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. *** P<0.01 vs. control; +++ P< 0.01 vs. adriamycin group. The presenting data are from paper modified from Agapito et al. Protective effect of melatonin against adriamycin toxicity in the rat. J Pineal Res 2001; 31: 23-30.

combination with hGH decreased the injured area after cardiac infarction by 86-87% and reduced the number of cardiac lesions by 75-80%.85 Also Morishima et al85a reported that melatonin protected against adriamycin (doxorubicin hydrochloride)-induced cardiomyopathy, the pathogenesis of which may involve free radical and lipid peroxidation. In that study, melatonin has been shown to affect zinc turnover, which acts as an antioxidant. Similar results were obtained by other authors; they found that melatonin was an effective antioxidant against cardiotoxicity of myocardium generated by this antibiotic86-89 (Fig. 3). This indole also suppresses the iron-induced lipid peroxidation in many tissues, icluding the heart.90 Arteaga et al91 compared the antioxidative effect of a few antioxidants. They showed that the antioxidative potency of estradiol in vitro was 10-100 times higher than that of α- and γ-tocopherol and melatonin in protection against the oxidation of LDL-cholesterol from postmenopausal women. Benot et al92 suggests that the antioxidative mechanism of melatonin plays a very important role in blood pressure reduction and in the protection against atherosclerosis. Clinical data concerning the relation between melatonin and cardiovascular system in humans are very scarce. Dominguez-Rodriguez et al26 examined serum levels of melatonin and some parameters of oxidative stress (glutathione peroxidase and lipid peroxidation levels) in light/dark period in patients with acute myocardial infarction. They showed that acute myocardial infarction is associated with a nocturnal melatonin deficit in serum and increased oxidative stress. They suggested that melatonin, at least in part, is depleted during night to reduce the free radicals formed in acute myocardial infarction. People with hypertension demonstrate lower melatonin levels than those with normal blood pressure but the administration of the hormone in question declines blood pressure to normal range. It has been shown that melatonin reduces blood pressure of both normo-93-95 and hypertensive96 persons. Additionally, melatonin influences the resistance of large arteries to blood flow in both men95 and young women.94 Cagnacci et al94 examined the influence of melatonin administration in a dose of 1 mg on the circulation of young, healthy women. They found that melatonin greatly influences artery blood flow, decreases blood pressure, and blunts noradrenergic activation. Compared to that, Arangino et al95 observed that melatonin, in a dose 1 mg, reduces blood pressure and decreases

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catecholamine level after 90 min in human subjects. Even a decrease of 5 to 10 mm Hg in blood pressure is very important. Rich-Edwards et al97 suggested that, in hypertensive subjects, a similar decrease in diastolic blood pressure is associated with a 20% reduction of cardiovascular mortality. Arangino et al95 indicated that endogenous melatonin contributes to the nocturnal decrease in blood pressure and of catecholamine levels. People with high levels of low-density lipoprotein (LDL)-cholesterol have low levels of melatonin. It has been shown that melatonin suppresses the formation of cholesterol by 38% and reduces LDL accumulation by 42% in freshly isolated human mononuclear leukocytes.98 Cohen99 observed a 10-20% reduction of cholesterol in women, using the B-oval pill. It is a very important fact because, for example, Angier100 suggested that even a 10-15% depletion in blood cholesterol results in a 20 to 30% reduction of the risk of coronary heart disease. On the other hand, no changes have been shown in total cholesterol, HDL-cholesterol, and triglycerydes concentration after 6 months of melatonin administration in insomniac patients101 and in women in age 64-80.102 Summarising, melatonin may exert its effect on circulation by: 1. 2. 3. 4. 5. 6.

interference with arterial response to catecholamines, reduction of norepinephrine efflux from perivascular nerves, decrease of noradrenergic activity, suppression of prostaglandin production, influence on nitric oxide production, via specific receptors.

Additionally, melatonin may reduce blood pressure via the following mechanisms: 1. 2. 3. 4.

by a direct effect on the hypothalamus; as an antioxidant which lowers blood pressure; by decreasing the levels of catecholamines, or by relaxing the smooth muscles, lining the aorta.

Concluding, as melatonin concentrations have been reported to decrease with age and in many cardiological diseases, melatonin replacement therapy may decrease the incidence of sudden cardiac death during acute myocardial infarction disease and hypertension especially in elderly patients.

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42. Seegar H, Mueck AO, Lippert TH. Effect of melatonin and metabolities on copper-mediated oxidation of low density lipoprotein. Br J Clin Pharmacol 1997; 44:283-284. 43. Abyja PM, Liebmann P, Hayn M et al. Antioxidant role of melatonin in lipid peroxidation of human LDL. FEBS Lett 1997; 413:289-293. 44. Wakatsuki A, Okatani Y. Melatonin protects against the free radical-induced impairment of nitric oxide production in the human umbilical artery. J Pineal Res 2000; 28:172-178. 45. Okatani Y, Wakatsuki A, Watanabe K et al. Melatonin inhibits vasospastic action of oxidized low-density lipoprotein in human umbilical arteries. J Pineal Res 2000; 29:74-80. 46. Holmes SW, Sugden D. The effect of melatonin on pinealectomy-induced hypertension in the rat. Br J Pharmacol 1976; 56:360-361. 47. Kawashima K, Miwa Y, Fujimoto K et al. Antihypertensive action of melatonin in the spontaneously hypertensive rat. Clin Exp Theo Pract 1987; A9:1121-1131. 48. Karppanen H, Airaksinen MM, Sarkimaki I. Effects in rats of pinealectomy and oxypertine on spontaneous locomotor activity and blood pressure during various light schedules. Ann Med Exp Biol Fenn 1973; 51:93-103. 49. Laflamme KA, Wu L, Foucart S et al. Impaired basal sympathetic tone and alpha 1-adrenergic responses in association with the hypotensive effect of melatonin in spontaneously hypertensive rats. Am J Hypertens 1998; 11:219-229. 50. Wu L, Wang R, de Champlain J. Enhanced inhibition by melatonin of α-adrenoceptor-induced aortic contraction and inositol phosphate production in vascular smooth muscle cells from spontaneously hypertensive rats. J Hypertens 1998; 16:339-347. 51. Vanecek J, Kosar E, Vorlicek J. Daily changes in melatonin binding sites and the effect of castration. Mol Cell Endocrinol 1990; 73:161-170. 52. Seltzer A, Viswanathan M, Saavedra JM. Melatonin-binding sites in brain and caudal arteries of the female rat during the estrous cycle and after estrogen administration. Endocrinology 1992; 130:1896-1902. 53. Cagnacci A, Zanni AL, Veneri MG et al. Influence of exogenous melatonin on catecholamine levels of postmenopausal women prior and during oestradiol replacement. Clin Endocrinol 2000; 53:367-377. 54. Cagnacci A, Arangino S, Angiolucci M et al. Effect of exogenous melatonin on vascular reactivity and nitric oxide in postmenopausal women: Role of hormone replacement therapy. Clin Endocrinol 2001; 54:261-266. 55. Verdecchia P, Schillaci G, Gatteschi C et al. Blunted nocturnal fall in blood pressure in hypertensive women with future cardiovascular morbid events. Circulation 1993; 88:986-992. 56. Doolen S, Krause DN, Duckles SP. Estradiol modulates vascular response to melatonin in rat caudal artery. Am J Physiol 1999; 276:H1281-H1288. 57. Doolen S, Krause DN, Dobocovich M et al. Melatonin mediates two distinct responses in vascular smooth muscle. Eur J Pharmacol 1998; 345:67-69. 58. Weekley LB. Melatonin-induced relaxation of rat aorta: Interaction with adrenergic agonists. J Pineal Res 1991; 11:28-34. 59. Yanagisawa M, Kurihara H, Kimura S et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332:411-415. 60. Svensson J, Strandberg K, Tuvemo T et al. Thromboxane A2: Effects of airway and vascular smooth muscle. Prostaglandins 1977; 74:425-236. 61. Weksler B, Marcus A, Jaffe E. Synthesis of prostaglandin I2 (prostacyclin) by cultured human and bovine endothelial cells. Proc Natl Acad Sci USA 1997; 74:3922-3926. 62. Fuechgott RF, Vanhoutte PM. Endothelium-derived relaxing and contracting factors. FASEB J 1988; 3:2007-2018. 63. Okatani Y, Watanabe K, Sagara Y. Effect of nitric oxide, prostacyclin, and tromboxane on the vasospastic action of hydrogen peroxide on human umbilical artery. Acta Obstet Gynecol Scand 1997; 76:515-520. 64. Pozo D, Reiter RJ, Calvo JP et al. Physiological concentrations of melatonin inhibit nitric oxide synthase in rat cerebellum. Life Sci 1994; 55:PL455-PL460. 65. Pozo D, Reiter RJ, Calvo JR et al. Inhibition of cerebellar nitric oxide synthase and cyclic GMP production by melatonin via complex formation with calmodulin. J Cell Biochem 1997; 65:430-442. 66. Cuzzoorea S, Constantino 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. 67. Wakatsuki A, Okatani Y, Ikenoue N et al. Melatonin inhibits oxidative modification of low-density lipoprotein particles in normolipidemic post-menopausal women. J Pineal Res 2000; 28:136-142. 68. Chen LD, Tan DX, Reiter RJ et al. In vivo and in vitro effects of the pineal gland and melatonin on [Ca2+ + Mg2+]-dependent ATPase in cardiac sarcolemna. J Pineal Res 1993; 14:178-183.

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69. Benitez-King G, Rios A, Martinez A et al. In vitro inhibition of Ca2+/calmodulin-dependent kinase II activity by melatonin. Biochim Biophys Acta 1996; 1290:191-196. 70. Anton-Tay F, Mortiney R, Tovar R et al. Modulation of subcellular distribution of calmodulin by melatonin in MDCK cell. J Pineal Res 1998; 24:35-42. 71. Mei YA, Lee PPN, Wie H. Melatonin and ist analogs potentiate the nifedipine-sensitive high-voltage-activated calcium current in chick embryonic heart cells. J Pineal Res 2001; 30:13-21. 72. Tan DX, Chen LD, Poeggeler B et al. Melatonin, a potent, endogenous hydroxyl radical scavenger. Endocrine Reg 1993; 1:57-60. 73. 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. 74. Stasica P, Ulanski P, Rosiak JM. Melatonin as a hydroxyl radical scavenger. J Pineal Res 1998; 25:65-66. 75. Sewerynek E, Melchiorri D, Chen LD et al. Melatonin reduces both basal and bacterial lipopolysaccharide-induced lipid peroxidation in vitro. Free Rad Biol Med 1995a; 19:903-909. 76. Sewerynek E, Poeggeler B, Melchiorri D et al. H2O2-induced lipid peroxidation in rat brain homogenates is greatly reduced by melatonin. Neurosci Lett 1995b; 195:203-205. 77. Sewerynek E, Reiter RJ, Melchiorri D et al. Oxidative damage in the liver induced by ischemia-reperfusion: Protection by melatonin. Hepatogastroenterology 1995c; 43:898-905. 78. Sewerynek E, Wiktorska J, Lewinski A. Effects of melatonin on the oxidative stress induced by thyrotoxicosis in rats. Neuroendocrinol Lett 1999; 20:157-163. 79. Marchioli R. Antioxidant vitamins and prevention of cardiovascular disease: Laboratory, epidemiological and clinical trial data. Pharmacol Res 1999; 40:227-238. 80. 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. 81. 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. 82. Sahna E, Acet A, Kaya Ozer M 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. 83. Lee Y-M, Chen H-R, Hsiao G et al. Protective effects of melatonin on cardial ischemia-reperfusion injury in vivo. J Pineal Res 2002; 33:72-80. 84. Salie R, Harper I, Cillie Ch et al. Melatonin protects against ischaemic-reperfusion myocardial damage. J Mol Cell Cardiol 2001; 33:343-357. 85. Castagnino HE, Lago N, Centrlla JM et al. Cytoprotection by melatonin and growth hormone in early rat myocardial infarction as revealed by Feulgen DNA stainging. Neuroendocrinol Lett 2002; 23:391-395. 85a. Morishima I, Okumura K, Matsui H et al. Zinc accumulation in adriamycin-induced cardiomyopathy in rats: Effects of melatonin, a cardioprotective antioxidant. J Pineal Res 1999; 26:204-210. 86. Agapito MT, Antoli Y, del Brio MT et al. Protective effect of melatonin against adriamycin toxicity in the rat. J Pineal Res 2001; 31:23-30. 87. Xu MF, Tang PL, Qian ZM et al. Effects by doxorubicin on the myocardium are mediated by oxygen free radicals. Life Sci 2001; 68:889-901. 88. Xu M, Ashraf M. Melatonin protection against lethal myocyte injury induced by doxorubicin as reflected by effects on mitochondrial membrane potential. J Mol Cell Cardiol 2002; 34:75-79. 89. Dziegiel P, Jethon Z, Suder E et al. M. Role of exogenous melatonin in reducing the cardiotoxic effect of daunorubicin and doxorubicin in the rat. Exp Toxicol Pathol 2002; 53:433-439. 90. Tang PL, Xu MF, Qian ZM. Different behaviour of cell membranes towards iron-induced oxidative damage and the effects of melatonin. Biol Signals 1997; 6:291-300. 91. Arteaga E, Rojas A, Villaseca P et al. The effect of 17β-estradiol and α-tocopherol on the oxidation of LDL cholesterol from postmenopausal women and the minor effect of γ-tocopherol and melatonin. Menopause 2000; 7:112-116. 92. Benot S, Goberna R, Reiter RJ et al. Physiological levels of melatonin contribute to the total antioxidative capacity of human serum. J Pineal Res 1999; 27:59-64. 93. Cagnacci A, Sodani R, Yen SSC. Melatonin enhances cortisol levels in aged women: Reversible by estrogens. J Pineal Res 1997; 22:81-85. 94. Cagnacci A, Arangino S, Angiolucci M et al. Influence of melatonin administration on the circulation of women. Am J Physiol 1998; 274:R335-R338. 95. Arangino S, Cagnacci A, Angiolucci M et al. Effects of melatonin on vascular reactivity, catecholamine levels, and blood pressure in healthy men. Am J Cardiol 1999; 83:1417-1419.

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96. Birau N, Peterssen U, Meyer C et al. Hypotensive effect of melatonin in essential hypertension. IRSC Med Sci 1981; 9:906-909. 97. Rich-Edwards JW, Manson JE, Hennekens CH et al. The primary prevention of coronary heart disease in women. N Engl J Med 1995; 332:1758-1766 98. Muller-Wieland D, Behnke B, Koopmann K et al. Melatonin inhibits LDL receptor activity and cholesterol synthesis in freshly isolated human mononuclear leukocytes. BBRC 1994; 203:416-421. 99. Cohen M, Josimovich J, Brzezinski A. Melatonin: From Contraception to Breast Cancer Prevention. Potomac: Sheba Press, 1995:76. 100. Angier N. Health Benefits from Soy Protein. New York Times. 1995, Aug. 3, p. A1. 101. Siegrist C, Benedetti, Orlando A. Lack of changes in serum prolactin, FSH, TSH, and estradiol after melatonin treatment in doses that improve sleep and reduce benzodoazepine consumption in sleep-disturbed, middle-aged, and erderly patients. J Pineal Res 2001; 30:34-42. 102. 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 somatostatuin C in erderly women. Neuroendocrinol Lett 2002; 23:17-19.

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CHAPTER 7

Pineal Gland and Cancer—An Epigenetic Approach to the Control of Malignancy: Evaluation of the Role of Melatonin Christian Bartsch and Hella Bartsch

Abstract

T

he secretion of the pineal hormone melatonin is under control of the hypothalamic suprachiasmatic nuclei, the seat of the central circadian clock, and conveys information concerning time of day as well as season to practically all parts of the body. This means that melatonin is an integral part of the circadian time-keeping system. According to the summarized findings a link exists between the pineal gland and cancer, a mutual and dynamic interaction between the secretion of melatonin and malignant growth. A fresh tumor is “sensed” by the pineal gland via neuroimmunoendocrine changes leading to a stimulation of melatonin secretion which in turn activates endogenous defence processes. At this stage of cancer development melatonin can exert a direct tumor-inhibitory activity. If the tumor increases in size the circulating levels of melatonin are depleted in many types of cancer being accompanied by progressing circadian neuroendocrine as well as vegetative disturbances. Such weakening of the temporal structure of the sub-systems of the host can be viewed as a preparatory step for a successful seeding of metastases. Evidence exists that melatonin is trapped by cancerous tissue which may even possess the feature of ectopic melatonin production from its precursor amino acid tryptophan which in turn limits pineal melatonin production further in the presence of big cancerous masses. Although melatonin does not directly inhibit advanced tumors its substitutional administration appears to be beneficial by overcoming sleep-disturbances as well as by fostering the endorphin system leading to a better quality of life. These favourable effects on the central nervous system seem to facilitate a mobilization of endogenous defence mechanisms against the malignant process improving survival. This means that melatonin via indirect systemic mechanisms is able to favourably affect even advanced forms of malignancy. These facts can be viewed as evidence for an involvement of the pineal gland in temporal epigenetic control processes of cancer. On the basis of the present findings it appears to be justified to advocate the development of new strategies for the treatment of solid tumors in which melatonin is combined with conventional therapies. In case of leukemias, however, melatonin should be avoided since it may, due to its stimulatory effects on the haematopoietic system, aggravate this disease. The dynamic changes of circulating melatonin occurring during different phases of malignant disease could be used for diagnostic purposes if intra-individual changes are specifically considered. Evidence exists that other low-molecular weight pineal substances may also play a role possessing a tumor-inhibitory activity even on undifferentiated tumor cells which are refractory to melatonin. Since there are indications that these new pineal substances may be regulated by melatonin the central role of the main pineal hormone in the link between the pineal gland and cancer is thus emphasized. 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

Introduction At a time when hopes are starting to fade again that a purely genetic approach in oncology will decisively and effectively control cancer alternative strategies based upon epigenetic mechanisms are being rediscovered.1,2 One of them is considering a link between the pineal gland,3 an unpaired diencephalic organ, and malignancy.4 Since the corpus pineale as it is also called is involved in the central control of temporal neuroimmunoendocrine processes5-7 this approach thus assumes a link between integrative neural processes and disintegrative tendencies manifesting in cancer. During the first part of the twentieth century Engel and Bergmann8,9 as well as Hofstätter10 performed pioneering experimental and clinical studies in Vienna dealing with the antineo-plastic activity of bovine pineal glands. After the discovery of melatonin by Lerner11 in 1958 these findings became almost forgotten. Vera Lapin working at the Vienna Cancer Research Center in the 1970s rediscovered them, reviewed the topic of pineal gland and cancer,12 and performed important experimental studies.13 A central finding was that surgical removal of the pineal gland (pinealectomy) stimulated both primary tumor growth and formation of metastases thus leading to reduced survival.14,15 This was in accordance to observations of other investigators16 including Bindoni17 who observed that pinealectomy even stimulates cell-division of normal tissues. The question arose how this general anti-proliferative effect of the pineal gland could be explained and whether it is mainly due to the pineal hormone melatonin or not. In order to find answers to this central question the current knowledge regarding the action of melatonin on experimental and on clinical cancers will be summarized in the following.

Effect of Melatonin on Tumor Growth The Action of Melatonin on Experimental in Vivo Tumors When surveying the available literature regarding the action of melatonin on experimental tumor growth in animals18 indications were found that the pineal hormone can exert divergent effects even within the same cancer model system. Based upon the fundamental findings of Reiter et al19 as well was Tamarkin et al20 with respect to the anti-reproductive effect of melatonin in rodents which can only be observed in case of late afternoon/evening injections to superimpose the secretory surge of endogenous melatonin (after the onset of darkness) it was tempting to assume that the same may also apply to the inhibitory action of melatonin on tumor growth. Using transplantable tumors in mice (the so-called Ehrlich tumor as well as a transplantable fibrosarcoma) as test systems it was demonstrated that late afternoon injections, preferably under long photoperiods (i.e., a light regimen of more than 12.5 hours per 24 hours lighting cycle), were able to inhibit whereas the same dose of melatonin given in the morning accelerated tumor growth or shortened survival.18 These findings thus demonstrated a circadian stage-dependent anti-tumor effect of melatonin which was subsequently confirmed by investigations of Wrba et al.21 On the basis of these results it became conventional that melatonin was administered in most subsequent experimental and clinical studies in the late afternoon or in the evening to achieve optimal tumor-inhibitory effects. A considerable number of investigations have dealt with the effect of melatonin on experimental mammary cancers in female Sprague-Dawley rats induced by 7,12dimethylbenz-[a]anthracene (DMBA)22 being one of the most important model systems for human breast cancer.23 These hormone-dependent tumors are under the control of gonadal steroids24 and particularly prolactin.25 Melatonin is known to inhibit prolactin secretion as well as to affect the endocrine balance which regulates the ovulatory cycle.26 This renders a plausible explanation why the pineal hormone is able to inhibit tumors of this type27,28 affecting both their promotion and initiation.29 The anti-initiational effect is due to a competitive interaction of melatonin with hepatic phase I enzymes (P450-monoxygenases)30,31 which hydroxylate melatonin to yield 6-hydroxymelatonin32,33 and DMBA leading to the formation of 3,4-dihydrodiol-1,2-epoxy derivative, the ultimal carcinogen.29,34 It can be assumed

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furthermore that metabolically activated intermediary free radical forms of DMBA are scavenged directly by melatonin which has been reported to possess a pronounced anti-oxidative activity.35 Apart from the DMBA model system for breast cancer melatonin has been successfully tested to inhibit rat mammary cancer induced by N-nitroso-N-methylurea (NMU).36,37 These findings could have considerable clinical relevance since NMU-induced tumors are more alike human breast cancers showing a stronger estrogen-dependency as well as the formation of metastases.38 Since melatonin administration during the promotional phase inhibits tumor growth in this model system without substantially affecting the concentrations of circulating estradiol and prolactin it is assumed that the oncostatic action of the pineal hormone is predominantly exerted directly at the cellular level. Melatonin has been tested on a considerable number of experimental in vivo tumors (for a review see ref. 38). It is evident that those tumors are inhibited most effectively which show hormone-dependency and/or a relatively high degree of cellular differentiation. Typical examples are the behavior of transplantable prostatic or mammary cancers. The growth of experimental prostatic cancers is effectively blocked by melatonin only if they are androgen-dependent or possess melatonin receptors,39,40 androgen-insensitive tumor sub-lines are refractory41,42 or may even be stimulated.43 Serial transplants of a DMBA-induced mammary tumor in inbred rats44,45 were found to be inhibited by melatonin only at an early and slow-growing passage of relatively high differentiation (carcinosarcoma) whereas a later and fast-growing passage of low differentiation (sarcoma) showed no response to chronic administration of the pineal hormone.46 Other undifferentiated hormone-independent in vivo tumors, such as the so-called Yoshida tumor, Walker 256 carcinosarcoma and others are not affected by melatonin.47 Since such tumors are on the other hand stimulated in their growth by pinealectomy16 it is obvious that the pineal gland contains other as yet unidentified anti-tumor substances48-54 with potentially important actions even on general development of multi-cellular organisms.55,56 Experiments with spontaneous endometrial carcinomas in BDII/Han rats57 illustrate complex neuroendocrine actions of melatonin during the development of sex-hormone-dependent tumors. The pineal hormone prolonged survival only if chronic night-time administration in drinking water was initiated by day 30 of life,58 i.e., shortly before beginning of puberty, whereas life-long treatment was ineffective if treatment was started on day 50, when animals had attained maturity.59 Since the pineal hormone delays pubertal development in both male and female rats60,61 it can be anticipated that the action of melatonin on endometrial cancer is exerted by delaying reproductive maturation. It is well conceivable that if melatonin treatment is started even earlier in postnatal life or if maternal melatonin secretion is modulated it could affect spontaneous endometrial cancer development even more profoundly. The validity of this assumption requires to be tested by further experiments in this as well as on other spontaneous hormone-dependent tumors such as breast cancer-prone C3H mice which are known to be inhibited by melatonin.62 The mechanism involved in the inhibition of well-differentiated tumors by melatonin not only consists of a neuroendocrine hormone-receptor-mediated component but is likely to include immune-mediated processes leading to tumor rejection. Melatonin possesses well-documented stimulatory effects on different parts of the haematopoietic and immune systems6,63 involving both membrane and nuclear melatonin receptors.64,65 This could also render an explanation why melatonin was found to stimulate the growth of virus-induced leukemia in mice whereas pinealectomy was inhibitory.66,67 According to recent experiments of D.E. Blask and his group evidence exists that melatonin controls tumor growth by inhibiting the metabolism of linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODE), 68,69 an important mitogenic signalling molecule which amplifies EGF-responsive mitogenesis and thus stimulates cancerous growth.70 The effect of melatonin on in vivo experimental tumor growth may thus be understood as a process involving both neuro-immunoendocrine as well as metabolic mechanisms.

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The Action of Melatonin on Cancer Cells under in Vitro Conditions Melatonin has been shown to inhibit the proliferation of a number of in-vitro cell lines at physiological concentrations including the human mammary cancer cell lines MCF-7,71,72 T47D and ZR75-1,47,73,74 the prostate cell-line LNCaP,75,76 biopsies derived from human melanomas77 as well as a murine adenohypophyseal prolactinoma.78 Studies on other cancer cell lines showed that melatonin inhibits only at pharmacological concentrations or has no effect. This included the human cell line HEp-2 originating from a laryngeal carcinoma, K562 being an erythroleukemia, EFO-27 of human ovarian origin as well as the mammary cell line EFM-1952,79 (for a detailed review see ref. 80). Also in case of MCF-7 cells the inhibitory action of melatonin is not always observable and appears to be confined to certain sub-clones.81 In some experiments even a tumor-stimulatory action of melatonin was detected such as in case of the breast cancer cell line MDA-MB-231 as well as on melanoma cells.47 In endometrial cancer cell-lines it was found that only estrogen receptor positive (SNG-II) but not estrogen receptor negative cells (Ishikawa) were inhibited by melatonin.82 Divergent effects of melatonin were also detected if the pineal hormone was given to primary cell cultures derived from different human mammary as well as ovarian cancer biopsies54 but no correlation with the presence of sex-steroid hormone receptors was detectable in this case. A missing inhibitory effect of melatonin on tumor cells can be explained by a progressing loss of differentiation leading to not only absence of receptors for sex-steroids but also for melatonin. Recent studies showed that both membrane (MT1)83-86 and nuclear melatonin receptors (RORα)87-89 are essential determinants for an inhibitory effect of the pineal hormone on tumor cells. The detailed mechanisms involved are under investigation by ongoing studies. In order to achieve a better predictability for an oncostatic effect of melatonin it would therefore be necessary to determine the levels of both sex-steroid hormone and melatonin receptors since the pineal hormone is apparently involved in the regulation of the estrogen-response system90,91 and thus codetermines functional sensitivity to circulating 17β-estradiol. According to findings of Gilad et al92 melatonin possesses only a transient inhibitory effect on androgen-dependent benign human prostatic epithelial cells since the pineal hormone inactivates its own receptors via a protein kinase C-mediated mechanism. This indicates that the growth-inhibitory action of melatonin on sex-steroid hormone dependent cells can be subtle and may be confined to certain phases of the cell cycle. From these findings it is plausible why a progressing loss of differentiation of cancer cells (affecting both melatonin and other hormone receptors) of cancer cells is bound to lead to insensitivity to melatonin. A typical example for this was found in case of a human melanoma cell line where the pineal hormone inhibited only early passages which were well-differentiated and slow-growing whereas undifferentiated and fast-growing late passages were refractory and were even stimulated at millimolar concentrations.93 Stimulation of cancer cells by melatonin does not appear to be an uncommon feature since the pineal hormone stimulated the growth of some primary cell cultures derived from human mammary as well as ovarian tumor biopsies.54 Such paradox reactions of cancer cells to hormone treatment have also been found in sub-clones of the mammary cell-line MCF-7 with respect to the antiestrogen tamoxifen.94 Such unexpected hormonal effects on cellular growth can be attributed to mutational processes in cancer cells leading to profound derangements within intracellular signalling cascades which in normal cells underlie a complicated and stringent fine tuning by both intra- and extra cellular signals.

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Clinical Experience with the Treatment of Melatonin in Oncological Patients Clinical trials have been confined to patients suffering from advanced or even terminal malignancies since melatonin is not an officially approved drug which underwent systematic studies to determine its efficacy as well as toxicity. Comprehensive reviews of the studies performed until now were published recently. 95,96 The studies performed by Lissoni and colleagues deserve special mention since they are the most detailed and best documented. From the experience of this group having treated several hundreds of cancer patients it appears that the pineal hormone may indeed possess favourable effects if given in the late afternoon or evening to superimpose the endogenous surge of the hormone. Melatonin was initially administered to patients with advanced malignancies that were refractory to other types of treatment.97 Among the 54 patients treated only one patient showed an objective tumor response, minor responses were found in two cases and disease stabilization occurred in 20 patients. Also in other related studies98,99 melatonin did not effectively stop or slow down the course of advanced malignant tumor processes underlining that the pineal hormone cannot not be viewed as a cytostatic agent. Lissoni, however, often had the impression that melatonin treatment led to an improved general condition of his patients. This observation encouraged him to perform further studies on more than 200 patients affected by terminal malignant disease. These studies were mostly performed under randomised conditions using melatonin at 10-30 mg per day and achieved the following results: in bronchial cancer patients with metastases daily melatonin treatment significantly elevated one-year survival (+20%) compared to controls;100 patients suffering from glioblastoma melatonin given with brain irradiation significantly elevated one-year survival (+37%) compared to irradiation alone;101 in patients with heavily pretreated metastatic breast cancer melatonin co-administered with tamoxifen highly significantly extended one-year survival (+39%) compared to antiestrogen alone;102 patients with resected melanoma plus lymph node involvement had significantly less relapses after one year under melatonin (-44%) compared to best supportive care alone,103 and in patients with different solid tumors with brain metastases melatonin plus irradiation significantly increased one-year survival (+25%) compared to irradiation alone.104 The greatest number of patients treated by Lissoni with melatonin received the pineal hormone in combination with interleukin-2 (IL-2), a lymphokine which is known to possess considerable side-effects leading among others to high fever as well as hypotension. This treatment if combined with melatonin (40-50 mg per day) was found to be better tolerated and the therapeutic results were improved: among more than 500 patients with locally advanced or metastasised solid tumors of different origins one-year survival was significantly elevated.105-108 Even in case of patients with advanced solid tumors having a life-expectancy of less than six months and not responding to either chemotherapy or any other types of adjuvant treatment melatonin combined with IL-2 not only improved survival but also their quality of life.109 A favourable effect upon the quality of life appears to be a particularly important feature of chronic late afternoon/evening melatonin treatment of cancer patients since, from ethical point of view, a mere life extension in agony does not appear to be a desirable therapeutic aim. This effect of melatonin is most probably due to the well-documented sleep-inducing effect110 of the pineal hormone as well as a supportive effect on the endorphin system leading to reduction of pain.111 It can be assumed that an improvement of the general well-being of cancer patients helps to foster endogenous defence mechanisms against tumor growth and would thus indirectly contribute to a longer survival although, according to the preceding chapters, melatonin is unlikely to directly inhibit the growth of advanced malignancies. According to the critical review of Hrushesky95 the clinical results obtained so far with melatonin by Lissoni and colleagues are encouraging and justify a further systematic verification under double-blind placebo controlled conditions. If these results will indeed be confirmed they would justify a use of melatonin in cancer patients as an effective supportive measure to optimise existing oncotherapeutic strategies.

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Analysis of Melatonin and of Its Metabolite 6-Sulfatoxymelatonin in Cancer Patients Patients with Tumours of the Reproductive System Breast Cancer Patients Initially, the circadian profiles of urinary melatonin excretion were analysed in untreated postmenopausal Indian patients suffering from breast cancer (mostly primary localized tumors) as well as in controls with uterovaginal prolapse. A 30% depression of the 24-hour excretion of melatonin among the cancer patients was accompanied by a phase delay of the circadian peak leading to higher levels in the early morning than at night.112 Almost parallel to this study, Tamarkin et al113 detected a significant depression of nocturnal serum melatonin in unoperated primary breast cancer patients of clinical stages I and II if estrogen receptor positive tumors were present. Bartsch et al114 subsequently analysed the circadian rhythm of serum melatonin in German breast cancer and found a 56% depression of the amplitude compared to age-matched controls with benign breast disease. This depression showed a tumor-size dependency being more pronounced if big tumors were present (T3: -73%; T2: -53%, T1: -27%), an observation which was also reported by Hoffmann et al.115 Patients with recurrent breast tumors on the other hand which appeared after surgical removal of the primary tumor did not exhibit any depression of melatonin. Since the main metabolite of melatonin, 6-sulfatoxymelatonin (aMT6s), was found to show parallel circadian changes to melatonin in serum among patients with primary breast cancer116 it was concluded that the observed changes of melatonin in these individuals were not due to a modified hepatic metabolism of the pineal hormone. This finding paved the way for further studies using the noninvasive measurement of nocturnal urinary aMT6s to estimate the levels of circulating melatonin. In a subsequent study on untreated German breast cancer patients with localized primary tumors the nocturnal urinary excretion of aMT6s was found to be significantly depressed (-48%) showing an inverse correlation with tumor size as in the preceding study.117

Patients with Endometrial, Cervical or Ovarian Cancer

Karasek et al118,119 detected a significant depletion of the nocturnal surge of circulating melatonin by around 50% in endometrial cancer patients compared to age-matched controls. Grin and Grünberger120 observed an even greater depletion of circulating melatonin by about 90% in such patients. In contrast to endometrial cancer it appears that the presence of cervical cancer hardly affects circulating melatonin.119 In a large-sized study on patients with ovarian cancer (n=119) a high variability of the no7cturnal urinary excretion of aMT6s was found— some of them showing very low levels whereas others exhibited exceedingly high values.121 A similar observation was made in French ovarian cancer patients for the levels of circulating melatonin (Touitou and Bartsch, unpublished results). Karasek et al,119 however, did not detect changes of the circadian profiles of serum melatonin in patients suffering from ovarian cancer. It is conceivable that intra-ovarian melatonin production122 may have contributed to the high levels of circulating melatonin observed in some of these patients.

Patients with Prostate Cancer In two consecutive studies performed under comparable clinical conditions using the same RIA-methodology the circadian rhythm of serum melatonin was determined in untreated patients with benign prostatic hypertrophy (BPH) or with primary localized malignant prostate (PC) tumors. Patients with PC showed extremely low levels of nocturnal melatonin.123-125 When pooling the results of the studies a 71% depression of the melatonin amplitude resulted compared to patients with BPH.126 A sub-division of these patients according to the stage of their tumors showed an inverse correlation with tumor-size. At the T1-stage the amplitude of

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melatonin was depressed by 28% compared to patients with small BPH but patients with T2and T3/T4-tumors exhibited a drastic depletion by almost 80% compared to patients with BPH of comparable size. Interestingly, patients with so-called incidental carcinomas (PCi) which are small foci of highly differentiated malignant cells detected during the histological examination of BPH showed nocturnal serum melatonin concentrations that were higher than in BPH patients.123,124 Since the melatonin metabolite aMT6s in both serum and urine showed parallel changes to melatonin in the different groups of patients studied125 it has to be concluded that the depression of circulating melatonin in primary prostate cancer patients is not due to a modified peripheral metabolism of the pineal hormone but may either be caused by a reduced pineal secretion or an enhanced binding/degradation by tumor tissue.

Patients with Cancer Outside of the Reproductive System Patients with Thyroid Cancer In female Russian patients with primary thyroid cancer before surgery a 56% lower nocturnal excretion of aMT6s was found than in controls of comparable age not affected by thyroid disease.127,128 A similar depression of aMT6s was also detected in controls suffering from different types of benign thyroid disease127,128 indicating that thyroid enlargement, irrespective whether it is of malignant or benign nature, negatively affects the levels of circulating melatonin. In contrast to Kvetnaia et al127 Karasek et al96 found highly significantly elevated levels of nocturnal serum melatonin in thyroid cancer patients compared to healthy age-matched controls. It could well be that this elevation was due to the presence of distant metastases since elevated levels of melatonin were also detected in breast and prostate cancer patients suffering from disseminated disease.129

Patients with Larynx Cancer Male patients with primary larynx cancer only showed a marginal increase of the average nocturnal urinary aMT6s-excretion (+12%) compared to age-matched healthy controls. Sub-division of these patients according the size of their primary tumor revealed that patients at the T2-stage exhibited a 125% increase compared to controls whereas patients with T3-tumors were at the level of controls and those with T4-tumors showed a 62% depression.127

Bronchial Cancer Patients In male patients with bronchial cancer of clinical stages T1-4N0-3M0-1, the majority being nonsmall cell cancers, a highly significantly depression of nocturnal aMT6s-excretion by 60% was observed.127 Viviani et al130 found obliterated day/night variations of circulating melatonin in patients with nonsmall cell bronchial cancer whereas Dogliotti et al131 reported very high early morning and night-time melatonin levels in blood of patients at clinical stages III and IV, however, very low concentrations if small cell lung cancers were present.

Stomach Cancer Patients In male patients with primary unoperated stomach cancer without metastases (T3-4N2-XM0) Kvetnaia et al127 found a 59% depletion of the nocturnal urinary excretion of aMT6s. In an earlier study Kvetnoi and Levin132 detected a phase delay of urinary melatonin excretion in such patients leading to depressed levels at night and elevated levels in the morning.

Colorectal Cancer Patients In patients with primary unoperated colorectal carcinoma, with or without metastases, Khoory and Stemme133 observed a very pronounced depletion of nocturnal plasma melatonin. In contrast to this, Kvetnaia et al127 found a 44% higher nocturnal urinary aMT6s-excretion in operated and untreated male patients who were mostly affected by large tumors of stages T3 and T4 and which in some cases showed a pronounced lymph node involvement and distant metastases.

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Patients with Other Types of Malignancies Incongruent changes were found for the 24h urinary excretion of aMT6s in patients with osteosarcoma:134 80% of them showed lower whereas the others had highly elevated levels compared to controls. In Hodgkin’s sarcoma Lissoni et al135observed clearly elevated concentrations of nocturnal circulating melatonin. From the above summarized results in patients with cancer of the reproductive tract or outside of the same it is obvious that melatonin can show considerable variations with respect to the levels of circulating melatonin even among patients affected by the same tumor type. Further studies are therefore required to better understand the mechanisms involved in such changes. For this purpose, studies analysing melatonin secretion and production in tumor-bearing animals are relevant.

Analysis of Melatonin in Tumor-Bearing Animals Initially, Vera Lapin observed a negative correlation between pineal melatonin content and tumor-size in rats bearing Yoshida tumors136 indicating an inhibition of pineal melatonin biosynthesis by cancer growth. This view was supported by studies of Leone and Skene137 as well as Schmidt et al138 who found that supernatants of cancer cells inhibit the production of pineal melatonin under in vitro conditions. As opposed to that, it was observed in F344 Fischer rats with DMBA-induced mammary tumors that their circannual rhythm of nocturnal urinary aMT6s-excretion was obliterated due to an elevated melatonin production.139 A similar observation was made in female BDII/Han rats during the development of spontaneous endometrial adenocarcinomas.139 F344 Fischer rats with an early tumor passage (derived from the above-mentioned DMBA-induced mammary tumors) showed an elevated melatonin production which was accompanied by an enhanced activity of arylalkylamine-N-acetyltransferase140 (AA-NAT, the rate-limiting step of pineal melatonin biosynthesis) which is under adrenergic control.141 These tumor-bearing animals also showed an activation of the sympathetic nervous system (elevated urinary excretion of norepinephrine but not of epinephrine) which in turn was due to a stimulation of cellular immunity (elevated urinary excretion of macrophage-derived biopterin and of γ-interferon in plasma).142 In contrast to that, rats with tumors of similar size but of a later passage, being fast growing and showing distant metastases, nocturnal peak plasma melatonin was depressed by almost 75%.142 This depression was not accompanied by either a reduced activity of AA-NAT or other steps of melatonin biosynthesis but circulating tryptophan, the precursor amino acid of melatonin, was drastically reduced. This reduction was not caused by cachexia since other amino acids remained unchanged (Bartsch C et al, unpublished results). Maestroni and Conti143 found that human mammary cancer tissue binds considerable amounts of melatonin. This renders an additional explanation for the observed depression of circulating melatonin in patients as well as animals with advanced tumors. It is conceivable that not only melatonin but also its precursor tryptophan may be trapped by cancer cells thus contributing to a deficiency of this amino acid in blood. Slominski et al144 recently reported that tryptophan is converted to melatonin within melanoma cells whereas Bartsch et al142 are assuming that melatonin may be catabolically cleaved to kynurenine derivatives within tumor tissue. This further adds to the complexity of the metabolism of melatonin in a cancer-affected organism. Further investigations are urgently needed to clarify details of these pathophysiological phenomena.

In Which Way Does the Depression of Circulating Melatonin in Cancer Patients Offer a Rationale for a Substitutional Therapy? If circulating melatonin is found to be depressed in patients with localized primary cancers it would appear logical to consider a substitutional therapy with the aim to control the malignant process. Such hopes, however, do not appear to be justified on the basis of the

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above-described experimental findings since tumor-bearing animals (e.g., with advanced serial transplants of DMBA-induced mammary cancers) showing a depression of circulating melatonin are totally refractory to a potential tumor-inhibitory effect of exogenous melatonin.145 Despite this, the clinical studies of Lissoni indicate that melatonin administration is apparently able to delay the course of advanced or even final malignant disease (see review ref. 95) leading to an extended survival. As mentioned before, it may be anticipated that this life-prolonging effect is probably due to favourable effects on the sub-systems of the body resulting in an improved neuroimmunological surveillance as well as endocrine balance which may help to control metastatic spread being a central determinant for the patients’ prognosis. An integral part of this so-called neuroimmunoendocrine effect of melatonin on malignancy could be to re-establish and -synchronize circadian disturbances affecting the autonomic nervous including the sleep-wake cycle146 as well as the neuroendocrine system,126,147,148 and perhaps even the central circadian clock in the suprachiasmatic nucleus.149 In addition, melatonin administration could positively affect the production and secretion of endogenous anti-tumor substances present in the pineal gland52-54,150,151 as well as in other organs48,152-154 helping to resist the formation of metastases. The observed elevated production of melatonin in patients with metastases and local recidives could therefore be viewed as an effort of the organism to resist the detrimental and destructive malignant process. The same could apply to the phase of early tumor development when endogenous melatonin secretion is apparently up-regulated.142 In this case it would be worth testing whether an additional administration of melatonin could inhibit tumor development and growth since experimental findings obtained both under in vivo and in vitro conditions indicate that melatonin is able to effectively control well-differentiated tumors. Finally, a word of caution regarding a potential administration of melatonin to patients suffering from haematopoietic neoplasias such as leukemia: experimental findings exist that the pineal hormone shortens the survival of mice with leukemia.66,67 It was also found that melatonin stimulated the growth of some primary cell lines derived from human mammary or ovarian biopsies.54 Therefore further systematic clinical studies on cancer patients are needed and it will not be advisable to advocate an uncontrolled self-treatment with melatonin by oncological patients.

Potential Diagnostic Relevance of Melatonin in Oncology A central question is whether changes of circulating melatonin in cancer patients could be used for diagnostic purposes. Reductions of melatonin are found in certain types of malignancy, such as breast and prostate cancer, but mainly if medium-sized or large primary tumors are present,121 i.e., at a time when the malignant process is clinically clearly evident. Therefore such changes of circulating melatonin will only have a limited diagnostic value compared to conventional tumor markers used in clinical chemistry. Endogenous melatonin on the other hand is up-regulated if local recidives or distant metastases develop.121 Relative to the depression of melatonin during the growth of the unoperated localized primary tumor this alteration is quite dramatic. If melatonin secretion was measured at regular intervals during the course of the malignant disease it would thus be possible to obtain indications for the growth of new cancer cells after surgical removal of the primary tumor. For this purpose noninvasive determinations of nocturnal urinary 6-sulfatoxymelatonin (being a reliable estimate of nocturnal melatonin production in cancer patients121) may be included in the monitoring program of oncological patients parallel to conventional tumor markers. A similar approach may also be used for the early diagnosis of cancer since the production of the pineal hormone is elevated by the growth of early stages of cancer, such as in case of patients with so-called incidental carcinoma124 as well as in animals bearing well-differentiated tumors.140,142 Since melatonin shows a considerable inter-individual variation but a high individual stability with properties of a personal marker rhythm155 it would be necessary to establish individual norms rather than normal ranges among healthy populations.

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Potential Significance of (Patho)Physiological Changes of Melatonin for the Aetiology of Cancer Recent studies revealed that the central circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus is involved in the control of cancer: destruction of the SCN149 as well as a deficit of the circadian Period2 gene156 accelerates tumor development. The circadian production and secretion of melatonin is driven by the SCN and it serves as an important output signal of the central clock conveying information regarding time of day to practically all parts of the body including the SCN itself.141 The endogenous circadian oscillation of the SCN and thereby the secretion of pineal melatonin is synchronized to environmental photoperiods by retinally perceived light.141 The SCN and the pineal gland together with the eyes are a functional unit serving as the central circadian time-keeping system of the body. This system is apparently negatively affected by cancer growth and the above-described increasing depression of circulating melatonin in cancer patients and tumor-bearing animals in the presence of localized primary tumors129 together with temporal neuroendocrine disturbances147,148 can be viewed as a weakening of central control mechanisms over malignancy to facilitate metastatic spread. The circadian time-keeping system including melatonin is profoundly influenced by shift-work and East-West travels. Recent publications indicate that nurses on rotating night shifts over prolonged periods of time as well as female flight attendants working on long-distance flights seem to possess an increased risk to develop breast cancer.157,158 Light at night is known to inhibit melatonin production3,141 which when given uninterruptedly leads to total suppression of pineal secretion and is therefore called “physiological pinealectomy”. An obliteration of the nocturnal surge of melatonin, fully or even partially, stimulates experimental cancers.38,159 Erren160 discusses that demographic differences of breast cancer could follow systematic geographic patterns connected with seasonally modulated circadian rhythms of melatonin at different latitudes. Due to the high state of industrialization in Western nations connected with an unlimited access to artificial light it, however, appears likely that such geographic differences no longer exist and that a chronic self-chosen overexposure to light at night leads to a suppression of nocturnal melatonin secretion. This so-called “light pollution” may not only have extinguished previous seasonal endocrine and behavioural patterns including reproduction in humans161 but could also contribute to a higher risk for the development of hormone-dependent cancers. This view is shared by Stevens162 who initially hypothesized that electric power via an inhibition of melatonin may stimulate breast cancer163 but he extended this theory to different components of the electromagnetic spectrum including light. The effects of extremely low frequency electric and magnetic fields due to alternating currents as well as of pulsed high frequency electromagnetic fields connected with mobile telecommunication on both melatonin164-166 and experimental tumor growth167,168 are still quite controversial but according to our present knowledge appear to have no grave general health hazards. It has also been hypothesized that drugs which inhibit melatonin secretion may lead to an enhanced risk for breast cancer.162 Epidemiological studies, however, have provided no sound evidence that β-blockers and certain benzodiazepines which suppress melatonin secretion169-171 lead to an elevated cancer risk.172,173 It is possible that absence of pineal melatonin circadian secretion could be of a different physiological significance in humans than in experimental animals. There is no evidence that patients after pinealectomy due to the presence of a pineal tumor exhibit an increased cancer risk. The same applies to those individuals who naturally show very low or even absent circadian amplitudes of the pineal hormone. In order to understand the real pathophysiological role of melatonin for the aetiology of cancer it may perhaps be necessary to correlate life-long individual patterns of melatonin secretion with the trend to develop neoplasias. This, however, is beyond the current scope of medical research. Findings in experimental animals with a tendency to develop spontaneous tumors indicate that there may be a decisive temporal biological window before the onset of puberty during which manipulations of circulating melatonin could decisively modulate the development of endometrial cancer in

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adulthood.59 Nocturnal serum melatonin is known to physiologically decline during human growth and puberty.174 Could there perhaps be a connection between the earlier onset of human puberty as well as growth acceleration in our days, “light pollution” (to further reduce melatonin secretion) and the elevated incidence of hormone-dependent cancers such as of breast and prostate?

