchical dependence within two main regulatory systems, viz. nervous and endocrine ones. It became more and more evident that the mechanism of biological regulation should be founded on the coordinated functional interaction between the endocrine system and the central and peripheral nervous system based on the common type of information perception and transmission at subcellular, cellular and tissue levels. Recent data on identification of the same and similar physiologically active substances, acting within the nervous system as neurotransmitters and neurohormones; and, locally or distantly as hormones within the endocrine system, enable us to incorporate both systems in the concept of the universal diffuse neuroendocrine system (DNES). Actually, it can be possible to unite in the organisms the structurally isolated nervous and endocrine systems by means of functional relationships between biogenic amines and regulatory peptides and, to a certain extent, to provide a basis for the concept of integrated functions.2,71 Peripheral “non-endocrine” cells of DNES take part in the immune response, inflammatory reactions, cell growth and proliferation and may play an important role in the control of normal and pathological processes in the organism. To ascertain of the morphologic fundamentals of the hormonal function in non-endocrine cells would enable to understand better the intercellular mechanisms of the adaptation and compensation of the functional disorders appearing permanently in the organism during the vital activity. Further research into the nature of synthesis and deposition of hormones in non-endocrine cells seems to be very promising. The list of the cells producing and storing melatonin indicates that melatonin has a unique position among the hormones of the DNES, being found in practically all organ systems. However, in spite of data showing an active participation of melatonin in adaptive response, as

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well as in pathophysiology, the functional significance of peripheral, extrapineal melatonin-producing cells remains practically unknown.

Extrapineal Melatonin and Pathological Processes Showing unique properties as a free radical scavenger, regulator of biological rhythms and cell proliferation, melatonin causes indefatigable interest of pharmacologists as a potential medicinal substance. The current literature contains a lot of data about successful prospects of melatonin’s clinical utilization.20 So, being a most powerful antioxidant it has been stated that melatonin has a great potential in treatment of Alzheimer’s and Parkinson’s diseases.105,116 Administration of low doses of exogenous melatonin may have a positive effect upon the establishment a normal sleep-wake cycle in children with neurologic syndromes, mental retardation, blindness or epilepsy.101,133 Melatonin and light treatment of patients suffering seasonal affective disorders and depressive disorders within population of European north is widely discussed as a therapeutic approach.88,107 Different mechanisms of melatonin action on the immune system have been proposed.49 Besides, in clinical and animal studies melatonin demonstrated to have antitumor activity especially in combination with immuno-therapy.14,77 During last ten years we have studied the functional morphology and behaviour of extrapineal melatonin-producing cells as well as other main APUD cells in different pathologies and environmental conditions (e.g., ionizing and non-ionizing radiation, tumour growth and cytostatic therapy, autoimmune and gastrointestinal diseases, pharmacological and toxicological influence, etc.). The data obtained testify an active participation of extrapineal melatonin, as well as of other hormones, in the pathogenesis of various diseases.63,69,70

Extrapineal Melatonin and Seasonal Rhythm Disorders It is well-known that synthesis of melatonin in retinal tissues and Harderian gland has a rhythmic mode of secretion and is coordinated by the central circadian clock. In the case of melatonin production in the periphery, its light dependence remains to be defined.. In young chickens raised under a 12L:12D light/dark cycle (12 h Light:12 h Dark) pinealectomy blunted plasma melatonin levels at mid-scotophase.37 Similar results were obtained in rats adapted to 12L:12D conditions, in which it was clearly demonstrated that circadian rhythms were not abolished by pinealectomy. However, although serum concentration of melatonin and N-acetylserotonin were greatly reduced, this did not happen in the gastrointestinal tract, the quantitatively most important organ of extrapineal melatonin production.22,129 Gastrointestinal melatonin release seems to be related to food intake periodicity. Thus, higher peripheral levels of melatonin were observed after food intake or long-term food deprivation.29 In histological studies the behaviour of extrapineal melatonin-producing cells in response to food deprivation was examined. Twenty-four hours after food deprivation the number of EC-cells rises approximately 2-fold as compared to control, accompanying the increase in blood plasma serotonin. The cells demonstrate intensification of the argentaffin reaction. On day 3 of fasting the number of EC-cells and intensity of argentaffin reaction decrease to normal, whereas on day 7 EC-cells and argentaffin reaction increase again.111,112 Peroral treatment of humans and animals with pharmacological doses of L-tryptophan at daytime produced a significant increase in the concentration of circulating melatonin, which was comparable with nocturnal melatonin peak. L-tryptophan administration to pinealectomized animals also increased plasma melatonin content.53,127 Thus the basal daytime melatonin level in gastrointestinal tract depends of nutritional factors—amount and composition of ingested food—and of availability of tryptophan as a precursor of melatonin formation.54 Although the pineal gland is the main source of melatonin synthesis during the night, during daytime a substantial portion of extrapineal melatonin is produced by enterochromaffin cells of the gastrointestinal tract in response of feeding.26