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150. Bartsch H, Bartsch C, Flehmig B. Pineal anti-tumor activity (PATA) of rats under different physiological conditions. In: Trentini GP, DeGaetani C, Pevet P, eds. Fundamentals and Clinics in Pineal Research. New York: Raven Press, 1987:381-384. 151. Ebels I, Benson B. A survey of the evidence that melatonin and unidentified pineal substances affect neoplastic growth. In: Bartsch C, Bartsch H, Blask DE et al, eds. The Pineal gland and Cancer: Neuroimmunoendocrine Mechanisms in Malignancy. Berlin: Springer, 2001:275-293. 152. Smith RC, Litwin MS, Lu Y et al. Identification of an endogenous inhibitor of prostatic carcinoma cell growth. Nat Med 1995; 1:1040-1045. 153. To CT, Tsao MS. The roles of hepatocyte growth factor/scatter factor and met receptor in human cancers (Review). Oncol Rep 1998; 5:1013-1024. 154. Calnan DP, Westley BR, May FE et al. The trefoil peptide TFF1 inhibits the growth of the human gastric adenocarcinoma cell line AGS. J Pathol 1999; 188:312-317. 155. Arendt J. Melatonin. Clin Endocrinol 1988; 29:205-209. 156. Fu L, Pelicano H, Liu J et al. The circadian gene Period2 plays an important role in tumor progression and DNA damage response in vivo. Cell 2002; 111:41-50. 157. Schernhammer ES, Laden F, Speizer FE et al. Rotating night shifts and risk of breast cancer in women participating in the nurses’ health study. J Natl Cancer Inst 2001; 93:1563-1568. 158. Rafnsson V, Tulinius H, Jonasson JG et al. Risk of breast cancer in female flight attendants: a population-based study (Iceland). Cancer Causes Control 2001; 12:95-101. 159. Dauchy RT, Sauer LA, Blask DE et al. Light contamination during the dark phase in “photoperiodically controlled” animal rooms: effect on tumor growth and metabolism in rats. Lab Anim Sci 1997; 47:511-518. 160. Erren TC. Does light cause internal cancers? The problem and challenge of an ubiquitous exposure. Neuroendocrinology Lettt 2002; 23(Suppl 2):61-70. 161. Roenneberg T, Aschoff J. Annual rhythm of human reproduction: II. Environmental correlations. J Biol Rhythms 1990; 5:217-239. 162. Stevens RG. Circadian disruption and breast cancer. In: Bartsch C, Bartsch H, Blask DE et al, eds. The Pineal Gland and Cancer: Neuroimmunoendocrine Mechanisms in Malignancy. Berlin: Springer, 2001:512-517. 163. Stevens RG, Davis S. The melatonin hypothesis: electric power and breast cancer. Environ Health Perspect 1996; 104(Suppl 1):135-140. 164. Selmaoui B, Touitou Y. Magnetic field exposure and pineal melatonin production. In: Bartsch C, Bartsch H, Blask DE et al, eds. The Pineal Gland and Cancer: Neuroimmunoendocrine Mechanisms in Malignancy. Berlin: Springer, 2001:534-540. 165. Fedrowitz M, Westermann, Löscher W. Magnetic field exposure increases cell proliferation but does not affect melatonin levels in the mammary gland of female Sprague Dawley rats. Cancer Res 2002; 62:1356-1563. 166. Radon K, Parera D, Rose DM et al. No effects of pulsed radiofrequency electromagnetic fields on melatonin, cortisol, and selected markers of the immune system in man. Bioelectromagnetics 2001; 22:280-287. 167. Anderson LE, Morris JE, Sasser LB et al. Effects of 50- or 60-hertz, 100 microT magnetic field exposure in the DMBA mammary cancer model in Sprague-Dawley rats: possible explanations for different results from two laboratories. Environ Health Perspect. 2000; 108:797-802. 168. Bartsch H, Bartsch C, Seebald E et al. Chronic exposure to a GSM-like signal (mobile phone) does not stimulate the development of DMBA-induced mammary tumors in rats: results of three consecutive studies. Radiat Res 2002; 157:183-190. 169. Arendt J, Bojkowski C, Franey C et al. Immunoassay of 6-hydroxymelatonin sulfate in human plasma and urine: abolition of the urinary 24-hour rhythm with Atenolol. J Clin Endocrinol Metab 1985; 60:1166-1173. 170. McIntyre IM, Burrows GD, Norman TR. Suppression of plasma melatonin by a single dose of the benzodiazepine alprazolam in humans. Biol Psychiatry 1988; 24:108-112. 171. Monteleone P, Forziati D, Orazzo C et al. Preliminary observations on the suppression of nocturnal plasma melatonin levels by short-term administration of diazepam in humans. J Pineal Res 1989; 6:253-258. 172. Felmeden DC, Lip GY. Antihypertensive therapy and cancer risk. Drug Saf 2001; 24:727-739. 173. Coogan PF, Rosenberg L, Palmer JR et al. Risk of breast cancer according to use of antidepressants, phenothiazimes, and benzdiazepines (United States). Cancer Causes Control 2000; 11:839-845. 174. Waldhauser F, Weiszenbacher G, Tatzer E et al. Alterations in nocturnal serum melatonin levels in humans with growth and aging. J Clin Endocrinol Metab 1988; 66:648-652.

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CHAPTER 8

Expression and Signal Transduction Pathways of Melatonin Receptors in the Pituitary Hana Zemkova, Ales Balik and Stanko S. Stojilkovic

Abstract

P

ituitary cells from neonatal animals express functional MT1 subtype of melatonin receptors that signal through pertussis toxin-sensitive G proteins. Their activation by melatonin leads to a decrease in cAMP production and activity of protein kinase A, and attenuation of gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion. Single cell calcium and electrophysiological recordings revealed that reduction in gonadotropin release results from melatonin-induced inhibition of GnRH-stimulated calcium signaling. Melatonin inhibits both components of calcium signaling in gonadotrophs, calcium influx through voltage-dependent calcium channels and inositol (1,4,5)-trisphosphate-mediated calcium release from intracellular stores. Inhibition of calcium influx and the accompanied calcium-induced calcium release from ryanodine-sensitive intracellular pools by melatonin results in a delay of GnRH-induced calcium signaling. On the other hand, attenuation in GnRH-induced calcium release affects the amplitude of calcium signals. The potent inhibition of GnRH-induced calcium signaling and gonadotropin secretion by melatonin provides an effective mechanism to protect premature initiation of pubertal changes that are dependent on gonadotropin plasma levels. During the development, the tonic inhibitory effects of melatonin on GnRH action gradually attenuate, due to a decline in expression of functional melatonin receptors and changes in GnRH receptor signaling pathways. In adult animals, melatonin does not affect pituitary functions directly, whereas the coupling between melatonin release and hypothalamic functions, including GnRH release, are preserved, and are critically important in synchronizing the external photoperiods and reproductive functions through still not well characterized mechanisms.

Introduction In seasonally breeding mammals, annual cycle of external light has important functions in the reproduction and causes seasonal changes in appetite, energy metabolism, and growth of fibers and horns.1 The production of melatonin plays a central role in this very complex process that reflects on activity of numerous pathways. The activity of melatonin-producing enzyme in pineal gland, arylalkylamine N-acetyltransferase (serotonin N-acetyltransferase, EC2.3.1.87), precisely reflects duration of the night (Fig. 1A). Melatonin is necessary and sufficient for entrainment of seasonal photoperiodic responses to the annual cycle of day lenght.2 In addition to controlling the seasonal biological rhythms in mammals, melatonin also participates in the coordination of circadian rhythms with external light-dark cycle.3,4 The extracellular messenger functions of melatonin are mediated by its plasma membrane receptors expressed in central and peripheral target tissues. In mammals, the high density of melatonin receptors are localized in the hypothalamic suprachiasmatic nuclei (SCN) 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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Figure 1. Characterization of melatonin production by pineal glan and expression of melatonin receptors in pituitary cells. A) The effects of long “summer-like” (ANAT) (upper trace) and short “winter-like” (bottom trace) day lengths on daily rhythm in N-acetyl transferase activity in pineal gland – schematic repersentation. B) Developmental decrease in concentration of melatonin receptors in pituitary rats – schematic repersentation. For experimental information illustrated in A and B, see Hoffmann et al, 1981113 and Vanecek 1988.9 C) RT-PCR analysis of transcripts for melatonin MT1 and MT2 receptor subtypes in pituitary from neonatal rats.

GnRH-secreting neurons within the preoptic area and/or the mediobasal hypothalamus, depending on the species. Melatonin receptors in high density are also present in pars tuberalis and pars distalis regions of anterior pituitary. Melatonin receptors in the SCN mediate melatonin feedback action and are responsible for the phase-shifting effects on the circadian rhythms.4-6

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Hypothalamic GnRH neurons and pituitary are the main sites of the reproductive actions of melatonin.7,8 However, the effects of melatonin in pituitary pars distalis are time-limited, as melatonin receptor expression progressively decreases over the perinatal period (Fig. 1B).9,10 The structures of two high-affinity melatonin receptor subtypes, termed MT1 and MT2, have been identified in mammalian brain.11,12 The MT1 subtype seems to be widely expressed and functionally important subtype, including hypothalamus and pituitary, whereas the expression of MT2 subtype is more localized,6,13 Other forms or states of the mammalian melatonin receptor may also exist in mammalian brain. This includes the expression of the third type of melanin receptor (MT3), identified in central and peripheral hamster tissues,14,15 and a melatonin receptor-related protein, which does not bind iodinated melatonin.16 The principal effects of native melatonin receptors in many cell types are mediated by inhibiting the activity of adenylyl cyclase, leading to a decrease in intracellular levels of cAMP17-19 and inhibition of cellular processes regulated by this intracellular messenger, including activation of protein kinase A.20,21 Functional studies with recombinant MT1 and MT2 receptors confirmed that melatonin inhibits agonist- and forskolin-stimulated adenylyl cyclase.6,11,22,23 In cells expressing MT1 receptors, inhibition of adenylyl cyclase pathway is occasionally accompanied with facilitation of phospholipase C activity, an enzyme that produces two intracellular messengers, inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG).24,25 Activation of the MT2 receptors also leads to the inhibition of soluble guanylyl cyclase activity,26 but to which extent this is a receptor-specific function is unclear. Melatonin receptors also inhibit voltage-dependent calcium influx and release of calcium from intracellular stores, as well as calcium-dependent hormone secretion, induced by other phospholipase C-coupled receptors. This phenomenon, well characterized in neonatal pituitary cells,27-30 will be discussed in detail in this chapter.

Photoperiods, Melatonin and Reproduction A majority of wild species have seasonal reproduction in order to give birth at the optimal time of year, usually spring, allowing the new-born to grow and develop under favorable temperature and food availability conditions. The seasonal variations of photoperiod (day length) serve as a signal to trigger changes in reproductive behavior, body fat storage, weight gain, or hibernation. Decreasing photoperiod lengths indicate that winter is approaching and allows species to prepare in advance. However, the same signal may trigger opposite reproductive actions, depending on species. In hamsters, for example, short photoperiods induce gonadal involution, decrease in circulating levels of gonadal and gonadotropic hormones, and inhibition of reproduction, whereas the gradual recovery of these functions occurs with extension of photoperiods.31 These changes ensure that hamsters, animals with a short gestation period, breed in the spring. In contrast, in sheep, which have a much longer gestation period, the shortening of days triggers breeding during the autumn and animals bear young during the spring.32 Humans also secrete melatonin in a pattern that reflects the environmental light-dark cycle, but the seasonal melatonin information is not an integral part of their reproductive cycle. The role of melatonin in animal reproduction was confirmed in experiments with its exogenous infusion, which successfully mimics the seasonal effects of changing photoperiod. For example, in Siberian hamster changes in reproduction that are adaptive for winter can be mimicked by administration of a short-day like melatonin signal directly to the SCN.33 In perinatal life in hamsters, melatonin may mimic maternal entraining signals.34 Removal of the pineal gland abolishes both the normal pattern of melatonin synthesis and seasonal changes in reproduction.35 Daily administration of melatonin delays sexual maturation in the male Wistar rats, and causes a pronounced decline in circulating gonadotropin level.36 Although rats are not photoperiod-sensitive animals, recently it has been found that melatonin mediates photo-responsiveness in F344 rats, which may be due to differences from other strains in the location, density, or affinity of their melatonin receptors.37

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Melatonin probably influences reproduction at three levels: the hypothalamic GnRH neurons, pituitary, and gonads and reproductive tissues. Melatonin microimplants into the area of preoptic and mediobasal hypothalamus of mice (areas that contain GnRH neurons) elicited complete gonadal involution, whereas its injection in other areas was ineffective.38 It has been hypothesized that melatonin acts directly on synapses of hypothalamic neurons and inhibits reproduction by decreasing GnRH synthesis and release.2,38 In accordance with this, it has been suggested recently that melatonin regulates GnRH gene transcription in the immortalized GnRH-secreting neurons in a cyclic manner.7,8 The possibility that melatonin directly modulates pituitary function was established in 1987 with finding that melatonin receptors are expressed in pituitary.9,39 The subsequent investigations in neonatal gonadotrophs revealed that melatonin inhibits GnRH-induced increase in several intracellular messengers, including cAMP,18 DAG40 and intracellular calcium concentrations ([Ca2+]i).41 Melatonin also acts at the level of the gonads, where it modulates androgen production by Leydig cells,42,43 and in prostate epithelial cells, where suppresses cGMP levels.44 Whereas the actions of melatonin in hypothalamus account for seasonal adaptation in reproductive cycle, the direct effects of this messenger in pituitary appear to be critical in controlling prepubertal hormonal milieu.

Localization of Melatonin Receptors

The radioligand125 I-melatonin has been commonly used to localize binding sites in central and peripheral tissues. Autoradiographic studies indicated that melatonin receptors are expressed in brain, with high density of receptors in hypothalamic SCN,39,45-47 as well as outside the brain, including retina,48 anterior pituitary,9,39,49 some arteries,50 and in cells of immune system.51 In mammals, the pars tuberalis of anterior pituitary contains the highest concentration of melatonin receptors, whereas in pars distalis melatonin binding is restricted to gonadotroph fraction of secretory cells.10,52,53 Melatonin receptors are expressed more widely in fetal and newborn animals, both in the SCN10 and pituitary.9,54 In the pars distalis, melatonin receptor number gradually declines over the perinatal period (Fig. 1B)9,54 to about 10% of the initial neonatal number of melatonin receptors.9 In the pars tubelaris, the expression of melatonin receptors does not change during the development.55 The structure of two melatonin receptor subtypes, called MT1 and MT2, has been identified in mammals by cloning.11,12,56,57 Both receptors belong to the seven-transmembrane-domain G protein-coupled receptor superfamily that signals through pertussiss toxin-sensitive Gi/Go pathways.17,19,27 Both receptor subtypes exhibit high affinity for melatonin; MT1 binds melatonin with Kd of 20-40 pM, whereas MT2 binds this agonist with Kd of about 200 pM.46 The pharmacology of MT1 and MT2 receptors is relatively poor58,59 and the lack of specific antagonists for these two receptors limits investigations on their expression and function. For example, luzindole is the only known effective antagonist that has slight selectivity for the MT2 receptor subtype.60 The mammalian MT1 receptor is expressed in hypothalamic SCN and hypophyseal pars tuberalis.11 Quantification of MT1 mRNA expression by PCR and melatonin binding revealed that the obvious developmental decrease in melatonin receptor number in pars tubelaris and SCN of Syrian hamster could not be attributed to the inhibition of mRNA expression, but rather could be related to the post-transcriptional blockade of the MT1 receptor expression.57 There are also circadian variations in melatonin receptor density in pars tubelaris, which is directly regulated by the daily variations of melatonin itself.61 During the long photoperiod, melatonin receptor density in Syrian hamster pars tubelaris reaches its maximum in the first half of the light period and its minimum at the end of the night.62 The mammalian MT2 melatonin receptor has been found in retina and brain.12 A third receptor subtype, called MT3 receptor, has been cloned from zebra fish, Xenopus, and chickens, but not from mammals.22,63 However, the existence of mammalian MT3-like receptor has been recently hypothesized in both central and peripheral hamster tissues.14,15 A protein containing the melatonin-binding sites with MT3 characteristics was sequenced and it has 95% amino acid identity with the human enzyme quinone reductase 2, known mainly for its

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detoxifying properties. The functional role of this putative melatonin receptor in mammals thus remains to be established. The MT1 melatonin receptors are believed to mediate the majority of responses to melatonin in the SCN, including feedback control of circadian rhythms and control of prolactin and gonadotropin release.6,11 The role of MT2 melatonin receptor is less clear and it appears to mediate the melatonin inhibition of dopamine release in retina.6,12,64 In MT1 receptor-deficient mice, the MT2 melatonin receptor substitutes the role for MT1 in the phase-shifting response.6 The MT1 melatonin receptor mRNAs has been found in pituitary pars tuberalis of the rat.11,57,65 During the attempt to clone melatonin receptors from the human pituitary, a melatonin receptor-related protein was identified. This protein contains 57% amino acid sequence identity with the transmembrane domain 1 of the melatonin MT1 receptor, does not bind iodo-melatonin and its endogenous ligand and physiological roles are to be established.16 In situ hybridization showed that gonadotropin-positive cells represent a very small fraction of pars tuberalis cells and do not express MT1 receptor.66 MT1 receptor is expressed in pars distalis and is localized in gonadotroph fraction of cells, whereas the expression of MT2 subtype in pituitary is more questionable. The presence of both subtypes was found in teleost fish pituitary.67 Our recent RT-PCR analysis, however, suggested that both subtypes of melatonin receptor mRNAs are also expressed in anterior pituitary from neonatal rats, but that the expression of MT1 melatonin receptor is more robust (Fig. 1C).

Melatonin Actions in Gonadotrophs In the anterior pituitary gland, melatonin acts primarily at two secretory cell types: lactotrophs that secrete prolactin, and gonadotrophs that secrete two gonadotropins: luteinizing hormone and follicle-stimulating hormone. The mechanisms of melatonin actions in lactotrophs are unknown at large,68 whereas its actions in gonadotrophs are well documented. Functional studies showed that gonadotrophs express melatonin receptors and their activation leads to inhibition of GnRH-controlled gonadotropin release.69-73 It appears that down-regulation of adenylyl cyclase activity by melatonin receptors represents the major pathway that accounts for inhibition of GnRH-induced calcium signaling and secretion. However, these effects were observed only in neonatal gonadotrophs.41,70-73 This coincides with an early expression of high-density melatonin receptors and their gradual decline in the anterior pituitary during the postnatal development (Fig. 1B). A small fraction of melatonin receptors persists in the adult pituitary9, but melatonin is ineffective in these cells. At the present time, it is not clear what underlines the lack of effects of melatonin in adult gonadotrophs: the low expression level of receptors, or the lack of effective coupling of residual receptors to intracellular signaling pathways.

GnRH-Induced Signaling Reproductive functions in vertebrates are controlled by neuropeptide GnRH, also known as luteinizing hormone-releasing hormone (LHRH). This neuropeptide is synthesized by hypothalamic GnRH neurons and is secreted in a pulsatile manner into the hypophyseal portal system. So far, sixteen forms of GnRH have been isolated from the brain of vertebrates. In the vast majority of species, several forms occur in anatomically and developmentally distinct neuronal populations. In mammalian brain, two GnRH forms, called GnRH-I and GnRH-II, coexist. GnRH-II is the most evolutionarily conserved form of GnRH,74 but its function is not yet known. GnRH-I binds with high affinity to plasma-membrane GnRH receptors in gonadotroph cells.75 Like melatonin receptors, GnRH receptor belongs to the rhodopsin-like family of seven transmembrane domain receptors.76,77 In contrast to melatonin receptor, GnRH receptor is coupled to a pertussis toxin-insensitive Gq/G11 proteins that stimulate phospholipase Cβ pathway, leading to the generation of IP3 and DAG production78,79 and activation of phospholipase D pathway.80 However, a growing number of information also indicates that GnRH receptor cross-couples to Gs and Gi/o-signaling pathway.81

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Figure 2. Typical patterns of calcium signaling in gonadotrophs from adult animals in response to GnRH, native agonist for these cells. Measurements of intracellular calcium concentration ([Ca2+]i) were performed in cells from ovariectomized rats loaded with indo-1AM. The subthreshold response was usually observed in 10 pM to 1 nM concentration range, the baseline oscillations in 0.1 to 10 nM concentration range, and the biphasic oscillatory and non-oscillatory responses in 10 to 1000 nM concentration range of GnRH.

The intracellular signaling by GnRH receptors is relatively well established in gonadotrophs from adult rats,82,83 and the oscillatory pattern of GnRH-induced [Ca2+]i responses in gonadotrophs from immature animals is comparable to that observed in gonadotrophs from adult animals.84 In adult gonadotrophs IP3 binds to specialized tetrameric IP3 receptor-channel complex that spans the endoplasmic reticulum membrane85 and triggers oscillatory release of Ca2+ from the endoplasmic reticulum. DAG activates Ca2+-dependent protein kinase which in turn affects several pathways, including the extracellular Ca2+ entry via voltage-dependent calcium channels. Such influx is necessary for the long-lasting maintenance of oscillations in [Ca2+]i.86,87 The cross-coupling of GnRH receptors to Gs signalling pathway may also participate in modulating voltage-dependent Ca2+ influx and Ca2+ release from IP3-sensitive intracellular pool. Increase in the [Ca2+]i can take several forms. Figure 2 illustrates typical patterns of calcium signals: 1. low frequency oscillatory [Ca2+]i signaling waves (subthreshold oscillations) induced by low GnRH concentrations; 2. transient oscillatory [Ca2+]i waves known as baseline oscillations, with a cycle frequency of 3-20 min-1 controlled by GnRH concentration;

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Figure 3. Electrophysiological monitoring of GnRH-induced [Ca 2+] i oscillations as changes in calcium-controlled potassium currents (IK(Ca)) in neonatal gonadotrophs. A) Effects of apamin, a blocker of SK-type of IK(Ca) on GnRH-induced current oscillations. B) Attenuation of agonist-induced IK(Ca) oscillations by melatonin. Horizontal bars indicate the time of GnRH, apamin, and melatonin application. C) Electrophysiologocal recording from neonatal gonadotrophs in culture.

3. biphasic responses, consisting of an initial non-oscillatory spike, which amplitude is controlled by GnRH concentration, followed by baseline oscillations, which frequency is controlled by GnRH; 4. non-oscillatory [Ca2+]i responses, induced by pharmacological GnRH concentrations. GnRH-stimulated and IP3-mediated signal transduction pathway increases the [Ca2+]i from ~100 nM to ~1µM, which is sufficient to trigger gonadotropin release. In cells exhibiting baseline [Ca2+]i oscillations, gonadotropin release is also oscillatory.88 The recovery to the baseline [Ca2+]i levels in oscillating cells is accomplished by Ca2+ sequestering into the endoplasmic reticulum and mitochondria and by efflux outside the cell by Ca2+ pump-ATPase and Na+/Ca2+ exchange system in the plasma membrane. GnRH-stimulated increase in [Ca2+]i together with activated protein kinase C regulates many other cellular functions, including the ion channel activity and gene expression.89

Electrophysiological measurements have revealed that GnRH-induced [Ca2+]i transients trigger oscillatory changes of membrane potential driven by rhythmic opening and closing of apamin-sensitive, Ca2+-activated K+ channels. In voltage-clamped cells, measurements of Ca2+-activated K+ current can substitute for calcium measurements (Fig. 3A) and were frequently used as an additional method in characterizing the nature of calcium signaling in immature and adult gonadotrophs.73,90,91 There are three obvious experimental advantages for such measurements. First, this current monitors the [Ca2+]i changes in the plasma membrane domain. Second, the possible chelating effects of calcium dyes are eliminated. Third, the

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Figure 4. Effects of extracellular calcium influx and calcium-induced calcium release on initiation of calcium oscillations. Left traces, 10 µM GnRH-induced current oscillations in cells bathed in calcium-containing medium. Right traces, Effects of removal of extracellular calcium A) and the addition of 10 µM nifedipine, a blocker of voltage-dependent L-type calcium channels B) and 10µM ryanodine, a blocker of intracellular calcium release channels C) on initiation of calcium responses.

membrane potential is controlled, which provides an elegant method to eliminate voltage-dependent calcium influx or to substitute the periodic with steady calcium influx. However, this current depends on [Ca2+]i in a nonlinear and saturable manner, indicating that the amplitude of responses should be interpreted with reservation. Using such measurements, we found that neonatal gonadotrophs exhibit some characteristics not present in gonadotrophs from adult animals.73,92,93 When stimulated with GnRH in physiological concentration range, neonatal gonadotrophs exhibit more often non-oscillatory responses. More importantly, the coupling of voltage-dependent calcium influx with Ca2+ release from ryanodine-sensitive intracellular stores plays a critical role in initiation of GnRH-induced [Ca2+]i oscillations in neonatal gonadotrophs, but was lost in gonadotrophs from adult animals. In cells with blocked voltage-dependent calcium influx (by removal of extarcellular calcium or by the addition of nifedipine, a blocker of L-type voltage-dependent calcium channels), the latency preceding the GnRH-induced response is prolonged more than 3 times (Fig. 4A, B). Similar effect has ryanodine in concentrations that blocks Ca2+ release from intracellular pools (Fig. 4C). In other cell types, both IP3 and ryanodine receptors co-exist and interact, and calcium released from the ryanodine receptor-controlled stores significantly contributes to the rise in [Ca2+]i94,95 However, that is not the case in neonatal gonadotrophs. In these cells, calcium-induced calcium release from ryanodine-sensitive intracellular calcium pool is very small to generate global calcium signals, but is sufficient to amplify voltage-dependent calcium influx to the level needed to influence IP3-controlled Ca2+ release. The ryanodine

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Figure 5. Inhibitory melatonin effects are independent of the pattern of GnRH-induced calcium signals. Effects of melatonin on GnRH-induced calcium signaling in gonadotrophs exhibiting baseline oscillations (left panels) and irregular calcium fluctuations (right panels). Effects of melatonin were independent of the time of its application (A and B) and were also observed in cells bathed in calcium-deficient medium (C). Notice that removal of extracellular calcium does not abolish ongoing GnRH-induced calcium oscillations (D), whereas the addition of melatonin does it (A).

receptor has been found in other anterior pituitary cells, including GH3 immortalized pituitary cells.96 Thus, the expression and functional coupling of ryanodine-sensitive channels in neonatal gonadotrophs probably coincides with expression and function of melatonin receptors in these cells. It is also known that GnRH-induced [Ca2+]i oscillations of neonatal gonadotrophs rapidly disappear in the absence of extracellular calcium,84 whereas they last for a prolonged period in adult gonadotrophs.90,91 The short-lasting Ca2+ oscillations in neonatal gonadotrophs may indicate that their intracellular stores contain less amount of Ca2+ and that their refilling is highly dependent on voltage-gated calcium influx. The mechanism by which GnRH receptor activation leads to opening of dihydropyridine-sensitive Ca2+ channels is not clear. L-type Ca2+ channels could be modulated directly by G protein in a membrane-delimited way by βγ dimer released from activated G protein.97 Alternatively, GnRH could stimulate Ca2+ influx by phosphorylation of channel protein via protein kinase C signaling pathway.98

Melatonin Effects on GnRH Signaling In picomolar to low nanomolar concentration range, melatonin attenuates or completely inhibits GnRH-induced [Ca2+]i increase and prolongs latency of responses (Figs. 3B and 5B). The inhibitory effects of melatonin were observed in 40-70% of rat neonatal gonadotrophs.41,71-73,92 Melatonin inhibitory effects were not dependent on the time of its application and the pattern of GnRH-induced responses (Fig. 5), but were dependent on GnRH

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Figure 6. Dependence of melatonin-induced latency prolongation on GnRH concentration. A) Concentration-dependent effects of GnRH on the pattern of calcium signals recorded in the absence (left) or presence (right) of 1 nM melatonin. B) The relationship between GnRH concentration and amplitude (upper panel), frequency (middle panel), and latency (bottom panel). Open circles, GnRH-treated cells; filled circles, GnRH + melatonin-treated cells.

concentration (Fig. 6). However, the percentage of cells with complete inhibition of calcium signaling was more frequently observed in cells responding to GnRH with non-oscillatory signals and low-frequency oscillations (Fig. 5, right). Melatonin prolongs latency in a manner comparable to that observed in experiments with Ca2+-deficient solution and the addition of nifedipine and ryanodine (Fig. 4). During the sustained stimulation (10-30 min), inhibitory effects of melatonin were observed also in previously melatonin-insensitive cells,92 indicating that the strength of coupling of melatonin receptors with its intracellular signaling pathways varies among neonatal gonadotrophs. Latency prolongation by melatonin seems to involve melatonin-induced inhibition of extracellular Ca2+ entry, since the same effect was observed in Ca2+-deficient medium or in the presence of nifedipine.41,72,73 The effects of melatonin and Ca2+-deficient media on GnRH-induced calcium signaling were not additive.92 Provided that GnRH-stimulated cAMP/ protein kinase A pathway100,101 in neonatal gonadotrophs activates Ca2+ entry through voltage-dependent L-type Ca2+ channels,99 melatonin could inhibit Ca2+ entry by inhibiting cAMP production, the later being demonstrated in neonatal gonadotrophs.18 This effect of melatonin is probably mediated via α-subunit of pertussis toxin-sensitive Gi proteins (see model in Fig. 7), as indicated in amphibian melanophores17 or pars tuberalis cells from Djungarian hamsters19 and confirmed in cells expressing recombinat receptor.25

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Figure 7. Model of gonadotropin-releasing hormone and melatonin actions in neonatal gonadotrophs. Stimulation of gonadotropin-releasing receptors (GnRH-R) causes activation of phospholipase C (PLC) through Gq-dependent signaling pathway, leading to the production of diacylglycerol (DAG) and inositol (1,4,5)-triphosphate (IP3). DAG together with Ca2+ stimulates protein kinase C (PKC). The cross-coupling of GnRH to Gs signaling pathway accounts for stimulation of adenylyl cyclase (AC), leading to increase in cAMP production and activation of protein kinase A (PKA). Both PKC and PKA are involved in control of voltage-gated calcium channels (VGCC). The influx of Ca2+ through VGCC triggers Ca2+ release from ryanodine (Ry)-sensitive calcium pool. Calcium and IP3 coordinately regulate opening of IP3 receptor-channels expressed in endoplasmic reticulum (IP3 store), leading to a massive Ca2+ release from this pool, which frequently occurs in an oscillatory manner. The rise in Ca2+ is sufficient to trigger release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Melatonin receptors (MT-R) signal through Gi pathway to inhibit AC. Melatonin also stimulates PLC, but only in high concentrations and βγ dimer of Gi or α subunit of Gq accounts for stimulation of PLC. PKA phosphorylates IP3 receptor-channels, an action that facilitates IP3-Ca2+-controlled calcium release. Such organization of calcium signaling provides an effective mechanism for amplification of signals from VGCC through Ry store to IP3 store (indicated by thickness of arrows), whereas down-regulation of AC activity by MT-R provides an effective mechanism for inhibition of this cascade.

However, the inhibitory effect of melatonin on Ca2+ oscillations was also observed during GnRH stimulation in cells bathed in Ca2+-deficient medium (Fig. 5).73,92,102 This indicates that melatonin not only inhibits voltage-dependent calcium influx, but also inhibits Ca2+ release from intracellular Ca2+ stores. The mechanism by which melatonin inhibits IP3-dependent calcium signaling is largely unknown. The finding that melatonin has no effect on Ca2+ oscillations evoked by intracellular injection of IP392,93 could indicate that MT receptor inhibits the GnRH-stimulated signaling pathway upstream of IP3 receptor activation. Melatonin has been

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shown to inhibit the GnRH-induced increase in DAG,40 suggesting that it may inhibit phospholipase C activity. However, this is an unlikely explanation, because potentiation rather than inhibition of phospholipase C has been found in cells transiently expressing melatonin receptor, 24,25 as well as in neonatal gonadotrophs (see below). Because the majority of GnRH-stimulated DAG production comes from phospholipase D pathway,80 it is likely that melatonin inhibits this pathway. An alternative explanation for the observed effects on GnRH-induced Ca2+ mobilization is inhibition of adenylyl cyclase pathway. A cross-talk between the cAMP and phosphoinositide signaling pathways is well documented. For example, in hepatocytes the frequency of calcium oscillations triggered by hormones linked to IP3 production is increased by activation of receptors positively coupled to adenylyl cyclase.103-105 Because both melatonin receptor subtypes expressed in neonatal gonadotrophs are negatively coupled to adenylyl cyclase, it is reasonable to speculate that sensitivity of IP3 receptors for IP3 is lowered in cells with activated melatonin receptors. Depending on melatonin and GnRH concentration, melatonin exhibits two different effects on GnRH-induced [Ca2+]i increases in neonatal rat gonadotrophs.93 In the physiological concentration range, melatonin consistently inhibits Ca2+-oscillations induced by lower GnRH concentrations (2 nM) or delays the responses, with half-maximal inhibition at about 30 pM. These values are in good agreement with the 60 pM dissociation constant of 125I-melatonin binding found in binding studies on neonatal gonadotrophs,106 as well as with the 20-40 pM Kd value reported for the recombinant MT1 melatonin receptor.11 At higher (nanomolar) concentrations, melatonin also enhanced GnRH-induced Ca2+-dependent K+ current, but only in a fraction of the neonatal gonadotrophs stimulated with 10 nM GnRH.92 Melatonin alone (in the absence of GnRH) was unable to trigger Ca2+ signals73 or to change the pattern of calcium signals triggered by intracellular introduction of IP3.93 Thus, the potentiating effect of melatonin could be mediated by cross-coupling of melatonin receptors to Gq signaling pathway, as suggested in cells transiently expressing the recombinant melatonin receptors,25 or more probably by coupling of βγ-subunits of pertussis toxin-sensitive Gi protein to phospholipase C.24 In general, the physiological relevance of such coupling is low, because melatonin-induced [Ca2+]i increase has only been observed in response to non-physiological concentrations of melatonin and in about 10% of cultured ovine pars tuberalis non-gonadotroph cells expressing endogenous MT1 receptor, as well as in about 10% of neonatal rat gonadotrophs.

Development and Receptor Expression It looks contradictory to have operative a very sophisticated mechanism for pulsatile GnRH release in hypothalamus and in the same time also to have an effective system to block the action of GnRH in target pituitary cells, as it demonstrated in neonatal animals. However, such a dual control of gonadotroph function is physiologically justified. It provides an effective mechanism to down regulate, but not to abolish, gonadotropin secretion. During development reproductive functions have to be arrested, but low levels of plasma gonadotropins are needed for keeping operative gonadal steroidogenesis that is critical for numerous cellular pathways. Thus, GnRH release is required in pre-pubertal period, but its action should be attenuated in order to keep plasma gonadotropin levels bellow the threshold for initiation of peripubertal changes. In general, rapid desensitization of GnRH receptors could provide the potential mechanism for down-regulation of gonadotropin secretion. However, that is not the case with mammalian GnRH receptors; this receptor lacks the C-terminal tail, which slows-down the rate of receptor desensitization.82 The transient expression of high-density melatonin receptors in gonadotrophs appears to be critical for the fetal programming of the reproductive axis,29 i.e., melatonin may affect the onset of puberty37 through its inhibitory effects on the pubertal activation of the reproductive functions.107 Furthermore, the developmental down-regulation of melatonin receptors could serve to gradually establish normal action of GnRH on gonads and seasonal effects of melatonin on reproduction. Finally, there is a decline of pineal melatonin production with age, as a consequence of a deficit in the pathway of serotonin utilization.108 which could partly account for less effectiveness of this agonist in adults.

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In contrast to the physiological relevance of transient expression of melatonin receptors in gonadotrophs, the mechanism by which the developmental down-regulation of melatonin receptor expression is mediated is not clear at the present time. During embryonic development, anterior pituitary cells express mRNAs for several hormones in various combinations. For example in mice at embryonic day 16 plurihormonal population accounts for more than 60% of cells, at postnatal day 1 it is approximately 35%, and at postnatal day 38 it is only about 25%.109 Thus, it appears that developmental disappearance of melatonin receptors in anterior pituitary cells parallels the development loss of plurihormonal population of cells, suggesting that both phenomena could be controlled by the same mechanism. One possibility is that melatonin receptors are only expressed in plurihormonal gonadotrophs and that programmed cell death selectively removes the majority of these cells. However, the speed of cell turnover rates in young and adult rat anterior pituitary pars distalis is 60-70 days,110 arguing against the hypothesis that melatonin-sensitive gonadotrophs disappear within 20 days, unless these cells have much higher turnover rate than other anterior pituitary cells.30 Another possibility is that melatonin-sensitive cells are not yet fully differentiated and melatonin receptors disappear as a consequence of programmed cell differentiation. This hypothesis suggests that a single population of gonadotrophs always exist within the anterior pituitary and that down-regulation of melatonin receptor expression is a feature of differentiating cells.30 It is supposed that during the process of fetal development pituitary cells differentiate in a specific temporal and spatial manner so that particular hormones are expressed due to a cascade of transient signaling events and expression of cell-type specific transcription factors.111 Distribution and appearance of specific transcription factors that precede terminal differentiation of pituitary cell types thus may also parallel developmental disappearance of melatonin receptor.

Perspectives Although the importance of melatonin in control of reproductive functions is well established, still very little is known about the mechanisms by which melatonin receptors contribute to this process. It is obvious that melatonin influences reproduction by acting at two effectors: extrapituitary regions and directly on pituitary cells. The actions of melatonin directly in pituitary pars distalis cells appears to be related to the control of hormonal status during the development rather than adapting the pituitary function to seasonal and daily light cycles. With respect to the extrapituitary actions of melatonin, there are several recent breakthrough developments, which could provide a solid base for further investigations on the mechanisms by which melatonin signals for changes in photoperiods by modulating the pattern of GnRH release. These include technical achievements in investigations with GnRH neurons, specifically the generation of immortalized GnRH-secreting neurons and mice with fluorescence green protein-tagged GnRH neurons. In pituitary, the actions of melatonin in lactotroph population of cells are purely characterized. At the present time, it is unknown do these cells express melatonin receptors or is the action of melatonin on lactotrophs mediated indirectly. It is also unknown which cellular mechanisms were triggered by melatonin in pars tubelaris cells, and how they are related to prolactin secretion. The actions of melatonin in gonadotrophs are better characterized. Activation of melatonin receptors in these cells leads to the attenuation of GnRH-induced calcium signaling and calcium-controlled gonadotropin secretion. In addition, it is well established that both pathways for calcium signaling in these cells are affected, voltage-dependent calcium influx and calcium mobilization from endoplasmic reticulum. It is also reasonable to speculate that the negative coupling of melatonin receptors to adenylyl cyclase accounts for down-regulation of GnRH-induced calcium signaling. However, the role of cAMP in GnRH-induced calcium signaling has not been clarified in these cells. Further studies are needed to identify plasma membrane channels affected by activated melatonin receptors, and messengers mediating the action of melatonin receptors on these channels. These include the potential role of βγ subunits of pertussis toxin-sensitive G proteins in activating inward rectifier potassium channels, and down regulation of adenylyl cyclase by α subunit, which could reflect on activity of cyclic

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nucleotide-gated channels, protein kinase A-controlled cationic channels, and/or IP3 receptor-channels. Work in neonatal gonadotrophs also revealed two additional aspects of melatonin actions; the gradual decline in expression of melatonin receptors, which temporally coincides with the loss of multi-hormonal cells, and the ineffectiveness of residual receptors in post-pubertal animals. Also, the expression and coupling of ryanodine receptors with voltage-gated calcium influx appears to be limited to pre-pubertal period. These observations raised the questions about the mechanism of gonadotroph differentiation and its physiological relevance. Furthermore, calcium signaling in neonatal gonadotrophs seems to be more dependent on voltage-gated calcium influx than in gonadotrophs from post-pubertal animals, and that differentiation of cells results in enlarging the intracellular calcium pool, which operate in a manner comparable to that observed in skeletal muscles. Additional studies should address this hypothesis and provide details about such a complex transition during pre- and peri-pubertal periods. Finally, it has been reported recently about the existence of two types of GnRH receptors in mammalian pituitary.112 The majority of gonadotrophs from adult animals coexpress both types of GnRH receptors and their presence could account for the differential secretion pattern of luteinizing hormone and follicle-stimulating hormone.112 Whether or not these two GnRH receptors also differ in intracellular calcium signaling and what are their roles during development is not yet clear. We speculated that there are two GnRH-receptor signaling pathways operative in neonatal gonadotrophs, and that only one pathway is sensitive to melatonin inhibition.92 These initial studies, however, should be extended to specifically account for the existence of two subtypes of GnRH receptors in neonatal gonadotrophs.

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CHAPTER 9

The Role of Thermoregulation in the Soporific Effects of Melatonin: A New Perspective Saul S. Gilbert, Cameron J. van den Heuvel, Drew Dawson and Kurt Lushington

Abstract

M

elatonin was first purified nearly fifty years ago and has since been identified as a sleep promoting, thermoregulatory and circadian agent in humans. In this chapter we examine the shifting perspective on the endogenous functions of melatonin, from evidence examining its exogenous effects on central circadian physiology to its action on sleep via peripheral thermoregulation. We discuss the possibility that a function of melatonin is to initiate peripheral vasodilatation which, in turn, may act as a physiological trigger for sleep. From this exploration, we conclude that melatonin’s capacity to both promote distal peripheral heat loss and, hence, sleep, alone or in combination, act to reinforce circadian adaptation.

Introduction Since Lerner’s initial discovery in 1956, the endogenous roles of melatonin and its mechanism of action in humans have been a focus of ongoing research interest to sleep and circadian physiologists. As such, our understanding of melatonin has increased substantially, especially regarding its role in coordinating circadian physiology. By contrast, although it is known to have sleep promoting properties, the precise role that melatonin plays in sleep and how this role is mediated remains unclear. This review will examine how the perception of melatonin’s physiological role has developed in the last decades with a special focus on sleep and melatonin’s mechanisms of action. Further, together with an examination of the relationship between thermoregulation and sleep, we will examine the interaction between melatonin, sleep and thermoregulation.

Melatonin Synthesized enzymatically from serotonin, melatonin is a neurohormone that is secreted from the pineal gland during the subjective night in humans. In normally entrained subjects, it shows a characteristic plasma profile with excretion rising in the early evening to a peak in the early morning and then declining to undetectable levels during the subjective day. While melatonin is produced in many organisms from algae to mammals, and its role varies considerably across the phylogenetic spectra, in humans it appears to play a major role in coordinating circadian rhythmicity and a yet-to-be fully defined role in sleep and thermoregulation (for review, see ref. 1).

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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Historical Overview: Sleep, Body Temperature and Melatonin The Soporific Effect of Melatonin The interest shown by sleep researchers in melatonin follows from the early realization that it plays an endogenous role in the sleep process. Lerner and Case first established that large pharmacological (80 mg) doses of intravenous melatonin increased self-reported sleepiness in two young adults.2 This was later supported using more objective measures by Anton-Tay and colleagues who recorded a significant reduction in polysomnographically determined sleep onset latency following 0.25-1.25 mg/kg (i.v.) melatonin in six young adults.3 Although only a small number of studies were conducted in humans in the 1970s, these typically confirmed the earlier melatonin results. However in the 1980s, there was a marked increase in the number of studies investigating many aspects of melatonin administration. A particular focus in the 1980-90s was an exploration into melatonin as a ‘natural’ hypnotic with various groups examining the effects of exogenous melatonin in normals and, in the early 1990s, as a treatment in insomniacs. Over this period, rapid-release oral preparations were typically employed with acute oral dosages in the supraphysiological range (for example, 2-80 mg). In general, the findings indicated that melatonin significantly increased sleepiness and shortened the latency to sleep onset but did not affect sleep architecture. In addition, greater effects were typically evident in normals compared with insomniacs. Despite the early positive findings, and a considerable number of studies examining optimal dose duration, delivery routes, dosages and release formulation, melatonin’s initial promise as a natural sedative/hypnotic for the treatment of insomnia has since proved disappointing (e.g., ref. 4). Further, despite initial suggestions that low endogenous melatonin levels may predispose some individuals to poor sleep,5 little evidence of a direct relationship between endogenous melatonin levels and sleep have been forthcoming.6,7 A second line of enquiry into the effects of endogenous melatonin on sleep followed on from earlier work in animals where its role as a marker of circadian timing,8,9 and its capacity to modulate the timing of the internal body clock,10 was highlighted. As a consequence of these findings, it was suggested this phase shifting effect of melatonin may explain its effect on sleep in humans.10 Indirect support for such a role came from clinical studies on blind subjects (for review, see ref. 11). More direct support for Redman’s hypothesis was provided by Lewy and colleagues,12 who established a phase response curve (PRC) for melatonin. Lewy and Sack found a robust 1-2 hour phase advance in the circadian rhythm of core body temperature following three days of 0.5 mg oral melatonin administered 5-7 hrs before the onset of the unmasked endogenous melatonin rhythm. It was, therefore, established that the melatonin PRC is opposite (i.e., 180 degrees out of phase) to that of light. As such, it was clear that the phase shifting effects of melatonin were sufficient to explain the re-entrainment of the sleep-wake cycle in blind subjects as well as its utility as a treatment for circadian-related sleep disorders such as jet lag.13-19 However, apparent to researchers by 1993, was the fact that the phase shifting effects of melatonin were not sufficient to explain the short-term sedative/hypnotic effects observed following daytime melatonin administration.20 In recognition of this, several researchers began to explore the possibility that melatonin may affect sleep by mechanisms other than direct phase resetting of the circadian system (e.g., refs. 20-23). At around this time, a possible role of thermoregulation in mediating the soporific actions of melatonin directly was beginning to be investigated.