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Binding sites for melatonin are detectable in the central nervous system as well as in a variety of peripheral tissues such as bone marrow, blood cells, brown fat, caudal artery, colon, duodenum, testis, heart, kidney, liver, lung, muscle, pancreas, prostate, skin, spleen, stomach, testis, thymus, thyroid and white fat.42,87 Besides, circadian expression of clock genes was also found in many peripheral tissues including liver, muscle, kidney, lung, mononuclear leukocytes and fibroblasts. Indeed, most mammalian cells seem to be have their own clock.8-10 Whether this fact has any relation to extrapineal melatonin production is not known yet. Diurnal melatonin level may be an important marker of seasonal affective disorder and depression. Seasonal affective patients show a diurnal elevation of serotonin and melatonin in blood during summer (on average 2.4 times as high as in control group of patients) in comparison with winter, but no significant differences in other circadian rhythms were found between ill and healthy persons.57 During winter patients also not showed significant diurnal variations in blood serotonin levels.40 It is possible to assume that extrapineal melatonin takes part in the pathophysiology and adaptive response of this disease. Therefore the investigation of extrapineal melatonin in this aspect may be of great importance.

Extrapineal Melatonin and Regulation of Gastrointestinal Functions There are few experimental and clinical data regarding the role of melatonin in the regulation of gastrointestinal tract functions. It is hypothesized that melatonin plays an important role in physiological activity of gastrointestinal tract. Disturbances in melatonin secretion may result in gastrointestinal tract diseases. Histochemical assay demonstrated the presence of melatonin in various organs of the gastrointestinal tract.95 Receptors for melatonin and enzymes involved in its synthesis from tryptophan were also detected in all gastrointestinal tract tissues. Maximum amounts of this hormone were found in the mucosa, while the submucosal and muscle layers contain the lowest concentrations of melatonin.28,87 The distribution of receptors for melatonin in gastrointestinal tract tissues follows a similar pattern: the density of melatonin receptors in the mucosa is much higher than that in the submucosal and muscle layers. Distribution of intracellular melatonin is as follows: nucleus > microsomes > mitochondria > cytoplasm.74 Similarly to various hormones, whose synthesis and presence were revealed in the central nervous system and gastrointestinal tract, the effects of melatonin can be mediated by the endocrine, neurocrine, paracrine, and autocrine mechanisms.74 Probably, the effects of melatonin synthesized in gastrointestinal tract are primarily mediated by a paracrine mechanism. Besides biorhythmic, antioxidant, and immunomodulating activities, melatonin may affect motor functions of gastrointestinal tract, microcirculation, and mucosal cell proliferation. In vitro and in vivo experiments with animals showed that melatonin inhibits motor activity of the gastrointestinal tract. The degree of inhibitory effects is directly proportional to the tone and intensity of contractions in the stomach, duodenum, and small and large intestines.24,52 Melatonin inhibits motor activity of gastrointestinal tract stimulated with various agents, e.g., serotonin and carbachol (a cholinoreceptor agonist). Feedback mechanisms underlying synthesis and secretion of melatonin and serotonin in animals seem to be involved in the regulation of gastrointestinal tract function.19,25 The inhibitory effect of melatonin on muscle contractions could be mediated by various mechanisms, including binding to specific, serotonin-inhibiting receptors and regulation of activity of Ca2+ channels and of Ca2+-activated K+ channels in cell membranes.24,30 Besides the direct effects of melatonin on muscle cell membranes, melatonin blocks nicotinic acetylcholine receptors on cells in the submucosal nervous plexus of the small intestine in guinea pigs.11 Collectively, the data indicate the possible participation of extrapineal melatonin in development of gastrointestinal diseases. Melatonin has a protective effect on the development of stress-induced, ischemia-induced, ethanol-elicited and acetylsalicylic-provoked gastric ulcers as well as on dextran- and dinitrobenzene, sulfonic acid-induced colitis in animal models.18,39,79,92 The mechanism of melatonin protective action is complex and may include the following:26,27

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1. Direct strong, melatonin antioxidant action, as it was demonstrated in the case of gastric ulcer and colitis induced by various factors. Intragastric administration of melatonin to rats with ischemic gastric ulcers decreased significantly the incidence of ulceration and the size of ulcerative lesions. Melatonin decreased the content of free radicals in the plasma and enhanced blood supply to the stomach wall.60 By Doppler ultrasonography it was found that rats with 40% ethanol-induced gastric ulcers and treated with melatonin showed decreased incidence of ulceration, increased blood flow in the stomach wall, and normalization of blood supply to the gastric mucosa inhibited by serotonin. Therefore, the anti-ulcer effect of melatonin is related not only to its antioxidant properties, but also to the improvement of microcirculation.36 2. Melatonin stimulation of antioxidative enzymes (superoxide dismutase and glutathione reductase) which protect the gastric mucosa against damage caused by ischemia-reperfusion.32 3. Melatonin modulation of proliferative activity of cells. Melatonin is probably one of the most potent regulators of cell proliferation in the gastrointestinal tract mucosa. Experiments in animals showed that pinealectomy stimulates proliferative activity of cells in various organs, including the gastrointestinal tract.17 It was shown that proliferative activity of mucosal cells in the small and large intestines of rats remained high for at least 6 months after pinealectomy (a considerable period of the rat’s life span). The effect of melatonin on proliferation of gastrointestinal tract mucosal cells involves endocrine, paracrine and neurocrine pathways: vagotomy and local sympathectomy attenuated pinealectomy-induced acceleration of proliferation of small intestine crypt cells. However, proliferative activity of these cells still remained above the control.33 Cell proliferation in pinealectomized animals was not normalized for a long period, which indicates an important role of melatonin in the regulation of proliferative processes in the gastrointestinal tract mucosa. The phenomenon of melatonin-induced inhibition of cell proliferation was studied in vitro and in vivo experiments in the field of oncology.5 4. A prostaglandin-mediated mechanism may be involved in the protection of gastric mucosa. Melatonin modulates cell proliferation probably by stimulating prostaglandin E2 production, which was demonstrated in experiments with gastric ulcers in rats induced by piroxicam administration (a non-steroid antiinflammatory drug).31 It was hypothesized that the mechanism underlying melatonin-induced stimulation of prostaglandin E2 synthesis involves the activation of cyclooxygenase, which catalyzes production of prostaglandins, prostacyclin, and thromboxane from polyunsaturated fatty acids.3 Since prostaglandin E and thromboxane inhibit secretion of hydrochloric acid and pepsin, and stimulate production of bicarbonates in the gastric mucosa, it can be suggested that endogenous melatonin synthesized in the gastrointestinal tract mucosa produces similar effects on gastric secretion.38,117 5. Gastrointestinal tract mucosa protection by melatonin-induced bicarbonate secretion. It was demonstrated that intestinal melatonin is involved in mediating central nervous stimulation on duodenal epithelial bicarbonate secretion via action on enterocyte MT2-receptors.109

Certainly, regulation of general immune response under action of melatonin may also take place in the regulation of gastrointestinal function and gastrointestinal diseases. Moreover, melatonin may interact with receptors and subsequently stimulate the synthesis of gastroprotective hormones exerting a direct defense effect on the epithelium, enhancing submucosal blood flow and preventing the damage induced by ischemia followed by reperfusion.81 Therefore, melatonin can be considered as a potential gastroprotective agent in various disturbances of the digestive tract.