The Thermoregulatory Effect of Melatonin The capacity of melatonin to affect thermoregulation was first reported by Carman and colleagues in 1976.24 These authors administered 150-1600 mg of melatonin to neurologic and psychiatric patients and noted a decrease in core body temperature. Subsequent investigation by Åkerstedt et al, revealed that the circadian rhythms of melatonin and core body temperature

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were negatively correlated in young adult males under ad lib sleep conditions, and that the acrophase of the melatonin rhythm was coincident with the nadir in rectal temperature.25 In addition, these authors found that a significant percentage of the circadian variation in rectal temperature was explained by melatonin. These results revealed that a. the two rhythms were closely coupled, and b. that the amplitude of the melatonin rhythm may modulate the amplitude of the temperature rhythm (or vice versa). Further evidence supporting these propositions was provided by Wever who demonstrated that melatonin and rectal temperature rhythms displayed an inverse temporal relationship in young adults under free-running conditions.26 Furthermore, Wever demonstrated (with a forced desynchrony protocol) that, even under a 29h day, the melatonin and core temperature rhythms showed similar free-running periods and remained in synchrony with each other. Similarly, Arendt and colleagues reported a close inverse relationship between melatonin and rectal temperature rhythms in young males under a fractional desynchronization protocol where the “day” was either progressively increased or decreased by ten-minute increments.27 Shanahan and Czeisler later investigated the relationship between plasma melatonin and rectal temperature following exposure to bright light in young males under constant routine conditions.28 They demonstrated that the melatonin and rectal temperature rhythms maintained their usual phase relationship even after circadian phase inversion. Finally, Kräuchi and Wirz-Justice, examined the circadian rhythm of skin and core body temperature as well as melatonin excretion in a 34h constant routine.29 They found that the melatonin rhythm showed an inverse relationship with core body temperature rhythm, although it was phase delayed by 4.5h from the time of the hand temperature minimum and 6h from the time of the hand temperature maximum.

More direct evidence of the thermoregulatory effects of melatonin were reported by Strassman and colleagues in 1991 who found that intravenous infusions of physiological levels of melatonin blocked the temperature increase associated with photic suppression of nocturnal melatonin.23 Around this period, several groups also reported findings that oral doses of melatonin (0.1-5.0 mg) significantly reduced daytime core temperature,20,22 while it was also demonstrated that exposure to bright light (>2500 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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21. Badia P, Myers B, Boecker M et al. Bright light effects on body temperature, alertness, EEG and behavior. Physiol Behav 1991; 50(3):583-8. 22. Cagnacci A, Elliott JA, Yen SS. Melatonin: A major regulator of the circadian rhythm of core temperature in humans. J Clin Endocrinol Metab 1992; 75(2):447-52. 23. Strassman RJ, Qualls CR, Lisansky EJ et al. Elevated rectal temperature produced by all-night bright light is reversed by melatonin infusion in men. J Appl Physiol 1991; 71(6):2178-82. 24. Carman JS, Post RM, Buswell R et al. Negative effects of melatonin on depression. Am J Psychiatry 1976; 133(10):1181-6. 25. Akerstedt T, Froberg JE, Friberg Y et al. Melatonin excretion, body temperature and subjective arousal during 64 hours of sleep deprivation. Psychoneuroendocrinology 1979; 4(3):219-25. 26. Wever RA. Light effects on human circadian rhythms: A review of recent Andechs experiments. J Biol Rhythms 1989; 4(2):161-85. 27. Arendt J, Bojkowski C, Folkard S et al. Some effects of melatonin and the control of its secretion in humans. Ciba Found Symp 1985; 117:266-83. 28. Shanahan TL, Czeisler CA. Light exposure induces equivalent phase shifts of the endogenous circadian rhythms of circulating plasma melatonin and core body temperature in men. J Clin Endocrinol Metab 1991; 73(2):227-35. 29. Krauchi K, Wirz-Justice A. Circadian rhythm of heat production, heart rate, and skin and core temperature under unmasking conditions in men. Am J Physiol 1994; 267(3 Pt 2):R819-29. 30. Campbell SS, Dawson D. Aging young sleep: A test of the phase advance hypothesis of sleep disturbance in the elderly. J Sleep Res 1992; 1(3):205-210. 31. Barchas J, DaCosta F, Spector S. Acute pharmacology of melatonin. Nature 1967; 214(91):919-20. 32. Kleitman N, Doktorsky A. The effect of the position of the body and sleep on rectal temperature in man. Am J Physiol 1933; 104:340-343. 33. Aschoff J. Wechselwirkung zwischen Kern und Schale im Wärmehaushalt. Physikalische Therapie 1956; 3:113-133. 34. Murphy PJ, Campbell SS. Nighttime drop in body temperature: a physiological trigger for sleep onset? Sleep 1997; 20(7):505-11. 35. Van Den Heuvel CJ, Kennaway DJ et al. Effects of daytime melatonin infusion in young adults. Am J Physiol 1998; 275(1 Pt 1):E19-26. 36. Aschoff J, Wever R. Spontanperidik des Menschen bei Ausschluss aller Zeitbeber. Natur-wissenschaften 1962; 49:337-342. 37. Mills JN, Minors DS, Waterhouse JM. The circadian rhythms of human subjects without timepieces or indication of the alternation of day and night. J Physiol 1974; 240(3):567-94. 38. Czeisler CA, Weitzman E, Moore-Ede MC et al. Human sleep: its duration and organization depend on its circadian phase. Science 1980; 210(4475):1264-7. 39. Strogatz SH, Kronauer RE, Czeisler CA. Circadian regulation dominates homeostatic control of sleep length and prior wake length in humans. Sleep 1986; 9(2):353-64. 40. Lack LC, Lushington K. The rhythms of human sleep propensity and core body temperature. J Sleep Res 1996; 5(1):1-11. 41. Cajochen C, Dijk DJ, Borbely AA. Dynamics of EEG slow-wave activity and core body temperature in human sleep after exposure to bright light. Sleep 1992; 15(4):337-43. 42. Edwards SJ, Montgomery IM, Colquhoun EQ et al. Spicy meal disturbs sleep: an effect of thermoregulation? Int J Psychophysiol 1992; 13(2):97-100. 43. Cagnacci A, Soldani R, Romagnolo C et al. Melatonin-induced decrease of body temperature in women: a threshold event. Neuroendocrinology 1994; 60(5):549-52. 44. Murphy PJ, Campbell SS. Enhanced performance in elderly subjects following bright light treatment of sleep maintenance insomnia. J Sleep Res 1996; 5(3):165-72. 45. Van Den Heuvel CJ, Reid KJ, Dawson D. Effect of atenolol on nocturnal sleep and temperature in young men: reversal by pharmacological doses of melatonin. Physiol Behav 1997; 61(6):795-802. 46. De Koninck J, Swingle PG, Herbert M et al. Self-regulation ofcore body temperature and sleep. Sleep Research 1993; 22:399. 47. Badia P, Murphy PJ, Myers BL et al. Alcohol ingestion and night time melatonin levels. J Sleep Res 1994; 23:477. 48. Gilbert SS, van den Heuvel CJ, Dawson D. Daytime melatonin and temazepam in young adult humans: equivalent effects on sleep latency and body temperatures. J Physiol 1999; 514(Pt 3):905-14. 49. Gilbert SS, Burgess HJ, Kennaway DJ et al. Attenuation of sleep propensity, core hypothermia, and peripheral heat loss after temazepam tolerance. Am J Physiol Regul Integr Comp Physiol 2000; 279(6):R1980-7. 50. Horne JA, Reid AJ. Night-time sleep EEG changes following body heating in a warm bath. Electroencephalogr Clin Neurophysiol 1985; 60(2):154-7.

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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.

References 1. United nations, department of economic and social affairs. World Population Ageing: 1950-2050. New York: United Nations, 2001. 2. United nations, population division, department of economic and social affairs. World Population Prospects: The 2000 Revision. Highlights. New York: United Nations, 2001. 3. Schulman C, Lunefeld B. The aging male. World J Urol 2002; 20:4-10. 4. Grimley Evans J. Aging and medicine. J Intern Med 2000; 247:159-167. 5. Rozencweig R, Grad BR, Ochoa J. The role of melatonin and serotonin in aging. Med Hypotheses 1987; 23:337-352. 6. Grad BR, Rozencweig R. The role of melatonin and serotonin in aging: Update. Psychoneuroendocrinology 1993; 18:283-295. 7. Maestroni GJM, Conti A, Pierpaoli W. Pineal melatonin, its fundamental immunoregulatory role in aging and cancer. Ann NY Acad Sci 1988; 521:140-147. 8. Pierpaoli W, Dall’Ara A, Pedrinis E et al. The pineal control of aging: The effects of melatonin and pineal grafting on survival of older mice. Ann NY Acad Sci 1991; 621:291-313. 9. Pierpaoli W, Regelson W. Pineal control of aging: Effects of melatonin and pineal grafting in aging mice. Proc Natl Acad Sci USA 1994; 91:787-791. 10. Reiter RJ. The pineal gland and melatonin in relation to aging: A summary of the theories and of the data. Exp Gerontol 1995; 30:199-212. 11. Reiter RJ. Melatonin and aging. In: Morley JE, Ambrecht HJ, Coe RM et al, eds. The Science of Geriatrics. New York: Springer, 2000:323-333. 12. Touitou Y. Human aging and melatonin. Clinical relevance Exp Gerontol 2001; 36:1083-1110. 13. Karasek M, Reiter RJ. Melatonin and aging. Neuroendocrinolo Lett 2002; 23(suppl. 1):14-16. 14. Pandi-Perumal SR, Seils LK, Kayumov L et al. Senescence, sleep, and circadian rhythms. Ageing Res Rev 2002; 1:559-604. 15. Arendt J. Melatonin and the mammalian pineal gland. London: Chapman and Hall, 1995. 16. Karasek M. Melatonin in humans: Where we are 40 years after its discovery. Neuroendocrinol Lett 1999; 20:179-188.

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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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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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69. Kvetnoy IM. Extrapineal melatonin in pathology: new perspectives for diagnosis, prognosis and treatment of illness. Neuroendocrinol Lett 2002; 23 Suppl 1:92-96. 70. Kvetnoy IM. Neuroimmunoendocrinology: Where is the field for study? Neuroendocrinol Lett 2002; 23:119-120. 71. Kvetnoy IM. The APUD system (structure-function organization and biological significance in normal and pathological states). Adv Physiol Sci 1987; 18:84-102. 72. Larsson L-I. On the possible existence of multiple endocrine, paracrine and neurocrine messengers in secretory system. Invest Cell Pathol 1980; 3:73-85. 73. Lee PP, Pang SF. Melatonin and its receptors in the gastrointestinal tract. Biol Signals 1993; 2:181-193. 74. Lerner A, Case J, Takahashi J. Isolation of melatonin, the pineal gland factor that lightens melanocytes. J Amer Chem Soc 1958; 80:2587-2589. 75. Lewinski A. Rybicka I, Wajs E et al. Influence of pineal indolamines on the mitotic activity of gastric and colonic mucosa epithelial cells in the rat: Interaction with omeprazole. J Pineal Res 1991; 10(2):104-108. 76. Lissoni P, Malugani F, Malysheva O et al. Neuroimmunotherapy of untreatable metastatic solid tumors with subcutaneous low-dose interleukin-2, melatonin and naltrexone: Modulation of interleukin-2-induced antitumor immunity by blocking the opioid system. Neuroendocrinol Lett 2002; 23(4):341-344. 77. Lissoni P. Is there a role for melatonin in supportive care? Support Care Cancer 2002; 10(2):110-116. 78. Mei Q, Yu JP, Xu JM et al. Melatonin reduces colon immunological injury in rats by regulating activity of macrophages. Acta Pharmacol Sin 2002; 23(10):882-886. 79. Menendez-Pelaez A, Reiter RJ. Distribution of melatonin in mammalian tissues: The relative importance of nuclear versus cytosolic localization. J Pineal Res 1993; 15:59-69. 80. Motilva V, Cabeza J, Alarcon de la Castra C. New issues about melatonin and its effects on the digestive system. Curr Pharm Des 2001; 7:909-931. 81. Nilsson AH. The gut as the largest endocrine organ in the body. Ann Oncol 2001; 12:Suppl2:S63-68. 82. Nowak JZ, Zurawska E, Zawilska J. Light-mediated regulation of serotonin synthesis and serotonin N-acetyltransferase (NAT) activity in the rabbit retina. Neurosci Res Commun 1988; 3:47-54. 83. Nowak JZ, Zurawska E. Serotonin N-acetyltransferase (NAT) activity in hen retina and pineal gland: in vivo pharmacological induction at noon and antagonism of the light-evoked suppression at night. Neurochem Int 1989; 15:567-573. 84. Olney RC, Tsuchiya K, Wilson DM et al. Chondrocytes from osteoarthritic cartilage have increased expression of insulin-like growth factor (IGF-I) and IGF-binding protein-3 (IGBP-3) and –5, but not IGF-II or IGFBP-4. J Clin Endocrinol Metab 1996; 81:1096-1103. 85. Pan QP, Fang ZP, Huang FJ. Identification, localization and morphology of APUD cells in gastroenteropancreatic system of stomach-conteining teleosts. World J Gastroenterology 2000; 6(6):842-847. 86. Pang SF, Dubocovich ML, Brown GM. Melatonin receptors in peripheral tissues: a new area of melatonin research. Biol Signals 1993; 2(4):177-180. 87. Partonen T. Extrapineal melatonin and exogenious serotonin in seasonal affective disorder. Med Hypotheses 1998; 51(5):441-442. 88. Pavlikowski M, Winczyk K, Karasek M. Oncostatic action of melatonin: Facts and question marks. Neuroendocrinol Lett 2002; 23 Suppl 1:24-26. 89. Pearse AGE. Common cytochemical and ultrastructural characteristics of cells producing polypeptide hormones (the APUD series) and their relevance to thyroid and ultimobranchial C-cells and calcitonin. Proc Roy Soc 1968; 170:71-80. 90. Pearse AGE. The cytochemistry and ultrastructure of polypeptide hormone-producing cells of the APUD series and the embryologic, physiologic and pathologic implications of the concept. J Histochem Cytochem 1969; 17:303-313. 91. Pentney PT, Bubenik GA. Melatonin reduces the severity of dextran-induced colitis in mice. J Pineal Res 1995; 19(1):31-39. 92. Pratt BL, Takahashi JS. Adrenergic regulation of melatonin release in chick pineal cell cultures. J Neurosci 1987; 7:3665-3674. 93. Quay WB, Ma YH. Demonstration of gastrointestinal hydroxyindol-O-methyltransferase. IRCS Med Sci 1976; 4:563. 94. Raikhlin NT, Kvetnoy IM, Tolkachev VN. Melatonin may be synthesised in enterochromaffine cells. Nature 1975; 255:344-345. 95. Raikhlin NT, Kvetnoy IM, Kadagidze ZG et al. Immunomorphological studies on synthesis of melatonin in enterochromaffine cells. Acta Histochem Cytochem 1976; 11(1):75-77.

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96. Raikhlin NT, Kvetnoy IM, Osadchuk MA. Diffuse endocrine system (APUD-system). Obninsk, MRRC Press, 1992. 97. Raikhlin NT, Kvetnoy IM. Lightening effect of the extract of human appendix mucosa on frog skin melanofores. Bull Exper Biol Med 1974; 8:114-116. 98. Raikhlin NT, Kvetnoy IM. Melatonin and enterochromaffine cells. Acta Histochem 1976; 55:19-25. 99. Ralph CL. Melatonin production by extrapineal tissues. In: Melatonin: Current status and perspectives. Oxford: Pergamon Press 1981: 7-19. 100. Ramstad K, Loge JH. Melatonin treatment of sleep disorders in disabled children. Tidsskr Nor Laegeforen 2002; 122(10):1009-1011. 101. Reiter RJ Melatonin synthesis: Multiplicity of regulation. Adv Exp Med Biol 1991; 294:149-148. 102. Reiter RJ, Richardson BA, Hurlbut EC. Pineal, retinal and Harderian gland melatonin in a diurnal species, the Richardson’s Ground Squirrel (Spermophillus richardsonii). Neurosci Lett 1981; 22:285-288. 103. Reiter RJ, Richardson BA, Matthews SA et al. Rhythms in immunoreactive melatonin in the retina and Harderian gland of rats: persistence after pinealectomy. Life Sci 1983; 32:1229-1236. 104. Reiter RJ. Oxidative damage in the central nervous system: protection by melatonin. Prog Neurobiol 1998; 56(3):359-384. 105. Ribelayga C, Pevet P, Simonneaux V. HIOMT drives the photoperiodic changes in the amplitude of the melatonin peak of the Siberian hamster. Am J Physiol Regul Integr Comp Physiol 2000; 278(5):R1339-1345. 106. Rohr UD, Herold J. Melatonin deficiencies in women. Maturitas 2002; 41 Suppl 1:S85-104. 107. Sagar SM, Klassen GA, Barclay KD et al. Tumour blood flow: measurement and manipulation for therapeutic gain. Cancer Treat Rev 1993; 19:299-349. 108. Sjoblom M, Jedstedt G, Flemstrom G. Peripheral melatonin mediates neuronal stimulation of duodenal mucosal bicarbonate secretion. J Clin Invest 2001; 108: 625-633. 109. Solcia E, Usellini L, Buffa R et al. Endocrine cells producing regulatory peptides. In: Polak JM, ed. Regulatory Peptides. Basel: Birkhauser Verlag. 1989: 220-246. 110. Solomatina TM, Volgarev MN, Bassalyk LS et al. Changes in the number of EC cells in the small intestine and the serotonin level in the blood plasma of fasting rats. Biull Eksp Med 1985; 100(8):162-164. 111. Solomatina TM, Volgarev MN. Enterochromaffin cells of the gastrointerstinal tract and their hormonal activity in response to various dietary factors. Patol Fiziol Eksp Ter 1982; 5:77-81. 112. Stefulj J, Hortner M, Ghosh M et al. Gen expression of the key enzymes of melatonin synthesis in extrapineal tissues in the rat. J Pineal Res 2001; 30(4):243-247. 113. Talley NJ. Review article: 5-hydroxytryptamine agonists and antagonists in the modulation of gastrointestinal motility and sensation: clinical implications. Aliment Pharmacol Ther 1992; 6:273-289. 114. Tan DX, Manchester LC, Reiter RJ et al. Identification of highly elevated levels of melatonin in bone marrow: its origin and significance. Biochimica et Biophysica Acta 1999; 1472;206-214. 115. 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 Top Med Chem 2002; 2(2):181-97. 116. Thomas FJ, Koss MA, Hogan DL et al. Enprostil, a synthetic prostaglandin E2 analogue, inhibits meal-stimulated gastric acid secretion and gastrin release in patients with duodenal ulcer. Amer J Med 1986; 81:44-49. 117. Vakkuri O, Rintamaki H, Leppaluoto J. Presence of immunoreactive melatonin in different tissues of the pigeon (Columba livia). Gen Comp Endocr 1985; 58:69-75. 118. Vaughan GM, Reiter RJ. Pineal dependence of the Syrian hamster’s nocturnal serum melatonin surge. J Pineal Res 1986; 3:9-14. 119. Verhofstad AAJ, Steinbusch HWM, Joosten HWJ et al. Immunocytochemical localization of noradrenaline, adrenaline and serotonin. In Immunocytochemitry. Practical Applications in Biology and Pathology / edited by J.M. Polak and S. Van Noorden. Bristol: Wright PSG. 1983: 143-168. 120. Vialli M. Histology of the enterochromaffin cells. In: Erspamer V, ed. 5-Hydroxytryptamine and Related Indolealkylamines. Handbook of Experimental Pharmacology. Vol. 19. Berlin: Springer Verlag, 1966:1-65. 121. Vivien-Roels B, Pevet P, Dubois MP et al. Immunohistochemical evidence for the presence of melatonin in the pineal gland, the retina and the Harderian gland. Cell Tissue Res 1981; 217:105-15. 122. Wainwright SD. Development of hydroxyindole-O-methyltransferase in the retina of chick embryo and young chick. J Neurochem 1979; 32:1099-1101. 123. Weissbluth L, Weissbluth M. Infant colic: The effect of serotonin and melatonin circadian rhythms on the intestinal smooth muscle. Med Hypotheses 1992; 39:164-167.

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124. Wiechmann AF, Bok D, Horwitz J. Localization of hydroxyindole-O-methyltransferase in the mammalian pineal gland and retina. Invest Ophthalmol Vis Sci 1985; 26:253-265. 125. Wiechmann AF, Hollyfield JG. Localization of hydroxyindole-O-methyltransferase-like immunoreactivity in photoreceptors and cone bipolar cells in the human retina: A light and electron microscope study. J Comp Neurol 1987; 258:253-266. 126. Yaga K, Reiter RJ, Richardson BA. Tryptophan loading increases daytime serum melatonin in intact and pinealectomized rats. Life Sci 1993; 52:123112-38. 127. Yu HS, Pang SF, Tang PL. Increases in the level of retinal melatonin and persistence of its rhythm in rats after pinealectomy. J Endocrinol 1981; 91:477-481. 128. Yu HS, Pang SF, Tang PL. Persistence of circadian rhythms of melatonin and N-acetylserotonin in the serum of rats after pinealectomy. Neuroendocrinology, 1981; 32(5):262-265. 129. Zabel M, Dietel M. Endocrine markers in malignant tumor cells producing parathyroid hormone-related protein. Exp Clin Endocrinol 1993;101(5):297-302. 130. Zawilska J, Nowak JZ. Regulation of melatonin biosynthesis in vertebrate retina: involvement of dopamine in the suppressive effects of light. Folia Histochem et Cytobiol 1991; 29:3-14. 131. Zawilska JB, Nowak JZ. Regulatory mechanisms in melatonin biosynthesis in retina. Neurochem Int 1992; 20:23-26. 132. Zucconi M, Bruni O. Sleep disorders in children with neurologic diseases. Semin Pediatr Neurol 2001; 8(4):258-275.

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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<0.05 (see ref. 9).

of sleep –wakefulness are poorly understood, reflecting our incomplete knowledge of the physiological significance of the sleep phenomenon per se. Melatonin treatment is typically found to increase sleepiness and to promote sleep in healthy humans, when administered during the day, or to improve overnight sleep in insomniacs9 (Fig. 1). These effects are observed soon after treatment (within about 30 min) and in response to melatonin doses that induce physiological (i.e., around 100 pg/ml) and pharmacological (over 200 pg/ml) circulating levels of the hormone in human blood. Technical and ethical issues preclude many types of studies that could help to elucidate the mechanisms of melatonin action on human sleep. It is, thus, necessary to conduct such studies using appropriate animal models. However, studies on the effects of melatonin on sleep in nocturnally active rodents produced inconsistent findings when the pharmacological doses were used, with no effect of physiological doses of the hormone detected.10-12 This is likely to be explained by differences in the predominant timing of sleep relative to the environmental light-dark cycle and synchronized with it circadian physiology in nocturnal and diurnal species. Since both types of species secrete melatonin only at night, the nocturnal animals sleep when their melatonin levels are low, while diurnal species, including humans, sleep when their melatonin production is high. Thus, it appears that nocturnal animals normally are not sensitive to the acute sleep-promoting effects of melatonin, and thus cannot be used to study the homeostatic mechanisms of melatonin action on sleep. An optimal animal for studying the mechanisms of melatonin action on human sleep would be a diurnal vertebrate, phylogenetically close to human, with robust circadian rhythms and consolidated nocturnal sleep episode, with the pineal gland secreting melatonin throughout the night and one being sensitive to the sleep-promoting effects of the hormone. Further significant benefits in elucidating the molecular mechanisms of melatonin action on sleep would also be warranted if the genetic code of such a species were available and its effective manipulation feasible. To have all these features in one laboratory animal species is problematic, however a combination of species can provide a satisfactory answer to a number of questions. So far, the effects of melatonin on sleep initiation and maintenance are being studied in diurnal non-human primates, macaques,13,14 and in a diurnal teleost, zebrafish.15 The clear advantages of the non-human primate model is in the phylogenetic proximity of these animals to humans, major similarities in human and macaque brain organization and functioning and, importantly, presence of a characteristic consolidated nocturnal sleep period, with several sleep cycles, consisting of quiet (NREM) and paradoxical (REM) sleep.17 Although lacking all these important advantages of a non-human primate model, the zebrafish also have robust circadian rhythm of activity and melatonin secretion,15,16 widely distributed functional melatonin receptors18 and nighttime rest behavior showing critical behavioral similarities with mammalian sleep.15

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Figure 2. Melatonin and diazepam affect locomotor activity in zebrafish via specific membrane receptors. Pretreatment with specific antagonist for melatonin receptors, luzindole, blocked the decline in locomotor activity induced by A) melatonin, but not by B) diazepam or C) pentobarbital. Pretreatment with specific benzodiazepine receptor antagonist, flumazenil, blocked reduction in locomotor activity following B) diazepam, but not A) melatonin or C) pentobarbital treatment. Control solutions are vehicles for each treatment used. Data are expressed as mean ± SEM group changes (%) in daytime locomotor activity, measured for 2-hours after treatment, relative to basal activity. N=30, each group; **p<0.01 (see ref. 15).

Furthermore, zebrafish model has additional and very significant advantages for deciphering the molecular mechanisms of sleep regulation by melatonin. Those include a small size and high reproductive rate, allowing to conduct high throughput behavioral assays. The transparency of zebrafish larvae allows direct non-invasive single-cell recording of neuronal activity in multiple brain areas, using calcium-sensitive dyes.19 The ability of zebrafish larvae to absorb different substances via skin permits non-invasive and/or long-term administration of pharmacological treatments. Most importantly, an enormous accumulated experience in conducting large-scale genetic screens and multiple mutant zebrafish phenotypes available,20 with the genetic and physical maps being near completion, make zebrafish a potentially outstanding model for sleep research. Studies conducted in many laboratories demonstrate that melatonin has specific binding sites in various central and peripheral tissues of different animal species.21 The cloning of a family of G protein-coupled melatonin receptors22 opened new possibilities for the understanding of melatonin’s action in various target cells. These receptors inhibit cAMP accumulation via a pertussis-toxin sensitive G-protein in most of the tissues tested but, in some tissues, can also affect other signal transduction pathways, e.g., those involving cGMP, diacylglycerol or GABA.23 Melatonin’s high lipid solubility also suggests that this hormone may well have some direct actions inside cells, in addition to its actions on cellular membrane receptors. The exact mechanisms underlying melatonin’s effects on homeostatic sleep regulation remain to be elucidated. A zebrafish model showed that acute effect of melatonin on sleep is mediated via specific melatonin receptors and can be blocked by specific melatonin receptor antagonists (Fig. 2). Interestingly, in both primate and teleost animal models, similar to that observed in humans, the effect of melatonin on sleep initiation show a distinct dose dependency, reaching peak activity at high physiological or low pharmacological concentrations (Fig. 3A). A further increase in dose used does not significantly change the magnitude of the effect (Fig. 4). In contrast, other hypnotic agents, none of which are part of a normal neurochemical system of sleep regulation, typically show a gradual dose-dependent increase in their hypnotic property, inducing general anesthesia in both animals and humans, when used at high concentrations (Fig. 3B,C). These differences between the effects of melatonin and those of the commonly used hypnotics might be explained by different mechanisms involved in melatonin’s sleep-promoting effects. Unlike most of the existing hypnotics, melatonin does not appear to act via GABA-ergic system, since in humans,24 macaques or zebrafish a sleep-promoting effect

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Figure 3. Melatonin and conventional sedatives promote rest behavior in larval zebrafish. Melatonin, diazepam and sodium pentobarbital (barbital) significantly and dose-dependently reduced zebrafish locomotor activity (A,C, E) and increased arousal threshold (B, D, F). Each data point represents mean ± SEM group changes in a 2-hour locomotor activity relative to basal activity, measured in each treatment or control group for 2 hours prior to treatment administration. Arousal threshold data are expressed as the mean ± SEM group number of stimuli necessary to initiate locomotion in a resting fish. Closed diamond—treatment, open square—vehicle control; N=20, each group (see ref. 15).

of melatonin is not attenuated by specific benzodiazepine receptor antagonist, flumazenil (Fig. 2). Pharmacological analysis using melatonin receptor ligands with different degree of affinity to MT1 and MT2 specific melatonin receptors suggest that the acute effect of melatonin on sleep is mediated through MT1 receptor (Zhdanova et al, in press). These receptors were documented in multiple brain areas, including those involved in sleep regulation, e.g., hypothalamus or thalamic nuclei. They are saturated at relatively low melatonin levels, close to those observed normally, which might explain why melatonin efficacy reaches plateau at near-physiological concentrations. Other lines of evidence suggest that the effect of melatonin on sleep in humans might also be related to the hormone’s ability to induce hypothermia.25 Indeed, the nocturnal decline in body temperature is concurrent with the increase in melatonin release from the pineal gland. Both of these rhythmic patterns are remarkably stable and maintain their characteristic phase relationships after light-induced circadian shifts. At present, the mechanisms through which melatonin can decrease core body temperature are not clear. The hypothermic effect of melatonin might result from an overall reduction in alertness or may entirely depend on the peripheral action of the hormone, since it has been shown to enhance heat loss, perhaps via peripheral vasodilation.26 However, the fact that zebrafish, a “cold-blooded” animal is sensitive to the effect of melatonin on sleep suggest that a thermoregulatory mechanisms might not be the primary one in the ability of melatonin to promote sleep behavior.

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Figure 4. Effects of a long-term melatonin treatment with escalating melatonin doses. Mean (SEM) sleep onset times in six monkeys during administration of escalating melatonin doses (5-320 µg/kg, 3 days each dose; black bars), compared to periods of placebo treatment (white bars). PLC1-basal placebo treatment; PLC2- washout placebo treatment. p<0.05 for all melatonin doses, relative to placebo (see ref. 14).

The use of currently available hypnotics has some drawbacks, including changes they induce in sleep architecture, development of tolerance, requiring increase in the dose used, and manifestation of a post-treatment grogginess in the morning. Human studies show that melatonin treatment does not appear to either significantly affect normal nighttime sleep or increase morning-after sleepiness. The effects of both physiological and pharmacological doses of melatonin are not accompanied by any dramatic changes in electrophysiological sleep architecture, i.e., temporal distribution and duration of different sleep stages. Similarly, we have reported earlier that an increase in circulating melatonin levels within the physiological range is not an imperative signal for sleep, but rather a gentle promoter of general relaxation and sedation, elements of sleepiness, which, in favorable conditions, might significantly facilitate sleep onset, and are typical of a period that is conventionally called “quiet wakefulness.”27 Interestingly, when a person is adequately motivated, he can readily overcome these “feelings” and be both alert and productive for some time, regardless of his blood melatonin level. No development of tolerance has been reported in humans and studies conducted in macaques show that a month of daily administration of the physiological (5 ug/kg) dose of melatonin does not change the efficacy of melatonin’s effect on nighttime sleep in rhesus monkeys (Fig. 5). In summary, the pineal hormone melatonin plays an important role in sleep regulation in diurnal species, acting via both circadian and homeostatic mechanisms of sleep control and, perhaps, being a natural link between these two regulatory systems. Although the exact mechanisms of melatonin effects on sleep remain to be elucidated, they appear to be mediated via specific melatonin receptors, located in the brain areas involved in sleep regulation and on the periphery. The combined sleep-promoting and chronobiological effects of melatonin treatment could be of substantial assistance to those suffering from insomnia of different origin, including age-related sleep disturbances or circadian rhythm sleep disorders.

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Figure 5. Earlier sleep onset during long-term treatment with a physiological dose of melatonin. Mean (SEM) sleep onset times in four monkeys during six consecutive one-week periods of treatment with placebo (PLC, white bars) or 5 µg/kg melatonin (black bars). PLC1-basal placebo treatment; PLC2-washout placebo treatment. p < 0.05 for all melatonin treatment weeks, relative to basal (see ref. 14).

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15. Zhdanova IV, Wang SY, Leclair OU et al. Melatonin promotes sleep-like state in zebrafish. Brain Res 2001; 903(1-2):263-8. 16. Balsamo E, Santucci V, Seri B et al. Nonhuman primates: laboratory animals of choice for neurophysiologic studies of sleep. Lab Anim Sci 1977; 27:879-86. 17. Cahill GM, Hurd, MW Batchelor et al. Circadian rhythmicity in the locomotor activity of larval zebrafish. Neuroreport 1998; 9:3445-9. 18. Reppert SM. Melatonin receptors: Molecular biology of a new family of G protein-coupled receptors. J Biol Rhythms 1997; 12(6):528-31. 19. O’Malley DM, Kao YH et al. Imaging the functional organization of zebrafish hindbrain segments during escape behaviors. Neuron 1996; 17(6):1145-55. 20. Talbot WS, Hopkins N. Zebrafish mutations and functional analysis of the vertebrate genome, Genes Dev 2000; 14:755-62. 21. Sugden D. Melatonin: Binding site characteristics and biochemical and cellular responses. Neurochem Int 1994; 24(2):147-157. 22. Reppert SM Melatonin receptors: Molecular biology of a new family of G protein-coupled receptors. J Biol Rhythms 1997; 12:528-531. 23. Vanecek J. Cellular mechanisms of melatonin action. Physiol Rev 1998; 78(3):687-721. 24. Nave R, Herer P, Haimov I 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. 25. Hughes RJ, Badia P. Sleep-promoting and hypothermic effects of daytime melatonin administration in humans. Sleep 1997; 20:124-131. 26. 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; 83:134-139. 27. Zhdanova IV, Wurtman RJ, Morabito C et al. Effects of low oral doses of melatonin, given 2-4 hours before habitual bedtime, on sleep in normal young humans. Sleep 1996; 19(5):423-431.

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CHAPTER 15

The Effect of Different Wavelengths of Light in Changing the Phase of the Melatonin Circadian Rhythm Helen R. Wright and Leon C. Lack

Abstract

S

ome sleep problems are due to abnormal timing of the circadian system. Circadian rhythm sleep disorders can be treated with appropriately timed bright light which normalizes circadian rhythm timing. Some evidence in animals and humans suggests that the circadian system may be differentially sensitive to colored light. We compared the efficacy of broad-band white light as well as short and long wavelength light in suppressing nocturnal melatonin and retiming the melatonin onset, a reliable time marker of the endogenous human circadian system. Results show the human melatonin circadian system is more responsive to shorter blue and green wavelengths of light than the longer amber and red wavelengths. These results suggest a more effective treatment for these sleep problems.

Introduction Sleep problems and resultant daytime tiredness affect approximately 20 to 40% of the adult population at some time.1,2 Certain sleep disorders are caused by mistiming of our circadian rhythm or ‘body clock’. A significant number of people are affected by some type of circadian rhythm sleep disorder, whether chronically as in Delayed and Advanced Sleep Phase Syndrome and Seasonal Affective Disorder (winter depression) or transiently as in shift work and jet lag. All of these disorders arise when individuals attempt to sleep at a suboptimal time according to their endogenous circadian rhythm. This results in poor and inadequate sleep, sleepiness and fatigue at inappropriate times, decreased motor and cognitive performance and overall impaired well-being. For each of these conditions, appropriately timed bright light therapy has been shown to be effective in altering the timing of the circadian rhythm and consequently improving sleep.3-5 The timing of our body clock can be reliably and conveniently indicated by the endogenous onset of melatonin excretion that usually occurs in the early evening. Studies have therefore tested the efficacy of light therapy in humans by the ability of light to suppress nocturnal melatonin and phase shift the melatonin circadian rhythm. Most past studies have used bright white (broad band) light as the light source. However, a small number of recent studies have indicated that the human circadian system may be more responsive to shorter wavelength light. Therefore we evaluated the effect of short and longer wavelengths of light in phase shifting the melatonin rhythm.

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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Circadian Rhythm Sleep Disorders Delayed Sleep Phase Syndrome (DSPS) In our insomnia clinic, we see a number of younger clients with DSPS. Typically, they have difficulty falling asleep at their desired bed time and an inability to wake spontaneously at the desired time in the morning (Fig. 1). This occurs due to a delay, in clock time, of their major sleep period. For example, individuals with DSPS may wish to sleep between the hours of 11:00 p.m. and 7:00 a.m., however, according to their delayed circadian rhythm, their best sleep period may actually occur between 2:00 a.m. and 10:00 a.m. If they go to bed at 11:00 p.m., sleep onset latencies could be up to three hours. Usually total sleep time is prematurely interrupted due to the need to wake for work/school/family commitments. This shortened sleep episode leads to daytime sleepiness, especially in the mornings, irritability, and a lack of concentration, all of which could subsequently affect school and work performances as well as family life. Sleep onset insomnia may ensue if long (> 30 minutes) sleep latencies continue over several weeks. A number of studies have found that the melatonin and core body temperature rhythms are delayed in those people with sleep onset insomnia and DSPS when compared to control groups.6-9 However, an advance(or earlier timing) of the circadian rhythm and the sleep/wake cycle can be attained by appropriately timed bright light administration. A limited number of studies have used bright light stimulation to advance the endogenous rhythm of people with DSPS and sleep onset insomnia.10-15 These have demonstrated that morning bright light exposure has been shown to effectively phase advance the melatonin rhythm and the sleep-wake cycle.

Advanced Sleep Phase Syndrome (ASPS) Conversely, Advanced Sleep Phase Syndrome (ASPS) is a disorder in which the major sleep period is early or advanced with respect to the desired sleep/wake period (Fig. 1). Individuals, typically the older age group, who have ASPS have overwhelming early evening sleepiness, an early sleep onset and morning awakening earlier than desired. They present with an inability to stay awake in the evening and/or with early morning awakening insomnia. For example, bedtimes could be as early as 7:00 p.m. and wake up times at 3.00 a.m. Although individuals with ASPS usually have no difficulty in initiating sleep, the tendency to feel sleepy and fall asleep early in the evening can be a social handicap. If the person attempts to delay bedtime by remaining active in the evening, awakening will still occur too early and, as a result, total sleep

Figure 1. Schematic diagram of the ‘normal’ sleep period and the typical sleep period of Advanced and Delayed Sleep Phase Syndrome. Reprinted and modified with permission from Ferber R. Solve your child’s sleep problem. New York: Simon and Schuster, 1985:119.

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time will be decreased. This will inevitably lead to excessive daytime sleepiness, fatigue, moodiness and other symptoms of sleep deprivation such as lack of motivation and concentration. Those who experience early morning awakening insomnia have advanced or early circadian temperature and melatonin rhythms compared to an aged matched control group of good sleepers.16 Again, bright light therapy has been found to be efficacious in phase delaying the circadian parameters, including the sleep/wake cycle, of individuals with early morning awakening insomnia and Advanced Sleep Phase Syndrome.17-23

Jet Lag Although a transient problem, jet lag and the concomitant sleep, alertness and performance problems can have an effect on the individual as well as on businesses, governments, and even sporting events.24 Travellers and flight crew experience jet lag as they cross several time zones in a short period of time and experience a desynchrony between their endogenous circadian rhythm and the clock time of their destination. One survey has found that up to 94% of passengers suffered jet lag with 45% stating they found these symptoms severely disturbing.25 The symptoms of jet lag due to this desynchrony include such sleep disturbances as difficulty initiating and maintaining sleep, and poor daytime functioning due to sleepiness, impaired alertness, fatigue, decrements in performance, lack of concentration, some gastrointestinal problems, and mood disturbances such as tenseness, anger, fatigue, confusion and lack of vigour.26-28 Several studies monitoring flight crew have been carried out before, during and following multiple long-haul flights. As the crew were unable to synchronise to the rapid time zone changes, the circadian nadir of alertness and performance could occur during flight.29,30 Although the severity of symptoms depends on the number of time zones crossed as well as the direction of the flight, appropriately timed exposure to bright light and darkness as well as exogenous melatonin has been found to be effective in treating jet lag. Exposure to daylight may be sufficient to entrain the circadian rhythms after eastward flights across four to eight time zones, however, artificial light would be necessary following westward flights when the most favourable time for light exposure would occur after sunset.31 Bright light may thus assist the traveller in alleviating the symptoms of jet lag however few studies have been conducted in this area. In fact, the evidence of the benefits of bright light exposure for the treatment of jet lag is not substantial and is sometimes conflicting.32 Case studies 33,34 and laboratory based studies35,36 suggest that evening bright light before and after arriving at the destination of a westward flight and conversely, morning bright light for an eastward flight should benefit the traveller to minimise the symptoms of jet lag.33,37

Shift Work Another internal desynchronization between the endogenous circadian rhythms and sleep occurs during shift work. Approximately 5-10% of the population work night shift, typically between the hours of 10:00 p.m. and 6:00 a.m.38 Of these night shift workers, approximately 60% experience some type of sleep disturbance. Night shift workers need to be alert during the time that their endogenous sleep propensity is actually highest. They then try to sleep during the day when circadian determined sleepiness is low. This results in the shift worker experiencing shortened and fragmented sleep, often with total sleep time reduced by two to four hours.2,39 The overall negative impact of night shift work incudes sleep disturbances, impaired physical and psychological health and disturbed social and family life.40 Furthermore, shift workers experience diminished alertness during the night work period and this can lead to poor performance, low productivity and fatigue related work accidents. It has been suggested that fatigue was a contributing causal factor in the major industrial accidents of the Exxon Valdez, Bhopal, Chernobyl and Three Mile Island which all occurred between midnight and 5:00 a.m.41 Simulated night shift studies and field studies have demonstrated that appropriately timed bright light therapy can improve the adaptation to shift work.42-50 Although there are few night shift studies in the field, it appears that bright light during the night shift plus darkness

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immediately following the shift, may result in a phase delay of the circadian rhythm, longer total sleep time during the day, and improvement in night time alertness.

Seasonal Affective Disorder (SAD) In 1984, Rosenthal and colleagues first reported symptoms of a syndrome they called Seasonal Affective Disorder (SAD).51 The predominant features of SAD are a depressed mood and hypersomnia occurring during the autumn and winter months when there are reduced hours of daylight. Besides a depressed mood and increased sleep, SAD is also characterised by carbohydrate craving, weight gain, lack of energy and motivation, anxiety, irritability and social withdrawal.51,52 A comprehensive review of survey studies estimating the prevalence of winter-SAD found the prevalence rates in North America and Europe range from 0.4% to 9.5%.53 Rosenthal originally hypothesised that changes in light exposure over the seasons was a major contributing factor in the development of winter depression.51 A phase delay hypothesis, a phase-instability hypothesis, a photoperiod hypothesis and a melatonin hypothesis have all been put forward to explain the pathogenesis of SAD but these have not been substantiated.54 It does appear that there is some involvement of the circadian system in the aetiology of SAD. In this way, bright light therapy may assist in relieving the symptoms of SAD. From reviews on the use of phototherapy for SAD, it appears that light of at least 2500 lux administered in the mornings for two hours, initially for one to two weeks then intermittingly over the season, is effective in alleviating some symptoms of SAD.55,56

Light Therapy There are certain characteristics of light therapy that play important roles in its efficacy as a therapeutic tool. These particular features include the timing, intensity and duration of the light pulse(s) as well as the wavelength (color) of light.

Timing According to the human phase response curve (PRC), to achieve a phase delay of the circadian rhythm a light stimulus needs to be presented before the endogenous core body temperature minimum, which usually occurs from 4-6 am. Thus a phase delay would be produced with evening light stimulation. To attain a phase advance the stimulus is to be presented after the temperature minimum, that is, in the morning.57-59

Intensity Intensity also affects the magnitude of melatonin suppression and phase change. Low (indoor) intensity light can suppress melatonin as well as phase change the melatonin rhythm.60-64 However, the degree of the resetting response of the circadian rhythm will increase with the intensity of illumination in a nonliner function.60-62

Wavelength There have been a number of animal and human studies that give support for the differential sensitivity of pineal activity to different colors or wavelengths of light. The melatonin output in animals has been shown to be more sensitive to shorter wavelength light than longer wavelengths.65-69 In humans, studies have also demonstrated that light of shorter wavelengths (blue and green) is more effective than longer wavelength light (amber and red) in suppressing nocturnal melatonin.70-74 Two recent studies have comprehensively investigated the differential ability of various wavelengths of light in suppressing nocturnal melatonin.72,74 In order to characterise the human circadian photoreceptor system the researchers established an action spectrum for nocturnal melatonin suppression. To ascertain an action spectrum, a comparison is made with the number of photons required for the same biological effect at different wavelengths. The researchers evaluated eight wavelengths from 426 to 600 nm. The shorter wavelengths (< 500 nm) were

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identified as the most potent wavelength region for regulating the human pineal gland. Therefore, the human ‘circadian system’, or more precisely, the pineal gland, appears to be more sensitive to shorter wavelength light than longer wavelengths. These extensive studies have demonstrated that the shorter wavelengths of light induce greater nocturnal melatonin suppression. However, of greater clinical importance is the differential measure of circadian rhythm phase change. As some studies have found no significant correlations between the amount of nocturnal melatonin suppression and phase advance or delay of the melatonin rhythm,75,76 it has been suggested that there may be a different mechanism that mediates the melatonin suppressing signal and the phase shifting signal to the pineal gland.77 Indeed, it has been shown in rats that the suppression of melatonin by propranolol does not induce a phase change in the onset of 6-sulphatoxymelatonin.78 In addition, a further experiment in rats found that a 5-HT2C antagonist attenuated the acute suppression of melatonin production following a light pulse, however, this had no effect on the subsequent phase delay in the onset of melatonin production.79 If there are perhaps different mechanisms underlying acute melatonin suppression to light and the subsequent phase shift of the melatonin rhythm, then it would be important to directly test the phase change capacity of different wavelengths of light. We have been the first to measure phase change and thus provide the first and important confirmation of the differential wavelength effects on circadian timing.

Light Administration Devices Using a portable light administration device, we have investigated the ability of different wavelengths of light in not only suppressing nocturnal melatonin but also in phase changing the melatonin rhythm, using the nocturnal melatonin onset as a phase marker of the circadian system. A portable light device overcomes the inherent problems associated with a fixed light source such as the traditional light box. Light boxes usually comprise either fluorescent or incandescent light sources mounted behind translucent screens. However, there are practical disadvantages in using this source of bright light. Firstly, the light box requires a mains power outlet nearby thus confining the individual to a particular location. Also, the amount of illuminance that the eye actually receives decreases with the square of the distance from the light source.80 Therefore, the actual amount of light exposure can be effectively reduced by either changing the distance of the light source from the eyes or by just altering the direction of gaze.81,82 Furthermore, compliance to light box treatment may be reduced by the inconvenience of maintaining a fixed location in front of the box for considerable periods of time. In our clinic it is not unusual to find that our clients with DSPS do not have sufficient time in the morning to sit in front of a light box to receive adequate bright light exposure. As an alternative to a light box we used a portable light device in our experimental studies. The light device used light emitting diodes (LEDs), a power efficient light source that provides high intensity light in the visible spectrum when the LEDs are close to the eyes. They also have the advantage of being able to be powered with small dry cell batteries and thus being portable. The lenses were removed from the frames of ordinary reading glasses and two LEDs per eye were attached to the lower rim of the frames. The light from each LED was directed at the center of the pupil of each eye at a distance of 12 mm from the corneal surface. This provided light to each eye comparable in expanse of the visual field and intensity to that provided by a typical light box. This provided continuous visual light stimulation regardless of physical location and direction of gaze.

Phase Change Studies In one study we compared these portable LED glasses with a conventional light box in suppressing nocturnal melatonin and delaying the melatonin rhythm.83 We administered a two-hour light pulse, commencing at midnight, to 66 healthy good sleepers. The volunteers were randomly allocated to either a control (no light) condition, a light box condition or one of two ‘LED’ conditions. One LED condition used white LEDs and the other used blue/green

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LEDs. The blue/green LEDs had a peak wavelength of 497 nm which was very close to the peak wavelength of light that Brainard and colleagues earlier found was most effective in suppressing melatonin.70,71 The light box produced 2000 lux measured at eye level at a distance of 90 cm and the combination of two LEDs per eye, at a distance of 12 mm from the eye, was the equivalent photopic lux to the light box (2000 lux). Saliva melatonin was assessed from saliva samples collected using polyester swab salivettes (Sarstedt, Germany). We found that the blue/green LED produced the greatest amount of melatonin suppression (70%) followed by the light box (65%) and then the white LEDs (50%). Of greater clinical importance, however, was the amount of phase delay produced. From just a single 2-hour light pulse, the blue/green LED produced a phase delay of the melatonin onset by 40 minutes while the white LED and light box produced phase delays of around 20 minutes. These results suggested a differential sensitivity of the circadian system timing to different wavelengths of light. Of particular interest was the lack of correlation between the amount of melatonin suppression and the amount of phase delay of the melatonin onset, perhaps indicating that the immediate suppression of pineal melatonin secretion and the change of circadian timing may be effected by different processes as proposed by Hashimoto and colleagues.77 Therefore, since circadian phase change, rather than melatonin suppression is required to treat circadian sleep disorders, it was necessary to evaluate different wavelengths for their phase change capability. To further explore the entrainment effects of light wavelength we then compared the effect of longer and shorter wavelength light stimuli in suppressing nocturnal salivary melatonin and in phase delaying the melatonin onset.85 The light was administered via light emitting diodes attached to the portable light device, with all LEDs equated for irradiance intensity level. We had fifteen healthy good sleepers participate in all light conditions and a no light control condition. There was at least a week between light conditions so the volunteers’ sleep pattern and circadian rhythm could return to normal if affected by the light stimuli. The wavelengths we compared were 660 nm (red), 595 nm (amber), 525 nm (green), 497 nm (blue/green), and 470 nm (blue). The spectral distribution of the colors were relatively monochromatic with approximate half peak bandwidths ranging between 10 nm and 18 nm (Fig. 2). The electrical input current was adjusted so that all LEDs were equated for irradiance value of 65 µW/cm2. Therefore each eye, irradiated with two LEDs, received 130 µW/cm2. Volunteers were exposed to a two-hour light stimulus starting from 24:00. Saliva was collected on the night of the light stimulus and the following night to assess melatonin suppression and phase delay. To illustrate some of the results (Fig. 3) A-C shows the mean melatonin concentrations (pM) for the control condition (A) and red (B) and blue (C) light conditions on night one (solid line) and night two (dotted line). For the control condition (Fig. 3A) and the longer wavelengths of light, 660 nm (red)(Fig. 3B), it can be seen that there is no difference between the slopes and timing of the night one and night two melatonin curves, indicating no phase delay of the melatonin onset. However, for the shorter 470 nm (blue) (Fig. 3C) there is a clear suppression of melatonin on night one and a phase delay of the melatonin curve on night two. We found that the shorter wavelengths of green, blue/green and blue light significantly suppressed nocturnal melatonin by approximately 70% compared to no melatonin suppression during the red and amber lights pulses and the no light control condition. Furthermore, after just two hours of light exposure, these shorter wavelengths also produced phase delays on the subsequent night of about 30 minutes (Fig. 4). These data are consistent with those of the earlier study of Brainard et al (1985) that showed the greatest melatonin suppression with peak wavelength of 509 nm, and little or no suppression from light with peak wavelengths of 574 nm and 604 nm. However our results are not consistent with those of Zeitzer who found circadian phase advance following morning red light exposure.76 This difference may be due to the length of light exposure, with the researchers exposing subjects to 5 hours of light over three consecutive mornings (total of 15 hours). Also timing of the light pulses was optimal for phase advance, being centred 1.5 hours after the temperature nadir. Moreover, the intensity of 200 photopic

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Figure 2. Spectral power distribution for each colour light emitting diode. Reprinted with permission from Wright H, Lack, L. Chronobiol Int 2001; 18:801-808. ©2001 Marcel Dekker Inc.

lux has been shown in previous experiments to be of sufficient light intensity to induce an advance of the melatonin onset.60-62 However, since Zietzer and colleagues did not compare other wavelengths using the same protocol, differential sensitivity of the circadian system to wavelength could not be explored. Alternatively, there is the possibility that longer wavelengths may effectively phase advance but not phase delay circadian rhythms. Perhaps there is a different mechanism within the circadian clock or within the retina for phase delay and phase advance of the circadian pacemaker. At the level of the circadian clock, animal researchers have proposed that the mammalian circadian clock has an M (morning) oscillator or a per1/cry1 oscillator and an E (evening oscillator) or per2/cry2 oscillator.86 Other researchers have found that the period genes mPer1 and mPer2 react differently to a light pulse.87 They found that the mPer1 is necessary to elicit a phase advance following morning light, and the mPer 2 to evoke a phase delay following light in the early subjective night. Therefore it is possible that the human circadian clock also has these period genes which not only react differentially to light exposure in the morning and the evening but also have differential sensitivities to light wavelength. Therefore, our next experiment further explored the differential effects of wavelength on the circadian system. This time, light pulses were administered in the morning. Two 2-hour pulses of light starting at 06:00 hours were administered to 42 healthy good sleepers who were randomly assigned to each light condition. There is sufficient evidence to suggest that for a given light pulse of equal duration and intensity, it is more difficult to effect a phase advance than a phase delay. Studies have found that after applying a single light pulse at different circadian times, melatonin phase delays were, on average, greater than phase advances on that first

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Figure 3A. Mean melatonin concentration of the control group showing no melatonin suppression on night 1 (solid line) and no phase delay of the rhythm on night 2 (dotted line).

Figure 3B. Mean melatonin concentration of red LED (660 nm) group showing melatonin suppression on night 1(solid line) from 24:00 to 02:00 and a phase delay of the melatonin rhythm on night 2 (dotted line).

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Figure 3C. Mean melatonin concentration for the blue LED (470 nm) group showing melatonin suppression on night 1 (solid line) from 24:00 t0 02:00 and a phase delay of the melatonin rhythm on night 2 (dotted line).

day/night after light exposure.88,89 Furthermore, smaller phase advances have been detected when measurements are taken on the first day after light exposure.89 It may be that phase advances of the circadian pacemaker require a few days to reach a steady-state relationship.90 Therefore following baseline assessment of the timing of the melatonin rhythm, 2-hour light pulses were administered on two consecutive mornings from 06:00. Assessment of phase change was made on the third night. Some volunteers completed more than one condition, and for those participants there was at least a week between each light condition so circadian rhythms and sleep could return to normal. Each condition was conducted over three consecutive nights, with saliva collected for melatonin assay on night one, before the first morning light stimulus, and on night three after the second morning of light exposure. After the total of four-hours of light stimulation we found the blue LED, with a peak wavelength of 470 nm, phase advanced the melatonin onset by over one hour (Fig. 4). Similarly, the blue/green and green LEDs induced a phase advance of almost 50 and 40 minutes respectively. The red and amber LED produced no significant phase advance compared to the control. Again, in regard to phase change, we have found the circadian system more responsive to shorter wavelength than to longer wavelength light.

Clinical Effectiveness When treating circadian rhythm sleep disorders the ‘clinical’ effectiveness of the portable light device is of great importance. When we defined clinical effectiveness as a phase change of 30 minutes or greater, in both phase advance and phase delay studies, between 60 to 87% of participants experienced phase delays or advances of 30 minutes or more following the light pulses. Therefore, the portable LED light glasses with the shorter wavelength light emitting diodes appear to be a clinically effective device to induce phase advances and phase delays. For individuals with circadian rhythm sleep disorders it is usual to be exposed to the light pulse

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Figure 4. Phase advance and delay (minutes) (means and SEM bars) for the no light control condition and each light condition. * significant (p < .05) difference to the control no light condition and longer wavelength amber (595 nm) and red (660 nm) LED conditions.

over a period of several days to weeks. Hence, one would expect greater phase shifts using these devices in a typical clinical setting than those found in the present studies, with only one or two exposures to the light

Photoreceptors Retinal stimulation appears to be critical for circadian phase change. Of growing interest in the circadian rhythm research area is that of photoreception and the determination of which photopigments, within the retina, are responsible for photic entrainment of the circadian system. When mice, lacking both rod and cone photoreceptors were exposed to a 15-minute pulse of monochromatic light (λmax 509 nm), they still showed circadian phase shifts.91-93 Possibilities for a mammalian putative circadian photoreceptor include cryptochrome proteins,94-99 a novel opsin-based photopigment such as melanopsin,100-104 or a nonopsin pigment.105 The photoreceptor mechanism that mediates circadian phase in humans has been widely considered. In initial human studies, Brainard and colleagues showed that the curve generated from their melatonin suppression data was similar to the scotopic visual curve and therefore suggested that rhodopsin may be the photopigment involved in circadian entrainment in humans.70 This has been more recently supported by Rea and colleagues.106 However, after inducing a phase advance following a red light pulse, Zeitzer and colleagues proposed that the photopic visual system, in particular, the long-wavelength cones, are also implicated in the mediation of human circadian transduction.76 More recently, studies have demonstrated that the photopic (long and medium wavelength-sensitive cones) system may not be as involved in circadian rhythm regulation and that the human circadian rhythm is mediated by a novel opsin photopigment in the retina.72,107 Similarly, Thapan and colleagues concluded that light induced melatonin suppression is induced by a novel photoreceptor in the retina, with a peak at around 460 nm, and not the cone and rod photopigments necessary for human vision.74 However, this has tentatively been disputed by a further study which took into account neuroanatomical and neurophysiological data, and suggested that the S-cone pigment of the photopic system (peak sensitivity about 440 nm) may play an important role in providing photic input to the human circadian system.108 From these conflicting conclusions it can be seen that

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further research needs to be conducted to ascertain which photopigments within the retina are mediators for the visual stimulation zeitgeber of the human circadian system. Whatever the case, the melatonin circadian parameters of suppression and phase delay appear to be more sensitive to shorter wavelength light.

Future Directions and Conclusions Future studies comparing the effectiveness of the portable light device comprising the shorter wavelength LEDs should include clinical populations. For example, it would be important to be able to phase delay the circadian rhythm and sleep-wake cycle of individuals with Advanced Sleep Phase Syndrome and conversely, phase advance individuals with Delayed Sleep Phase Syndrome. It would also be of interest to use the LED glasses, in simulated and field studies, to alleviate jet lag following eastward and westward flights. It would be envisaged that for this situation, the LED glasses could be used prior to departure and then once on board the actual flight. In addition, simulated and field studies involving night shift workers may show that the portable light device could benefit these workers by increasing nighttime alertness and daytime sleepiness.

Summary Our studies have demonstrated that, using a novel portable light device using LED light sources, the human melatonin circadian system is more responsive to the shorter blue, blue/ green and green wavelengths of light than the longer amber and red wavelengths. Not only were the shorter wavelengths more effective in suppressing nocturnal melatonin, but they were also more effective in phase advancing and phase delaying the melatonin rhythm and, therefore, are of potential clinical benefit.

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16. Lack L, Mercer J, Wright H. Circadian rhythms of early morning awakening insomniacs. J Sleep Res 1996; 5:211-219. 17. Campbell SS, Dawson D, Anderson MW. Alleviation of sleep maintenance insomnia with timed exposure to bright light. J Am Geriatric Soc 1993; 41(8):829-836. 18. Lack L, Wright H. The effect of evening bright light in delaying the circadian rhythms and lengthening the sleep of early morning awakening insomniacs. Sleep 1993; 16(5):436-443. 19. Lack LC, Gibbon S, Schumaker K et al. Comparison of bright and placebo light treatment for morning insomnia. Sleep Res 1994; 23:278. 20. Lack LC, Schumacher K. Evening light treatment of early morning insomnia. Sleep Res 1993; 22:225. 21. Singer CM, Lewy AJ. Case-report: Use of the dim light melatonin onset it the treatment of ASPS with bright light. Sleep Res 1989; 18:445. 22. Suhner AG, Stauble TN, Murphy PJ et al. Sleep maintenance insomnia - How effective is intermittent bright light treatment at home? Sleep 2000; 23:A123. 23. Terman M. Light treatment. In: Kryger M, Roth T, Dement W, eds. Principles and Practice of Sleep Medicine, 2nd ed. Philadelphia: WB Saunders, 1994: 1012-1029. 24. Richardson G, Tate B. Hormonal and pharmacological manipulation of the circadian clock: Recent developments and future strategies. Sleep 2000; (Suppl 3):S77-85. 25. Suhner A, Schlagenhauf P, Johnson R et al. Comparative study to determine the optimal melatonin dosage form for the alleviation of jet lag. Chronobiol Int 1998; 15(6):655-666. 26. American sleep disorders association. The international classification of sleep disorders: Diagnostic and coding manual. Rochester, MN: Allen & Lawrence, 1990. 27. Czeisler CA, Allan JS. Pathologies of the sleep-wake schedule. In: Williams R, Karacan I, Moore C, eds. Sleep disorders: Diagnosis and treatment 2nd ed. NY: John Wiley, 1988: 109-129. 28. Waterhouse J, Reilly T, Atkinson G. Jet-lag. Lancet 1997; 350:1611-1616. 29. Gander P, Rosekind M, Gregory K. Flight crew fatigue VI: A synthesis. Aviat Space Environ Med 1998; 69(9 Suppl):B49-60. 30. Gander PH, Gregory KB, Connell LJ et al. Flight crew fatigue IV: Overnight cargo operations. Aviat Space Environ Med 1998; 69(suppl 9):B26-36. 31. Boulos Z. Bright light treatment for jet lag and shift work. In: Lam R. ed. Seasonal Affective Disorder and Beyond. Washington, DC: American Psychiatric Press, 1998: 253-287. 32. Chesson AL, Littner M, Davila D et al. Practice parameters for the use of light therapy in the treatment of sleep disorders. Sleep 1999; 22(5):641-648. 33. Czeisler CA, Allan JS. Acute circadian phase reversal in man via bright light exposure: Application to jet lag. Sleep Res 1987; 16:605. 34. Sasaki M, Kurpsaki Y, Onda M et al. Effects of bright light on circadian rhythmicity and sleep after transmeridian flight. Sleep Res 1989; 18:442. 35. Boivin DB, James FO. Phase-dependent effect of room light exposure in a 5-h advance of the sleep-wake cycle: Implications for jet lag. J Biol Rhythms 2002; 17(3):266-276. 36. Burgess HJ, Crowley SJ, Gazd CJ et al. Get a jump on jet lag. Sleep 2002; 25:A182. 37. Kripke DE, Loving RT. Bringing therapy to light. Sleep Rev 2001; Winter:46-50. 38. Åkerstedt T. Shift work and disturbed sleep/wakefulness. Sleep Med Rev 1998; 2(2):117-128. 39. Dawson D, Armstrong S. Chronobiotics - drugs that shift rhythms. Pharmacol Ther 1996; 69(1):15-36. 40. Barton J, Folkard S, Smith L et al. Effects on health of a change from a delaying to an advancing shift system. J Occup Environ Med 1994; 51(11):749-755. 41. Van Reeth O. Sleep and circadian disturbances in shift work: Strategies for their management. Horm Res 1998; 49:158-162. 42. Crowley SJ, Lee C, Tseng CY et al. Circadian adaptation to night shift work: Daytime dark is good, adding light during the night shift is better. Sleep 2002; 25:A155-156. 43. Czeisler CA, Johnson MP, Duffy JF et al. Exposure to bright light and darkness to treat physiologic maladaption to night work. N Eng J Med 1990; 322(18):1253-1259. 44. Dawson D, Campbell SS. Timed exposure to bright light improves sleep and alertness during simulated night shifts. Sleep 1991; 14(6):511-516. 45. Dawson D, Encel N, Lushington K. Improving adaptation to simulated night shift: Timed exposure to bright versus daytime melatonin administration. Sleep 1995; 18(1):11-21. 46. Eastman CI, Boulos Z, Terman M et al. Light treatment for sleep disorders: Consensus report. VI. Shift work. J Biol Rhythms 1995; 10(2):157-164. 47. Eastman CI, Martin SK. How to use light and dark to produce circadian adaptation to night shift work. Ann Med 1999; 31(2):87-98. 48. Eastman CI, Stewart KT, Mahoney MP et al. Dark goggles and bright light improve circadian rhythm adaptation to night-shift work. Sleep 1994; 17(6):535-543.

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49. James FO, Chevrie E, Boivin DB. Improvement of daytime sleep in shift workers by judicious light exposure. Sleep 2002; 25(Abstract Supplement):A156. 50. Yoon I-Y, Jeong D-U, Kwon K-B et al. Bright light exposure at night and light attenuation in the morning improve adaptation of night shift workers. Sleep 2002; 25(3):351-356. 51. Rosenthal NE, Sack DA, Gillin JC et al. Seasonal affective disorder: A description of the syndrome and preliminary findings with light therapy. Arch Gen Psychiatry 1984; 41:72-80. 52. Terman M, Terman JS, Quitkin FM et al. Light therapy for seasonal affective disorder: A review of efficacy. Neuropsychopharmacology 1989; 2(1):1-22. 53. Mersch PPA. Prevalence from population surveys. In: Patonen T, Magnusson. A, eds. Seasonal Affective Disorder: Practice and Research. NY: Oxford University Press, 2001: 121-141. 54. Boivin D. Circadian clock. In: Partonen T, Magnusson. A, eds. Seasonal Affective Disorder: Practice and research. NY: Oxford University Press, 200:247-258. 55. Partonen T. Light Therapy. In: Partonen T, Magnusson A, eds. Seasonal Affective Disorder: Practice and Research. NY: Oxford University Press, 2001:65-78. 56. Partonen T, Magnusson A. Guidelines for management. In: Partonen T, Magnusson A, eds. Seasonal Affective Disorder: Practice and Research. NY: Oxford University Press, 2001:113-118. 57. Czeisler CA, Kronauer RE, Allan JS et al. Bright light induction of strong (Type 0) resetting of the human circadian pacemaker. Science 1989; June:1328-1333. 58. Jewett ME, Rimer DW, Duffy JF et al. Human circadian pacemaker is sensitive to light throughout subjective day without evidence of transients. Am J Physiol 1997; 273(5 Pt2):R1800-1809. 59. Minors DS, Waterhouse JM, Wirz-Justice A. A human phase-response curve to light. Neurosci Lett 1991; 133:36-40. 60. Boivin DB, Duffy JF, Kronauer RE et al. Sensitivity of the human circadian pacemaker to moderately bright light. J Biol Rhythms 1994; 9:315-331. 61. Boivin DB, Duffy JF, Kronauer RE et al. Dose-response relationship for resetting of human circadian clock by light. Nature 1996; 379(6565):540-542. 62. Boivin DD, Brown EN, Yuan A et al. The onset and offset of melatonin secretion is equally sensitive to the intensity-dependent resetting effect of light in humans. Sleep 1999; 22(Supplement):S138. 63. Cajochen C, Zeitzer JM, Czeisler CA et al. Dose-response relationship for light intensity and ocular and electroencephalographic correlates of human alertness. Behavior Brain Res 2000; 115:75-83. 64. Zeitzer JM, Dijk DJ, Kronauer RE et al. Sensitivity of the human circadian pacemaker to nocturnal light: Melatonin phase resetting and suppression. J Physiol 2000; 526(3):695-702. 65. Benshoff HM, Brainard GC, Rollag MD et al. Suppression of pineal melatonin in Peromyscus leucopus by different monochromatic wavelengths of visible and near-ultraviolet light (UV-A). Brain Res 1987; 420(2):397-402. 66. Brainard GC, Richardson BA, King TS et al. The influence of different spectra on the suppression of pineal melatonin content in the Syrian hamster. Brain Res 1984; 294(2):333-339. 67. Cardinali DP, Larin F, Wurtman RJ. Control of the rat pineal gland by light spectra. Proc Natl Acad Sci USA 1972; 69:2003-2005. 68. Takahashi JS, De Coursey PJ, Bauman L et al. Spectral sensitivity of a novel photoreceptive system mediating entrainment of mammalian circadian rhythms. Nature 1984; 308(5955):186-188. 69. Pu M. Physiological response properties of cat retinal ganglion cells projecting to suprachiasmatic nucleus. J Biol Rhythms 2000; 15(1):31-36. 70. Brainard G, Lewy A, Menaker M et al. Effect of light wavelength on the suppression of nocturnal plasma melatonin in normal volunteers. Ann NY Acad Sci 1985; 453:376-378. 71. Brainard GC, Lewy AL, Menaker M et al. Dose-response relationship between light irradiance and the suppression of plasma melatonin in human volunteers. Brain Res 1988; 454:212-218. 72. Brainard GC, Hanifin JP, Greeson JM et al. Action spectrum for melatonin regulation in humans: Evidence for a novel circadian photoreceptor. J Neurosci 2001; 21(16):6405-7412. 73. Morita T, Tokura H, Wakamura T et al. Effects of the morning irradiation of light with different wavelengths on the behavior of core temperature and melatonin in humans. Appl Human Sci 1997; 16(3):103-105. 74. Thapan K, Arendt J, Skene D. An action spectrum for melatonin suppression: Evidence for a novel nonrod, noncone photoreceptor system in humans. J Physiol 2001; 535(1):261-267. 75. Kubota T, Uchiyama M, Suzuki H et al. Effects of nocturnal bright light on saliva melatonin, core body temperature and sleep propensity rhythms in human subjects. Neurosci Res 2001; Supplement 42(2):115-122. 76. Zeitzer JM, Kronauer RE, Czeisler CA. Photopic transduction implicated in human circadian entrainment. Neurosci Lett 1997; 232:135-138. 77. Hashimoto S, Nakamura K, Honma S et al. 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78. Kennaway DJ, Rowe SA. Effect of stimulation of endogenous melatonin secretion during constant light exposure on 6-sulphatoxymelatonin rhythmicity in rats. J Pineal Res 2000; 28:16-25. 79. Kennaway DJ, Moyer RW, Voultsios A et al. Serotonin, excitatory amino acids and the photic control of melatonin rhythms and SCN c-FOS in the rat. Brain Res 2001; 897:36-43. 80. Wibom R. Light-definitions and measurements. In: Wetterberg L, ed. Light and Biological Rhythms in Man. New York: Pergamon, 1993: 23-28. 81. Dawson D, Campbell SS. Bright light treatment: Are we keeping our subjects in the dark? Sleep 1990; 13(3):267-271. 82. Gaddy JR. Sources of variability in phototherapy. Sleep Res 1990; 19:394. 83. Wright HR, Lack LC, Partridge KJ. Light emitting diodes can be used to phase delay the melatonin rhythm. J Pineal Res 2001; 31:350-355. 84. Czeisler CA, Brown EN, Ronda JM et al. A clinical method to assess the endogenous circadian phase (ECP) of the deep circadian oscillator in man. Sleep Res 1985; 14:295. 85. Wright HR, Lack LC. Effect of light wavelength on suppression and phase delay of the melatonin rhythm. Chronobiol Int 2001; 18(5):801-808. 86. Daan S, Albrecht U, Van der Horst GTJ. et al. Assembling a clock for all seasons: Are there M and E oscillators in the genes? J Biol Rhythms 2001; 16(2):105-116. 87. Albrecht U, Zheng B, Larkin D et al. mPer1 and mPer2 are essential for normal resetting of the circadian clock. J Biol Rhythms 2001; 16(2):100-104. 88. Dawson D, Lack L, Morris M. Phase resetting of the human circadian pacemaker with use of a single pulse of bright light. Chronobiol Int 1993; 10(2):94-102. 89. Van Cauter E, Sturis J, Byrne MM et al. Demonstration of rapid light-induced advances and delays of the human circadian clock using hormonal phase markers. Am J Physiol 1994; 266:E953-963. 90. Lewy AJ, Sack RL. The use of melatonin as a marker for circadian phase and as a chronobiotic in blind and sighted humans. In: Wetterberg L, ed. Light and Biological Rhythms in Man. New York: Pergamon, 1993: 173-185. 91. Freedman MS, Lucas RJ, Soni B et al. Regulation of mammalian circadian behavior by nonrod, noncone, ocular photoreceptors. Science 1999; 284:502-504. 92. Lucas RJ, Foster RG. Neither functional rod receptors nor rod or cone outer segments are required for the photic inhibition of pineal melatonin. Endocrinol 1999; 140:1520-1524. 93. Lucas RJ, Freedman MS, Munoz M et al. Regulation of the mammalian pineal by nonrod, noncone, ocular photoreceptors. Science 1999; 284:505-507. 94. Devlin PF, Kay SA. Cryptochromes - bringing the blues to circadian rhythms. Trends Cell Biol 1999; 9:295-298. 95. Griffin EA, Stanknis D, Weitz CJ. Light-independent role of CRY1 and CRY2 in the mammalian circadian clock. Science 1999; 286(5440):768-771. 96. Lucas RJ, Foster RG. Photoentrainment in mammals: A role for cyrptochrome? J Biol Rhythms 1999; 14:4-10. 97. Miyamoto Y, Sancar A. Vitamin B2-based blue-light photoreceptors in the retinohypothlamic tract as ther photoactive pigments for setting the circadain clock in mammals. Proc Natl Acad Sci 1998; 95:6097-6102. 98. Sancar A. Cryptochrome: The second photoactive pigment in the eye and its role in circadian photoreception. Ann Rev Biochem 2000; 69:31-67. 99. Thresher RJ, Viataterna MH, Miyamoto Y et al. Role of mouse cryptochrome blue-light photoreceptors in circadian photoresponses. Science 1998; 282:1490-1494. 100. Berson DM, Dunn FA, Takao M. Phototranduction by retinal ganglion cells that set the circadian clock. Science 2002; 295(5557):1070-1073. 101. Hattar S, Liao H-W, Takao M et al. Melanopsin-containing retinal ganglion cells: Architecture, projections, and intrinsic photosensitivity. Science 2002; 295(5557):1065-1070. 102. Provencio I, Rodriguez IR, Jiang G et al. A novel human opsin in the inner retina. J Neurosci 2000; 20(2):600-605. 103. Roberts JP. What sets the biological clock? The Scientist 2002; 16(28):1-4. 104. von Schantz M, Provencio I, Foster RG. Recent developments in circadian photoreception: More than meets the eye. Invest Ophthalmol Vis Sci 2000; 41(7):1605-1607. 105. Thompson CL, Blaner WS, Van Gelder RN et al. Preservation of light signaling to the suprachiasmatic nucleus in vitamin A-deficient mice. PNAS 2001; 98(20):11708-11713. 106. Rea MS, Bullough JD, Figueiro MG. Human melatonin suppression by light: A case for scotopic efficiency. Neurosci Lett 2001; 299:45-48. 107. Brainard GC, Hanifin JP, Rollag MD et al. Human melatonin regulation is not mediated by the three cone photic visual system. J Clin Endocrinol Metabol 2001; 86(1):433-436. 108. Rea MS, Bullough JD, Figueiro MG. Phototransduction for human melatonin suppression. J Pineal Res 2002; 32:209-213.

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CHAPTER 16

Clinical Utility of the Antioxidant Melatonin in the Newborn Eloisa Gitto, Russel J. Reiter, Aurelio Amodio and Ignacio Barberi

Introduction on Oxidative Stress

R

eactive oxygen species (ROS) are considered to play a major role in the pathogenesis of a wide range of human disorders. This may be a particularly important pathogenetic mechanism in the newborn nursery. The phrase “oxygen radical disease of prematurity” has been coined to collectively describe a wide range of neonatal disorders based on the belief that premature newborns are deficient in antioxidant defenses at a time when they are subjected to acute and chronic oxidant stresses.1 Experimental and clinical studies have shown that any harmful tissue event (infections, trauma, anoxia) is perceived mainly by the macrophage and monocyte cells which secrete cytokines among which are interleukin-1 (IL1) and tumor necrosis factor (TNF). These agents stimulate stromal cells with the additional production on cytokines by fibroblasts, endothelium, epithelium and mast cells. The second wave of cytokine production brings about the synthesis of IL1, IL2, IL6, and IL8 which allow for the progression and the amplification of the inflammatory response. Due to the interaction of these mediators, the inflammatory cells activate polymorphonuclear leucocytes (PMN), macrophages/monocytes, platelets, mast-cells as well as a variety of humoral immuno-systems including complement, coagulation-fibrinolysis and arachidonic acid.2 The activation of the above-mentioned mechanisms leads to the formation of toxic substances derived from oxygen, e.g., free radicals and ROS. These oxidizing agents have important effects on a variety of cells as regulators of signal transduction, activators of key transcription factors, and modulators of gene expression and apoptosis. Oxygen-derived free radicals, collectively termed reactive oxygen species (ROS), are normally produced in living organisms. When overproduced, they are important mediators of cell and tissue injury. There is therefore a critical balance between free radical generation and antioxidant defenses.3,4 Oxidative stress in vivo is a degenerative process caused by the overproduction and propagation of free radical reactions. Free radical reactions lead to the oxidation of lipids, proteins and polysaccharides and to DNA damage (fragmentation, apoptosis, base modifications and strand breaks), and therefore have a wide range of biologically toxic effects.4,5 Newborns and particularly preterm infants are at high risk of oxidative stress and they are very susceptible to free radical oxidative damage.6 Indeed, there is evidence of an imbalance between antioxidant and oxidant generating systems which causes oxidative damage.7 In comparison with healthy adults, lower levels of plasma antioxidants such as vitamin E, β-carotene, and sulfhydryl groups, lower levels of plasma metal binding proteins such as ceruloplasmin and transferrin, and reduced activity of erythrocyte superoxide dismutase are typical of newborn infants. Furthermore, infants frequently have higher plasma levels of non-transferrin-bound iron and higher erythrocyte free iron than adults.8 The brain may be especially at risk of free radical-mediated injury, because neuronal membranes are rich in polyunsaturated fatty acids and because the human newborn, especially if 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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preterm, has a relative deficiency of superoxide dismutase and glutathione peroxidase.9 Excess free iron and deficient iron-binding protein and metabolizing capacity are additional features favoring oxidant stress in premature infants.10,11 Free radicals may be generated by different mechanisms, such as ischemia-reperfusion, neutrophil and macrophage activation, Fenton chemistry, endothelial cell xanthine oxidase, free fatty acid and prostaglandin metabolism and hypoxia.12,13

Oxidative Stress and Perinatal Asphyxia Out of the approximately 130 million annual births worldwide, it has been calculated that 4 million suffer from birth asphyxia, and of these, 1 million dies and a similar number develops some sequela.14 The incidence of birth asphyxia is higher in developing than in so-called developed countries. Still, in the latter, 2-6 births per thousand develop hypoxic ischemic encephalopathy,15 representing between 8,000 and 25,000 infants in the EU area. Of these, many develop severe injuries. One definition of birth asphyxia in use is based on the finding of three of the four following criteria: 1. 2. 3. 4.

pH in umbilical arterial cord blood < 7.00; apgar score < 4 more than 5 minutes; multiple organ failure, and hypoxic ischemic encephalopathy.16 Is therefore has become clear that the diagnosis of birth asphyxia can only be made retrospectively;16 it is the sequence of symptoms and signs and how the brain and other organs react over time that indicate the diagnosis. Therefore, neither the diagnosis nor prognosis can be decided until some time after birth. To make the matter even more complicated, it was recently suggested that only 20% of babies developing neonatal encephalopathy suffered from birth asphyxia.18

Hypoxia and ischemia during perinatal asphyxia give rise to an inadequate substrate supply to brain tissue, resulting in damage of neuronal cells. At cell level, cerebral hypoxia-ischemia sets in motion a cascade of biochemical events commencing with a shift from oxidative to anaerobic metabolism, which leads to an accumulation of NADH, FADH and lactic acid and H+ ions.19 If the asphyxia insult persists, the fetus is unable to maintain circulatory centralization, and the cardiac output and extent of cerebral perfusion fall. Owing to the acute reduction in oxygen supply, oxidative phosphorylation and ATP production in the brain are diminished.20,21 As a result, the Na+/K+ pumps in the cell membranes are deprived of the needed energy to maintain ionic gradients. With a reduced membrane potential, large numbers of calcium ions flow through voltage-dependent ion channels, down an extreme extra-/intracellular concentration gradient, into the cell. Intracellular accumulation of Na+ and Cl- ions leads to swelling of the cells as water enters by osmosis (cytotoxic cell edema).22 This cell damage is thought to be caused by the post ischemic production of oxygen radicals, synthesis of NO, inflammatory reactions and an imbalance between the excitatory and inhibitory neurotransmitter systems. Part of the secondary neuronal cell damage may be caused by induction of a well-known cellular suicide program referred to as apoptosis.23 Production of reactive species in the early reperfusion phase plays a substantial role in the resulting brain cell damage. Among the toxicants generated are the superoxide anion radical (O2˙-) and hydrogen peroxide (H2O2). The latter agent can be converted to the highly reactive hydroxyl radical by transition metals, in particular free iron, ultimately leading to lipid peroxidation of the brain cell membranes as well as other macromolecular damage.24

Respiratory Distress Syndrome and Oxidative Stress Hyperoxic exposure itself, although essential for survival of respiratory distress syndrome (RDS) infants, probably induces excessive production of reactive oxygen metabolites in the respiratory system. There exist, however, several potential causes of intracellular and extracellular

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oxidant stress in the preterm newborns with RDS. The high inspiratory concentrations of oxygen required to achieve adequate arterial oxygenation, prooxidant drugs and infections or extrapulmonary inflammation can all promote ROS accumulation and the utilization and depletion of antioxidative factors. Exposure of premature newborns to hyperoxia is a factor in the development of chronic disease. Chronic lung disease (CLD) of the newborn is one of the definitive factors influencing the mortality and morbidity of very low birth–weight infants.25,26 The etiology of CLD is unknown, but many investigators have suggested that free radicals could have a key role in its development. The exposure of immature lungs to prolonged periods of high levels of inspired oxygen is accepted as an important contributor to the development of CLD through both free radical effects on endothelial and epithelial cell barriers that lead to pulmonary edema and trigger mechanisms that lead to activation and accumulation of inflammatory cells. Ogihara et al27 have suggested a role for oxygen radicals as the trigger for CLD. In addition, their data also indicate that the plasma allantoin concentrations and the allantoin/urate ratio may be useful early predictors of the development of CLD. Exposure to hyperoxia commonly occurs during mechanical ventilation of the premature newborn and is a factor in the development of CLD28 The most common reason neonates require respiratory support is because of RDS. It is being increasingly realized that modes of mechanical ventilation that result in end-inspiratory alveolar over-stretching and/or repeated alveolar collapse and re-expansion disturb the normal fluid balance across the alveolo-capillary membrane. The effects of this include disturbance in the integrity of the endothelium and epithelium and impairment of the surfactant system; these changes are similar to those seen in acute RDS.29,30 G. Vento et al31 evaluated the effect of O2 exposure during the first 6 days of life in the tracheobronchial aspirate fluid of 16 mechanically ventilated preterm infants in terms of both antioxidant response and oxidative damage, by measuring total antioxidant activity, uric acid concentrations and protein carbonyl content. Total antioxidant activity was not detectable or was very low in the babies not requiring O2 therapy. The highest value of uric acid was found in the baby ventilated with 100% oxygen. Yigit et al32 demonstrated that serum malondialdehyde (MDA) levels were higher in infants requiring mechanical ventilation compared to those breathing spontaneously, but the difference was not statistically significant. There exist, however, several potential causes of intracellular and extracellular oxidant stress in the preterm newborns with RDS.

Oxidative Stress and Neonatal Sepsis Sepsis represents a serious problem in newborns with an incidence of 1 to 10 cases per 1000 live births, with even higher rates in low-birth-weight neonates. Hospital acquired infections in neonatal intensive care units may also occur as frequently as 30 infections per 100 patients. Mortality rates in septic newborns are 30% to 50%.33 Sepsis is characterized by alterations in body temperature, hypotension, hypoperfusion with cellular injury and organ failure, often resulting in death.34 There are several reports which suggest that reactive oxygen species (ROS) play a significant role in the pathogenesis of neonatal sepsis and its complications.35,36 Batra et al36 have documented increased production of ROS in septic neonates. Similarly, Seema et al35 found in newborns with sepsis significantly higher levels of TNF-alpha and increased activity of antioxidative enzymes, superoxide dismutase and glutathione peroxidase.

Antioxidant Therapy

Compounds that prevent cellular damage caused by ROS may act in numerous ways,37 ranging from prevention of their formation (such as chelators of metal ions and anti-inflammatory agents) to their interception once formed. Most antioxidants used clinically fall into the latter category. Antioxidants may be broadly classified into enzymatic (SOD, catalase, and glutathione peroxidase) or nonenzymatic (vitamins C, E, β-carotene, and allopurinol).

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Enzymatic antioxidants have been well characterized for some time. As these enzymes are already present in cells, it appears logical to supplement them, especially in the premature neonate, who may be deficient. Superoxide dismutase (SOD) and catalase are naturally occurring antioxidant enzymes whose therapeutic potential deserves investigation. SOD protects against oxidative damage by catalyzing the dismutation of the O2 to H2O2. The biological relevance of SOD in reducing oxidative damage has been demonstrated under many experimental conditions. In some exceptional cases, e.g., Down syndrome patients, overexpression in SOD activity leads to increased oxidative damage, a likely result of increased generation of the OH.38 The naturally occurring forms of these enzymes are large molecules that do not easily penetrate cell membranes, including the blood-brain barrier. Studies in animals suggest that supplementation with a single antioxidant enzyme, such as SOD, is not protective; it must be used in combination with another enzyme, such as catalase.38,39 Furthermore, these enzymes, when administered systemically, do not readily enter cells unless conjugated to polyethylene glycol or encapsulated in liposomes.40 To date, the use of catalase has not been investigated in humans. An alternative approach is delivery of large doses directly to the target organ, for which the lung is ideally suited. Benefits from exogenously administered natural surfactant may at least partly relate to its antioxidant properties.41,42 Intratracheal delivery of recombinant human SOD has also shown promise in a piglet model of pulmonary O2 toxicity.41,43 The dietary antioxidant, vitamin E (α-tocopherol), was among the first to be used in the hope of preventing neonatal disease. The use of vitamin E was based on evidence that neonates are deficient and on the actions of this compound in preventing membrane lipid peroxidation, which is purposed to contribute to disease. It has since been recognized that measurement of plasma vitamin E as a measure of sufficiency is seriously flawed.44 In addition, prolonged pharmacological dosage of vitamin E has been linked to bacterial killing by neutrophils and mononuclear cells, another theoretical hazard of antioxidant therapy. Recent clinical increased incidence of sepsis and necrotizing enterocolitis in neonates.45 This may have been caused by interference with oxidant-dependent mechanisms of trials in adults have also emphasized a troubling reality that has already been described in vitro; exogenously administered dietary antioxidants in high doses (vitamin C and β-carotene in particular) may act as pro-oxidants.46 and actually increase mortality in some groups of patients.46,47 Additives to pharmacological preparations of antioxidants may also cause harm, as was thought to be the case with E-Ferol, an i.v. preparation of vitamin E (α-tocopherol acetate) that led to a number of deaths in neonatal nurseries during the early 1980s.47

Melatonin as Antioxidant Melatonin, an endogenously produced indoleamine formed in adult humans, but only minimally so in neonates, is a highly effective antioxidant and free radical scavenger. That melatonin is a free radical scavenger was first suggested by Ianas et al.48 Tan et al were the first to document that melatonin detoxifies the hydroxyl radical.49 The hydroxyl radical (˙OH) is generally considered destructive to cells because of its very high reactivity with any molecule it encounters. As noted above, Tan et al49 were the first to document that melatonin detoxifies the ˙OH. The approaches used to demonstrate this interaction included the direct scavenging of ˙OH by melatonin after the photolysis of H2O2 with 254 nm ultraviolet light and studies in which melatonin competed with the spin trap, 5,5-dimethylpyrroline-N-oxide (DMPO), for the ˙OH. In this competition study melatonin, in increasing concentrations (from 1 to 100 µM) dose-dependently reduced the formation of DMPO-˙OH adducts which were estimated and identified by high performance liquid chromatography with electrochemical detection (HPLC-EC) and electron spin resonance spectroscopy (ESR). ESR is considered the most definitive test to identify spin trap-˙OH adducts. The dose-response study showed that the concentration of melatonin required for neutralizing 50% of the ˙OH generated, i.e., the IC50, was 21 µM. This value proved to be roughly 5 and 10 times lower than that for two known ˙OH scavengers, glutathione and mannitol, respectively.

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Since these reports, there have been a number of confirmatory studies using a wide variety of methodologies. Without exception, the investigations have shown melatonin to be an efficient ˙OH scavenger. While most of these studies have been conducted in pure chemical, cell-free systems, animal studies have also shown melatonin to scavenge the ˙OH in vivo.50,51 The average calculated rate constant for the scavenging of the ˙OH by melatonin is similar to that of other known efficient ˙OH scavengers.52 According to Zang et al,53 melatonin interacts with H2O2 as indicated by the dose-response reduction in its concentration in a mixture to which increasing amounts of melatonin were added. Details of the interaction mechanisms of melatonin with H2O2 were, however, not provided. According to Tan et al,54 melatonin scavenges H2O2 with the formation of N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK); this molecule may also possess significant scavenging activity. Pieri et al55,56 concluded that melatonin may be a more efficient scavenger of the peroxyl radical than is trolox (water-soluble-vitamin E). Nitric oxide (NO˙), a nitrogen-based radical, is believed to cause significant macromolecular destruction under certain experimental circumstances, e.g., cerebral ischemia/reperfusion injury. In the one study where it has been tested, melatonin was found to scavenge NO˙.57 The coupling of NO˙ with O2˙-, a reaction that occurs at a diffusion-controlled rate, results in the generation of the peroxynitrite anion (ONOO-). Although not a free radical, this species is highly destructive to nearby macromolecules and has been implicated as an agent contributing to the loss of neurons in amyotrophic lateral sclerosis.58 That melatonin neutralizes the ONOOwas originally demonstrated in a cell-free system which depended on the ONOO- induced oxidation of dihydrorhodamine 123 to rhodamine, a reaction that was reduced in a concentration-dependent manner when melatonin was included in the mixture.59 In an ancillary in vitro study, this group also reported that melatonin prevented DNA strand breaks normally caused by ONOO-. These in vitro observations have been exploited in whole animal studies by the group of Cuzzocrea et al.60-62 who have repeatedly shown melatonin to reduce immunocytochemically-detectable nitrotyrosine, a molecule which represents the nitration of tyrosine by ONOO-, in models of inflammation. These studies are consistent with melatonin scavenging of the ONOO- and are supported by the recent observations of Blanchard et al.63 who described the in vitro nitrosation of melatonin by peroxynitrite. Several clinical studies on melatonin showed that this antioxidant may be able to reduce oxidative stress in newborns with sepsis, distress or other conditions where there is ROS production. Severe asphyxia results in tissue damage and cell death in the brain and in many other organs as well. One fundamental aim of treatment of perinatal asphyxia is to prevent injury of the central nervous system. Neuronal survival depends, among other factors, on the duration of a reduced cerebral blood flow. At very low flow, neurons can survive for only 6-9 min.64 Under conditions of global hypoxemia normal autoregulation may be impaired.65,66 which compromises adequate brain blood flow. Restoration of microcirculation and oxygen delivery is necessary to enhance an optimal cerebral outcome but the injury also may be amplified during reoxygenation. Our study, the first where melatonin was given to human newborns,67 measured a product of lipid peroxidation, malondialdehyde, and the nitrite + nitrate levels in the serum of asphyxiated newborns before and after treatment with the antioxidant melatonin given within the first 6 hours of life. Ten asphyxiated newborns received a total of 80 mg melatonin (8 doses of 10 mg each separated by a two hour intervals) orally. One blood sample was collected before melatonin administration and two additional blood samples (at 12 and 24 hours) were collected after giving melatonin. A third group of healthy newborn children given diluent served as controls. Plasma MDA levels were significantly higher in 0, 12 and 24 hour blood from the asphyxiated newborns than in healthy babies (control). Following treatment of asphyxiated newborns with melatonin, there was a significant reduction in the products of lipid peroxidation at both 12

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and 24 hours after treatment (p<0.05) but these values were still much higher than in the control newborns. Three of 10 asphyxiated newborns who were not treated with melatonin died by 72 hours after birth; none of the asphyxiated infants given melatonin died within this interval. All the control newborns survived. High nitrite/nitrate levels, stable metabolites of NO, were measured in the 0, 12 and 24 hour blood samples. At all time points these were higher in the asphyxiated newborns than in the healthy babies (p<0.001). Following melatonin administration, nitrite + nitrate levels dropped significantly while they remained high and even further increased in the asphyxiated infants not given melatonin. The results indicate that the melatonin may be beneficial in the treatment of newborn infants with asphyxia. The protective actions of melatonin in this study may relate to the antioxidant properties of the indole as well as to the ability of melatonin to increase the efficiency of mitochondrial electron transport. Another study68 was conducted to determine the changes in the clinical status and the serum levels of lipid peroxidation products [malondialdehyde (MDA) and 4-hydroxylalkenals (4-HDA)] in 10 septic newborns treated with the antioxidant melatonin given within the first 12 hours after diagnosis. Ten other septic newborns in a comparable state were used as “septic” controls, while 10 healthy newborns served as normal controls. All septic patients were diagnosed as having highly probable sepsis (HPS) or probable sepsis (PRS) and were treated with antibiotics according to standard protocols. A total of 20 mg melatonin was administered orally in two doses of 10 mg each, with a 1 hour interval. One blood sample was collected before melatonin administration and two additional blood samples (at 1 and 4 hours) were collected after melatonin administration to assess serum levels of lipid peroxidation products. Serum MDA + 4-HDA concentrations in newborns with sepsis were significantly higher than those in healthy infants without sepsis; in contrast, in septic newborns treated with melatonin there was a significant reduction (p<0.05) of MDA + 4-HDA to the levels in the normal controls at both 1 and 4 hours (p<0.05). Melatonin also improved the clinical outcome of the septic newborns as judged by measurement of sepsis-related serum parameters after 24 and 48 hours. Three of 10 septic children who were not treated with melatonin died within 72 hours after diagnosis of sepsis; none of the 10 septic newborns treated with melatonin died. In conclusion another recent study was conducted to determine if the treatment with the antioxidant melatonin would lower IL-6, IL-8, TNFα and nitrite/nitrate levels in seventy-four newborns with RDS of III or IV grade (radiographically confirmed) diagnosed within the first 6 hours of life. The comparison of serum parameters between melatonin-treated and untreated RDS newborns indeed confirms the anti-inflammatory effects of melatonin. Compared with the melatonin treated RDS newborns, in the untreated infants the concentrations of IL-6, IL-8 and TNFα were significantly higher at 24h, 72 h and at 7 days after start melatonin treatment. The data confirm that the serum interleukins levels were similar before melatonin administration in the 74 RDS newborns enrolled in this study and also show that a significant increment in these parameters was already seen after 24 hours in those who didn’t receive melatonin. Our finding of increased cytokines early in the course of Respiratory Distress Syndrome supports the hypothesis that they are important mediators in the early inflammatory response in the preterm lung. We have also demonstrated increased concentrations of proinflammatory cytokines in blood from infants with RDS during the first days of life. The values correlate with gestational age and iatrogenic damage in the form of oxygen exposure and mechanical ventilation. Therefore, increased concentrations of proinflammatory cytokines can be the most valuable early indicator of grade of Respiratory Distress Syndrome and need of higher concentrations of oxygen and duration of ventilation and suggest that melatonin treatment can determine selective blockage of components of inflammation. In view of these findings and considering its virtual absence of toxicity,69-71 additional trials with melatonin treatment should be conducted in newborns where oxidative stress is elevated.