Extrapineal Melatonin: Oncological Aspects of Biological Significance During ontogenesis biogenic amines are identified at very early embryogenetic stages and play a peculiar role as intracellular hormones which control cell division processes. The function of controlling cell proliferation rates (especially those “out of step” with the biological

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rhythms of organism development) seems to be of special importance for some biogenic amines in general as well as for melatonin in particular, also at later postnatal stages or organism life. Participation of melatonin in the regulation of cellular division is relevant in oncology. The antitumoral effects of melatonin and its clinical application are widely discussed in the current literature as well as in the present book. The anticarcinogenic potential of melatonin has been demonstrated both in vivo and in vitro studies in relation to different types of carcinomas.15,58,78 The mechanism of melatonin oncostatic action is complex and in addition to the direct modulation of mitotic activity includes regulation of endocrine and immune systems and the anti-oxidant action.15,78,89 In spite of many studies devoted to unraveled the inhibiting effect of exogenous (or pineal) melatonin on tumour growth, the role of extrapineal melatonin remains unclear. In our own experiments we have addressed the subject of the behaviour of melatonin producing cells in tumor growth.61 A number of hormones (including melatonin) synthesized by APUD cells can affect the proliferation and differentiation of tumour cells.72 The data show an important participation of APUD cells in the endogenous mechanisms of tumour growth. Generally, the “functional depletion” of APUD cells, which are the site of production of hormones with antiproliferative activity as well as the increase of the secretion of hormones which are able to stimulate cell proliferation may arrange the conditions which are favourable for fast tumour growth and metastases formation. For example, we were able to induce a functional modification of mast cells forming an endogenous “radioprotective shield” around the tumour by accumulating melatonin and serotonin.61 In particular, we observed an increase in tumour cell sensitivity to ionizing radiation after administration of ketotifen, a drug which prevents the release of histamine and other mediators from mast cells.59 Ketotifen injections before radiation therapy of tumours increased radiosensitivity by 26% in terms of growth rate and 20% in terms of proliferative activity.69 Besides, it is well-known that there exists a special type of tumors—apudomas—which develops from APUD cells.41,43 In gastrointestinal tract, most apudomas are carcinoids-the typical neoplasms from EC cells. The presence of hormone-producing cells in the non-endocrine carcinomas has a great theoretical and applied significance.130 By using immunohistochemical methods, it was shown that about 30% of all non-endocrine carcinomas of different histological types and localization contain endocrine cells and about 60% of such tumors have melatonin-producing cells in their composition.64 Indeed, we showed an increase of the number of EC and other melatonin-producing cells for initial stages and a decrease of the number of these cells for late stages of carcinogenesis. These data are in good agreement with the depression in melatonin plasma level found in cancer-positive patients during the phase of primary tumour growth as compared to early stages of tumour development.14,48 Concerning other APUD cells, their behaviour and functional morphology also change during tumour growth. For example, in the case of non metastatic tumours hypoplasia and decreased functional activity of ECL-cells (histamine) and of G-cells (gastrin) of stomach as well as of A- cells (glucagon) of pancreas were noted with increases in number at advanced stages of cancer. The proliferative activity of tumor cells plays a key role in neoplastic growth, invasiveness and metastatic formation.108 Therefore assessment of proliferation can be an effective index to judge the malignant potential of various carcinomas. PCNA (proliferative cell nuclear antigen), which is synthesized in cells in S-phase of cell cycle, is one of the most suitable markers of proliferation. PCNA is an immunohistochemical marker of proliferative activity and its determination is possible only in tissue specimens of tumors obtained during surgery. Together with Drs. C. and H. Bartsch from Tubingen University, using both immunohistochemical analysis and radioimmunoassay we studied the excretion of 6-sulphatoxymelatonin (aMT6s) in urine, the expression of PCNA and the number of melatonin immunopositive cells in primary human gut and lung tumors (cancer of colon, rectum, stomach and lung) without metastases. Our results showed strong positive correlations between the expression of PCNA in tumors