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References 1. Sullivan JL. Iron, plasma antioxidants and the “oxygen disease of prematurity”. Am J Dis Child 1988; 142:1341-4. 2. Esteban J, Morcillo JE, Cortjo J. Oxidative stress and pulmonary inflammation: Pharmacological intervention with antioxidants. Pharmacol Res 1999; 40:393-404. 3. Bhandari V, Mauli KN, Kresch M. Hyperoxia causes an increase in antioxidant enzyme activity in adult and fetal rat type II pneumocytes. Lung 2000; 178:53-60. 4. Gutteridge JMC, Mumby S, Quinlan GJ et al. Pro-oxidant iron is present in human pulmonary epithelial lining fluid: Implications for oxidative stress in the lung. Biochem Biophys Res Commun 1996; 220:1024-7. 5. Kelly FJ, Lubec G. Hyperoxic injury of immature guinea pig lung is mediated via hydroxyl radicals. Ped Res1995; 38:286-291. 6. Saugstad OD. Mechanisms of tissue injury by oxygen radicals: Implications for neonatal disease. Acta Paediatr 1996; 85:1-4. 7. Phylactos AC, Leaf AA, Costeloe K et al. Erythrocyte cupric/zinc superoxide dismutase exhibits reduced activity in preterm and low-birth-weight infants at birth. Acta Paediatr 1995; 84:1421-5. 8. Ogihara T, Okamoto R, Kim H et al. New evidence for the involvement of oxygen radicals in triggering neonatal chronic lung disease. Ped Res 1996; 39:117-9. 9. Inder TE, Graham P, Sanderson K et al. Lipid peroxidation as a measure of oxygen free radical damage in the very low birthweight infant. Arch Dis Child Fetal Neonatal Ed 994; 70(2):F107-11. 10. Sullivan JL. Iron metabolism and oxygen radical injury in prematurte infants. Boca rato, CRC 1992 in press. 11. Evans PJ, Evans R, Kovar IZ et al. Bleomycin-detectable iron in the plasma of premature and full-term neonates. FEBS Lett 1992; 303:210-2. 12. McCord JM. Oxygen-derived free radicals in post-ischemic injury. N Engl J Med 1985; 312:159-63. 13. Mishra OP, Delivoria-Papadopoulos M. Cellular mechanisms of cerebral injury in the developing brain. Brain Res Bull 1999; 48:233-8. 14. World Health Organisation. Child Health and Development: Health of the newborn. Geneva, 1991. 15. Levene ML, Kornberg J, Williams THC. The incidence and severity of post-asphyxial encephalopaty in full-term infants. Early Hum Dev 1985; 11:21-6. 16. Use and abuse of the Apgar Score. Committee on Fetus and newborn, American Academy of Pediatrics, and Committee on Obstetric Practice, American college of Obstetrician and gynecologists. Pediatrics 1996; 98:141-2. 17. Nelson KB, Emery ES. Birth asphyxia and the neonatal brain: What do we know and when do we know it? Clin Perinatol 1993; 20:327-44. 18. Adamson SJ, Alessandri LM, Badawi N et al. Predictors of neonatal encephalopaty in full term infants. BMJ 1995; 311:598-602. 19. Palmer C, Brucklalcher RM, Christensen MA et al. Carbohydrate and energy metabolism during the evolution of hypoxic-ischemic brain damage in the immature rat. J Cereb Blood Flow Metab 1990; 10:227-35. 20. Yager JY, Brucklalcher RM, Vannucci RC. Cerebral energy metabolism during hypoxia-ischemia and early recovery in immature rats. Am J Physiol 1992; 262:H672-7. 21. Berger R, Gjedde A, Heck J et al. Extension of the 2-deoxyglucose methiod to the fetus in utero: Theory and normal values for the cerebral glucose consuption in fetal guinea pigs. J Neurochem 1994; 63:271-9. 22. Vannucci RC, Christensen MA, Yager JY. Nature, time-course and extent of cerebral edema in perinatal hypoxic-ischemic brain damage. Pediatr Neurol 1993; 9:29-34. 23. Berger R, Garnier Y. Perinatal brain injury. J Perinatol Med 2000; 28:261-85. 24. Halliwell B, Gutteridge JC. Role of free radicals and catalytic ions in human disease: An overview; in Packer AN (ed): Methods in Enzymology: San Diego, Academic Press, 1990:1-85. 25. Banks BA, Ischiropoulos H, McClelland M et al. Plasma 3-nitrotyrosine is elevated in premature infants who develop bronchopulmonary dysplasia. Pediatrics 1998; 101:870-4. 26. Saugstad OD. Chronic lung disease. The role of oxidative stress. Biol Neonate 1998; 74 (Suppl.1):21-8. 27. Ogihara T, Okamoto R, Kim H et al. New evidence for the involvement of oxygen radicals in triggering neonatal chronic lung disease. Ped Res 1996; 39:117-9. 28. Ikegami M, Kallapur S, Michna J et al. Lung injury and surfactant metabolism after hyperventilation of premature lambs. Ped Res 2000; 47:398-404.

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29. Verbrugge SJ, Lachmann B. Mechanism of ventilation- induced lung injury: physiological rationale to prevent it. Monaldi Arch Chest Dis1999; 54:22-37. 30. Verbrugge SJ, Uhlig S, Negger SJ et al. Different ventilation strategies affect lung function but do not increase tumor necrosis factor- alpha and prostacyclin production in lavaged rat lungs in vivo. Anesthesiology 1999; 91:1834-43. 31. Vento G, Mele MC, Mordente A et al. High total antioxidant activity and uric acid in tracheobronchial aspirate fluid of preterm infants during oxidative stress: an adaptive response to hyperoxia? Acta Paediatr 2000; 89(3):336-42. 32. Yigit S, Yurdakok M, Kilinc K et al. Serum malondialdehyde concentration as a measure of oxygen free radical damage in preterm infants. Turk J Pediatr 1998; 40(2):177-83. 33. Perez E M, Weisman LE. Novel approaches to the prevention and therapy of neonatal bacterial sepsis. Clin Perinatol 1997; 24: 213-225. 34. Antonielli M. Sepsis and septic shock: pro-inflammatory or anti-inflammatory state? J Chemother 1999; 6: 536-540. 35. Seema KR, Mandal RN, Tandon A et al. 1999 Serum TNF-alpha and free radical scavengers in neonatal septicemia. Indian J Pediatr 66: 511-516. 36. Batra S, Kumar R, Seema et al. Alterations in antioxidant status during neonatal sepsis. Ann Trop Paediatr 2000; 20: 27-33 37. Halliwell B. Reactive oxygen species in living systems: Source, biochemistry, and role in human disease. Am J Med 1991; 91:14S-22S. 38. Mao GD, Thomas PD, Lopaschuk GD et al. Superoxide dismutade (SOD)-catalase conjugates. J Biol Chem 1993; 268:416-620. 39. Crapo JD, DeLong DM, Sjostrom K et al. The failure of aerosolized superoxide dismutase to modify pulmonary oxygen toxicity. Am Rev Respir Dis 1977; 115:1027-33. 40. Turrens JF, Crapo JD, Freeman BA. Protection against oxygen toxicity by intravenous injection of liposome-entrapped catalase and superoxide dismutase. J Clin Invest 1984; 73:87-95. 41. Thibeault DW, Rezaiekhaligh M, Mabry S et al. Prevention of chronic pulmonary oxygen toxicity in young rats with liposome-encapsulated catalase administered intratracheally. Pediatr Pulmonol 1991; 11:318-27. 42. Matalon S, Holm BA, Baker RR et al. Characterization of antioxidant activities of pulmonary surfactant mixtures. Biochim Biophys Acta 1990; 1035:121-7. 43. Davis JM, Rosenfeld WN, Sanders RJ et al. Prophylactic effects of recombinant humane superoxide dismutase in neonatal lung injury. J Appl Physiol 1993; 74:2234-41. 44. Karp WB, Robertson AF. Vitamin E in neonatology. Adv Pediatr 1986; 33:127-47. 45. Johnson L, Bowen Jr FW, Abbasi S et al. Relationship of prolonged pharmacologic serum levels of vitamin E to incidence of sepsis and necrotizing enterocolitis in infants with birth weight 1,500 grams or less. Pediatrics 1985; 75:619-38. 46. Podmore ID, Griffiths HR, Herbert KE et al. Vitamin C exhibits pro-oxidant properties. Nature 1998; 392:559. 47. Saldanha RL, Cepeda EE, Poland RL. The effect of vitamin E prophylaxis on the incidence and severity of bronchopulmonary dysplasia. J Pediatr 1982; 101:89-93. 48. Ianas O, Olivescu R, Badescu I. Melatonin involvement in oxidative processes. Rom J Endocrinol 1991; 29:117-23. 49. Tan DX, Chen LD, Poeggeler B et al. Melatonin: A potent, endogenous hydroxyl radical scavenger. Endocrine J 1993; 1:57-60. 50. Li XJ, Zhang LM, Gu J et al. Melatonin decreases production of hydroxyl radical during ischemia-reperfusion. Acta Pharmacol Sin 1997; 18:394-6. 51. Tan DX, Manchester LC, Reiter RJ et al. A novel melatonin matabolite, cyclic 3-hydroxymelatonin: A biomarker of in vivo hydroxyl radical generation. Biochem Biophys Res Commun 1998; 253:614-20. 52. Reiter RJ, Tan DX, Manchester LC et al. Biochemical reactivity of melatonin with reactive oxygen and nitrogen species: A review of the evidence. Cell Biochem Biophys 2001; 34(2):237-56. 53. Zang LY, Cosma G, Gardner H et al. Scavenging of reactive oxygen species by melatonin. Biochim Biophys Acta 1998; 1425:467-77. 54. Tan DX, Manchester RJ, Reiter RJ et al. Significance of melatonin in antioxidative defense system: Reactions and products. Biol Signals Recept 2000; 9:137-59. 55. Pieri C, Marra M, Moroni F et al. Melatonin, a peroxyl radical scavenger more efficient than vitamin E. Life Sci 1994; 55:PL271-6. 56. Pieri C, Moroni F, Marra M et al. Melatonin is an efficient antioxidant. Arch Gerontol Geriatrics 1995; 20:159-65.

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57. Noda Y, Mori A, Liburty R et al. Melatonin and its precursors scavenge nitric oxide. J Pineal Res 1999; 27:159-63. 58. Beckman JS, Chen J, Ischiropoulos H et al. Oxidative chemistry of peroxynitrite. Methods Enzymol 1994; 233:229-40. 59. Gilad E, Cuzzocrea S, Zingarelli B et al. Melatonin as a scavenger of peroxynitrite. Life Sci 1997;60:PL169-74. 60. 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. 61. Cuzzocrea S, Zingarelli B, Costantino G et al. Protective effect of melatonin in non-septic shock model induced by zymosan in the rat. J Pineal Res 1998; 25:24-33. 62. El-Sokkary GH, Reiter RJ, Cuzzocrea S et al. Role of melatonin in reduction of lipid peroxidation and peroxynitrite formation in non-septic shock induced by zymosan. Shock 1999; 12:402-8. 63. Blanchard B, PompomD, Ducrocq C. Nitrosation of melatonin by nitric oxide and peroxynitrite. J Pineal Res 2000; 29:184-93. 64. Carter BS, Haverkamp AD, Merestein GB. The definition of acute asphyxia. Clin Perinatol 1993; 20:287-303. 65. Martin E, Barkovich AJ. Magnetic resonance imaging in perinatal asphyxia. Arch Dis Child 1995; 72:F62-70. 66. Rutherford M, Pennock J, Schwieso J et al. Hypoxic-ischaemic encephalopathy: early and late magnetic resonance imaging findings in relation to outcome. Arch Dis Child 1996; 75:F145-51. 67. Fulia F, Gitto E, Cuzzocrea S et al. Increased levels of malondialdehyde and nitrite/nitrate in the blood of asphyxiated newborns: reduction by melatonin. J Pineal Res 2001; 31:343-9. 68. Gitto E, Karbownik M, Reiter RJ et al. Effects of Melatonin Treatment in Septic Newborns. Ped Res 2001; 50No6:756-60. 69. Jahnke G, Marr M, Myers C et al. Maternal and developmental toxicity evaluation of melatonin administration orally to pregnant Sprague-Dawley rats. Toxicol Res 1999; 50:271-9. 70. Jan JE, Hamilton D, Seward N et al. Clinical trials of control release melatonin in children with sleep-wake cycle disorders. J Pineal Res 2000; 29:34-9. 71. Seabra M de LV, 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.

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CHAPTER 17

Diurnal 5-HT Production and Melatonin Formation Jimo Borjigin and Jie Deng

W

e have provided evidence that pineal 5-hydroxytryptomine (5-HT or serotonin) production is up regulated at night, and is controlled by beta-adrenergic signaling.1 In this paper, we demonstrate that the increased 5-HT synthesis is due to increased protein expression of tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT synthesis. Melatonin is synthesized from dietary tryptophan through actions of four enzymes (see Fig. 3 and ref. 2). Tryptophan hydroxylase (TPH) controls the first step of the pathway in which tryptophan is converted to 5-hydroxytryptophan (5-HTP). Aromatic amino acid decarboxylase (AADC) catalyzes the conversion of 5-HTP to 5-hydroxytryptamine (5-HT, serotonin). Melatonin formation from 5-HT requires two pineal/retina specific enzymes: serotonin N-acetyltransferase (NAT), which forms N-acetylserotonin (NAS) from serotonin, and hydroxyindole-O-methyltransferase (HIOMT), which produces melatonin from NAS. Of the four enzymes involved, NAT has long been viewed as the ‘rate-limiting’ enzyme for melatonin production, due to its diurnal pattern of activity, and has been extensively analyzed.2 In comparison, the regulation of TPH and its contribution in melatonin production is less well understood. A few decades ago, TPH, the rate-limiting enzyme for 5-HT production, was found to be diurnally regulated in the rat pineal with a two-fold increase in activity at night.3,4 Recently, we have shown that 5-HT synthesis is increased early at night, and that the increase in 5-HT production is abolished when beta-adrenergic signaling is blocked.1 In this study, we analyze TPH mRNA and protein expression in the rat pineal gland and compare it with the expression of two rhythmically expressed messages patched 15 and PL22 (manuscript submitted), normalized to GAPDH control. We find no detectable difference in TPH mRNA levels throughout a diurnal cycle (Fig. 1). In contrast, TPH protein levels vary diurnally as shown in Figure 2. The fact that TPH protein levels increase immediately after the lights are turned off (Fig. 2) and that 5-HT production increases within 20 min of lights-off1 supports the idea that post-transcriptional mechanisms are responsible for the increased 5-HT production at night in the rat pineal gland. A number of studies have demonstrated an increase in TPH activity in rat pineals at night.3,4,6 It is also well established that beta-adrenergic signaling activates the increase in TPH activity,4,7 and 5-HT synthesis.1 Furthermore, purified rat brain TPH is phosphorylated in vitro by cAMP-dependent protein kinase (PKA),6,8 and beta-adrenergic signaling leads to an increase in intracellular cAMP levels in night pineal gland.2 On the other hand, alpha-adrenergic signaling has been shown to increase 5-HT secretion in vitro9,10 and in vivo.1 These data support the model of intracellular control of 5-HT synthesis and release shown in Figure 3. Nighttime release of norepinephrine activates both alpha- and beta-adrenergic receptors. Beta-adrenergic receptor, upon stimulation induces an increase in intracellular cAMP level that serves to stimulate NAT transcription and increase NAT protein stability, which leads to increased metaboMelatonin: 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. TPH mRNA expression in the rat pineal assayed by Northern blot analysis. Pineal glands of adult (8 wks) male Sprague Dawley rats, housed in a temperaturecontrolled room under 14:10 h light/dark cycle, were harvested at the indicated times. Total RNA from single pineals was loaded in each lane, electrophoresed, blotted, and probed as indicated.

Figure 2. TPH protein expression in the rat pineal assayed by Western analysis. Pineals at indicated times were harvested as in Figure 1. Electroblotted protein extracts from 1/5 of a single pineal per lane were probed with anti-TPH antibody (Calbiochem, Cat#OP71L). Nighttime levels of TPH were normalized against the daytime average. The data is representative of five independent experiments.

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Figure 3. Scheme of signal transduction pathways for pineal 5-HT and melatonin synthesis and release.

lism of 5-HT. In addition to the beta-receptor mediated increase in 5-HT consumption, activated alpha-adrenoceptor increases 5-HT secretion.1 Both types of adrenergic signaling effectively lower the intracellular concentration of 5-HT. It is no wonder, then that the pineal has developed a simultaneous mechanism to increase 5-HT production required for melatonin biosynthesis. Studies mentioned above and presented in this paper suggest that cAMP signaling increases 5-HT synthesis via either stimulation of TPH protein synthesis or stabilization of TPH protein level.

References 1. Sun X, Deng J, Liu T et al. Circadian 5-HT production regulated by adrenergic signaling. Proc Natl Acad Sci USA 2002; 99:4686-4691. 2. Borjigin J, Li X, Snyder SH. The pineal gland and melatonin: Molecular and pharmacologic regulation. Annu Rev Pharmacol Toxicol 1999; 39:53-65. 3. Sitaram BR, Lees GJ. Diurnal rhythm and turnover of tryptophan hydroxylase in the pineal gland of the rat. J Neurochem 1978; 31:1021-1026. 4. Shibuya H, Toru M, Watanabe S. A circadian rhythm of tryptophan hydroxylase in rat pineals. Brain Res 1978; 138:364-368. 5. Borjigin J, Deng J, Wang MM et al. Circadian rhythm of patched1 transcription in the pineal regulated by adrenergic stimulation and cAMP. J Biol Chem 1999; 274:35012-35015. 6. Ehret M, Pevet P, Maitre M. Tryptophan hydroxylase synthesis is induced by 3',5'-cyclic adenosine monophosphate during circadian rhythm in the rat pineal gland. J Neurochem 1991; 57:1516-1521. 7. Toru M, Watanabe S, Nishikawa T et al. Physiological and pharmacological properties of circadian rhythm of tryptophan hydroxylase in rat pineals. In: Passouant P, Oswald I, eds. Advances in Biosciences, Vol. 21: Pharmacology of the States of Alertness. Oxford: Pergamon Press, 1979:253-255. 8. Johnansen PA, Jennings I, Cotton RGH et al. Tryptophan hydroxylase is phosphorylated by protein kinase A. J Neurochem 1995; 65:882-888. 9. Aloyo VJ, Walker RF. Alpha-adrenergic control of serotonin release from rat pineal glands. Neuroendocrinology 1988; 48:61-66. 10. Miguez JM, Simonneaux V, Pevet P. The role of the intracellular and extracellular serotonin in the regulation of melatonin production in rat pinealocytes. J Pineal Res 1997; 23:63-71.

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CHAPTER 18

Melatonin and Mitochondrial Respiration Yuji Okatani, Akihiko Wakatsuki and Russel J. Reiter

Abstract

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n the last decade, numerous publications have documented the protective actions of melatonin against a vast array of conditions in which free radical damage is a component (e.g., ischemia/reperfusion injury, aging and age-associated diseases, toxin exposure, lipopolysaccharide exposure). Melatonin is a highly ubiquitous direct free radical scavenger and indirect antioxidant. Mitochondria appear to constitute the greatest source of oxidants. Oxidatively damaged mitochondria are unable to maintain the energy demands of the cell, which leads to further production of free radicals. Cellular energy deficits caused by declines in mitochondrial function can impair normal cellular activities and compromise the cell’s ability to adapt to various physiologic stresses. This review summarizes the role of melatonin in mitochondrial physiology and describes the beneficial actions of melatonin against mitochondrial dysfunction in conditions such as ischemia/reperfusion injury and aging, in which oxygen free radicals have a major role. In addition, recent observations documenting the ability of melatonin to stimulate electron transport and ATP production in the inner mitochondrial membrane also have relevance for melatonin as an agent that could alter the processes of aging. Finally, this report describes data showing that melatonin is not only a pharmacologically useful free radical scavenger but that it also functions in this capacity at physiologic concentrations. The discovery of new actions of melatonin in mitochondria supports a novel mechanism that explains the protective effects of melatonin on cells.

Introduction Melatonin, the chief secretory product of the pineal gland, is a direct free radical scavenger and indirect antioxidant. In terms of its scavenging activity, melatonin has been shown to quench the hydroxyl radical (•OH), superoxide anion radical (O2-•), singlet oxygen (1O2), peroxyl radical (LOO-), nitric acid (NO•), and the peroxynitrite anion (ONOO-). One of the products of melatonin’s interaction with H2O2, N1-acetyl-N2-formyl-5-methoxykynuramine, is also a highly efficient radical scavenger.1-5 Additionally, melatonin’s direct antioxidant actions may be derived from its stimulatory effect on superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GRd), and glucose-6-phosphate dehydrogenase and from its inhibitory action on nitric oxide synthase.1,2,6 Mitochondria constitute the greatest source of oxidants on the basis of the following evidence:7 (1) The mitochondrial electron transport system consumes abundant oxygen utilized by the cell. (2) In contrast with other oxidant-producing systems of the cell, mitochondria are required for the production of ATP and are present in relatively high numbers in essentially all cells of the body. Oxidants generated by mitochondria appear to be the major source of the oxidative lesions that accumulate with age.7,8 Numerous reports also suggest that oxidation is a major contributor to cellular aging and the degenerative diseases that accompany aging, such as cancer, cardiovascular disease, immune system decline, brain dysfunction, and cataracts.7-10 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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Oxidants, such as O2-•, H2O2, and •OH, are produced continuously at a high rate as a by-product of aerobic metabolism.11,12 They damage cellular macromolecules, including DNA, proteins, and lipids. Accumulation of such damage may contribute to aging and age-associated degenerative diseases, and pathologic conditions, such as ischemia/reperfusion injury and sepsis.1,2 In this chapter, we argue for a role of melatonin in mitochondrial physiology and for the beneficial actions of melatonin against mitochondrial dysfunction in pathologic conditions, such as ischemia/reperfusion injury, as well as in normal aging where oxygen free radicals have a major role.

Mitochondria and Oxygen Free Radicals In the cells of higher animals, ~95% of the cellular energy denominator, ATP, is produced by oxidative phosphorylation in mitochondria, the remainder being synthesized by glycolytic phosphorylation. The mitochondrial electron transport system consumes approximately 90% of the oxygen utilized by the cell.7,8 Since the mitochondria are the site of utilization of the bulk of the inspired oxygen (O2), oxidants are produced continuously at a high rate as a by-product of aerobic metabolism. These oxidants include O2-•, H2O2, •OH, and possibly 1 O2.11,12 They damage cellular macromolecules, including DNA, proteins and lipids.3,4 Oxidative phosphorylation involves a multienzymatic process that permits the transfer of electrons through the electron transport chain. Complexes I - IV are involved in the oxidation of NADH, electron transport, and the generation of an electrochemical gradient. This electrochemical gradient, which is created by pumping protons across the inner mitochondrial membrane, is utilized by ATP synthase (Complex V) as a source of energy. Relevant to mitochondrial function is the efficiency of electron movement through the electron transport chain and its coupling to oxidative phosphorylation to produce ATP. The coupling efficiency can be measured experimentally by determining the ratio of ATP production to molecular oxygen consumed (ADP/O), and the respiratory control index (State 3 respiration/State 4 respiration).13 Most O2 taken into cells is reduced to water, a process that requires the addition of four electrons. The intermediate steps results in the formation of O2-•, H2O2, and •OH, corresponding to the reduction of O2 by one, two, or three electrons, respectively. Damage to inner membrane proteins comprising the electron transport chain can alter the efficiency of electron transport. Imbalance in the stoichiometry of functional electron transport proteins is proposed to lead to leakage in the flow of electrons to the terminal acceptor, cytochrome oxidase. This would increase the likelihood of O2-• formation. Crosslinks of inner mitochondrial membrane proteins by oxidation, or reactive aldehydes generated from lipid peroxidation, may also result in increased O2-•, H2O2, and •OH production, thus further increasing the damage that can lead to mitochondrial dysfunction. There are so far several observations that make the potential presence of melatonin in mitochondria likely and suggest a role for melatonin in mitochondrial physiology. Mitochondria do not produce glutathione (GSH), but rather they take it up from the cytoplasm. Melatonin reportedly stimulates cytoplasmic GSH synthesis and maintains this antioxidant in mitochondria.14 Binding experiments with 125Iodmelatonin also showed most of the specific binding to be present in the mitochondrial fraction of the cell.15 A metabolic effect of melatonin was initially described by Gilad and coworkers,16 who found that melatonin added to cultured 5774 macrophages reversed the inhibition of mitochondrial respiration caused by ONOO-. In addition, melatonin was shown to reduce the NADPH-dependent peroxidation of lipids in mitochondria derived from human placenta in vitro.17 Additionally, evidence shows that melatonin directly influences mitochondrial energy metabolism. Recently, Martin and Acuna-Castroviejo and colleagues18 demonstrated that melatonin (10 mg/kg, i.p.) can increase the activities of Complex I and Complex IV from rat brain and liver in a time-dependent manner. Additional investigations also showed that melatonin increased the activities of Complex I and Complex IV in a dose-dependent manner.18 Melatonin also prevented the decreases in the activities of these respiratory chain complexes induced

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by ruthenium red, a potent noncompetitive inhibitor of the mitochondrial Ca2+ uniport uptake system.19 The protective effects of melatonin were also documented in isolated mitochondria from rat brain and liver. Oxidative stress induced by tert-butyl-hydroperoxide reduced GPx and GRd activities in the mitochondria and increased oxidized GSH. Each of these changes was reversed when mitochondria were treated with melatonin, but was not affected by vitamins C and E.18 Collectively, these findings suggest that melatonin may have a significant role in maintaining mitochondrial homeostasis and in increasing the efficiency of electron transport.

Melatonin and Ischemia/Reperfusion-Induced Oxidative Damage to Mitochondria Against the above-mentioned background, we examined the protective effect of melatonin against oxidative damage to mitochondria further, using a model of ischemia/reperfusion. Considerable evidence suggests that oxygen-derived free radicals are involved in the pathogenesis of ischemia/reperfusion injury in various organs.20,21 Pharmacologic evidence, such as the beneficial effects of the xanthine oxidase inhibitor allopurinol,22 and of enzymes that metabolize reactive oxygen, such as SOD and catalase,23 supports the oxygen radical hypothesis. Also, other antioxidants, such as vitamin E and coenzyme Q10, have been used to protect against ischemia/reperfusion injury.24,25

Hepatic Ischemia/Reperfusion The ability of melatonin to influence mitochondrial respiration was examined in rat liver in vivo.26 Mature male rats were divided into four groups:the control group; hepatic ischemia (70 min); hepatic ischemia plus 2 h reperfusion; hepatic ischemia plus 2 h reperfusion and two injections of melatonin (10 mg/kg, i.p.); the first injection 15 min before ischemia and the second before reperfusion. All vessels (hepatic artery, portal vein) and the bile duct to the left and median liver lobes were occluded for 70 min with a vascular clamp. Thereafter, the clamp was removed and the liver was reperfused for 2 h in each group. Mitochondria from liver tissue were prepared, and the respiratory activity and ability of ATP synthesis were measured. Oxygen consumption measured in the presence of added ADP, inorganic phosphate and glutamate was defined as State 3 respiration, while that measured following the consumption of ADO was defined as State 4 respiration. The respiratory control index (RCI) was calculated as the ratio of State 3 respiration to State 4 respiration, and used a marker of mitochondrial respiratory activity (Fig. 1).13 The ADP/O was calculated as the ratio of the added ADP concentration to the consumption of oxygen during State 3 respiration. Uncoupled respiration was induced by adding dinitrophenol (DNP) in the presence of ADP, inorganic phosphate and glutamate. The capacity for ATP synthesis was measured by the pH change in the incubation medium initiated by adding succinate and ADP.27 The hydrogen ion concentration in the mitochondrial suspension deflected to alkaline stoichiometry with ATP formation coupled to succinate oxidation (ADP +Pi + nH+ ↔ ATP + H2O). In this study, when rats were subjected to ischemia and reperfusion, a marked reduction in the RCI, ADP/O, State 3 respiration, and DNP-induced uncoupled respiration was measured; these changes were significantly restored in those rats also treated with melatonin (Table 1). Similarly, the increase in pH coupled with mitochondrial energy transfer was suppressed by ischemia/reperfusion; this change was also reduced by melatonin treatment (Fig. 2). Mitochondrial lipid peroxidation was elevated and GPx activity was decreased during ischemia/ reperfusion; these changes were counteracted in rats treated with melatonin (Table 1). Additionally, electron microscopic observation demonstrated that mitochondria from rats that underwent ischemia/reperfusion only lost their swelling-contraction cycle and were in a swollen state. In contrast, some of the mitochondria from melatonin-treated animals were in a swollen state and others were in a relatively contracted state.

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Figure 1. Oxidative phosphorylation and dinitophenol (DNP)-induced uncoupled respiration of liver mitochondria. Liver motochondria (Mt) were incubated in a medium of 0.1 M sucrose, 10 mM KCl, 2 mM sodium phosphate (Pi), 10 mM Tris-HCL (pH 7.4) and 3 mM glutamate (Glu) at 25ºC. Respiration was induced by adding 150µM ADP and 25µM DNP. Oxygen consumption was polarographically recorded using Clark-type oxygen electrode fitted to a 2 mL water-jacketed closed chamber.

Figure 2. Effect of melatonin on the pH change of the incubation medium (mitochondria; 3 mg protein/ ml) coupled with the mitochondrial energy transfer reaction after 70 min liver ischemia followed by 2 h reperfusion. Melatonin (10 mg/kg BW) was injected intraperitoneally at 15 min prior to ischemia and at reperfusion. The pH change coupled with ATP formation was initiated by adding 10 mM succinate and 450 µM ADP. The results are expressed as the increment of pH change/ min/mg protein. Data are mean ± SE. Asterisks designate significant difference: *** P < 0.001.

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Table 1. Functional characteristics of rat liver mitochondria during ischemia and subsequent reperfusion

Ischemia/ Reperfusion

Melatonin + Ischemia/ Reperfusion

Characteristic

Control

Ischemia

RCI ADP/0 State 3 respiration State 4 respiration DNP uncoupled respiration TEARS GPx activity

4.13 ± 0.15 (11) 2.55 ± 0.06 (11) 24.76 ± 0.99 (11)

1.22 ± 0.04 (11)** 1.55 ± 0.18 (11) 2.55 ± 0.26 (11)†† 1.44 ± 0.19 (11)** 1.71 ± 0.07 (11) 2.14 ± 0.09 (11)†† 11.14 ± 1.13 (11)** 12.89 ± 0.91 (11) 18.29 ± 1.98 (11)†

6.24 ± 0.16 (11)

8.95 ± 0.25 (11)*

21.05 ± 0.49 (11)

10.54 ± 0.63 (11)** 9.70 ± 0.94 (11)

1.481 ± 0.07 (11) 5.65 ± 0.36 (11)

1.52 ± 0.07 (11) 4.86 ± 0.44 (11)

7.83 ± 0.42 (11)

6.86 ± 0.29 (11) 14.45 ± 1.88 (11)†

2.10 ± 0.13 (11)** 1.66 ± 0.08 (11)† 3.49 ± 0.26 (11)* 4.43 ± 0.44 (11)

All data are given as means ± SEM. * P < 0.01, ** P < 0.0001, vs control rats . † P < 0.05, †† < 0.0001 vs rats with ischemia/reperfusion. Number of animals used are given in parentheses. RCI: respiratory control index. DNP: dinitrophenol. TBARS: thiobarbituric acid reactive substance (nM/mg protein). GPx activity: glutathione peroxidase activity (U/min/mg protein). Respiration of State 3. State 4. and DNP uncounted are calculated as nM O2/min/mg protein.

Fetal Ischemia and Reperfusion We also documented the protective effect of melatonin against ischemia/reperfusion-induced oxidative damage to fetal cerebral tissue and mitochondria.28,29 In this study, the utero-ovarian arteries were occluded bilaterally for 20 min in female rats on Day 19 of pregnancy to induce fetal ischemia. Reperfusion was achieved by releasing the occlusion and restoring circulation for 30 min. Melatonin (10 mg/kg body weight) or vehicle was injected i.p. 60 min prior to occlusion. Ischemia/reperfusion significantly elevated mitochondrial lipid peroxidation and significantly reduced RCI as well as ADP/O; these changes were significantly blocked in those rats treated with melatonin (Fig. 3). Melatonin also reduced the ischemia/reperfusion-induced increases in the levels of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage, as well as in the amount of lipid peroxidation in the fetal brain. Melatonin crosses the placenta readily, and the same dose of melatonin given to pregnant rats significantly increased the activities of GPx and SOD in the fetal rat brain. 30,31 Collectively, these findings indicate that melatonin protects against ischemia/ reperfusion-induced impairment of mitochondrial respiration, ATP synthesis, swelling, and oxidative lipid and DNA damage. They also suggest that mitochondria are probably the main pharmacological targets of melatonin and that the effect of melatonin may be related to its direct potent free radical scavenger action or/and inhibition of the opening of the permeability transition pore (PTP).31,32 Additionally, melatonin’s antioxidant actions may, in part, be derived from its stimulatory effects on GPx and SOD. PTP opening is triggered by the association of calcium overload with an inducer, such as oxidative stress or high phosphate concentration, conditions encountered during ischemia/reperfusion. The opening of this pore leads to the destruction of the mitochondrial membrane potential and mitochondrial swelling, resulting in mitochondrial uncoupling and inhibition of ATP synthesis. Although the mitochondrial membrane potential has not been measured to date, these actions, coupled with melatonin’s ability to maintain the stability of the inner mitochondrial membrane33 and to restore the ischemia/reperfusion-induced disorganization of mitochondrial structures indicate that the indole may be important for optimal electron transport and energy production.

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Figure 3. Respiratory control index (RCI, open columns) and ADP/O (checkered columns) in fetal cerebral mitochondria following ischemia (20 min) and reperfusion (30 min) with or without melatonin treatment. Melatonin was injected to pregnant rat intraperitoneally at dose of 10 mg/kg 1 h before occlusion. Data are mean ± SD. Asterisks designate significant difference: * P < 0.05, ** P < 0.01.

Potential Links between Melatonin and Aging Despite extensive study of possible etiologic factors of aging, no definitive consensus has been reached on the causes of age-related degenerative conditions. However, considerable experimental evidence supports the idea that aging in general, and aging of the central nervous system, in particular, may be related in part to damage inflicted by oxygen free radicals and their intermediates.7,10,34 Free radical generation may increase as a consequence of normal aging, or alternatively, the defense system evolved to combat oxidative stress, including antioxidative enzymes, may diminish in effectiveness.35,36 The resulting free radical predominance in the internal environment may accelerate cell damage and associated pathophysiology with aging. In the last two decades, much has been written about the potential central role of mitochondrial dysfunction in the processes of aging.7,8 Since the mitochondria are the site of utilization of the bulk of the inspired oxygen, they are also the site of abundant free radical generation.11,12 This is reflected in the high rate of mitochondrial DNA damage relative to the damaged products measured in nuclear DNA.7,8 Thus, cellular energy deficits caused by declines in mitochondrial function may impair normal cellular activity and lead to the death of cells via apoptosis or necrosis. Since the discoveries of the age-associated loss of melatonin and its free radical scavenger action, many theories relating melatonin to aging have been put forward. Although interest is great in the potential causal association between the drop in melatonin in the elderly and degenerative signs of aging, the research findings are suggestive but incomplete.1 Also, there are so far a only few reports examining the relationship between melatonin and mitochondrial function. This chapter summarizes what is known about changes in melatonin and mitochondrial function with age, the potential role of oxygen-derived radicals in aging, and melatonin’s ability to function as an antioxidant based on our recent results from senescence-accelerated mouse (SAM) studies.

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Figure 4. Age-related changes in mid-dark concentrations of serum melatonin (open columns) and cerebral 8-hydroxydeoxyguanosine (8-OHdG, diagonally hatched columns) in female senescence-accelerated mouse (SAMP6). Melatonin-treated animals were given melatonin in the drinking fluid (2 µg/ml) from 8 to 12 months of age. Data are mean ± SE. Asterisks designate significant difference: ** P < 0.01, *** P < 0.001.

The SAM was developed by Takeda and colleagues37 as a murine model of accelerated aging. The SAM model comprises two strains:one prone to accelerated senescence (SAMP), and one resistant to accelerated senescence (SAMR1). SAMP8, a substrain of SAMP, tends to show deterioration of memory and ability to learn in addition to characteristics of accelerated aging.38 The SAMP6 strain of SAMP was reported to be a spontaneous experimental model of osteoporosis.39

Age-Related Changes in Peroxidation Products of Lipids, Proteins and DNA in SAM Initially, we examined the age-related changes in neural oxidative DNA damage in SAMP6 mice and oxidative damage of neural lipids and proteins in SAMP8 mice in the middle of the dark period of the daily light:dark cycle.40,41 Some mice were given melatonin in the drinking water (2 µg/ml) from 7 or 8 months of age to 12 months of age. As shown in Figure 4, the serum melatonin concentration in the SAMP6 mice decreased markedly between 8 and 12 months of age, and this dose of melatonin inhibited this age-related physiologic decline in melatonin concentration. 8-OHdG and 8-OHdG/dG, markers of free radical-mediated DNA base modification, exhibited significant age-related increases in SAMP6 mice. Thiobarbituric acid reactive substances (TBARS), and oxidized protein (protein carbonyl) in SAMP8 mice accumulated more rapidly than in SAMR1 mice (Fig. 5). Each of these age-related increases in oxidation products was corrected by long-term, orally administered physiologic levels of melatonin. Also, this melatonin treatment significantly corrected the age-related decrease in neural GPx activity. These results suggest an age-related increase in cerebral tissue vulnerability to oxidation in SAMP mice that can be modified by melatonin, most likely through the ability of melatonin to scavenge oxygen free radicals and to stimulate antioxidant enzyme activity.

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Figure 5. Age-related changes in concentration of lipid peroxidation product (TBARS, A), oxidized protein (protein carbonyl, B) and glutathione peroxidase (GPx) activity (C) in SAMR1 mice (open columns) or SAMP8 mice (closed columns). Melatonin-treated animals were given melatonin in the drinking fluid (2 µg/ml) from 7 to 12 months of age. Data are mean ± SD. Asterisks designate significant difference: * P < 0.05, ** P < 0.01, *** P < 0.001.

Age-Related Changes in Hepatic Mitochondrial Function The protective effects of melatonin on the age-related decrease in mitochondrial function were also documented in another series of SAM experiments.42,43 In these studies, we investigated whether long-term administration of physiological levels of melatonin influences hepatic mitochondrial respiratory activity in SAMP8 and SAMR1 mice. As shown in Figure 6, RCI, ADP/O, State 3 respiration, and DNP-dependent uncoupled respiration exhibited age-associated decreases in SAMP8 mice. SAMP8 mice also showed significant age-associated reductions in respiratory complex I and IV activities (Fig. 7). In contrast, no age-related effects were found in these parameters in SAMR1 mice. While no age effect was found in TBARS in both mouse strains, TBARS levels in SAMP8 mice were significantly more abundant than in SAMR1 mice.

Figure 6. Age-related changes in respiratory control index (RCI) (A), ADP/O (B) and dinitrophenol (DNP)-induced uncoupled respiration (C) in hepatic mitochondria from SAMR1 mice (open columns) or SAMP8 mice (closed columns). Melatonin-treated animals were given melatonin in the drinking fluid (2 µg/ml) from 7 to 12 months of age. Data are mean ± SE. Asterisks designate significant difference: * P < 0.05, ** P < 0.01, *** P < 0.001.

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Figure 7. Age-related changes in activity of complex I (A) and complex IV (B) in hepatic mitochondria from SAMR1 mice (open columns) or SAMP8 mice (closed columns). Melatonin-treated animals were given melatonin in the drinking fluid (2 µg/ml) from 7 to 12 months of age. Data are mean ± SE. Asterisks designate significant difference: * P < 0.05, ** P < 0.01.

While GPx activity SAMR1 mice did not change during the period tested, SAMP8 mice showed a significant decrease in GPx activity by 12 months. Daily oral melatonin administration significantly increased RCI, State 3 respiration, DNP-dependent uncoupled respiration, and complex I and IV activities in both mouse strains at 12 months of age. In addition, this melatonin treatment also decreased TBARS and increased GPx activity in both mouse strains at 12 months of age. The stimulatory effect of melatonin on mitochondrial respiratory activity was also demonstrated in a study of administration of an acute pharmacologic dose of melatonin.44 In this investigation, melatonin was administered to SAMP8 mice and SAMR1 mice intraperitoneally at a dose of 10 mg/kg body weight 1 h prior to sacrifice, when peak serum melatonin was achieved. Melatonin administration significantly increased RCI, ADP/O, State 3 respiration and DNP-dependent uncoupled respiration in SAMP8 mice, while also significantly reducing State 4 respiration in SAMP8 mice. The injection of melatonin also significantly increased complex I activity in both mouse strains and complex IV activity in SAMP8 mice. The earlier reduction in hepatic mitochondrial RCI from SAMP8 mice appears to be the result of an increased State 4 respiration and a reduced State 3 respiration. Similarly, the reduction in RCI by 12 months of age in SAMP8 mice was demonstrated by other investigators.45 The reduction in ADP/O in SAMP8 mice may be explained by their age-related uncoupling, and may also explain the functional decline in SAMP8 mice, because less ATP is made per unit of oxygen consumed. The reduction of DNP-induced uncoupling of respiration in SAMP8 mice suggests that the coupling mechanism for energy transfer reactions of the electron transport system may be altered in this mouse strain. Thus, age-associated decreases in State 3 respiration and DNP-induced uncoupled respiration in SAMP8 mice suggest that the proton electrochemical gradient of the mitochondrial inner membrane might be altered in this strain. The mechanism underlying the age-associated decline in the mitochondrial respiratory function in SAMP8 mice may be related, in part, to damage inflicted by oxygen-based radicals. Multiple lines of evidence, including the present results, suggest an increased susceptibility to oxidative deterioration in SAMP8 mice, which shows accelerated aging.34 The increase in TBARS and a low GPx activity in SAMP8 mice suggest that oxidative stress because of free radical generation combined with a less effective system of defense against oxidative stress may cause

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Figure 8. Schematic diagram of the hypothesized melatonin involvement in mitochondrial electron transport system and oxidative phosphorylation. Melatonin easily crosses biological membranes to reach the mitochondrial matrix. Melatonin can scavenge oxygen free radicals directly. Additionally, melatonin’s antioxidant action may derive from its stimulatory effect on glutathione redox cycling. Possible mechanism for the protective effect of melatonin on mitochondrial respiratory function may involve the stimulation of respiratory chain complex I and IV and the stabilization of mitochondrial membranes in addition to the ability to scavenge oxygen free radicals. As a consequence of the melatonin’s action at the level of mitochondria, electron transfer and oxidative phosphorylation are enhanced preserving energy status.

the alteration in mitochondrial function seen in this strain. Thus, the accumulated defects in mitochondrial function observed with age may reflect changes in the proton electrochemical gradient across the inner mitochondrial membrane. Such alterations influence many activities that depend on the membrane potential. These changes in the proton electrochemical gradient may reflect accumulated oxidative damage to the inner membrane. The results indicate that melatonin protects against the age-related decline in mitochondrial respiratory function. They also suggest that the usual age-related reduction in endogenous melatonin synthesis may contribute to the rise in DNA, lipid, and protein oxidation, while exogenously administered melatonin at essentially physiologic levels decreases oxidative stress. The findings are also consistent with observations in the aging rat, showing that pinealectomy, a procedure that induces a relative melatonin deficiency, accelerates the accumulation of oxidatively damaged lipid, protein, and DNA products in many organs.46 The protective effects of melatonin on the age-related decline in mitochondrial function are most likely achieved through its ability to scavenge oxygen free radicals and to stimulate antioxidant enzyme activity. Additionally, the direct stimulatory effect of melatonin on respiratory chains complex I and complex IV may involve another possible mechanism for the protective effect of melatonin on mitochondrial function. The mechanism underlying this actions is unknown. However, inhibition of complex I reportedly augment production of O2-•, H2O2, and • OH, which trigger cell damage.47 Thus, stimulation of the respiratory chain complex by melatonin may be another indirect way by which melatonin limits molecular destruction of essential molecules by reactive oxygen species.

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Another possible mechanism for the protective effects of melatonin on mitochondrial function may involve the stabilization of mitochondrial membranes. Melatonin reportedly stabilizes cell membrane fluidity, thereby preserving the dysfunction of these structures.47,48 Melatonin is highly lipophilic, which enables it to cross a variety of membranes readily and enter both the cytoplasmic and nuclear compartment of cells.1,2 When melatonin enters cellular membranes, it becomes situated mainly in a superficial position in the lipid bilayer, near the polar heads of membrane phospholipids. However, further studies are needed to document this possibility.