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and aMT6s excretion in urine. Strong negative correlations were also observed between melatonin-immunoreactivity and proliferative activity of tumor cells. These parameters were independent of the age of the patients as well as of the histological type and localization of tumor. Thus a new non-invasive method allowing a determination of the degree of tumor proliferation at different stage of malignant disease in daily clinical practice was established.16,62 In spite of many studies of the inhibiting effect of melatonin on tumour growth the mechanism of melatonin role in regulating proliferative activity of tumor cells remains unclear. Taking into account the direct connection between the contents of melatonin in blood and of aMT6s in urine, the determination of the latter appears to represent a reliable marker of the degree of melatonin synthesis in the organism. Therefore it is possible to entertain the following two hypotheses on two variants for of the participation of melatonin in tumour development, which have a great significance for prognosis in cancer patients. The first variant. A high urinary excretion of aMT6s is an evidence of an increase in melatonin secretion by pinealocytes and extrapineal melatonin sources in blood that in turn leads to a decrease of binding of melatonin in the tumor. Due to a deficiency of melatonin in the tumor the proliferative activity of tumour cells increases and the metastatic potential becomes stronger. The second variant. A decrease of urinary aMT6s excretion parallels a reduced secretion of melatonin from cellular sources into blood. Melatonin binding in the tumor increases under such conditions and via paracrine mechanisms results in suppression of tumor cell proliferation. As compared with the first variant the second one is more favourable for the prognosis of the patient. Hence it follows that the maintenance of urinary aMT6s within normal limits or above in cancer patients could be regarded as an unfavourable sign for prognosis which may gives evidence for defects within endogenous adaptative mechanisms. Collectively, the data discussed above open promising perspectives for the elaboration of new approaches for improvement of antitumor therapy, using the drugs which could change the level of biologically active substances into tumours, particularly melatonin.

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CHAPTER 14

Sleep and Melatonin in Diurnal Species Irina V. Zhdanova

Abstract

M

elatonin secretion, occurring at night in both diurnal and nocturnal species, provides an important circadian signal for initiating different types of behavior. In diurnal vertebrates, e.g., humans, macaques, zebrafish, this pineal hormone also has a pronounced acute effect on sleep. This effect on homeostatic sleep regulation is mediated via specific melatonin receptors and has a distinct dose-dependency, reaching plateau at near-physiological concentrations.

Introduction A 24-hour period of rotation of our planet on its axis and its 12-month period of rotation around the Sun determine two major environmental rhythms, the daily and annual variations in ambient light, temperature and solar radiation. Since life on Earth depends on the energy coming from the Sun, these regular variations in energy flow require specific adaptive mechanisms to provide for critical chemical reactions and defend from deadly overheating, freezing or radiation damage. Predicting the major changes in the environment helps to adjust the physiological mechanisms in advance and, thus, increase the probability of the organism’s survival. To anticipate changes in the environmental light and temperature, the organisms developed a “clock” system, which allows to properly time the physiological events. The daily period of the “circadian clock” is close to 24 hours (circa – near; dia – day) and can be precisely entrained to 24-hour period by light perceived through the photoreceptors. One of the intrinsic features of the photoreceptors is their ability to synthesize melatonin (N-acetyl-5-methoxytryptamine). The circulating amino acid L-tryptophan is the precursor for melatonin synthesis; it is converted to serotonin (5HT) by a two-step process, catalyzed by the enzymes tryptophan hydroxylase and 5-hydroxytryptophan decarboxylase. This process involves serotonin’s N-acetylation, catalysed by N-acetyltransferease (NAT), and then its methylation by hydroxyindole- O-methyltransferase (HIOMT) to produce melatonin. Melatonin production is strictly periodic, occurring only at night, and is acutely suppressed by nighttime light exposure. Apparently, photoreceptors use melatonin for their local purposes, i.e., as a paracrine agent. However, the evolution of one of the major photoreceptory organs, the pineal gland (epiphysis cerebri), led it to produce the excessive amounts of melatonin and release it into the blood stream and cerebrospinal fluid. Due to high lipid solubility and, thus, ability to cross the cell membranes, melatonin can be rapidly distributed throughout the entire organism. As a result, a periodic production of melatonin by the pineal gland became a universal endocrine message of nighttime. Numerous studies on the role of melatonin in different physiological processes suggest that this nocturnal hormone plays an important role in diverse tissues and organs. However, due to different physiological and behavioral processes occurring at daytime and nighttime in diurnal

Melatonin: Biological Basis of Its Function in Health and Disease, edited by S.R. Pandi-Perumal and Daniel P. Cardinali. ©2006 Eurekah.com.

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and nocturnal species, melatonin may signal the onset of different activities and can affect similarly or differently the biological events occurring in these two major types of organisms. For example, for diurnal species, increase in melatonin production would signal the onset of their nighttime rest. In contrast, for nocturnal animals, melatonin production would coincide with the onset of their daytime activity. Humans, being diurnal, secrete melatonin during their habitual hours of sleep and the onset of melatonin secretion correlates with the onset of their evening sleepiness.1-3 The earliest studies, conducted in 1950s by Aaron Lerner and his associates showed that melatonin administration could increase sleepiness in humans and further studies have substantiated this finding (for review see ref. 4). They also showed that circulating melatonin levels, similar to those observed under physiological conditions, can induce sleepiness, strongly suggesting that melatonin has a physiological role in human sleep regulation. It is now well accepted that melatonin has two major ways of affecting sleep. It can act acutely on the mechanisms involved in sleep homeostasis, thereby making one sleepy, or it can modulate the mechanisms that impart circadian rhythmicity to the temporal pattern of sleep propensity. The contribution of each of these actions to the hormone’s net effect depends on the time of its administration, the nocturnal or diurnal lifestyle of the species studied and the individual’s sensitivity to melatonin.

Melatonin and Circadian Regulation of Sleep The circadian effects of melatonin appear to be almost universal and, largely, similar among the diurnal and nocturnal species. This can be explained by the similarities in the temporal organization of their circadian system. Indeed, in both diurnal and nocturnal animals, the neurons of the major circadian clock, the suprachiasmatic nuclei (SCN) of the hypothalamus, are normally active during the day and slow down at night. The activation of SCN neurons has an inhibitory effect on the pineal gland, defining a nocturnal pattern of melatonin secretion. If SCN neurons are activated at night, e.g., by environmental light perceived by the retina, melatonin production declines. Melatonin, in turn, can acutely attenuate the activity of SCN. This melatonin action is likely to support a normal decline in the activity of the SCN at night, further promoting melatonin secretion and contributing to an overall increase in the amplitude of circadian body rhythms. A temporal and functional interplay between melatonin and SCN, and their response to environmental light, promote a temporal alignment of multiple circadian body rhythms with each other (internal synchronization) and with the periodic changes in the environment (external synchronization). In addition to an acute inhibition of SCN activity, melatonin administration can also produce a shift in the circadian phase of SCN activity, either advancing or delaying its onset. The direction of the phase-shift depends on the time of melatonin treatment, i.e., administration of melatonin in the late afternoon can advance the circadian clock, while early-morning treatment can cause a phase delay.5 Studies conducted in vitro suggest that a chronobiological effect of melatonin, i.e., the induction of circadian phase shift, is likely to be explained by its direct effect on SCN neurons via specific, most likely, MT2 receptor.6,7 Although the magnitude of the melatonin-induced phase shifts can vary between the species, the overall phenomenon appears to be well conserved. Such phase shifts in the circadian oscillation of SCN activity may change the physiological and behavioral rhythmicity of the entire organism, including the sleep-wake cycle, and can significantly affect the sleep quality in both nocturnal and diurnal species. In humans suffering from circadian sleep disorders, daily melatonin treatment can help to reinforce the circadian synchronization with the environment and entrain the physiological rhythms to a 24-hour cycle.8

Melatonin and Homeostatic Regulation of Sleep A common phenomenon that the increase in sleepiness is roughly proportional to the duration of prior wakefulness is termed “sleep homeostasis”, reflecting the need to balance time spent asleep and time spent awake. Currently, the processes involved in homeostatic regulation

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Figure 1. Sleep efficiency in subjects with age-related insomnia following melatonin or placebo treatment. *p