Concluding Remarks Knowledge concerning the interaction between free radicals and the chief secretory product of the pineal gland, melatonin, has advanced rapidly in the last decade.49-51 Many of the mechanism by which melatonin detoxifies oxygen and nitrogen-based reactants have been defined. In the bulk of the studies where melatonin has been tested in experimental models of diseases, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, its efficacy in reducing neuronal degradation and loss has been attributed to its ability to function as a ubiquitous and multifaceted free radical scavenger and antioxidant.52-55 The discovery of new actions of melatonin in mitochondria may support a novel mechanism, which may explain some of the protective effects of melatonin on cell survival (Fig. 8). Although the data are suggestive of an association between melatonin and longevity, more thorough investigations must be carried out to prove or disprove this relationship.

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45. Nakahara H, Kanno T, Inai Y et al. Mitochondrial dysfunction in the senescence accelerated mouse (SAM). Free Radical Biol Med 1994; s16:621-626. 46. Reiter RJ, Tan DX, Tan SJ et al. Augmentation of indices of oxidative damage in life-long melatonin-deficient rats. Mech Aging Dev 1999; 110:157-173. 47. Garcia JJ, Reiter RJ, Ortiz GG et al. Melatonin enhances tamoxifen’s ability to prevent the reduction in microsomal membrane fluidity induced by lipid peroxidation. J Membr Biol 1998; 162:59-65. 48. Garcia JJ, Reiter RJ, Guerrero JM et al. Melatonin presents changes in microsomal membrane fluidity during induced lipid peroxidation. FEBA Lett 1997; 408:297-308 49. Pierpaoli W, Dall’ara A, Pedrininis E et al. The pineal control of aging. The effects of melatonin and pineal grafting on the survival of older mice. Ann NY Acad Sci USA 1991; 91:291-313. 50. Lenz S, Izumi S, Benediktsson H et al. Lithium chloride enhances survival of N2B/W lupus mice: Influence of melatonin and timing of treatment. Int J Immunopharmacol 1995; 17:581-592. 51. Oaknin-Bendahan S, Anis Y, Nir I et al. Effects of long-term administration on melatonin and a putative antagonist on the aging rats. Neuro Report 1995; 6:785-788 52. Acuna-Castroviejo D, Aoto-Montes A, Monti MG et al. Melatonin is protective against MPTP-induced striatal and hippocampal lesions. Life Sci 1997; 60:PL23-PL29. 53. Kim YS, Joo WS, Jin BK et al. Melatonin protects against 6- OHDA-induced neuronal death of nigrostriatal dopaminergic system. Neuro Report 1998; 9:2387-2390. 54. Reiter RJ, Cabrera J, Sainz RM et al. Melatonin as a pharmacological agent against neuronal loss in experimental models of Huntington’s disease, Alzheimer’s disease and Parkinsonism. Ann NY Acad Sci 1999; 890:471-485. 55. 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.

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CHAPTER 19

Melatonin Use As a Bone-Protecting Substance Daniel P. Cardinali, Marta G. Ladizesky, Verónica Boggio, Rodolfo A. Cutrera, Ana I. Esquifino and Carlos Mautalen

Abstract

T

his chapter discusses early studies on melatonin-bone relationships and recent data that suggest a direct effect of melatonin on bone. Suppression of melatonin secretion lowered serum calcium concentration, an effect prevented by melatonin administration. Treatment of ovariectomized rats with melatonin prevents bone loss by an effect partly dependent on residual estradiol levels. Melatonin presumably acts as an autacoid in bone cells since it is present in high quantities in bone marrow, where bone cell precursors are located. Melatonin dose-dependently augments proteins that are incorporated into the bone matrix, like procollagen type I c-peptide. Osteoprotegerin, an osteoblastic protein that inhibits the differentiation of osteoclasts is also augmented by melatonin in vitro. Another possible target cell for melatonin is the osteoclast, which degrades bone partly by generating free radicals. Melatonin through its free radical scavenger and antioxidant properties may impair osteoclast activity and bone resorption. Additionally, melatonin could impair osteopenia by improving slow sleep and restoring growth hormone secretion. The feasibility of the use of melatonin in therapy for augmenting bone mass in osteopenic diseases is discussed.

Mammalian Bone Is Continuously Remodeled Resorption of old bone by osteoclasts followed by formation of new bone by osteoblasts is a central event in bone physiology. These two closely coupled events are crucial for keeping anatomical and structural integrity of the skeleton.18 Bone remodeling runs cyclically with osteoclasts adhering to bone to remove it by acidification and proteolytic digestion. Then osteoblasts invade the resorption site and form new bone by secreting and mineralizing osteoid. After this, another type of cells, the lining cells, cover the area to restart the cycle again.18 Since levels of melatonin decline with age4,7,11,19,31 the hypothesis that melatonin is involved in age-related disorders like postmenopausal osteoporosis was entertained. This chapter will review the accumulating evidence indicating that the bone is a target for melatonin activity.

Early Studies Indicated an Effect of Melatonin on Bone Melatonin injection augmented serum calcium in rats through an effect best explained by changes in the secretion of PTH and calcitonin.3,13 In newborn rats, suppression of melatonin secretion by white light lowered serum calcium concentration.8 Occipital shielding or treatment of newborn rats with melatonin, prevented serum calcium decrease.10 That melatonin has a direct effect on bone was indicated by the demonstration that melatonin inhibited in vitro the increased calcium uptake in bone samples of rats treated with corticosterone.9 Indeed, 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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bone marrow cells, among which osteoblast and osteoclast precursors are found, are capable of synthesizing melatonin.2 Another indication that melatonin could act as an autacoid for bone cells was the finding of high concentrations of melatonin in bone marrow samples.33

Melatonin Acts on Both Osteoblasts and Osteoclasts in Vitro A direct activity of melatonin was demonstrated in rat preosteoblast and osteoblast-like osteosarcoma cell lines.29 Preosteoblast cells in the presence of nanomolar concentrations of melatonin underwent cell differentiation. After melatonin exposure both cell lines increased gene expression of bone matrix protein sialoprotein, as well as other bone marker proteins, like alkaline phosphatase, osteopontin and osteocalcin. The effect of melatonin was counteracted by the melatonin receptor antagonist luzindole, a suggestion of the involvement of melatonin receptors.29 In another study on human bone cells and osteoblastic cell lines exposed to melatonin, the methoxyindole increased cell proliferation in a dose-dependent way (maximal effect at 50 µM melatonin). In these cells melatonin increased procollagen type I c-peptide production without modifying alkaline phosphatase or osteocalcin.21 Several osteoblast-derived local factors control osteoclast activity. One of them is the osteoclast differentiation factor, a transmembrane protein that binds to the receptor activator of nuclear factor-κB (RANK) on the surface of the osteoclast to activate bone resorption. This effect is diminished by osteoprotegerin, which is an osteoblast-derived protein that inhibits the binding of osteoclast differentiation factor to its target cells.15 In mouse osteoblastic cell lines melatonin increased mRNA and protein levels of osteoprotegerin while decreasing RANK mRNA at micromolar concentrations. These observations indicate that melatonin can cause inhibition of bone resorption and augmentation of bone mass by down-regulating RANK-mediated osteoclast activation.14 In studies on goldfish scale, melatonin (10-100 nM) suppressed tartrate-resistant acid phosphatase and alkaline phosphatase activities, markers of osteoclastic and osteoblastic activity, respectively.32 Moreover, melatonin inhibited the stimulatory effect of estradiol as well as mRNA expression of estrogen receptor and of insulin-like growth factor, both related to osteoblastic growth and differentiation.32 The data indicate that melatonin may act directly on osteoclastic and osteoblastic cells by suppressing their differentiation. This is the single observation indicating an inhibitory effect of melatonin on osteoblasts. Since melatonin also inhibited osteoclasts, the final outcome of the effect in goldfish scale remains undefined. Osteoclasts generate high levels of superoxide anions during bone resorption and this may contribute to the degradative process. Melatonin is a significant free radical scavenger and antioxidant at both physiological and pharmacological concentrations.27,28 Besides its ability to directly neutralize a number of free radicals and reactive oxygen and nitrogen species, melatonin stimulates several antioxidative enzymes that increase its efficiency as an antioxidant. Melatonin protects macromolecules in all compartments of the cell making them more resistant to oxidative attack.27,28 Therefore, the effect of melatonin in preventing osteoclast activity in bone may depend in part on the free radical scavenging properties of melatonin. Experimental studies to test this hypothesis are lacking.

Low Melatonin Levels Correlate with Osteoporosis

Melatonin levels declines during menopause,30 diminishes with immobility36 and augments after exercise.1 These observations suggested the involvement of melatonin in age-related bone disease. Postmenopausal osteoporosis is the most common metabolic bone disease and a heterogeneous condition of skeletal fragility that leads to increased fracture risk. Although there is evidence that estrogen deficiency is an important contributory factor in the pathogenesis of osteoporosis,6,12 heredity, hormonal status, age, and various environmental factors exert modulating effects on bone and contribute to the etiology of this condition.18 A negative correlation between changes in 24-h profile of serum melatonin levels and circadian metabolism of type I

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collagen (an index of bone turnover) in postmenopausal, obese women was found.24 Hence the decreased melatonin levels can be an aggravating factor for postmenopausal loss of bone mass. In a similarly designed experimental study in ovariectomized rats, serum levels of melatonin at death correlated negatively with biochemical markers of bone resorption like cross-linked of type I collagen in serum or hydroxyproline and total calcium in urine, but not with biochemical markers of bone formation in serum (alkaline phosphatase; carboxy-terminal propeptide and carboxy-terminal telopeptide of type I procollagen).26

Melatonin Decreases Bone Loss in Vivo The first indication that melatonin administration is effective to decrease bone loss was provided by us using ovariectomized rats.17 Urinary deoxypyridinoline (a marker of bone resorption) and calcium excretion, circulating levels of calcium, phosphorus and bone alkaline phosphatase activity (a marker of bone formation), and bone mineral density and content, and bone area of total skeleton, were measured in adult rats for up to 60 days after ovariectomy. Rats received melatonin in the drinking water (25 µg/ml water) or drinking water alone. Urinary deoxypyridinoline increased significantly after ovariectomy an effect prevented by melatonin 30 but not 60 days after surgery. Fifteen days after surgery, a significant increase in serum phosphorus and bone alkaline phosphatase levels occurred in ovariectomized rats receiving melatonin as compared to their controls. Sixty days after surgery bone mineral density and content, and bone area decreased significantly in ovariectomized rats, an effect not modified by melatonin administration. Serum estradiol decreased significantly by 30 days after ovariectomy to attain values close to the limit of detection of the assay by 60 days after ovariectomy. Our results supported the conclusion that treatment with melatonin modifies bone remodeling after ovariectomy providing a given concentration of estradiol was present.17 Two subsequent studies corroborated the in vivo preventing effect of melatonin on bone loss. Ostrowska et al23 examined whether pinealectomy and melatonin administration could affect the post-ovariectomy osteoporotic process in rats. Melatonin (50 µg/100 g b.w.) was administered i.p. during a 4-week period. Alkaline phosphatase activity, carboxy-terminal propeptide of type 1 procollagen and cross-linked carboxy-terminal telopeptide of type 1 collagen concentrations, as well as the urinary excretion of calcium and hydroxyproline were measured. The study demonstrated that pinealectomy augmented bone resorption while melatonin prevented such an effect. In rats with a preserved pineal gland the effect of melatonin on bone turnover markers was less pronounced and transient. These observations agreed with our own data17 in that the effect of melatonin in ovariectomized, pineal-intact, rats was transient and that it was presumably related to the circulating estrogen levels. Treatment of mice with 5 mg/kg per day or 50 mg/kg per day of melatonin for 4 weeks significantly increased bone mineral density and bone mass. The treatment significantly reduced bone resorption parameters without affecting bone formation. Therefore, although melatonin increases the proliferation, differentiation, and bone nodule formation activity of osteoblasts in vitro21,29 it may not be osteogenic in vivo, at least in young growing mice.14 We recently assessed the effect of melatonin on bone metabolism in ovariectomized rats receiving or not estradiol replacement therapy.16 Melatonin was administered in the drinking water (25 µg/ml water) and estradiol (10 µg/kg body weight) or vehicle was given s.c. 5 days/ week for up to 60 days after surgery.16 Ovariectomy augmented, and melatonin or estradiol lowered, urinary deoxypyridinoline excretion (a marker of bone resorption). This effect of melatonin was seen mainly in ovariectomized rats. The efficacy of estradiol to counteract ovariectomy-induced bone resorption was increased by melatonin. On day 60 after surgery, bone mineral density and content decreased after ovariectomy and augmented after estradiol injection. Melatonin augmented bone area of spine and bone mineral content of whole skeleton and tibia. The highest values observed were those of rats treated conjointly with estradiol and melatonin. Therefore, post-ovariectomy disruption of bone remodeling could be prevented in rats by administering a pharmacological amount of melatonin (in terms of circulating

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melatonin levels), providing that appropriate levels of circulating estradiol were present. Moreover, the efficacy of estradiol to counteract ovariectomy-induced effect on bone increased by the concomitant administration of melatonin.16

Promotion of Growth Hormone (GH) Release Could Partly Explain Melatonin Effect on Bone It is generally accepted that GH is the most important hormone for normal longitudinal bone growth.22 GH stimulates growth of cartilage and other tissues by increasing the number of cells rather than by increasing cell size. Melatonin administration releases GH both in rats25 and man.5,34 The administration of a 3 mg dose of melatonin during 14 nights to elderly patients with chronic primary insomnia brought about a significant reduction in wake time after sleep onset, and an increase in sleep efficiency and stage 2 sleep.20 It must be noted that stage 2-4 sleep is associated with GH release in normal subjects.35 Thus an improvement of non REM sleep after melatonin in aged subjects is presumably producing an increase of GH release, with an indirect effect on bone loss during senescence.

Conclusions Melatonin can positively influence age-associated bone loss in a number of ways. One is direct on the bone by acting on osteoclasts and perhaps on osteoblasts and turning the calcium balance positive. The effect of melatonin needs adequate amounts of estrogen to become manifested.16 At least in the case of osteoclasts, melatonin activity could be associated with the potent antioxidant properties melatonin has (see refs. 27, 28). Another way melatonin can act is indirect, via the release of GH associated with the improvement of non REM sleep in old subjects.20,35 Collectively these observations open the possibility for a novel use of melatonin in human therapy to augment bone mass in diseases characterized by low bone mass and increased fragility, like osteoporosis. Controlled clinical trials to assess this possibility are being carried out.

Acknowledgments Work in authors’ laboratories was supported in part by the University of Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina, Fundación Bunge y Born, Buenos Aires, Fundación Antorchas, Buenos Aires, and Agencia Nacional de Promoción Científica y Tecnológica, Argentina.

References 1. Carr DB, Reppert SM, Bullen B et al. Plasma melatonin increases during exercise in women. J Clin Endocrinol Metab 1981; 53:224-225. 2. 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. 3. Csaba G, Barath P. The effect of pinealectomy on the parafollicular cells of the rat thyroid gland. Acta Anat (Basel) 1974; 88:137-146. 4. Dori D, Casale G, Solerte SB et al. Chrono-neuroendocrinological aspects of physiological aging and senile dementia. Chronobiologia 1994; 21:121-126. 5. Forsling ML, Wheeler MJ, Williams AJ. The effect of melatonin administration on pituitary hormone secretion in man. Clin Endocrinol (Oxf) 1999; 51:637-642. 6. Gaumet N, Braillon P, Seibel MJ et al. Influence of aging on cortical and trabecular bone response to estradiol treatment in ovariectomized rats. Gerontology 1998; 44:132-139. 7. Girotti L, Lago M, Ianovsky O et al. Low urinary 6-sulphatoxymelatonin levels in patients with coronary artery disease. J Pineal Res 2000; 29:138-142. 8. Hakanson DO, Bergstrom WH. Phototherapy-induced hypocalcemia in newborn rats: Prevention by melatonin. Science 1981; 214:807-809. 9. Hakanson DO, Bergstrom WH. Pineal and adrenal effects on calcium homeostasis in the rat. Pediatr Res 1990; 27:571-573. 10. Hakanson DO, Penny R, Bergstrom WH. Calcemic responses to photic and pharmacologic manipulation of serum melatonin. Pediatr Res 1987; 22:414-416.

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11. Iguchi H, Kato KI, Ibayashi H. Age-dependent reduction in serum melatonin concentrations in healthy human subjects. J Clin Endocrinol Metab 1982; 55:27-29. 12. Kalu DN, Liu CC, Hardin RR et al. The aged rat model of ovarian hormone deficiency bone loss. Endocrinology 1989; 124:7-16. 13. Kiss J, Banhegyi D, Csaba G. Endocrine regulation of blood calcium level. IInd ed. Relationship between the pineal body and the parathyroid glands. Acta Med Acad Sci Hung 1969; 26:363-370. 14. Koyama H, Nakade O, Takada Y et al. Melatonin at pharmacologic doses increases bone mass by suppressing resorption through down-regulation of the RANKL-mediated osteoclast formation and activation. J Bone Miner Res 2002; 17:1219-1229. 15. Krane SM. Genetic control of bone remodeling — Insights from a rare disease. N Engl J Med 2002; 347:210-212. 16. Ladizesky MG, Boggio V, Albornoz LE et al. Melatonin increases oestradiol-induced bone formation in ovariectomized rats. J Pineal Res 2003; 34:143-151. 17. Ladizesky MG, Cutrera RA, Boggio V et al. Effect of melatonin on bone metabolism in ovariectomized rats. Life Sci 2001; 70:557-565. 18. Manolagas SC. Birth and death of bone cells: Basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocrine Rev 2000; 21:115-137. 19. 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. 20. Monti JM, Alvarino F, Cardinali D et al. Polysomnographic study of the effect of melatonin on sleep in elderly patients with chronic primary insomnia. Arch Gerontol Geriatr 1999; 28:85-98. 21. Nakade O, Koyama H, Ariji H et al. Melatonin stimulates proliferation and type I collagen synthesis in human bone cells in vitro. J Pineal Res 1999; 27:106-110. 22. Ohlsson C, Bengtsson B, Isaksson OGP et al. Growth hormone and bone. Endocr Rev 1998; 19:55-79. 23. Ostrowska Z, Kos-Kudla B, Marek B et al. The influence of pinealectomy and melatonin administration on the dynamic pattern of biochemical markers of bone metabolism in experimental osteoporosis in the rat. Neuroendocrinol Lett 2002; 23 Suppl 1:104-109. 24. Ostrowska Z, Kos-Kudla B, Marek B et al. Assessment of the relationship between circadian variations of salivary melatonin levels and type I collagen metabolism in postmenopausal obese women. Neuroendocrinol Lett 2001; 22:121-127. 25. Ostrowska Z, Kos-Kudla B, Swietochowska E et al. Influence of pinealectomy and long-term melatonin administration on GH-IGF-I axis function in male rats. Neuroendocrinol Lett 2001; 22:255-262. 26. Ostrowska Z, Kos-Kudla B, Swietochowska E et al. Assessment of the relationship between dynamic pattern of nighttime levels of melatonin and chosen biochemical markers of bone metabolism in a rat model of postmenopausal osteoporosis. Neuroendocrinol Lett 2001; 22:129-136. 27. 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. 28. Reiter RJ, Tan DX, Qi W et al. Pharmacology and physiology of melatonin in the reduction of oxidative stress in vivo. Biol Signals Recept 2000; 9:160-171. 29. Roth JA, Kim BG, Lin WL et al. Melatonin promotes osteoblast differentiation and bone formation. J Biol Chem 1999; 274:22041-22047. 30. Sack RL, Lewy AJ, Erb DL et al. Human melatonin production decreases with age. J Pineal Res 1986; 3:379-388. 31. 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. 32. Suzuki N, Hattori A. Melatonin suppresses osteoclastic and osteoblastic activities in the scales of goldfish. J Pineal Res 2002; 33:253-258. 33. Tan DX, Manchester LC, Reiter RJ et al. Identification of highly elevated levels of melatonin in bone marrow: Its origin and significance. Biochim Biophys Acta 1999; 1472:206-214. 34. Valcavi R, Dieguez C, Azzarito C et al. Effect of oral administration of melatonin on GH responses to GRF 1-44 in normal subjects. Clin Endocrinol (Oxf) 1987; 26:453-458. 35. Van Cauter E, Plat L, Copinschi G. Interrelations between sleep and the somatotropic axis. Sleep 1998; 21:553-566. 36. Vaughan GM, McDonald SD, Jordan RM et al. Melatonin, pituitary function and stress in humans. Psychoneuroendocrinology 1979; 4:351-362.

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CHAPTER 20

Melatonin, Light and Migraine Bruno Claustrat, Christophe Chiquet, Jocelyne Brun and Guy Chazot

Abstract

M

igraine can be viewed as a transient disturbance of the body adaptive response to internal or external environmental changes. Among these factors, light is a major precipitating or aggravating factor of attacks. The few reports on migraine melatonin (MLT) relationship are concordant with an MLT secretion defect. Several mechanisms, which are not exclusive, might be put forward: local sympathetic abnormality, hypersensitivity of the retino-hypothalamic pathway, functional disturbance at the level of the suprachiasmatic nucleus. Since the pineal gland plays a role in the homeostatic equilibrium of the organism, low MLT could reinforce vulnerability of the rhythmic organization of the central nervous system in migraine and facilitate the cascade of events related to perivascular inflammation in the trigeminovascular system.

Introduction Although significant progress in the understanding of the pathophysiology of migraine has been made in recent years, major mechanisms are still unknown. There is a controversy whether migraine is a primary neurogenic disorder or vascular functional headache. It has been suggested that a periodic central disturbance of hypothalamic activity could account for the periodicity of the migraine attack and also provide a mechanism by which emotional disturbances could be mediated by pathways from the limbic system to the hypothalamus.1 Several data support the hypothesis that the suprachiasmatic nucleus of the hypothalamus might be the initial site of migraine attacks.2 Finally, migraine can be viewed as a transient disturbance of the body adaptive response to internal or external environmental changes.3 There is no doubt that migraine is commoner in women but this sexual predilection occurs only after puberty. Many women experience increased frequency and intensity at times of hormonal changes, i.e., menstruation, ovulation, the first trimester of pregnancy and the early postpartum period.4 Stress can be also a precipitating factor and affects pain control mechanisms. Migraine patients are sensitive to light, even at the interictal period, and display photophobia. As early as the second century A.D., Aretae Cappadocis wrote: “fugiunt enim quodam modo lucem, tenebraeque his aegritudinem solentur” (they avoid light by all possible means and the dark subsides their feeling of sickness).5 Changes in the life-routine (week-end or rest-day, shift-work, jet-lag) can also facilitate the onset of attacks. Sensitivity to smells and noise often increases in migraine.6 Strong odours may trigger attacks, sensitivity to odours and olfactory hallucinations sometimes develop during migraine and headaches attributed to diet might involve olfaction. Among the other external factors to be considered as environmental trigger factors, geomagnetic activity (G.M.A.), which is able to modify the pineal secretion, could be a candidate: there is a linear correlation of G.M.A. with the severity of the migraine attacks.7 This phenomenon could be related to the magnetic sensitivity of the pineal gland: in

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rats, pineal MLT content as well as N-acetyl transferase are inhibited by nocturnal exposure to a short-term magnetic field; their intact visual system is necessary for pineal response. Since the pineal gland plays an important role in homeostatic equilibrium, in close relationship with changing environmental conditions, the possibility that the pineal hormone MLT is deficient in migraine has been proposed and we therefore undertook investigations on MLT secretion in migraine some years ago.8

The Regulating System of Melatonin Secretion Like some circadian rhythms in mammals (drinking and feeding, wake-sleep cycle, temperature, cortisol or corticosterone....), the MLT rhythm is generated by an endogenous clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus. In fact, there are no anatomical or pathophysiological data reported in humans.9 Results have been obtained in animals, mainly in rodents and monkey, and extrapolated to humans.10 The photic information is transmitted to the central pacemaker via retino-hypothalamic fibers. The neural pathway from the SCN to the pineal gland passes first through the upper part of the cervical spinal cord, where synaptic connections are made with preganglionic cell bodies of the superior cervical ganglia (SCG) of the sympathetic chains.11 Then, neural cells in the SCG send projections to the pineal gland. The main neurotransmitter regulating the pineal gland is norepinephrine, which is released at night, in response to stimulatory signals originating in the SCN. In addition to norepinephrine, the sympathetic neurons of the SCG contain neuropeptide Y. Nerve fibers innervating the pineal gland also originate in perikarya located in the parasympathetic sphenopalatine and otic ganglia and the trigeminal ganglion which is the sensory ganglion of the fifth cranial nerve.12 The trigeminal ganglion also projects, via the ophthalmic division, to pial and dural arteries, venal sinuses and circle of Willis. The existence of sensory pathways surrounding large cerebral arteries provides a neuroanatomical explanation for the hemicranial distribution of migraine. With regard to the parasympathetic innervation, two peptides appear to be important: vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI), whereas substance P (SP), calcitonin gene-related peptide (CGRP), and pituitary adenylate cyclase-activating peptide (PACAP) are present in cell bodies of the trigeminal ganglion. These peptides with vasoactive properties are responsible for the vasomotor response and/or pain following activation of trigeminovascular fibers which contributes to migraine attacks.13 The light/dark cycle is the main Zeitgeber of the regulation system: in the presence of light, the output from the retinohypothalamic tract inhibits the MLT synthesis, whereas darkness stimulates it. The hormone is released from the pinealocytes in venous blood and can modulate the brain activity after passage across the blood-brain barrier.14 There is a possibility of a feed-back of MLT on the clock:15 MLT binding sites have been revealed at this level and MLT alters electrical or metabolic activity of the suprachiasmatic nuclei.16,17 An abnormality at any level of the regulation system unspecifically modifies MLT secretion, especially in patients with sympathalgia or dysautonomia.18 For example, in cluster headache, both a decrease of the amplitude and a phase advance of the MLT rhythm have been observed.19,20 Light displays a synchronising or an inhibitory effect on MLT secretion. The MLT rhythm is entrained to the dark period or it can be acutely interrupted by light exposure during the night; however, the intensity of light required to suppress the MLT production is higher in humans than in most animal species; in 1980, Lewy et al21 demonstrated that MLT secretion could be inhibited in humans if an artificial light of sufficient intensity and duration was applied (a dose-dependent effect is observed between 500 and 2500 lux given for 2 hours from 2 a.m. to 4 a.m.). This inhibitory effect shows a spectral sensitivity: green light is the most active whereas red light is unefficient.22 Further, after exposure to light on several consecutive nights, the MLT secretion escapes the inhibitory effect and progressively shifts to the morning (phase delay).

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Artificial bright light is able to entrain the MLT secretion: in subjects exposed to light at 3000 lux between 3 and 9 a.m., a phase-advance is observed.23 Reversely, a phase delay is observed after evening administration of the light area. Do the seasonal alterations of the natural photoperiod have a repercussion on the MLT secretion? The data show some discrepancies in the healthy subject, probably because artificial light or social events display a masking effect on the natural synchronisation. At a high degree of latitude in Finland, Kauppila et al, however, observed a two-hour enlargement of the melatonin secretion in winter, compared with the summer period.24 A decrease in plasma ovarian steroids runs parallel with the winter increase of the MLT secretion.

Migraine and Light There are between 30% and 57% of patients whose attacks may be precipitated or aggravated by visual stimuli ranging from falling snow to sunlight, flicker of moving pictures, defined patterned stimuli, colour and fluorescent lights, television or sun. Sensitivity to glare persists between episodes of headache. Fox and Davis were not able to demonstrate a seasonal variation in frequency of attacks in Californian patients with migraine, although they reported a tendency to more attacks during the summer compared to the winter months.25 On the contrary, such a variation has been reported in patients living in the Arctic area who are more likely to have headache during the bright summer season.26 Visual symptoms are prominent in attacks of migraine. In addition to the characteristic hallucinations of the visual aura, photophobia is a feature of most attacks either with or without aura (to the extent that it is a diagnostic criterion in the 1988 classification of the International Headache Society).27 Between 66% and 88% of patients report the occurrence of photophobia which is more marked on the side of headache. Some of the abnormalities in visual function indicate hyperexcitability of the visual pathways.28 Electrophysiological investigations suggest that it occurs at the cortical level. The observed hyperexcitability may reflect a direct augmentation of excitatory mechanisms, either at the level of the glutaminergic thalamocortical synapse, or at the level of recurrent excitatory circuits known to operate within the visual cortex. Finally, the visual field of many patients is abnormal between migraine events, with deficits demonstrated for achromatic perimetry and temporal modulation (flicker) perimetry.29 In addition, approximately 50% of patients display short-wavelength sensitivity deficit, using short-wavelength automated perimetry (SWAP).30

Melatonin and Migraine There are few reports on MLT and migraine, a consequence of methodological and ethical problems which hamper the development of the protocols; especially, patients should be free, at any time, of drugs which could alter MLT secretion. We first described a decrease in the levels of plasma MLT determined at 23 h in a large sample of patients (n = 93).31 Alteration of the urinary MLT excretion throughout the ovarian cycle was also recorded in menstrual migraine. Decrease of MLT levels was shown to be reinforced at the time of menses by Murialdo et al,32 whereas we only detected a larger dispersion of the levels at this time.33 These findings are in agreement with the global concept of cervical sympathetic hypofunction in migraine. In two studies, one found no significant differences in pupillary function between migraine and control, the other concluded that parasympathetic deficiency and/or sympathetic hyperfunction occurs in migraine, but the majority of investigations, however, reach the consensus that sympathetic hypofunction is likely.34 This may be secondary to carotid artery dilation and subsequent compression of the sympathetic plexus surrounding the artery, or could reflect impairment in central aminergic neuronal systems. Recently, we observed a hypersensitivity to moderate light levels in migraine at the interictal period, as expressed by an alteration of the plasma MLT profiles following light exposure in migraine patients compared with controls.35 During the placebo sessions, we observed plasma MLT levels significantly higher in patients than in controls, which is opposite to the above-reported results. In these reports, however, patients were much older, their attacks more

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frequent and seniority of illness was higher. In the latter case, infraclinical inflammation could have injured the traversing sympathetic fibres of the intracranial internal carotid artery, especially those projecting to the pineal gland, with as a consequence an alteration of the MLT secretion. The hypersensitivity of patients to light we have reported concords with the concept of central neuronal excitability in migraine.36 Since MLT secretion is inhibited during the day through the retinohypothalamic pathway (of which a main transmitter is glutamate) and the SCN is also involved in rapid changes of the light-induced pineal metabolism, we suggest that hypersensitivity of MLT suppression to light in migraine is at least in part the result of an increase of glutamate transmission of the input pathway. This observation is in agreement with results showing that GABA agonists which modulate glutamate-mediated neural transmission are effective in the treatment of migraine.37

Conclusion There is evidence of impaired MLT secretion in migraine. Several mechanisms which are not mutually exclusive might be involved: sympathetic abnormality, a consequence of local vasodilation; hypersensitivity to light of the retinohypothalamic pathway; finally, the SCN, which also drives the MLT secretion, could be the initial site of migraine attacks. Recently, retinal ganglion cells innervating the SCN were shown to intrinsically respond to light.38,39 These melanopsin containing cells are candidate photoreceptors for the photic entrainment of circadian rhythms, because the sensitivity and slow kinetics of the light response are compatible with those of the photic entrainment mechanism.39 Furthermore, this system appears to send photic information not only to the endogenous clock in the SCN, but also to other brain areas involved in irradiance detection, such as light activated pupil response. An abnormality at the level of this irradiance detection system could thus be suspected as a contributing factor in migraine. Given that migraine attacks are triggered by both light and changes in living habits, these data suggest a pivotal role of the retinohypothalamic tractus which includes SCN involved both in long-term phase-adjustments and rapid changes of light-induced pineal metabolism.40 Since the pineal gland maintains the organism in proper synchrony with the prevailing environmental conditions, these data support the existence of vulnerability of the rhythmic organisation of the central nervous system in migraine. In that sense, recurrent hemicranial headache and unilateral orbital cephalalgia with or without sympathetic symptoms have been described in pinealectomized subjects.41 Taking into consideration the antioxidative properties of MLT, the altered MLT secretion could also contribute to reinforce vulnerability to oxidative stress which has been reported to play a role in the etiology of migraine.42-44 Free radicals, mainly activating lipoxygenases, may cause unbalance of prostacyclin thromboxane pathways in blood vessels, platelets and white cells, thereby stimulating leukotrienes to the setting up of painful inflammatory reactions. The question remains whether MLT administration could be effective in the prophylaxis of migraine or as an attack treatment. Within the CNS, melatonin is a putative modulator of GABA function.45 Decreased levels of MLT in migraine might therefore reduce the activation threshold of GABAergic pain circuits. In addition, MLT inhibits the synthesis of prostaglandin E2 which activates sterile perivascular inflammation in the trigeminovascular system, inhibits NO synthesis and depresses calcium uptake.46-49 Further, MLT binding sites have been localised in cerebral blood vessels of primates, especially the circle of Willis, and MLT reduces cerebral blood flow in rats.50,51 Finally, although vasodilation or vasoconstriction has been reported, there is evidence that low (nmolar) MLT doses potentiate contractile responses to adrenergic nerve stimulation in isolated ring segments of rat caudal artery.52-54 On the other hand, headache occurs after oral administration of several mgs of MLT.55,56 In addition, an inappropriate time of administration of this hormone which works as a chronobiotic drug, able to advance or to delay the endogenous rhythms, could reinforce endogenous dyschronia.57,58 In that sense, MLT treatment in cluster headache was not very demonstrative.59 Control trials in this area would be needed to confirm the potential interest of MLT in migraine treatment.

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References 1. Welch KMA. Migraine. A biobehavioral disorder. Arch Neurol 1987; 44:323-327. 2. Zurak N. Role of the suprachiasmatic nucleus in the pathogenesis of migraine attacks. Cephalalgia 1997; 17:723-728. 3. Nappi G, Micieli G, Sandrini G et al. Headache temporal patterns: Towards a chronobiological model. Cephalalgia 1983; Suppl 1:21-30. 4. Edelson RN. Menstrual migraine and other hormonal aspects of migraine. Headache 1985; 25:376-379. 5. Aretaei Cappadocis. De causis et signis acutorum et diuturnorum morborum. Liber IV Grasset Lausanne 1772. 6. Snyder RD, Drummond PD. Olfaction in migraine. Cephalalgia 1997; 17:729-732. 7. Kuritzky A, Zoldan Y, Hering R et al. Geomagnetic activity and the severity of the migraine attack. Headache 1987; 27:87-89. 8. Toglia JU. Is migraine due to a deficiency of pineal melatonin? Ital J Neurol Sci 1986; 7:19-23. 9. Lydic R, Schoene WC, Czeisler CA et al. Suprachiasmatic region of the human hypothalamus: homolog to the primate circadian pacemaker? Sleep 1980; 2:355-361. 10. Edgar DM, Dement WC, Fuller CA. Effect of SCN lesions on sleep in squirrel monkeys: Evidence for opponent processes in sleep-wake regulation. J Neurosci 1993; 13:1065-1079. 11. 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. 12. Moller M, Baeres FMM. The anatomy and innervation of the mammalian pineal gland. Cell Tissue Res 2002; 309:139-150. 13. Moskowitz MA. Neurogenic inflammation in the pathophysiology and treatment of migraine. Neurology 1993; 43:16-20. 14. Pardridge WM, Mietus LJ. Transport of albumin-bound melatonin through the blood-brain barrier. J of Neurochemistry 1980; 34:1761-1763. 15. Reppert SM, Weaver DR, Rivkees SA et al. Putative melatonin receptors in a human biological clock. Science 1988; 242:78-91. 16. McArthur AJ, Gillette MU, Prosser RA. Melatonin directly resets the rat suprachiasmatic circadian clock in vitro. Brain Research 1991; 565:158-161. 17. Shibata S, Cassone VM, Moore RY. Effects of melatonin on neuronal activity in the rat suprachiasmatic nucleus in vitro. Neuroscience Letters 1989; 97:140-144. 18. O’Brien IAD, Lewin IG, O’Hare JP et al. Abnormal circadian rhythm of melatonin in diabetic autonomic neuropathy. Clinical Endocrinology 1986; 24:359-364. 19. Waldenlind E, Gustafsson SA, Ekbom K et al. Circadian secretion of cortisol and melatonin in cluster headache during active cluster periods and remission. J Neurol Neurosurg Psychiatry 1987; 50:207-213. 20. Chazot G, Claustrat B, Brun J et al. A chronobiological study of melatonin, cortisol growth hormone and prolactin secretion in cluster headache. Cephalalgia 1984; 4:213-220. 21. Lewy AJ, Wehr TA, Goodwin FK et al. Light suppresses melatonin secretion in humans. Science 1980; 210:1267-1269. 22. Horne JA, Donlon J, Arendt J. Green light attenuates melatonin output and sleepiness during sleep deprivation. Sleep 1991; 14:233-240. 23. Buresova M, Dvorakova M, Zvolsky P et al. Early morning bright light phase advances the human circadian pacemaker within one day. Neuroscience Letters 1991; 121:47-50. 24. Kauppila A, Kivela A, Pakarinen A et al. Inverse seasonal relationship between melatonin and ovarian activity in humans in a region with a strong seasonal contrast in luminosity. J Clin Endocrinol Metabolism 1987; 65:823-828. 25. Fox AW, Davis RL. Migraine chronobiology. Headache 1998; 38:436-441. 26. Salvesen R, Bekkelund SI. Migraine, as compared to other headaches, is worse during midnight-sun summer than during polar night. A questionnaire study in an arctic population. Headache 2000; 40:824-829 27. Headache Classification Committee of the International Headache Society. Classification and diagnostic criteria for headache disorders, cranial neuralgia and facial pain. Cephalalgia 1988; 7(suppl.):13-73. 28. McColl SL, Wilkinson F. Visual contrast gain control in migraine: Measures of visual cortical excitability and inhibition. Cephalalgia 2000; 20:74-84. 29. Mc Kendrick AM, Vingrys AJ, Badcock DR et al. Visual field losses in subjects with migraine headaches. Invest Ophthalmol Vis Sci 2000; 41:1239-1247. 30. Mc Kendrick AM, Cioffi GA, Johnson CA. Short-wavelength sensitivity deficits in patients with migraine. Arch Ophthalmo 2002; 120:154-161.

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31. Claustrat B, Loisy C, Brun J et al. Nocturnal plasma melatonin levels in migraine: A preliminary report. Headache 1989; 29:242-244. 32. Murialdo G, Fonzi S, Costelli P et al. Urinary melatonin excretion throughout the ovarian cycle in menstrually related migraine. Cephalalgia 1994; 14:205-209. 33. Brun J, Claustrat B, Saddier P et al. Nocturnal melatonin excretion is decreased in patients with migraine without aura attacks associated with menses. Cephalalgia 1995; 15:136-139. 34. Drummond PD. Pupil diameter in migraine and tension headache. Journal of Neurology Neurosurgery and Psychiatry 1987; 50:228-230. 35. Claustrat B, Brun J, Chiquet C et al. Melatonin secretion is hypersensitive to light (300 lux) in migraine patients. Cephalalgia 2004. 36. Chronicle EP, Mulleners WM. Visual system dysfunction in migraine: A review of the clinical and psychophysical findings. Cephalalgia 1996; 16:525-535. 37. Cutrer FM. Antiepileptic drugs: How they work in headache. Headache 2001; (suppl 1):S3-10. 38. Provencio I, Rollag MD, Castrucci AM. Photoreceptive net in the mammalian retina. Nature 2002; 415:493. 39. Berson DM, Dunn FA, Takao M. Phototransduction by retinal ganglion cells that set the circadian clock. Science 2002; 295:1070-1073. 40. Mikkelsen JD, Larsen PJ, Mick G et al. Gating of retinal inputs through the suprachiasmatic nucleus: Role of excitatory neurotransmission. Neurochem Int 1995; 27:263-272. 41. Chazot G, Claustrat B, Broussolle E et al. Headache and depression: Recurrent symptoms in adult pinealectomized patients. In: Nappi G, ed. Headache and depression. New York: Raven Press, 1991; 299-303. 42. Reiter RJ, Poeggeler B, Chen LD et al. Melatonin as a radical scavenger: Implications for aging and diseases. Ann NY Acad Sci 1994; 719:1-12. 43. Shimomura T, Kowa H, Nakano T et al. Platelet superoxide dismutase in migraine and tension-type headache. Cephalalgia 1994; 14:215-218. 44. Tozzi-Ciancarelli MG, De Matteis G, Di Massimo C et al. Oxidative stress and platelet responsiveness in migraine. Cephalalgia 1997; 17:580-584. 45. Cardinali DP, Golombek DA. The rhythmic GABAergic system. Neurochem Res 1998; 23:607-614. 46. Gimeno MF, Ritta MN, Bonacossa A et al. Inhibition by melatonin of prostaglandin synthesis in hypothalamus, uterus and platelets. In: Birau N, Schloot W, eds. Melatonin: Current status and perspectives. Oxford and New York: Pergamon Press, 1980:147-150. 47. Del Zar MM, Martinuzzo M, Falcon C et al. Inhibition of human platelet aggregation and thromboxane-B2 production by melatonin: Evidence for a diurnal variation. J Clin Endocrinol Metab 1990; 70:246-250. 48. Pozo D, Reiter RJ, Calvo JR et al. Physiological concentrations of melatonin inhibit nitric oxide synthase in rat cerebellum. Life Sciences 1994; 55:455-460. 49. Vacas MI, Keller Sarmiento MI, Cardinali DP. Pineal methoxyindoles depress calcium uptake by rat brain synaptosomes. Brain Research 1984; 294:166-168. 50. Stankov B, Capsoni S, Lucini V et al. Autoradiographic localization of putative melatonin receptors in the brains of two old world primates: Cercopithecus aethiops and Papio ursinus. Neuroscience 1993; 52:459-468. 51. Capsoni S, Stankov BM, Fraschini F. Reduction of regional cerebral blood flow by melatonin in young rats. NeuroReport 1995; 6:1346-1348. 52. Weekley LB. Effects of melatonin on isolated pulmonary artery and vein: role of the vascular endothelium. Pulmonary Pharmacol 1993; 6:149-154. 53. Evans BK, Mason R, Wilson VG. Evidence for direct vasoconstrictor activity of melatonin in « pressurized » segments of isolated caudal artery from juvenile rats. Arch Pharmacol 1992; 346:362-365. 54. 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; 276:207-213. 55. Claustrat B, Brun J, David M et al. Melatonin and jet lag : Confirmatory result using a simplified protocol. Biol Psychiatry 1992; 32:705-711. 56. Ellis CM, Lemmens G, Parkes JD. Melatonin and insomnia. J Sleep Res 1996; 5:61-65. 57. Lewy AJ, Ahmed S, Latham Jackson JM et al. Melatonin shifts human circadian rhythms according to a phase-response curve. Chronobiology International 1992; 9:380-392. 58. Zaidan R, Geoffriau M, Brun J et al. Melatonin is able to influence its secretion in humans: Description of a phase-response curve. Neuroendocrinology 1994; 60:105-112. 59. Leone M, D’Amico D, Moschiano F et al. Melatonin versus placebo in the prophylaxis of cluster headache: A double-blind pilot study with parallel groups. Cephalalgia 1996; 16:494-496.

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CHAPTER 21

Melatonin in Protection against Oxidative Damage Caused by Potential Carcinogens Malgorzata Karbownik

Abstract

O

xidative stress participates in the complex process of carcinogenesis. Any carcinogen may initiate the process of cancer development by generating free radicals. Numerous indicators of oxidative damage are enhanced in result of this carcinogenic action. Melatonin (N-acetyl-5-methoxytryptamine), an antioxidant and free radical scavenger, can potentially protect against oxidative damage and cancer initiation. Indeed, in numerous studies, examining several parameters of oxidative damage and using a number in vitro and in vivo models, this indoleamine has been shown to protect DNA and cellular membranes from carcinogen-induced oxidative abuse. The protection, provided by melatonin against cellular damage, due to carcinogens, makes it a potential therapeutic supplement in conditions of increased cancer risk.

Introduction Involvement of Oxidative Stress in the Process of Carcinogenesis Reactive oxygen species (ROS) are generated in aerobic organisms under physiological conditions; they are, however, effectively counteracted by natural defense mechanisms.1 Any internal or external pathological factor, carcinogen included, may disrupt the balance between the production and the detoxification of ROS, leading to oxidative stress. Oxidative stress plays a significant role in the pathogenesis of cancer.2 It participates in all steps of carcinogenesis; at the first step—the initiation—free radicals damage different molecules, leading either directly or indirectly to mutations and, consequently, to cancer initiation.2 The products of oxidative damage to DNA, lipid, and protein constitute markers of this process3 but, at the same time, they may contribute per se to DNA damage and, in consequence, to cancer development.4-7 One of the most frequently measured parameters of DNA damage is 8-oxo-2'-deoxyguanosine (8oxodGuo), which is highly mutagenic.4,5 In turn, oxidative damage to membranes results in lipid peroxidation; the latter one is a chain reaction, involving numerous by-products, which are damaging to DNA via direct or indirect mechanisms.5,6 The present proteins are also easily mutilated by free radicals, with consequential changes in enzyme activities and in some properties of membranes, like permeability, fluidity, signaling pathway, etc.7 The disruption of membrane function contributes to the process of carcinogenesis. Both endogenous and exogenous antioxidants can prevent the formation of early metabolites of the damage to macromolecules and, in this way, protect against cancer.1

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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Exogenous or endogenous factors are classified as carcinogens on the basis of epidemiological studies in humans and of experimental studies in animals. Most of the factors, presented in this chapter, have been selected from the list of hazards issued by the International Agency for Research on Cancer (Monographs on the evaluation of Carcinogenic Risks to Humans, IARC, Lyon, France).

Potential Mechanisms of the Protective Action of Melatonin against Toxic Effects of Carcinogens Melatonin (N-acetyl-5-methoxytryptamine) is one of the well documented antioxidants and free radical scavengers. The indoleamine has been found to effectively scavenge the highly toxic hydroxyl radical (.OH) and directly or indirectly detoxify the following free radicals or ROS: peroxynitrite anion (ONOO-), the superoxide anion radical (O2-.), nitric oxide (NO.), hydrogen peroxide (H2O2), singlet oxygen (1O2), guanosine radical (G.) (for a review see refs. 8-13). Melatonin is known to stimulate the activities od several antioxidant enzymes, like superoxide dismutase (SOD), γ-glutamylcysteine synthetase, glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rd), glucose-6-phosphate dehydrogenase (6-GPD), and catalase (CAT); melatonin also inhibits the activity of a pro-oxidative enzyme, i.e., nitric oxide synthase (NOS) (for a review see refs. 8-12). Much evidence has already been accumulated for the preventive action of melatonin against cancer (for a review see refs. 13-19). The anticarcinogenic action of melatonin results—to a large extent—from its antioxidative properties and free radical scavenging ability (for a review see refs. 13-16). Thus, melatonin, as an antioxidant and free radical scavenger, can prevent cancer initiation. Antioxidative effects of melatonin and its ability to scavenge free radicals are discussed in detail in another chapter of this book: “Antioxidant Properties of Melatonin”, by Reiter et al.

Oxidative Damage Caused by Potential Carcinogens—Protective Effects of Melatonin Iron Iron is a common heavy metal that is widely distributed in organisms. Iron is a cofactor for many biological reactions, but—when in excess—it can be damaging to cells. It is known that increased iron stores in the organism are associated with an increased risk of cancer.21 In turn, oxidative stress is said to be involved in the pathomechanism of cancer.3 The most basic reaction of oxidative stress is Fenton reaction (Fe2+ + H2O2 → Fe3+ + .OH + OH-); in this reaction both iron ions participate, i.e., ferrous ion (Fe2+) and ferric ion (Fe3+). Resulting from Fenton reaction .OH, being the most harmful free radical, may contribute to cancer at each step of carcinogenesis.3 It is worth stressing that not only iron but also other transition metals may participate in Fenton reaction, producing oxidative stress (some examples are discussed below). The mechanism of Fenton reaction is used to experimentally induce lipid peroxidation. In this model, tissues are usually incubated in the presence of two substrates for Fenton reaction—Fe 2+ and H 2O 2—and two products of lipid peroxidation—malondialdehyde + 4-hydroxyalkenals (MDA+4-HDA)—are measured. In an in vitro study, Fe2++H2O2-induced lipid peroxidation in rat liver homogenates was inhibited by melatonin; additionally, melatonin revealed synergistic effects with other antioxidants—vitamin E, vitamin C, glutathione (Table 2).22 Similarly, when monkey liver was used in such a study, melatonin effectively reduced the level of lipid peroxidation products, due to Fe2+ and H2O2 (Table 2).23 In turn, Fe2++H2O2-related lipid peroxidation in homogenates of hamster testes was effectively reduced by melatonin (Table 2).24 That finding suggests that a supplementation with melatonin may prevent iron-induced lipid peroxidation and iron-related sperm abnormalities, both factors contributing to carcinogenesis in male gonads.6,7,25

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It has recently been shown that in vivo treatment with melatonin protects against in vitro iron-induced lipid peroxidation in liver homogenates; however, when ascorbic acid was used for comparison in that model, no protective effect was observed (Table 1).26 Thus, an administration of melatonin to organisms decreases organ susceptibility to oxidative stress after tissues are oxidatively challenged in vitro. In the in vivo study, coinfusion of melatonin with ferrous citrate into rat substantia nigra or chronic systemic treatment with melatonin prevented iron-induced lipid peroxidation and

Table 1. Studies in vivo supporting the protective effects of melatonin against oxidative damage caused by different carcinogens

Species/Organ/ Tissue/Cellular Compartment Male Sprague Dawley rats; hepatic homogenates exposed to FeSO4 (15 µM) + H2O2 (0.1 mM) Male Sprague Dawley rats; substantia nigra Male Sprague Dawley rats; kidney

Male Syrian hamsters Brain, heart, lung kidney Male Wistar rats; kidney Male Sprague Dawley rats; liver Male Syrian hamsters Kidney collected 5 h after E2 treatment Liver collected 3 h after E2 treatment Male Syrian hamsters Kidney collected 5 h after E2 treatment

Dose of Melatonin Which Reduced or Prevented the Effect of Carcinogen

Carcinogen/Dose

Effect of Carcinogen

No treatment

↑ MDA+4-HDA

15 mg/kg b.w., 2x daily, 8 days

26

Ferrous citrate, 3.4 mM of iron infused into substantia nigra Ferric nitrilotriacetate (Fe-NTA), 15 mg Fe/kg b.w.

↑ MDA ↓glutathione

60 µg infused into substantia nigra or 10 mg/kg b.w., systemically, 7 days 25 mg/kg b.w., 30 min before Fe-NTA 50 mg/kg b.w., 30 min before Fe-NTA 6 x 15 mg/kg b.w. 6 x 5 mg/kg b.w. 1 x 15 mg/kg b.w. 1 x 15 mg/kg b.w. 10 mg/kg b.w., every 6 h for 24 h before KBrO3 inj. 4 x 50 mg/kg b.w., every 30 min before exposure to IR 15 mg/kg b.w., 0.5 h before and 2 h after E2 treatment

27

Cadmium chloride (CdCl2), 1 mg/kg b.w. Potassium bromate (KBrO3), 80 mg/kg b.w. Ionizing radiation (IR), 800 cGy 17β-estradiol (E2), 75 mg/kg b.w.

↑ nuclear 8oxodGuo ↑ MDA+4-HDA ↑ MDA+4-HDA

↑ MDA+4-HDA ↑ nuclear 8oxodGuo ↑ nuclear 8oxodGuo ↓ microsomal MF ↑ nuclear 8oxodGuo

Ref.

32

39

41

47

51

↑ nuclear 8oxodGuo 17β-estradiol (E2), 75 mg/kg b.w.

↑ nuclear 8oxodGuo

15 mg/kg b.w., 2 h and 0.5 h before and 2 h and 4 h after E2 treatment

52

Table continued on next page

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223

Table 1. Continued

Species/Organ/ Tissue/Cellular Compartment

Carcinogen/Dose

Male Sprague Dawley rats Liver, kidney, lung, spleen Liver, kidney

δ-Aminolevulinic acid (ALA), 40 mg/kg b.w. every second day for 15 days

Hepatic microsomal membranes, lung spleen, blood serum Male Sprague Dawley rats Spleen, blood serum Liver Male Sprague Dawley rats; liver

Phenylhydrazine (PHZ) 15 mg/kg b.w., 7 days Safrole, 300 mg/kg b.w.

Male Sprague Dawley Safrole, 100 mg/kg rats; liver from b.w. pinealectomized animals

Effect of Carcinogen

2-Nitropropane (2-NP), 4 mmol/ kg b.w. Phosphine (PH3), 2 mg/kg b.w. 7,12-dimethylbenz (a)anthracene (DMBA), 20 mg/kg b.w., 21 days

Ref.

**10 mg/kg b.w., 3x daily (15 days)

55 56 57

15 mg/kg b.w., 2 x daily, 8 days

26

0.2 mg/kg or 0.4 mg/kg b.w., 15 min before, 3 h and 20 h after safrole inj. 4 x 0.15 mg/kg b.w., s.c., every 2 h, first dose 15 min before safrole Physiological concentration of melatonin due to its higher night secretion by the pineal gland 2.5, 5.0, 10.0 mg/kg b.w., 0.5 h before 2-NP treatment 10 mg/kg b.w., 30 min before PH3 injection 4.2 mg/kg b.w., 21 days, simultaneously with DMBA

61

↑ nuclear 8oxodGuo ↓ microsomal and mitochondrial MF ↑ MDA+4-HDA

↑ MDA+4-HDA ↓ microsomal MF ↑ DNA adducts

↑ DNA adducts

↑ DNA adducts

Male Sprague Dawley rats; liver, lung, kidney Male Wistar rats; Brain and liver Brain, lung and liver Female mice, liver

Dose of Melatonin Which Reduced or Prevented the Effect of Carcinogen

↑ MDA+4-HDA

↑ nuclear 8oxodGuo ↑ MDA+4-HDA ↓ GSH-Px, SOD, ↓ CAT

62

65

69

71

Note: ↑ = increase; ↓ = decrease; 8oxodGuo = 8-oxo-2'-deoxyguanosine; MF = membrane fluidity; MDA+4-HDA = malondialdehyde + 4-hydroxyalkenals; GSH-Px = glutathione peroxidase; SOD = superoxide dismutase; CAT = catalase. * Other studies, concerning the protective effects of melatonin against oxidative damage, due to ionizing radiation, have recently been summarized (for a review see ref. 13). ** Other studies, concerning the protective effects of melatonin against oxidative damage, due to δ-aminolevulinic acid, have recently been summarized (for a review see ref. 16).

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glutathione depletion in substantia nigra (Table 1); these protective effects of melatonin was confirmed in the in vitro study (Table 2).27 In another experimental model of oxidative damage to membranes, ferric ions are used. The incubation of microsomal membranes in the presence of FeCl3, ADP and NADPH caused a decrease in membrane fluidity (the inverse of membrane rigidity), accompanied by an increased amount of lipid peroxidation products; a preincubation with melatonin protected against those oxidative changes (Table 2).28-30 It is worth mentioning that melatonin enhanced the protective effect of tamoxifen, an antiestrogenic drug, used in the treatment of breast cancer, against Fe3+ induced membrane oxidative damage.30

Ferric Nitrilotriacetate (Fe-NTA) Nitrilotriacetic acid is widely used as a substitute in detergents for household and hospital use, manifesting low toxicity in experimental animals; the ferric chelate—ferric nitrilotriacetate (Fe-NTA), has been reported to cause a high incidence of renal adenocarcinoma in animal models.31 Fe-NTA, applied in vitro, increased lipid peroxidation in rat kidney homogenates; similarly, Fe-NTA, injected to animals, increased the levels of MDA+4-HDA and of 8oxodGuo.32 As expected, both in vitro and in vivo effects of Fe-NTA were prevented by melatonin (Tables 1 and 2).32

Chromium (Cr) Chromium (Cr), an environmental pollutant, is used in occupational settings like, e.g., the production of chromates, chromium plating, chromate pigment manufacture, and in the production of cement and stainless steel. The primary toxic form, to which organisms are exposed, is hexavalent Cr (Cr6+).33 The carcinogenic potential of Cr is likely due to macromolecular damage caused by reactive intermediates, arising in the course of intracellular reduction of Cr6+ to trivalent Cr (Cr3+) and/or by Cr3+ itself; Cr3+ does not cross cellular membranes and accumulates within cells.33 Both chromium ions—Cr6+ and Cr3+—were used in in vitro studies to induce oxidative stress. When primary cultures of rat hepatocytes were incubated in the presence of Cr6+, DNA single-strand breaks, cellular toxicity, measured by the leakage of lactate dehydrogenase (LDH) from cells, and an increased level of lipid peroxidation products were observed.34 Melatonin prevented Cr6+-related oxidative changes and restored the levels of antioxidants—vitamins C and E, and the activity of CAT (Table 2).34 Trivalent Cr was applied to induce oxidative damage to purified DNA. Incubation of purified calf thymus DNA in the presence of Cr3+ plus H2O2 resulted in an increased 8oxodGuo formation, this effect was prevented by melatonin, applied in micromolar concentrations (Table 2).23,35-37 It is worth mentioning that ascorbic acid and trolox (a water soluble form of vitamin E) were much less effective than melatonin in reducing DNA oxidative damage in that in vitro model.36

Cadmium (Cd) Cadmium (Cd), a toxic transition metal, is widely used in occupational settings, such as smelting, refining of zinc, electroplating, galvanizing, nickel-cadmium battery production, welding, and it is also present in tobacco. Cadmium-induced lipid peroxidation is generally considered an important event in the carcinogenic potential of this metal.38 Cadmium, applied in a single injection, induced lipid peroxidation in different hamster organs—brain, heart, kidney, and lung; those damaging effects were prevented by a cotreatment with melatonin (Table 1).39

Bromium Potassium bromate (KBrO3), which is used as a food additive, is recognized as a renal carcinogen in animal models.40 An increased level of 8oxodGuo in rat kidney, due to bromium treatment, was effectively reduced by melatonin (Table 1).41

Melatonin in Protection against Oxidative Damage Caused by Potential Carcinogens

225

Mercury

Mercury—a heavy metal—reveals numerous toxic effects to organisms.42 It is likely involved in the pathomechanism of Alzheimer’s disease. Its cytotoxic effects in SHSY5Y neuroblastoma cells were accompanied by a reduction in intracellular glutathione; a preincubation with melatonin protected cells from mercury-induced GSH loss (Table 2).43

Cobalt (Co) Cobalt—a positively charged transition metal—reveals numerous toxic effects in humans and in animal models. Human cobalt intoxication has primarily been reported as a result of industrial exposure.44 Incubation of SHSY5Y neuroblastoma cells in the presence of cobalt resulted in neurotoxic effects and decreased intracellular concentration of glutathione; those damaging effects were prevented by melatonin (Table 2).45

Ionizing Radiation (IR)

Radiation injury to living cells is, to a large extent, due to free radical generation.46 Numerous studies have been performed, revealing protective effects of melatonin against oxidative abuse, due to ionizing radiation. Total body exposure of rats to γ-irradiation resulted in an increased formation of 8oxodGuo and in a decreased membrane fluidity in the liver; a cotreatment with melatonin completely prevented those oxidative changes (Table 1).47

Table 2. Studies in vitro supporting the protective effects of melatonin against oxidative damage caused by different carcinogens Concentration of Melatonin Which Reduced or Prevented the Effect of Carcinogen

Species/Tissue/ Cell/Compartment

Carcinogen/ Concentration

Effect of Carcinogen

Rat liver homogenates

Ferrous sulfate (FeSO4) (15 µM) + H2O2 (0.1 mM) Ferrous sulfate (FeSO4) (15 µM) + H2O2 (0.1 mM) Ferrous sulfate (FeSO4) (30 µM) + H2O2 (0.1 mM) Ferrous citrate, 1.0 µM

↑ MDA+4-HDA

0.4, 0.8, 1.2 and 1.6 mM

22

↑ MDA+4-HDA

0.4, 0.5, 0.6, 0.8 and 1.0 mM

23

↑ MDA+4-HDA

1.0, 2.0, 2.5, 5.0 mM

24

↑ MDA

0.5, 1.0, 2.0, 3.0 and 4.0 mM

27

Ferric nitrilotriacetate ↑ MDA+4-HDA (Fe-NTA) (50 µM)

0.5, 1.0, 2.0, 4.0 mM

32

Ferric chloride FeCl3 (0.2 mM)+ ADP (1.7 mM)+ NADPH (0.2 mM)

0.3, 1.0, 3.0 mM

28 29 30

Monkey liver homogenates Hamster testis homogenates Male Sprague Dawley rats, cortical homogenates Male Sprague Dawley rats, kidney homogenates Male Sprague Dawley rats Hepatic microsomes

↓ MF ↑ MDA+4-HDA

Ref.

Table continued on next page

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Table 2. Continued

Species/Tissue/ Cell/Compartment

Carcinogen/ Concentration

Wistar rat hepatocytes

Potassium dichromate [K2Cr2O7; Cr6+] 2.5 µM ↑ DNA singlestrand breaks 0.5 µM ↑ LDH leakage from cells 0.5 µM ↑ MDA 0.5 µM ↓ Vit. C, E 0.5 µM ↓ CAT Chromium chloride ↑ 8oxodGuo (CrCl3) [Cr(III)] (0.5 mM) + H2O2 (0.5 mM) Chromium chloride ↑ 8oxodGuo (CrCl3) (Cr3+) (0.5 mM) + H2O2 (0.5 mM) Chromium chloride ↑ 8oxodGuo (CrCl3) (Cr3+) (0.5 mM) + H2O2 (0.5 mM) Mercury chloride ↓ glutathione (HgCl2) (180 nM) Cobalt chloride (CoCl2) (0.3 mM) X-ray irradiation, 8 Gy

Purified calf thymus DNA

Purified calf thymus DNA

Purified monkey liver DNA

SHSY5Y neuroblastoma cells SHSY5Y neuroblastoma cells Human skin fibroblasts

Effect of Carcinogen

Concentration of Melatonin Which Reduced or Prevented the Effect of Carcinogen Ref. 34 0.5, 1.0 mM 0.5, 1.0 mM 0.5, 1.0 mM 1.0 mM 0.5 mM 0.25, 0.5, 1.0, 2.5, 5.0, 10.0 µM

35 36

0.05, 0.1, 0.5, 1.0, 10.0 µM

37

10.0 µM

24

1.0 µM (30-minpreincubation)

43

↓ glutathione

1.0 µM (12-hour preincubation)

45

↑ MDA

0.1 mM

48

Note. ↑ = increase; ↓ = decrease; 8oxodGuo = 8-oxo-2'-deoxyguanosine; MF = membrane fluidity; MDA+4-HDA = malondialdehyde + 4-hydroxyalkenals; LDH = lactate dehydrogenase; CAT = catalase.

Preincubation with melatonin reduced IR-related cell death and decreased IR-induced lipid peroxidation in cultured human skin fibroblasts (Table 2).48 The results of other studies on melatonin and its protective effects against oxidative damage caused by IR, have recently been reviewed in ref. 13.

17β-Estradiol 1,3,5[10]-Estratriene-3,17β-diol (17β-estradiol; E2), a natural estrogen, is classified as a carcinogen (for a review see refs. 49,50). The induction of renal tumors in Syrian hamsters, due to chronic exposure to estrogens, has extensively been examined as a model of hormonal carcinogenesis; this animal model shares numerous mechanistic features with estrogen-related tumors in human females, making its use appropriate for investigating the mechanisms of

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estrogen-related carcinogenesis.49 In hamster kidney model of E2-induced DNA damage, an increased oxidation of guanine bases is commonly observed; in this experimental model, E2 produces liver DNA damage as well. Estradiol, applied in a single injection, resulted in an increased level of 8oxodGuo in kidneys (at 5 hours) and in the liver (at 3 hours); those changes were prevented when the animals were cotreated with melatonin (Table 1).51 In another similar study, we found that—in contrast to melatonin—its precursor, N-acetylserotonin (NAS), did not reveal any protective effect against DNA damage.52 Thus, melatonin can be considered as a pharmacological agent for the use in cotreatment with estrogens.

δ-Aminolevulinic Acid δ-Aminolevulinic acid (ALA) is a precursor of haem synthesis; its increased blood concentration is found in patients suffering from inherited or acquired porphyrias—acute intermittent porphyria (AIP), hereditary tyrosinemia and lead poisoning.53 An increased incidence of cancer, especially in the liver, is observed in patients suffering from porphyrias.54 The accumulation of porphyrins or their precursors, followed by free radical generation and the release of iron from its storage sites, are assumed to be responsible for the higher incidence of cancer in porphyric patients. ALA is used in an experimental model of porphyria-related oxidative damage and carcinogenesis. In several studies, ALA has been shown to induce oxidative damage to macromolecules, while melatonin has been found to prevent these effects. Melatonin, when injected to rats, protected against the formation of 8oxodGuo in the liver, the kidney, the lung, and the spleen, resulting from a chronic treatment with ALA.55-57 Similarly, melatonin in vivo prevented from decreased membrane fluidity in hepatic and renal microsomes and mitochondria caused by ALA55,56 and from the formation of ALA-induced lipid peroxidation products in hepatic microsomal membranes,55 in lung and spleen homogenates57 and in blood serum.56 The results of other studies on the protective effects of melatonin against ALA-induced oxidative damage are reviewed in ref. 17.

Phenylhydrazine (PHZ) Phenylhydrazine (PHZ), belonging to the hydrazine family, is one of the most potent toxins used in experimental models of carcinogenesis.58 PHZ intoxication leads, among others, to hepatic and spleen iron overload, free iron release, followed by free radical generation.59 A chronic treatment with PHZ resulted in a pronounced increase in lipid peroxidation products in the spleen and in serum; those changes were prevented by melatonin but not by ascorbic acid.26 Additionally, the pronounced decrease in hepatic membrane fluidity was reduced by melatonin, whereas a cotreatment with ascorbic acid even enhanced the damaging effect of PHZ, resulting in a further decrease in membrane fluidity (Table 1).26

Safrole Safrole is a constituent of several essential oils and is used in perfumery, fat denaturation in soap manufacture, and in the production of heliotropin. Safrole is a complete hepatocarcinogen for rats and mice.60 When used in animal models, that toxin caused DNA damage. Safrole, applied in vivo, increased DNA adduct formation in rat liver; melatonin, in both pharmacological and physiological concentrations, protected—in dose dependent manner—against safrole-caused DNA damage.61,62 Safrole, injected at night, when the blood concentration of melatonin is physiologically higher, caused weaker DNA damage than when injected during the day; conversely, pinealectomy, which eliminates the night-time rise in melatonin concentration, enhanced the formation of DNA adducts.62 Blood concentration of melatonin was inversely related to the degree of DNA adduct formation induced by safrole (Table 1).61,62 Thus, melatonin, in physiological concentrations, prevented the oxidative damage, exerted by the carcinogen, when used in pharmacological concentrations.

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2-Nitropropane (2-NP) 2-nitropropane (2-NP), the secondary nitroalkane, widely used as an intermediate in chemical syntheses, in formulation of inks, paints, varnishes, adhesives, and other coatings, is a potent hepatocarcinogen in rodents.63 Additionally, leukemia and nonHodgkin’s lymphoma have been described among farmers exposed to solvents including 2-NP.64 Melatonin significantly reduced the level of lipid peroxidation products in rat liver, lung, and kidney and decreased the activity of sorbitol dehydrogenase (related to hepatic damage)— the changes caused by an earlier single intraperitoneal injection of 2-NP (Table 1).65 Similarly, melatonin reduced cellular proliferation, DNA synthesis, and histopathological changes in rat liver caused by 2-NP.66

Phosphine (PH3) Phosphine (PH3), generated by hydrolysis of metal phosphides (AlP, Mg3P2), is an important dopant in electronic industry. Genotoxic effects of PH3 have been described in mice67 and humans.68 In animal models, PH3 increased the level of MDA+4-HDA and decreased glutathione concentration in the brain, the lung, and the liver and, additionally, increased the level of 8oxodGuo in the brain and the lung; melatonin effectively prevented those changes (Table 1).69

7,12-Dimethylbenz(a)Anthracene (DMBA) 7,12-dimethylbenz(a)anthracene (DMBA) is a member of the polycyclic aromatic hydrocarbons with a severe carcinogenic effect. Melatonin was found to effectively reduce tumor incidence and tumor volume, due to DMBA treatment in rats.70 The indoleamine also prevented DMBA-related inhibition of GSH-Px activity and fully reversed the inhibition of CAT and SOD activity caused by DMBA (Table 1).71

Concluding Remarks The literature on the protective effects of melatonin against different carcinogens is much more abundant than it has been presented in this survey, but not in all of these studies parameters related to oxidative stress were avaluated. It is highly probable, however, that most of the anticarcinogenic effects of melatonin result—at least partially—from its antioxidative properties. Although the mechanism of anticarcinogenic action of melatonin is undoubtedly complex and comprises its effects not only on oxidative stress,19 still this mechanism seems to be the most important, concerning the prevention of cancer. Whereas most studies on melatonin and cancer are devoted to the application of the indoleamine in advanced steps of carcinogenesis,20 the results, summarized in this survey, speak in favour for the use of melatonin in healthy subjects under conditions related to the increased risk of cancer. The fact that in tumor bearing animals, thus in the course of tumor development, 6-sulphatoxymelatonin excretion is abolished,72 constitutes an additional piece of evidence for melatonin treatment under conditions of increased exposure to carcinogens—to prevent cancer initiation.

References 1. Sies H. Strategies of antioxidant defense. Eur J Biochem 1993; 215:213-219. 2. Dreher D, Junod AF. Role of oxygen free radicals in cancer development. Eur J Cancer 1996; 32A:30-38. 3. De Zwart LL, Meerman JHN, Commandeur JNM et al. Biomarkers of free radical damage applications in experimental animals and in humans. Free Radic Biol Med 1999; 26:202-226. 4. Floyd RA. The role of 8-hydroxydeoxyguanosine in carcinogenesis. Carcinogenesis 1990; 11:1447-1450.

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5. Marnett LJ. Oxyradicals and DNA damage. Carcinogenesis 2000; 21:361-370. 6. Burcham PC. Genotoxic lipid peroxidation products: Their DNA damaging properties and role in formation of endogenous DNA adducts. Mutagenesis 1998; 13:287-305. 7. Kasprzak KS. Oxidative DNA and protein damage in metal-induced toxicity and carcinogenesis. Free Radic Biol Med 2002; 32:958-967. 8. Reiter RJ. Oxidative damage in the central nervous system: Protection by melatonin. Prog Neurobiol 1998; 56:359-384. 9. Reiter RJ. Oxidative damage to nuclear DNA: Amelioration by melatonin. Neuroendocrinol Lett 1999; 10:145-150. 10. Reiter RJ, Tan DX, Qi W et al. Pharmacology and physiology of melatonin in the reduction of oxidative stress in vivo. Biol Signals Recept 2000; 9:160-171. 11. 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. 12. Reiter RJ, Tan DX, Karbownik M. Cholelithiasis, oxidative stress and melatonin. J Pineal Res 2001; 30:127-128. 13. 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. 14. 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. 15. Karbownik M. Potential anticarcinogenic action of melatonin and other antioxidants mediated by antioxidative mechanisms. Neuroendocrinol Lett 2002; 23 (suppl 1):39-44. 16. Karbownik M, Reiter RJ. Melatonin protects against oxidative stress caused by δ-aminolevulinic acid: Implication for cancer reduction. Cancer Invest 2002; 20:276-286. 17. Bartsch C, Bartsch H. Melatonin in cancer patients and in tumor-bearing animals. Adv Exp Med Biol 1999; 467:247-264. 18. Karasek M, Pawlikowski M. Pineal gland, melatonin and cancer. Neuroendocrinol Lett 1999; 20:139-144. 19. 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. 20. Lissoni P. Is there a role for melatonin in supportive care? Supp Care Med 2002; 10:110-116. 21. Stevens RG, Jones DY, Micozzi MS et al. Body iron stores and the risk of cancer. N Engl J Med 1988; 319:1047-1052. 22. Gitto E, Tan DX, Reiter RJ et al. Individual and synergistic antioxidative actions of melatonin: Studies with vitamin E, vitamin C, glutathione and desferrioxamine (desferoxamine) in rat liver homogenates. J Pharm Pharmacol 2001; 53:1393-1401. 23. Cabrera J, Burkhardt S, Tan D-X et al. Autoxidation and toxicant-induced oxidation of lipid and DNA in monkey liver: Reduction of molecular damage by melatonin. Pharmacol Toxicol 2001;89:225-230. 24. Karbownik M, Gitto E, Lewinski A et al. Relative efficacies of indole antioxidants in reducing autoxidation and iron-induced lipid peroxidation in testes: Implications for cancer initiation. J Cell Biochem 2001; 81:693-699. 25. Möller H, Skakkebaek NE. Risk of testicular cancer in subfertile men: Case-control study. Brit Med J 1999; 318:559-562. 26. Karbownik M, Reiter RJ, Garcia JJ et al. Melatonin reduces phenylhydrazine-induced oxidative damage to cellular membranes: Evidence for the involvement of iron. Int J Biochem Cell Biol 2000; 32:1045-1054. 27. Lin AM-Y, Ho L-T. Melatonin suppresses iron-induced neurodegeneration in rat brain. Free Radic Biol Med 2000; 28:904-911. 28. Garcia JJ, Reiter RJ, Guerrero JM et al. Melatonin prevents changes in microsomal membrane fluidity during induced lipid peroxidation. FEBS Lett 1997; 408:297-300. 29. Garcia JJ, Reiter RJ, Ortiz GG et al. Melatonin enhances tamoxifen’s ability to prevent the reduction in microsomal membrane fluidity induced by lipid peroxidation. J Membrane Biol 1998; 162:59-65. 30. Garcia JJ, Reiter RJ, Pie J et al. Role of pinoline and melatonin in stabilizing hepatic microsomal membranes against oxidative stress. J Bioenerg Biomembr 1999; 31:609-616. 31. Li J, Okada S, Hamazaki S et al. Subacute nephrotoxicity and induction of renal cell carcinoma in mice treated with ferric nitrilotriacetate. Cancer Res 1987; 47:1867-1869. 32. Qi W, Reiter RJ, Tan DX et al. Inhibitory effects of melatonin on ferric nitriloriacetate-induced lipid peroxidation and oxidative DNA damage in the rat kidney. Toxicology 1999; 139:81-91. 33. Snow ET. Metal carcinogenesis: Mechanistic implications. Pharmacol Ther 1992; 53:31-65.

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34. Susa N, Ueno S, Furukawa Y et al. Potent protective effect of melatonin on chromium(VI)-induced DNA single-strand breaks, cytotoxicity, and lipid peroxidation in primary cultures of rat hepatocytes. Toxicol Appl Pharmacol 1997; 144:377-384. 35. Qi W, Reiter RJ, Tan DX et al. Increased levels of oxidatively damaged DNA induced by chromium(III) and H2O2: Protection by melatonin and related molecules. J Pineal Res 2000; 29:54-61. 36. Qi W, Reiter RJ, Tan DX et al. Chromium(III)-induced 8-hydroxydeoxyguanosine in DNA and its reduction by antioxidants: Comparative effects of melatonin, ascorbate, and vitamin E. Environ Health Persp 2000; 108:399-402. 37. Burkhardt S, Reiter RJ, Tan D et al. DNA oxidatively damaged by chromium(III) and H2O2 is protected by the antioxidants melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine, resveratrol and uric acid. Int J Biochem Cell Biol 2001; 33:775-783. 38. Stohs SJ, Bagchi D, Hassoun E et al. Oxidative mechanisms in the toxicity of chromium and cadmium ions. J Environ Pathol Oncol 2000; 19:201-213. 39. Karbownik M, Gitto E, Lewinski A et al. Induction of lipid peroxidation in hamster organs by the carcinogen cadmium: Amelioration by melatonin. Cell Biol Toxicol 2001; 17:33-40. 40. Kurokawa Y, Maekawa A, Takahashi M et al. Toxicity and carcinogenicity of potassium bromate— a new renal carcinogen. Environ Health Perspect 1990; 87:309-335. 41. Cadenas S, Barja G. Resveratrol, melatonin, vitamin E, and PBN protect against renal oxidative DNA damage induced by the kidney carcinogen KBrO3. Free Radic Biol Med 1999; 26:1531-1537. 42. Boening DW. Ecological effects, transport, and fate of mercury: A general review. Chemosphere 2000; 40:1335-1351. 43. Olivieri G, Brack C, Muller-Spahn F et al. Mercury induces cell cytotoxicity and oxidative stress and increases beta amyloid secretion and tau phosphorylation in SHSY5Y neuroblastoma cells. J Neurochem 2000; 74:231-236. 44. Linnainmaa M, Kiilunen M. Urinary cobalt as a measure of exposure in the wet sharpening of hard metal and stellite blades. Int Arch Occup Environ Health 1997; 69:193-200. 45. Olivieri G, Hess C, Savaskan E et al. Melatonin protects SHSY5Y neuroblastoma cells from cobalt-induced oxidative stress, neurotoxicity and increased β-amyloid secretion. J Pineal Res 2001; 31:320-325. 46. Wallace SS. Enzymatic processing of radiation-induced free radical damage in DNA. Radiat Res 1998; 150:S60-S79. 47. Karbownik M, Reiter RJ, Qi W et al. Protective effects of melatonin against oxidation of guanine bases in DNA and decreased microsomal membrane fluidity in rat liver induced by whole body ionizing radiation. Mol Cell Biochem 2000; 211:137-144. 48. Kim BC, Shon BS, Ryoo YW et al. Melatonin reduces X-ray irradiation-induced oxidative damages in cultured human skin fibroblasts. J Dermatol Sci 2001; 26:194-200. 49. Roy D, Liehr JG. Estrogen, DNA damage and mutations. Mutat Res 1999; 424:107-115. 50. Liehr JG. Is estradiol a genotoxic mutagenic carcinogen? Endocrine Rev 2000; 21:40-54. 51. Karbownik M, Reiter RJ, Burkhardt S et al. Melatonin attenuates estradiol-induced oxidative damage to DNA: Relevance for cancer prevention. Exp Biol Med 2001; 226:707-712. 52. Karbownik M, Reiter RJ, Cabrera J et al. Comparison of the protective effect of melatonin with other antioxidants in the hamster kidney model of estradiol-induced DNA damage. Mutat Res 2001; 474:87-92. 53. Kappas A, Sassa S, Anderson KE. The porphyrias. In: Stanbury JB, Wyngaarden JB, Fredrickson DS, Goldstein JL, Brown MS, eds. The Metabolic Basis of Inherited Diseases. New York: McGraw-Hill, 1983; 1300-1384. 54. Linet MS, Gridley G, Nyrén O et al. Primary liver cancer, other malignancies, and mortality risks following porphyria: A cohort study in Denmark and Sweden. Am J Epidemiol 1999; 149:1010-1015. 55. Karbownik M, Reiter RJ, Garcia JJ et al. Melatonin reduces rat hepatic macromolecular damage due to oxidative stress caused by δ-aminolevulinic acid. Biochim Biophys Acta 2000; 1523:140-146. 56. Karbownik M, Tan DX, Manchester LC et al. Renal toxicity of the carcinogen δ-aminolevulinic acid: Antioxidant effects of melatonin. Cancer Lett 2000; 161:1-7. 57. Karbownik M, Tan DX, Reiter RJ. Melatonin reduces the oxidation of nuclear DNA and membrane lipids induced by the carcinogen δ-aminolevulinic acid. Int J Cancer 2000; 88:7-11. 58. Parodi S, De Flora S, Cavanna M et al. DNA-damaging activity in vivo and bacterial mutagenicity of sixteen hydrazine derivatives as related quantitatively to their carcinogenicity. Cancer Res 1981; 41:1469-1482. 59. Ferrali M, Signorini C, Sugherini L et al. Release of free, redox-active iron in the liver and DNA oxidative damage following phenylhydrazine intoxication. Biochem Pharmacol 1997; 53:1743-1751.

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60. IARC. Safrole, Isosafrole, and Dihydrosafrole. In: Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man. Lyon: IARC Press; 1976:10:231-300. 61. Tan DX, Poeggeler B, Reiter RJ et al. The pineal hormone melatonin inhibits DNA-adducts formation induced by the chamical carcinogen safrole in vivo. Cancer Lett 1993; 70:65-71. 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. Fiala ES, Czerniak R, Castonguay A et al. Assay of 1-nitropropane, 2-nitropropane, 1-azoxypropane and 2-azoxypropane for carcinogenicity by gavage in Sprague-Dawley rats. Carcinogenesis 1987; 8:1947-1949. 64. Petrelli G, Siepi G, Miligi L et al. Solvents in pesticides. Scand J Work Environ Health 1993; 19:63-65. 65. Kim SJ, Reiter RJ, Garay MVR et al. 2-Nitropropane-induced lipid peroxidation: Antitoxic effects of melatonin. Toxicology 1998; 130:183-190. 66. El-Sokkary GH. Inhibition of 2-nitropropane-induced cellular proliferation, DNA synthesis and histopathological changes by melatonin. Neuroendocrinol Lett 2002; 23:335-340. 67. Barbosa A, Rosinova E, Dempsey J et al. Determination of genotoxic and other effects in mice following short term repeated-dose and subchronic inhalation exposure to phosphine. Environ Mol Mutagen 1994; 24:81-88. 68. Garry VF, Griffith J, Danzl TJ et al. Human genotoxicity: Pesticide applicators and phosphine. Science 1989; 246:251-255. 69. Hsu C-H, Han B-C, Liu M-Y et al. Phosphine-induced oxidative damage in rats: Attenuation by melatonin. Free Radic Biol Med 2000; 28:636-642. 70. Kubatka P, Kalicka K, Chamilowa M et al. Nimesulide and melatonin in mammary carcinogenesis prevention in female Sprague-Dawley rats. Neoplasma 2002; 49:255-259. 71. Batcioglu K, Karagozler AA, Genc M et al. Comparison of the chemopreventive potentials of melatonin and vitamin E plus selenium on 7,12-dimethylbenz(a)anthracene-induced inhibition of mouse liver antioxidant enzymes. Eur J Cancer Prev 2002; 11:57-61. 72. Bartsch H, Bartsch C, Deerberg F et al. Seasonal rhythms of 6-sulphatoxymelatonin (aMT6s) excretion in female rats are abolished by growth of malignant tumors. J Pineal Res 2001; 31:57-61.

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CHAPTER 22

Influence of Melatonin on the Health and Diseases of the Retina Allan F. Wiechmann

Abstract

M

elatonin released from the pineal gland acts as an endocrine hormone on many distant target cells. Melatonin is also produced in the retina of most vertebrates, including humans, but its most likely role is to serve as a paracrine and intracrine signal of darkness to retinal cells. Melatonin produced by the retinal photoreceptors is thought to diffuse into the inner retina and bind to specific receptors on specific retinal neurons perhaps to modulate neuronal activity in response to signals from the photoreceptors. The retinal photoreceptors themselves express melatonin receptors and are therefore target cells for melatonin action. It is proposed that the circadian synthesis of melatonin reflects a beneficial role to the retina at nighttime, but if present in the retina during the light period, melatonin would have toxic effects on retinal cells. Melatonin appears to modulate the cyclic shedding and phagocytosis of photoreceptor outer segment disks as part of a renewal process, modulate neurotransmitter release from inner retinal neurons, and modulate retinal sensitivity to light as part of a dark adaptation process. These processes likely contribute to the survival and optimal performance of photoreceptor cells during the night. However, when melatonin is experimentally administered to the retina during the daytime, it increases the degree of light-induced photoreceptor cell death in animal models. This supports the hypothesis that disruption of retinal circadian rhythms may contribute to some forms of retinal dystrophies.

Introduction Melatonin is synthesized at night by retinal photoreceptors (Fig. 1) and appears to be involved in dark adaptation. The sites of action of retinally-synthesized melatonin are the photoreceptor cells and many neurons in the inner retinal layers. The concept that photoreceptors are targets for melatonin is relatively new and has the potential to lead to the discovery of new mechanisms by which melatonin influences circadian processes in the retina. There are many studies that offer compelling evidence that melatonin is intimately involved in circadian retinal processes that are essential for photoreceptor cell survival. Conversely, there are equally compelling studies that indicate that melatonin treatment is detrimental to photoreceptor survival. The reason for this dichotomy of detrimental versus beneficial effects of melatonin is not well understood. It is hypothesized that the diurnal rhythm of melatonin synthesis reflects a beneficial role during the dark period but if disrupted could present a hazard during exposure to light, and thereby contribute to the etiology of some retinal diseases. Because one function of melatonin may be to increase the sensitivity of the retina to light as part of a dark-adaptation mechanism, a consequence of this may be an increased sensitivity to the damaging effects of light.

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. A schematic section through the human retina with a schematic enlargement of the retina. Reproduced with permission from Webvision (http://webvision.med.utah.edu).

Sites of Retinal Melatonin Synthesis and Action Melatonin Synthesis in the Retina The retina synthesizes melatonin by the same pathway as in the pineal gland. It is synthesized from tryptophan in a series of four enzymatic steps. The mRNAs encoding the first enzyme, tryptophan hydroxylase (TPH), and the third enzyme, N-acetyltransferase (NAT), display a circadian rhythm of expression, with highest levels occurring at night.1,2 A diurnal rhythm of melatonin has been measured in the retina of several vertebrates,1,3-5 with peak levels occurring during the dark period. Modest cyclic changes in the melatonin-synthesizing enzyme, hydroxyindole-O-methyltransferase (HIOMT) mRNA levels and protein activity have also been reported.6,7 There is compelling evidence for the identification of the photoreceptors as the probable sites of retinal melatonin synthesis: (1) The RNA encoding HIOMT has been localized to the retinal photoreceptors in the chicken,8,9 (2) HIOMT immunoreactivity has been localized to photoreceptors of chicken retina,6 (3) a cyclic rhythm of NAT activity persists following kainic acid-induced lesioning of the inner retina,10 (4) the photoreceptor layer of the Xenopus retina continues to produce melatonin in darkness after isolation from the inner retina,3 (5) TPH mRNA has been localized to the photoreceptor layer in Xenopus,2 and (6) mRNA encoding NAT has been localized to the retinal photoreceptors in the rat retina.11

Melatonin Receptors in the Retina Earlier studies have shown that a nonhydrolyzable GTP analog specifically inhibits radioactive melatonin binding to its receptor,12-15 suggesting that the melatonin receptor belongs to the superfamily of G protein-coupled seven-pass transmembrane receptors. Since melatonin inhibits cyclic AMP accumulation in most tissues,16-18 the G protein coupled to the melatonin receptor is thought to be an inhibitory (Gi) G protein. In cultures of chicken retinal neurons, melatonin inhibits forskolin-induced cAMP accumulation and the effect is blocked by pertussis toxin.19 Cultures of human and rat retinal pigment epithelial (RPE) cells appear to express melatonin receptors, since melatonin inhibits forskolin-stimulated cyclic AMP synthesis in

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these cells.20 Also, melatonin affects the RPE membrane potentials and resistances at the apical or basal membrane either directly or indirectly via effects on cells of the chick neural retina.21 The cloning of the melatonin receptors represented an enormous advancement towards the elucidation of the function of melatonin. The melatonin receptor cDNA was first cloned from cultured Xenopus melanophores,22 and then from mammalian tissues.23,24 As predicted from earlier studies, the cDNAs encode a seven-pass transmembrane G-protein coupled receptor. Also, several types of the melatonin receptor have been identified in several species.24,25 Three melatonin receptors (Mel1a, Mel1b, and Mel1c) are expressed in the neural retina and RPE of Xenopus laevis, zebrafish, and chickens.25-27 In mammals, only two receptor types have been discovered, and are named MT1 and MT2, which are homologous to Mel1a and Mel1b, respectively. The existence of receptor subtypes may confer some advantages to target cells, such as (1) differential temporal regulation of receptor expression, (2) cell specificity, (3) selective intracellular signaling due to coupling to different effectors, and (4) selective regulation of signaling by desensitization or sensitization. In the human retina, MT1 receptors are located in photoreceptors (Fig. 2), dopaminergic and GABA-ergic amacrine cells, specific classes of horizontal cells, and in ganglion cells.28-30 Similar results have been reported in the retina of Xenopus laevis (African clawed frog; Fig. 3).31,32 Although the Mel1a, Mel1b, and Mel1c receptors are all expressed in the Xenopus RPE, melatonin receptors in the human RPE are yet to be identified. In situ hybridization studies of melatonin receptor RNA expression support the immunocytochemical results on receptor protein localization. In the Xenopus laevis retina, Mel1c and Mel1b mRNA is robustly expressed in the photoreceptor inner segments, and is also expressed in various cells of the inner nuclear layer (amacrine, horizontal, and bipolar cells), and in the ganglion cell layer (GCL). In the chicken retina, a broad band of receptor RNA (mostly Mel1a, and very little Mel1b) hybridization has been observed in the GCL and inner nuclear layer (INL),25 which is more suggestive of GABA-ergic amacrine and horizontal cell localization in the INL, rather than only dopaminergic cells. Further in situ hybridization studies have demonstrated the expression of melatonin receptor mRNA in chicken photoreceptors,33 which confirms the previous reports in frogs and human. The localization of melatonin receptors in horizontal cells and in dopaminergic and GABA-ergic amacrine cells was predicted in light of previous physiological and pharmacological studies that demonstrate that (1) melatonin increases the sensitivity of horizontal cells to light in salamander retina,34 (2) melatonin inhibits dopamine release in the mammalian retina,35 and (3) melatonin increases GABA release in the Xenopus retina.36 The localization of melatonin receptors in photoreceptors however, was surprising, and offers intriguing new avenues of research into the role of melatonin on photoreceptor physiology.

Putative Functions of Melatonin in the Retina Since the rate of retinal melatonin synthesis occurs on a cyclic or circadian rhythm, investigators have long suspected that melatonin plays a role in the regulation of some other key processes in the retina which also display cyclic rhythms of activity. Studies have suggested that melatonin is involved in photoreceptor outer segment disc shedding and phagocytosis,37-39 photomechanical movements,40-42 modulation of neurotransmitter release,3,35,43 and sensitivity to light.35 Some functions of melatonin in the retina appear to be mediated through antagonism of dopamine release from the amacrine cells of the inner retina. Melatonin inhibits the release of dopamine in the retina,35,43 and dopamine blocks the melatonin-induced cone elongation in amphibians.42 Furthermore, high levels of retinal dopamine in the light period inhibit the activity of NAT, a rate-limiting enzyme in melatonin synthesis in the photoreceptors.44 It is widely considered that melatonin and dopamine act as chemical messengers of night and day, respectively, and exert some of their effects by a mutual antagonism. Since melatonin is produced by the photoreceptor cells, and diffuses to the inner retina to bind to specific target cell receptors, melatonin produced in the retina is considered to act as a paracrine signal of

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Figure 2. Confocal images of MT1 receptor immunoreactivity in human retinal photoreceptors. Upper panel: MT1 immunoreactivity is present in the inner segments (IS) of photoreceptor cells (arrows). Both thin (arrows) and rounded (arrowheads) inner segments are present, and likely represent rod and cone photoreceptors, respectively. Lower panel: Control section treated with the MT1 receptor antibody preabsorbed with the immunogen peptide shows complete loss of reaction in rod inner segments but nonspecific signal remains, presumably in the cone inner segments (arrowheads). Reproduced with permission from Scher et al30 Invest Opthalmol Vis Sci 2002; 43:889-897, ©2002 Association for Research in Vision and Ophthalmology.

darkness. A signaling molecule is classified as paracrine when its site of action is in the same tissue or organ where it is produced. This is in contrast to the endocrine function of melatonin that is produced in the pineal gland and released into the circulation. The discovery that melatonin receptors are expressed on retinal photoreceptors suggests that the photoreceptors themselves may be direct targets of melatonin action.27,30-32 The localization of MT1 (Mel1a) melatonin receptors in photoreceptor cells has been observed in the human and rodent retina,28-30 and in chicken retina (Mel1c)34 thus confirming the original reports of Mel1a and Mel1b melatonin receptor expression in photoreceptors of Xenopus laevis.27,31,32 Since the photoreceptors produce retinal melatonin and also express melatonin receptors, a direct action of melatonin on the photoreceptors can be classified as intracrine or autocrine signaling. Melatonin appears to be involved in dark adaptation. Melatonin alters the sensitivity of the central visual system to light,45,46 and it increases the sensitivity of horizontal cells to light.35

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Figure 3. Immunocytochemistry of Xenopus laevis retina with melatonin receptor antibody. Fixed cryostat sections of Xenopus retina were incubated with the Mel1c receptor antibody and was labeled with a green fluorescent marker-antibody conjugate, and counter stained with a blue nuclear dye. Specific immunolabeling is observed in the inner segments (IS) of the photoreceptors and inner plexiform layer (IPL). The photoreceptor outer segments (OS) are devoid of immunolabeling.

Horizontal cells are located in the inner nuclear layer and send synaptic processes to the outer plexiform layer and are involved in transmission of lateral signaling. Also, horizontal cells express melatonin receptors.29-32 By binding to receptors in the horizontal cells of the inner retina or photoreceptors, melatonin likely influences cellular processes that result in an increased responsiveness to light, which would provide adaptation to darkness. Stimulation of dopamine receptors on horizontal cells47,48 induces uncoupling of horizontal cell gap junctions,49 which results in a decreased receptive field and lower sensitivity of the retina to light.50 It is well accepted that melatonin binds to melatonin receptors on amacrine and/or interplexiform cells to inhibit dopamine release at night. However, an alternative or additional mechanism is that melatonin may also bind to receptors on GABA-ergic amacrine cells, stimulate the release of GABA, which would then bind to receptors on dopaminergic amacrine cells, and inhibit their release of dopamine.36 The resulting lower dopamine levels may then cause an increase in receptive field size due to horizontal cell coupling since the dopamine-induced uncoupling of horizontal cell gap junctions would be inhibited. Although this would potentially result in lower visual acuity, it would presumably increase the sensitivity of the retina to light during the dark period, since more second-order neurons would respond to a light stimulus.50-52 Melatonin could also bind to receptors on horizontal cells to directly influence horizontal cell coupling. Furthermore, melatonin may regulate horizontal cell activity postsynaptically by inhibiting the increase in cyclic AMP due to D1 receptor activation.53 Melatonin may therefore modulate the actions of dopamine by inhibiting dopamine release from amacrine cells and inhibiting postsynaptic responses to D1 receptor activation on horizontal cells. These observations, combined with the report that melatonin increases horizontal cell sensitivity to light,35 support a role for a direct action of melatonin on horizontal cells. There is some evidence to support the hypothesis that melatonin has a direct action on retinal photoreceptors. Melatonin receptor proteins (Mel1c) are expressed on inner segment

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membranes,32 and Mel1b and Mel1c mRNA27 is expressed in photoreceptor cells of the frog Xenopus laevis. This photoreceptor localization of melatonin receptor protein was later confirmed in humans (MT1),30 and Mel1a receptor mRNA was localized to chicken photoreceptors.34 The distribution of melatonin receptors in Xenopus and human retina appears to be identical or very similar, further supporting the use of the Xenopus retina as a good model for the study of the role of melatonin in human retinal physiology. Although signals from the inner retina undoubtedly play a major role in the circadian activities of retinal photoreceptors,10,36,44,54,55 intracrine melatonin signaling in photoreceptors likely contributes substantially to circadian regulation in the retina.

Potential Role of Melatonin in Photoreceptor Cell Death The discovery that retinal photoreceptor cells express melatonin receptors provides new clues to the mechanisms by which melatonin enhances the degree of light-induced photoreceptor cell death in an animal model.56,57 We reported that a subcutaneous injection of melatonin immediately prior to continuous light exposure increases the degree of light-induced photoreceptor cell death in albino rats (Fig. 4).56 The superior quadrant of the retina is the most severely affected, which is not unexpected since the superior quadrant in general is more sensitive to light damage than other regions of the retina.58 These results confirmed and extended early reports that suggested that melatonin increases the degree of light-induced photoreceptor damage, but has no deleterious effect in the absence of the high intensity illumination used to induce light damage.59,60 This work was further confirmed and extended by demonstrating that luzindole, a melatonin receptor antagonist, protects photoreceptors from light-induced damage (Fig. 5). This observation indicates that the effect of melatonin on photoreceptor cell death is mediated through a retinal melatonin receptor.57 Together these studies suggest that melatonin, acting via specific retinal melatonin receptors, is involved in the mechanism of photoreceptor sensitivity to light damage. Because one function of melatonin appears to be to increase the sensitivity of the retina to light as part of a dark-adaptation mechanism, a consequence of this may be that if exposed to light during periods of high melatonin levels in the eye, there is also an increased sensitivity to the damaging effects of light. The Equivalent Light Hypothesis61 suggests that some forms of human blindness are due to the chronic activation of rod photoreceptors by constitutively active mutant phototransduction proteins. Many forms of inherited retinal degeneration are known to be due to mutated photoreceptor-specific proteins, such as opsin.62 The mutated proteins are hypothesized to produce a constant ‘equivalent light’ signal to the photoreceptor cells, which leads to apoptosis and photoreceptor degeneration. This hypothesis further predicts that the mechanism by which continuous real or equivalent light produces photoreceptor degeneration is by interfering with circadian processes, such as synthesis of specific proteins and outer segment disc shedding, both of which appear to be regulated by melatonin.37,63,64 This would lead to the death of photoreceptors including those not expressing the mutant gene. A human case study has reported that melatonin, combined with the serotonin uptake inhibitor Zoloft™, results in an optic neuropathy that is alleviated after discontinuation of the dietary melatonin.65 Serotonin is an intermediate product in the melatonin biosynthetic pathway, and disruption of proper relative levels of serotonin and melatonin may be the cause of the optic neuropathy. In contrast, melatonin delays photoreceptor degeneration in a mouse model of retinal degeneration,66 and it is generally accepted that melatonin is involved in the regulation cyclic shedding of photoreceptor outer segments,37 which is crucial for the survival of the photoreceptors. This apparent dichotomy of beneficial versus detrimental effects of melatonin is unknown, but may be a reflection of the circadian rhythm of melatonin synthesis. We suggest that melatonin has a beneficial role in the retina at nighttime and contributes to increased sensitivity to light, whereas during the light period, the presence of high levels of melatonin would continue to increase photoreceptor sensitivity to light, making the photoreceptors more susceptible to the damaging effects of light.

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Figure 4. Outer nuclear layer (ONL) thickness of rats exposed to high-intensity illumination (HII) for 24 hours. Measurements were made at 12 different loci of the superior (sup.) and inferior (inf.) peripheral and central retina. A single subcutaneous injection of melatonin or vehicle (sham) was given just prior to exposure to HII. A 10 µg melatonin injection results in considerable thinning of the ONL, especially in the superior region. Each group consisted of four animals. Reproduced with permission from Wiechmann and O’Steen56 Invest Opthalmol Vis Sci 1992; 33:1894-1902, ©1992 Association for Research in Vision and Ophthalmology.

It has been reported that in addition to the receptor-mediated actions of melatonin, it acts as an antioxidant to scavenge free radicals in the retina presumably to protect photoreceptor outer segment membranes from damage by hydrogen peroxide generated by light.67,68 Melatonin appears to be 100 times more potent than vitamin E in inhibiting light-induced oxidative processes in living rod photoreceptors.67 Some studies have suggested that cumulative effects of solar radiation and various drugs and other chemicals that generate free radicals may contribute to the etiology of age-related macular degeneration.69,70 Melatonin produced by the photoreceptors at nighttime may perhaps have an antioxidative protective function at night, but a receptor-mediated detrimental effect during the day. The concept that melatonin directly scavenges cytotoxic hydroxyl radicals has been challenged recently.71,72 Incubation of melatonin with a free radical-generating system results in the generation of a mixture of products, each of which can have different biological effects.71 Additionally, Melatonin appeared to have no effect on hydrogen peroxide levels in a metal ion-free in vitro assay.72 An interesting new facet to this controversy is the observation that melatonin increases the mRNA levels of the antioxidant enzymes superoxide dismutase and

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Figure 5. A graph of the number of photoreceptor nuclei in histologic sections at locations along the vertical meridian of rat retinas. Cell counts were made on 100 –µm lengths of retina at the specified location. Although protection is seen at most locations, except in the superior retina adjacent to the optic nerve, there is a greater difference between the inferior retinas of the two eyes. DMSO, dimethyl sulfoxide; ONL, outer nuclear layer. Reproduced with permission from Sugawara et al57 Invest Opthalmol Vis Sci 1998; 39:2458-2465, ©1998 Association for Research in Vision and Ophthalmology.

glutathione peroxidase in neuronal cell cultures, by altering the mRNA stability.73 Based on the time course of melatonin treatment, the alterations on antioxidant enzyme mRNA levels are thought to be receptor-mediated. Melatonin may therefore have both direct antioxidant properties as well as receptor-mediated effects on free radical scavenging. The effect of melatonin on antioxidant enzyme expression in retinal photoreceptors may represent a significant role of melatonin in the health of the photoreceptors, and is worthy of further study.

Concluding Remarks Melatonin is an output of the endogenous retinal circadian clock, and may be the signal that prepares the photoreceptors for the arrival of potentially destructive radiant energy that occurs at dawn. We have hypothesized that melatonin, synthesized by photoreceptors at night, acts both as an intracrine and paracrine circadian signal of darkness, and binds to specific receptors in photoreceptors and other retinal cells to increase visual sensitivity, thus facilitating dark adaptation and other circadian events that occur in the retina, such as expression of specific genes and proteins, and photoreceptor outer segment disc shedding. The distal tips of photoreceptor outer segments are shed on a daily rhythm as part of a renewal process, and are subsequently phagocytized by the adjacent retinal pigment epithelial (RPE) cells. Melatonin is thought to be involved in this proces,37 but the molecular mechanism is poorly understood and is a fundamentally important problem in retinal cell biology. Conversely, inappropriate (i.e., daytime) exposure of retinal cells to melatonin may be detrimental to photoreceptor survival.56,57,59,60 We suggest that melatonin is a potential hazard if present during exposure to light. Understanding the molecular mechanisms that convey the actions of melatonin may help to identify defects of the melatonin signaling pathway in some diseases of vision, and to assess

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whether alterations in normal melatonin levels may contribute to a disruption of normal retinal function. For example, age-related macular degeneration (ARMD) is likely the result of cumulative effects of genetic, environmental and chemical factors.74 Chronic exposure to melatonin at inappropriate times of day and lighting conditions may contribute to an increased risk of susceptibility to this debilitating disease. Since many individuals self-administer dietary melatonin, they may potentially be at a higher risk for developing age-related retinal degenerations that ensue from the death of photoreceptors. We propose that endogenous retinal melatonin is synthesized only at nighttime for a specific reason; chronic exposure of the retina to melatonin during intense environmental lighting may increase the risk of susceptibility of photoreceptors to light-induced damage.

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21. Nao-i N, Nilsson SEG, Gallemore RP et al. Effects of melatonin on the chick retinal pigment epithelium: Membrane potentials and light-evoked responses. Exp Eye Res 1989; 49:573-589. 22. Ebisawa T, Karne S, Lerner MR et al. Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores. Proc Natl Acad Sci USA 1994; 91:6133-6137. 23. Reppert SM, Weaver DR, Ebisawa T. Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses. Neuron 1994; 13:1177-1185. 24. 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; 92:8734-8738. 25. Reppert SM, Weaver DR, Cassone VM et al. Melatonin receptors are for the birds: Molecular analysis of two receptor subtypes differentially expressed in chick brain. Neuron 1995; 15:1003-1015. 26. Wiechmann AF, Campbell LD, Defoe DM. Melatonin receptor RNA expression in Xenopus retina. Mol Brain Res 1999; 63:297-303. 27. Wiechmann AF, Smith AR. Melatonin receptor RNA is expressed in photoreceptors and displays a cyclic rhythm in Xenopus retina. Mol Brain Res 2001; 91:104-111. 28. Fujieda H, Scher J, Hamadanizadeh SA et al. Dopaminergic and GABAergic amacrine cells are direct targets of melatonin: Immunocytochemical study of mt1 melatonin receptor in guinea pig retina. Vis Neurosci 2000; 17:63-70. 29. Fujieda H, Hamadanizadeh SA, Wankiewicz E et al. Expression of mt1 melatonin receptor in rat retina: Evidence for multiple cell targets for melatonin. Neuroscience 1999; 93:793-799. 30. Scher J, Wankiewicz E, Brown GM et al. MT(1) melatonin receptor in the human retina: expression and localization. Invest Ophthalmol Vis Sci 2002; 43:889-897. 31. Wiechmann AF, Wirsig-Wiechmann CR. Multiple cell targets for melatonin in Xenopus laevis retina: Distribution of melatonin receptor immunoreactivity. Vis Neurosci 2001; 18:1-8. 32. Wiechmann AF. Differential distribution of melatonin Mel1a and Mel1c receptors in Xenopus laevis retina. Exp Eye Res 2003; 76:99-106. 33. Natesan AK, Casonne VM. Melatonin receptor mRNA localization and rhythmicity in the retina of the domestic chick, Gallus domesticus. Vis Neurosci 2003; 19:265-274. 34. Wiechmann AF, Yang X-L, Wu SM et al. Melatonin enhances horizontal cell sensitivity in salamander retina. Brain Res 1988; 453:377-380. 35. Dubocovich ML. Melatonin is a potent modulator of dopamine release in the retina. Nature 1983; 306:782-784. 36. Boatright JH, Rubim NM, Iuvone PM. Regulation of endogenous dopamine release in amphibian retina by melatonin: The role of GABA. Vis Neurosci 1994; 11:1013-1018. 37. Besharse JC, Dunis DA. Methoxyindoles and photoreceptor metabolism: Activation of rod shedding. Science 1983; 219:1341-1342. 38. Ogino N, Matsumura M, Shirakawa H et al. Phagocytic activity of cultured retinal pigment epithelial cell from chick embryo: Inhibition by melatonin and cyclic AMP, and its reversal by taurine and cyclic GMP. Ophthalmic Res 1983; 15:72-89. 39. White MP, Fisher LJ. Effects of exogenous melatonin on circadian disc shedding in the albino rat retina. Vision Res 1989; 29:167-179. 40. Chéze G, Ali MA. Rôle de l’épiphésye dans la migration du pigment épithélial rétinien chez quelques Téléostéens. Can J Zool 1976; 54:475-481. 41. Kraus-Ruppert R, Lembeck F. Die Wirkung von Melatonin auf die Pigmentzellen der Retina von Fröschen. Pflüegers Arch 1965; 284:160-168. 42. Pierce ME, Besharse JC. Circadian regulation of retinomotor movements. I. Interaction of melatonin and dopamine in the control of cone length. J Gen Physiol 1985; 86:671-689. 43. Dubocovich ML, Takahashi JS. Use of 2-[125]iodomelatonin to characterize melatonin binding sites in chicken retina. Proc Natl Acad Sci USA 1987; 84:3916-3920. 44. Iuvone PM, Besharse JC. Dopamine receptor-mediated inhibition of serotonin N-acetyltransferase activity in retina. Brain Res 1986; 369:168-176. 45. Semm P, Vollrath L. Alterations in the spontaneous activity of cells in the guinea pig pineal gland and visual system produced by pineal indoles. J Neural Trans 1982; 53:265-275. 46. Reuss S, Kiefer W. Melatonin administered systematically alters the properties of visual cortex cells in cat: Further evidence for a role in visual information processing. Vision Res 1989; 29:1089-1093. 47. Krizaj D, Witkovsky P. Effects of submicromolar concentrations of dopamine on photoreceptor to horizontal cell communication. Brain Res 1993; 627:122-128. 48. Zarbin MA, Wamsley JR, Palacios JM et al. Autoradiographic localization of high affinity GABA, benzodiazepine, dopaminergic, adrenergic and muscarinic cholinergic receptors in rat, monkey, and human retina. Brain Res 1986; 374:75-92.

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49. Lasater EM, Dowling JE, Ripps H. Pharmacological properties of isolated horizontal and bipolar cells from the skate retina. J Neurosci 1984; 4:1966-1975. 50. Witkovsky P, Stone S, Besharse JC. Dopamine modifies the balance of rod and cone inputs to horizontal cells of the Xenopus retina. Brain Res 1988; 449:332-336. 51. Dowling JE. Retinal neuromodulation: The role of dopamine. Vis Neurosci 1991; 7:87-97. 52. Witkovsky P, Schutte M. The organization of dopaminergic neurons in vertebrate retinas. Vis Neurosci 1991; 7:113-124. 53. Iuvone PM, Gan J. Functional interaction of melatonin receptors and D1 dopamine receptors in cultured chick retinal neurons. J Neurosci 1995; 15:2179-2185. 54. Cahill GM, Besharse JC. Resetting the circadian clock in cultured Xenopus eyecups: Regulation of retinal melatonin rhythms by light and D2 dopamine receptors. J Neurosci 1991; 11:2959-2971. 55. Harsanyi K, Mangel SC. Activation of a D2 receptor increases electrical coupling between retinal horizontal cells by inhibiting dopamine release. Proc Natl Acad Sci USA 1992; 98:9220-9224. 56. Wiechmann AF, O’Steen WK. Melatonin increases photoreceptor susceptibility to light-induced damage. Invest Opthalmol Vis Sci 1992; 33:1894-1902. 57. Sugawara T, Sieving PA, Iuvone PM et al. The melatonin antagonist luzindole protects retinal photoreceptors from light damage in the rat. Invest Ophthalmol Vis Sci 1998; 39:2458-2465. 58. Rapp LM, Williams TP. A parametric study of retinal light damage in Albino and pigmeted rats. In: Williams TP, Baker BB, eds. The Effects of Constant Light on Visual Processes. New York: Plenum Press, 1980:135-159. 59. Bubenik GA, Purtill RA. The role of melatonin and dopamine in retinal physiology. Can J Physiol Pharmacol 1980; 58:1457-1462. 60. Leino M, Aho IM, Kari E et al. Effects of melatonin and 6-methoxy-tetrahydro-beta-carboline in light induced retinal damage: A computerized morphometric method. Life Sci 1984; 35:1997-2001. 61. Lisman J, Fain G. Support for the equivalent light hypothesis for RP. Nature Med 1995; 12:1254-1255. 62. Dryja TP. Molecular genetics of Oguchi disease, fundus albipunctatus, and other forms of stationary night blindness: LVII Edward jackson memorial lecture. Am J Ophthalmol 2000; 130:547-563. 63. Wiechmann AF, Komori N, Matsumoto H. Melatonin induces alterations in protein expression in the Xenopus laevis retina. J Pineal Res 2002; 32:270-274. 64. Wiechmann AF. Regulation of gene expression by melatonin: A microarray survey of the rat retina. J Pineal Res 2002; 33:178-185. 65. Lehman NL, Johnson LN. Toxic optic neuropathy after concomitant use of melatonin, Zoloft, and a high-protein diet. J Neuroophthalmol 1999; 19:232-234. 66. Liang F-Q, Aleman TS, Yang Z et al. Melatonin delays photoreceptor degeneration in the rds/rds mouse. Neuroreport 2001; 12:1011-1014. 67. Marchiafava PL, Longoni B. Melatonin as an antioxidant in retinal photoreceptor. J Pineal Res 1999: 26:184-189. 68. Sui AW, Reiter RJ, To CH. Pineal indolamines and vitamin E reduce nitric oxide-induced lipid peroxidation in rat retinal homogenates. J Pineal Res 1998; 24:239-244. 69. Gerste H. Review: Antioxidant protection of the ageing macula. Age Ageing 1991: 20:60-69. 70. Christe WG. Antioxidants and eye disease, Am J Med 1994; 97:14S-17S. 71. Horstman JA, Wrona MW, Dryhurst G. Further insights into the reaction of melatonin with hydroxyl radical. Bioorg Chem 2002; 30:371-381. 72. Fowler G, Daroszewska M, Ingold KU. Melatonin does not “directly scavenge hydrogen peroxide”: Demise of another myth. Free Radic Biol Med 2003; 34:77-83. 73. Mayo JC, Sainz RM, Antoli I et al. Melatonin regulation of antioxidant enzyme gene expression. Cell Mol Life Sci 2002; 59:1706-1713. 74. Evans JR. Risk factors for age-related macular degeneration. Prog Retin Eye Res 2001; 20:227-253.

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CHAPTER 23

Melatonin Synchronizes Cell Physiology through Cytoskeletal Rearrangements Gloria Benítez-King, Gerardo Ramírez-Rodríguez, David García and Fernando Antón-Tay

Abstract

M

elatonin is a lipophilic hormone that causes a broad spectrum of metabolic and physiological effects in the central nervous system and peripheral organs. It has been described that the hormone modulates kidney physiology, since modifies urine production and osmolarity in rats. MDCK cells are derived from a canine kidney that in culture form functional monolayers similar to the natural epitheliums. These cells form tight junctions and transport water that accumulate between the basolateral domain and the solid surface of the petri dishes forming blisters or domes. Both tight junction sealing and dome formation depend on microfilament rearrangements. It has been shown that melatonin causes cytoskeletal reorganization in cultured cells through both calmodulin and protein kinase C interactions. Current evidence indicates that cytoskeletal organization participates in structural polarity and cell shape maintenance, as well as in a broad spectrum of cell functions. In this paper we will review the recent evidence on the melatonin effects on microfilament organization in the kidney derived epithelial MDCK cells that occur concomitant with an increase in water transport in culture conditions that resembles the cyclic changes of melatonin plasma circulating levels. It is proposed that the hormone may synchronize renal cell physiology with the photoperiod through cyclic cytoskeletal reorganization. Also, the participation of PKC in the mechanism by which melatonin causes a cyclic increased water transport and microfilament reorganization is discussed.

Introduction Cyclic production of melatonin by the pineal gland synchronizes body rhythms with the photoperiod and its administration to mammals is followed by diverse effects in the central nervous system and peripheral organs.1 The mechanisms by which melatonin synchronizes the biological rhythms with the dark-light cycle have been proposed. Melatonin receptors coupled to a Gi protein have been identified and cloned.2,3 These receptors specifically bind melatonin and mediate inhibition of adenylate cyclase in the pars tuberalis and suprachiasmatic nucleus.2 Also, melatonin binds to orphan nuclear receptors of the retinoid family RZRα and RZRβ at nanomolar concentration and regulates gene transcription through the hormone binding to the DNA promoter sequences.4 It has been also suggested that melatonin modulates the activity of the Ca2+ dependent intracellular proteins calmodulin and protein kinase C (PKC).5,6 Finally it has been described that melatonin also is a free radical scavenger.7 It has been shown that melatonin binds to a purified liposome-incorporated calmodulin8 as well as to membrane-bound,9 and cytoplasmic calmodulin10 with high affinity (180 pM).8 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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Moreover, the hormone inhibits calmodulin-dependent enzyme activities such as cAMP phosphodiesterase activity in vitro,11 Ca2+ Mg2+-ATPase,12 nitric oxide synthase13 and calmodulin kinase II.14 Evidence about the melatonin-PKC interactions has been obtained in cultured cells and in vitro assays. The hormone activates a purified bovine brain PKC in vitro with a half-maximal activation of 1 nM6 and in the presence of Ca2+. In addition, the hormone increase by 30% the phorbol ester stimulated PKC activity and augment [3H]PDBu binding to the kinase.6 Also, stimulation of PKC by melatonin was demonstrated by measuring PKC activity in the membrane-cytoskeletal fraction obtained from N1E-115 cells cultured with 1 nM of this hormone.15 Melatonin increased PKC activity in this fraction by 71% after 5 min, and by 100% after either 15 or 25 min of incubation. Moreover, melatonin selectively activates the alpha isoform of PKC.16 This conclusion is supported by the general accepted notion that PKC translocation can be used as an index of the enzyme activation,17 and by the fact that incubation of N1E-115 cells with 1 nM melatonin is followed by a selective PKC alpha translocation from the cytosol to the membrane-cytoskeletal fraction.16 In recent years we have been interested in the cytoskeletal rearrangements caused by melatonin as a physiological model for the study of the interaction of melatonin with calmodulin and PKC. In N1E-115 cells, melatonin causes microtubule enlargements by preventing the tubulin polymerization inhibition through an antagonism of the Ca+2-Calmodulin complex.18 Furthermore, PKC activation by melatonin in these cells is followed by a twice increase in vimentin phosphorylation and by a transient and reversible vimentin intermediate filament reorganization.15 Cytoskeletal organization is involved in both the highly structural polarity and cell shape maintenance, as well as in a broad spectrum of cell functions.19 In this paper we will review the current evidence that support that melatonin, besides to produce metabolic changes, it may synchronize cell physiology with the photoperiod through cyclic cytoskeletal reorganization.5 We will describe the melatonin effects on microfilament organization in the kidney derived epithelial MDCK cells that occur concomitant with an increase in water transport in culture conditions that resembles the cyclic changes of melatonin plasma circulating levels. In addition, we will review the evidence that supports the PKC participation in the mechanisms by which a cyclic melatonin signal caused an increased water transport and microfilament reorganization in synchrony with the melatonin signal.

Melatonin Synchronizes Dome Formation in MDCK Cells Evidence obtained in cultured cells, concerns mainly with the melatonin effects in melanophores;20 PC12 cells,21 MCF-7 cells22 among other cell lines. Also, intracellular melatonin distribution in fibroblasts; bovine granulosa cells, neuroblastoma cells, etc, has been described.10,23 Kidney derived MDCK cells has been used to study the melatonin effects on cytoskeletal organization as well as the intracellular distribution of the hormone.10,24 Melatonin is a lipophylic molecule that crosses the plasmatic membrane and it has been found distributed in the cytosol and associated with the cytoskeleton in MDCK cells.10,23 Moreover, it has been shown that MDCK cell monolayers exposed to 1 nM melatonin for 4 days form an increased number of domes24 and that chronic exposure of MDCK cells to 1 nM melatonin causes thicker microfilament stress fibres and enhanced actin staining at the cell borders.24 MDCK cells in culture maintain biochemical, physiological and structural features of kidney transporting epithelia since they form epithelial cell monolayers that resemble the intercalated cells of renal cortical collecting ducts and respond to hormones and naturally occurring activators.25,26 MDCK cell monolayers transport water and electrolytes from the apical to the basolateral side through both the paracellular and transcellular routes and when they are cultured on nonpermeable supports, vectorial transported water is accumulated between the basolateral domain and the surface of the culture dishes forming blisters or domes.27 The capability of melatonin to cause both cytoskeletal rearrangements and an increased physiological response evidenced as an increased dome formation in MDCK cells, pointed out that MDCK

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Figure 1. Pattern of 3H-melatonin in the culture media of MDCK cells. Confluent MDCK cell monolayers were incubated with DMEM containing either the vehicle (
) for 12 h followed by 30 nM 3H-melatonin () for 12 h periods in a cyclic pattern. Culture media was taken and radioactivity counted by liquid spectrometry. Each count was done in 4 different Petri dishes. Results represent the mean of one out of three determinations.

cell monolayers are a useful model to study the cellular mechanisms by which melatonin synchronizes a specific physiology response with the photoperiod. In vitro, it is possible to reproduce the cyclic changes of melatonin plasma circulating levels, and therefore to reproduce the rhythmic metabolical and structural cell changes. Figure 1 shows the cyclic melatonin profile obtained in the culture media of MDCK cell monolayers after the vehicle or melatonin addition. Cells were incubated with the vehicle for a 12 h cycle followed by 30 nM 3H-Melatonin addition. After a 12 h cycle 3H-melatonin was withdraw and the vehicle was added (Fig. 1). Thin layer chromatography showed that melatonin was not degraded after this period of incubation and both melatonin in the cell culture media and that accumulated in the intracellular compartment migrated with a similar Rf (0.625) as the melatonin standard (Fig. 2). MDCK cells incubated for three consecutive cycles of 12h with melatonin followed by a 12 h without melatonin, showed a cyclic pattern of dome formation in synchrony with the melatonin signal.28 Increase in dome formation caused by melatonin was clearly observed in each of the three cycles. A gradual increase reaching a maximum after 6 h of melatonin incubation, was followed by a decrease in dome formation even in the presence of melatonin. The hormone increased dome number per field in the range between 58% and 72%, compared with monolayers cultured only with the vehicle.28 By 9 and 12 h after melatonin incubation, dome formation was decreased by 25%, and 30%, respectively, compared to the optimal effect observed after 6h of melatonin incubation. After hormone withdrawal, number of domes per field dropped even further, to 21, which is the average basal number observed in monolayers cultured in

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Figure 2. Identification of 3H-melatonin by thin layer chromatography. MDCK cell monolayers were incubated with 30 nM of 3H-melatonin for 6 h. Cell homogenates (left panel) or culture media (right panel) were precipitated with perchloric acid. After centrifugation, supernantants were extracted with chloroform and 3H-melatonin separated by thin layer chromatography. Melatonin (MEL) and 6-hydroxymelatonin (6OHMEL) standards were viewed by U.V. light.

regular media or in the presence of the vehicle.28 The results show that dome formation in the presence of melatonin followed a cyclic pattern with a similar profile to that of melatonin circulating in plasma. Moreover, in a dose-response experiment it was demonstrated that melatonin induced an optimal increase in dome formation at a concentration similar to circulating levels of the hormone in plasma. 10-9 M melatonin was the optimal concentration to induce the highest increase in dome formation. An increase of 82% was obtained after 6 h of incubation with 10-9 M melatonin. While increases of 50%, 66%, and 36%, were observed at 10-11, 10-7, and 10-5 M melatonin, respectively.28 MDCK cell monolayers cultured on semipermeable filters confirmed that vectorial water transport is increased in monolayers incubated with melatonin. [3H]-H2O flux measured every 3 h showed no differences in the initial 3 h of melatonin exposure, while a significant increase of 10% in the [3H]-H2O flux from the apical to the basal compartment was observed after 6 h of melatonin incubation. The radioactive water accumulation in this compartment decreased gradually after 9 or 12 h of melatonin treatment. No significant differences were found in the vehicle incubated cells cultured for a 12 h cycle.28 The results indicate that water flux from the apical to the basolateral domain in MDCK cell monolayers incubated with melatonin correlates with the temporal course of dome formation induced by the hormone.

Melatonin Synchronizes Microfilament Reorganization in MDCK Cells Actin filament integrity is necessary for modulation of transepithelial permeability through both paracellular and transcellular pathways. Microfilament association with membrane components regulates the sealing of the tight junctions and the paracellular pathway of ion transport.29 In the presence of cytochalasin B, cytoplasmic and cortical microfilaments are disrupted, tight junctions lose their organization and remain opened and transepithelial resistance (TER) is abolished.29,30 Also, actin cytoskeleton has been implicated in the transcellular pathway of ion and water transport. Microfilaments interact with transmembrane proteins involved in vectorial transport such as the band 3 anion exchanger,31 the epithelial Na+ K+ adenosine

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Figure 3. Effect of melatonin on microfilament organization in MDCK cell cortical rings. MDCK cell monolayers grown on glass cover slips were incubated with either A) the vehicle or B) 1nM melatonin for 6 h. Microfilaments were stained with rhodamine-phalloidin. Images were obtained with a digital camera and thickness of cortical rings measured with an image analyzer (C). Cortical rings are marked by arrows. Photomicrographs represent results obtained in three experiments done by duplicate. Bar= 10 µm.

triphosphatase (Na+ K+ ATPase),32 the Na+ K+ Cl- cotransporter33 and the Na+ channels.34 Actin filaments stimulate Na+ K+ ATPase activity by a mechanism that implicates a direct binding of actin to the enzyme.35 Cells forming a dome are detached from the surface and do not show an organized basal cytoskeleton.36 However, cells surrounding the domes showed at the basal side microfilaments organized in stress fibers and focal adhesion contacts.36 Incubation of MDCK cell monolayers in cycles of 12 h with the vehicle followed by a cycle of 12 h with 10-9 M melatonin showed that microfilament organization changes at both the apical and the basal sides of the cells surrounding the domes.28 Actin microfilaments in cells incubated with the vehicle during 6 and 12 h were organized in typical cortical rings at the apical levels and in stress fibres at the basal level. The pattern remained unchanged during the entire cycle. Cells incubated with melatonin for 3 h showed a similar pattern to that observed in cells cultured with the vehicle. However, after 6 h of incubation with the hormone, abundant and thicker stress fibres were observed in cells not forming a dome.28 In addition, the cortical ring in these cells were thicker when compared with the vehicle incubated cells (Fig. 3). Although in the presence of melatonin there is no increase in water transport trough the paracellular pathway, a significant increase of 0.3 µm was observed in the cortical ring wide of cells incubated with 10-9 M of the hormone. The stress fibres on the other hand showed a clear thickening at the focal contacts.28 This effect of melatonin on microfilament organization was reversible, since after 9 and 12 h in the presence of the hormone the actin microfilaments recover basal organization seen in the vehicle incubated cells.28 Thus, the gradual reversal at longer time of exposure to melatonin suggest that these changes can be induced again after one cycle of 12h without melatonin. Microfilament reorganization elicited by melatonin, correlates with the cyclic pattern of dome formation produced in synchrony with the melatonin signal. Altogether, these data suggest that melatonin effects on microfilament organization are related to the melatonin action on vectorial water transport in MDCK cells.

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Characterization of the Cellular Pathway by Which Melatonin Increases Ion and Water Transport Epithelial cells develop tight junctions in the basolateral domain that regulate paracellular ion diffusion from the lumen to the interstitial side.37 The Na+K+ ATPase located in the basolateral domain, creates a driving force for sodium vectorial water transport from the apical to the basolateral domain through the transcellular pathway.37,38 The vectorial flux of water and ions across an epithelial monolayer can be evaluated by measuring the TER across the monolayers. MDCK cells shown a stable TER of 650 Ω/cm2. MDCK cell monolayers incubated in cycles of 12 h in the presence of the vehicle followed by a 12 h of incubation with 1 nM melatonin showed that TER did not change after 12 h of incubation in the presence of the vehicle.28 While after 6 h of incubation with 10-9 M melatonin, the TER value decreased by 28% and remained low for up to 12 h suggesting water and ion passage throughout the paracellular route. However, FITC-dextran flux does not change in monolayers incubated for 6 h, 9 h, or 12 h in the presence of 10-9 M melatonin and the permeability values were similar to those obtained for monolayers incubated with the vehicle alone.28 Basal FITC-dextran flux observed in this conditions could be due to selective low passage through the tight junctions. These results discard the selective ion and water transport across the paracellular pathway and suggest that melatonin can increase epithelial permeability through the transcellular pathway. In fact, the effects of melatonin on permeability were evaluated by inhibiting the Na+ K+ ATPase with 10 µM ouabain, and assesing the formation of domes in the presence of melatonin. Ouabain abolished dome formation when added to confluent monolayers for 1 h. Furthermore, a complete blockage in the increased dome formation was observed in MDCK cell monolayers preincubated with ouabain and then treated with 10-9 M melatonin for 6 h.28 These results support that the increased water and ion flux is induced by melatonin and takes place through the transcellular pathway.

Role of Protein Kinase C in the Mechanism by Which Melatonin Induces Microfilament Reorganization and Dome Formation Besides actin microfilament regulation of vectorial ion and water transport in epithelial cells, it has been proposed that PKC also participates in this process. 1,2-dioctanoylglycerol, a PKC activator, increases TER by 100%, while the PKC inhibitors H-7 and polymyxin B blocked TER development in MDCK cells.39 Furthermore, treatment of cultured proximal tubule cells with the PKC agonist phorbol 12-myristate 13-acetate (PMA) is followed by an increased phosphorylation of the Na+ K+ ATPase, and by translocation of the enzyme to the plasma membrane.40,41 Phosphorylation is proportional to the increased Na+ K+ ATPase activity observed.41 These data indicates that PKC participates in the modulation of both paracellular and transcellular pathways for ion and water transport. Therefore, participation of PKC in the mechanisms by which melatonin causes an increase in vectorial water transport was explored by measuring dome formation in the presence of the PKC agonist, PMA , or the PKC inhibitors bisindolylmaleimide or calphostin C.28 PMA, the PKC agonist increased dome formation similarly to melatonin.28 While, both PKC inhibitors, calphostin C and bisindolylmaleimide, inhibited the increase in dome formation caused by melatonin. Similarly, both PKC inhibitors decreased dome formation elicited by PMA. Thus, data indicate that PKC activation is involved in the mechanism by which melatonin increases dome formation in MDCK cells. The precise mechanism by which PKC participates in dome formation elicited by melatonin is unknown. However, it is known that PKC is activated by melatonin in MDCK cell homogenates with an EC50 of 1 nM6. Moreover, in MDCK cells a gradual increase in PKC activity was observed in the first 6 hr of 10-9 M melatonin incubation followed by a decrease after 9 hr of treatment.42 The pattern of PKC activity in MDCK cells incubated with melatonin correlates with the cyclic pattern of dome formation produced in synchrony with the melatonin signal. Since PKC α was selectively activated and translocated from the membrane-cytoskeletal fraction to the cytosol in an initial period of melatonin incubation, and decreased levels were

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Figure 4. Effect of melatonin on cortical ring microfilament protein phosphorylation. A) Cortical rings were characterized by rhodamine-phaloidine staining. Cortical rings are marked by arrows. B) PKC association to microfilaments were characterized by staining with the PKC specific fluorescent ligand RIM-1. Fluorescent PKC is marked by arrows. MDCK cells were incubated with the vehicle or 10-9 M melatonin for 6 h and labelled with 32P-orthophosphate during 4 h. Forty µg of protein from isolated cortical rings were separated by SDS-PAGE. C) Densitometric analysis of the autoradiograms of the 42 Kd (A, B), the 56 Kd (C, D), and the 84 Kd (E, F) protein phosphorylation in MDCK cells incubated with the vehicle (A,C,E) or 1 nM melatonin (B, D, F). Results represent the mean of three densitometric scannings obtained from one representative experiment of three. *p < 0.05.

observed after 6 hr of MDCK cell incubation with the hormone.42 Then, data suggest that PKC may be initially activated and then down regulated after melatonin exposure. In addition, it is known that Na+ K+ ATPase is activated by PKC40 and that a recruitment of this enzyme to the plasma membrane occurs in the presence of the PKC agonist, PMA.41 This evidence together with the fact that the effect of melatonin on dome formation is abolished by the Na+ K+ ATPase inhibitor, ouabain,28 suggest that the interaction of melatonin with PKC6 may increase the Na+ K+ ATPase activity.41 Since, phosphorylation of several actin binding proteins by PKC participates in actin microfilament rearrangements to form both stress fibres and focal adhesion contacts.17 Thus, participation of PKC in the mechanism by which melatonin causes an increase and thickening of stress fibres was explored in MDCK cells cultured with the hormone in the presence of the PKC agonist, PMA, or specific PKC inhibitors. 43 Rhodamine-phalloidin microfilament staining of MDCK cells showed that microfilaments in cells cultured with PMA were longer an thicker with a similar distribution pattern to those observed in melatonin incubated cells.43 While in the presence of the PKC inhibitors, calphostin C and bisindolylmaleimide, microfilament enlargement and thickening elicited by PMA or melatonin were abolished.43 In addition, thickening of cortical microfilaments observed in the presence of 10-9 M melatonin was prevented by both PKC inhibitors.43 Furthermore, it was demonstrated that PKC is associated with microfilament cortical rings purified from MDCK cells by [3H]PDBu binding44 and by RIM-1 staining. Cortical rings were characterized by rhodamine-phalloidin staining as shown in Figure 4A. Microfilaments showed a polyhedrical arrangement. The PKC specific fluorescent ligand RIM-1 stained the isolated cortical rings as shown in Figure 4B. Densitometric analysis of cortical ring phosphoproteins, previously

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Figure 5. Schematic representation of the mechanisms by which melatonin causes microfilament reorganization and an increase in water transport in MDCK cells. Melatonin (MEL) enters the cell and interacts with protein kinase C (PKC). This interaction is followed by an increase in PKC activity. Activated PKC may phosphorylate actin binding proteins and thus microfilament reorganization takes place. Also, PKC may increase the Na+ K+ ATPase activity through recruitment of the enzyme to the basolateral membrane. Both microfilament reorganization and Na+ K+ ATPase increased activity caused and increased ion and water transport in MDCK cell monolayers.

separated by SDS-PAGE, showed that melatonin increased the 84 kDa, 57 kDa protein band phosphorylation. These bands correspond to the molecular weights of PKC and vimentin a substrate for PKC, respectively. No changes were detected in a 42 kDa protein band phosphorylation that corresponds possibly to a cytokeratin (Fig. 4C). Together, data supports that PKC participates in the mechanism by which melatonin causes microfilament reorganization (Fig. 5). Moreover, since it is known that PKC interacts with the small GTPase protein Rho and this protein modulates microfilament organization it is possible that an interaction between a PKC activated by melatonin and Rho,45 may cause an increase in stress fibres formation46 and the modifications in actin dynamics that would induce the formation of domes (Fig. 5).

Concluding Remarks In mammals, melatonin is produced by the pineal gland with a circadian rhythm synchronized with the photoperiod. A nocturnal rise of melatonin in the plasma is derived primarily from the melatonin synthesized in the pineal gland and released to the general circulation where it reaches a nanomolar concentration.1 In this review we presented evidence that kidney

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epithelial cells in culture respond to nanomolar concentrations of melatonin following a cyclic pattern that resembles the cyclic variation of melatonin levels in the plasma. A cyclic increase in vectorial water transport through the transcellular route, implicating reorganization of the actin cytoskeleton and participation of PKC, occurs in synchrony with the melatonin signal. These data suggest that modifications in urine production and osmolarity observed in the kidney during the dark phase in which melatonin reaches highest concentration may be related to cytoskeletal and permeability changes elicited by the hormone.47,48 Evidence presented in this review, support the proposed hypothesis that plasma circulating levels of melatonin may synchronize cell physiology with the photoperiod by changing cytoskeletal organization.24 In addition, data presented here support that melatonin regulation of the Ca2+-dependent PKC α isoform may represent a fine tunning of the transient intracellular Ca2+ that in turn modulates cell physiology through cytoskeletal rearrangements.

Acknowledgments This work was supported in part by CONACYT, México grant No 31782-N and a fellowship from CONACYT for GRR.

References 1. Reiter RJ. The melatonin rhythm: Both a clock and a calendar. Experientia 1993; 49:654-664. 2. Masana MI, Dubocovich ML. Melatonin receptor signaling: Finding the path through the dark. Sci STKE 2001; E39. 3. 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; 92:8734-8738. 4. Carlberg C. Gene regulation by melatonin. Ann NY Acad Sci 2000; 917:387-396. 5. Benítez-King G, Antón-Tay F. Calmodulin mediates melatonin cytoskeletal effects. Experientia 1993; 49:635-641. 6. Antón-Tay F, Ramírez G, Martínez I et al. In vitro stimulation of protein kinase C by melatonin. Neurochem Res 1998; 23:605-610. 7. 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 Top Med Chem 2002; 2:181-197. 8. Benítez-King G, Huerto-Delgadillo L, Antón-Tay F. Binding of 3H-Melatonin to calmodulin. Life Sci 1993; 53:201-207. 9. Romero MP, García-Pergañeda A, Guerrero JM et al. Membrane-bound calmodulin in Xenopus Laevis oocytes as a novel binding site for melatonin. FASEB J 1998; 12:1401-1408. 10. Antón-Tay F, Martínez I, Tovar R et al. Modulation of the subcellular distribution of calmodulin by melatonin in MDCK cells. J Pineal Res 1998; 24:35-42. 11. Benítez-King G, Huerto-Delgadillo L, Antón-Tay F. Melatonin modifies calmodulin cell levels in MDCK and N1E-115 cell lines and inhibits phosphodiesterase activity in vitro. Brain Res 1991; 557:289-292. 12. Antón-Tay F, Huerto-Delgadillo L, Ortega-Corona B et al. Melatonin antagonism to calmodulin may modulate multiple cellular functions. In: Toitou Y, Arendt J, Pevet P, eds. Melatonin and the pineal gland from basic science to clinical application. Amsterdam: Elsevier, 1993:41-46. 13. Pozo D, Reiter RJ, Calvo JR et al. Inhibition of cerebellar nitric oxide synthase and cyclic GMP production by melatonin via complex formation with calmodulin. J Cell Biochem 1997; 65:430-442. 14. Benítez-King G, Ríos A, Martínez I et al. In vitro inhibition of Ca2+/calmodulin dependent protein kinase II activity. Biochem Byophys Acta 1996; 1290:191-196. 15. Benítez-King G. PKC activation by melatonin modulates vimentin intermediate filament organization in N1E-115 cells. J Pineal Res 2000; 29:8-14. 16. Benítez-King G, Hernández ME, Tovar R et al. Melatonin activates PKC-α but not PKC-ε, in N1E-115 cells. Neurochem Int 2001; 39:95-102. 17. Liu JP. Protein kinase C and its substrates. Mol Cell Endocr 1996; 116:1-29. 18. Huerto-Delgadillo L, Antón-Tay F, Benítez-King G. Effects of melatonin on microtubule assembly depend on hormone concentration: Role of melatonin as a calmodulin antagonist. J Pineal Res 1994; 17:55-62. 19. Cid-Arregui A, De Hoop M, Dotti CG. Mechanism of neuronal polarity. Neurobiol Aging 1995; 16:239-243.

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20. Reed BL, Finnin BC, Ruffin NE. The effect of melatonin and epinephrine on the melanophores of freshwater teleosts. Life Sci 1969; 8:113-120. 21. Roth JA, Rabin R, Angello K. Melatonin suppression on PC12 cell growth and dead. Brain Res 1997; 768:63-70. 22. Blask DE, Wilson ST, Zalatan F. Physiological melatonin inhibition of human breast cancer cell growth in vitro: Evidence for a gluthathione-mediated pathway. Cancer Res 1997; 57:1909-1914. 23. Finocchiaro LM, Glikin GC. Intracellular melatonin distribution in cultured cell lines. J Pineal Res 1998; 24:22-34. 24. Benítez-King G, Huerto-Delgadillo L, Antón-Tay F. Melatonin effects on the cytoskeletal organization of MDCK and neuroblastoma N1E-115 cells. J Pineal Res 1990; 9:209-220. 25. Taub M, Chuman L, Saier MH et al. Growth of madin-darby canine kidney epithelial cell (MDCK) line in hormone-supplemented, serum-free medium. Proc Natl Acad Sci USA 1979; 76:3338-3342. 26. Lever JE. Inducers of mammalian cell differentiation stimulate dome formation in a differentiated kidney epithelial cell line (MDCK). Proc Natl Acad Sci USA 1979; 76:1323-1327. 27. Cereijido M, Ehrenfeld J, Fernández-Castelo S et al. Fluxes, junctions, and blisters in cultured monolayers of epithelioid cells (MDCK). Ann NY Acad Sci 1981; 372:422-441. 28. Ramírez-Rodríguez G, Meza I, Hernández ME et al. Melatonin induced cyclic modulation of vectorial water transport in kidney derived MDCK cells. Kidney Int 2003; 63:1356-1364. 29. Meza I, Ibarra G, Sabanero M et al. Occluding junctions and cytoskeletal components in a cultured transporting epithelium. J Cell Biol 1980; 87:746-754. 30. Meza I, Sabanero M, Stefani E et al. Occluding junctions in MDCK cells: Modulation of transepithelial permeability by the cytoskeleton. J Cell Biochem 1982; 18:407-421. 31. Drenckhahn D, Schluter K, Allen DP et al. Colocalization of band 3 with ankyrin and spectrin at the basal membrane of intercalated cells in the rat kidney. Science 1985; 230:1287-1289. 32. Nelson WJ, Veshnock PJ. Ankyrin binding to (Na+ + K+) ATPase and implications for the organization of membrane domains in polarized cells. Nature 1987; 328:533-536. 33. Jorgensen PL, Petersen J, Rees WD. Identification of a Na+, K+, Cl- cotransport protein of Mr 34 000 from kidney by cytoskeleton components. Biochim Biophys Acta 1984; 775:105-110. 34. Edelstein NG, Catterall WA, Moon RT. Identification of a 33- kilodalton cytoskeletal protein with high affinity for the sodium channel. Biochemistry 1988; 27:1818-1822. 35. Cantiello HF. Actin filaments stimulate the (Na+ + K+) ATPase. Am J Physiol 1995; 269:F637-F643. 36. Castillo AM, Reyes JL, Sánchez E et al. 2,3-Butanedione monoxime (BDM), a potent inhibitor of actin-myosin interaction, induces ion and structure fluid transport in MDCK monolayers. J Muscle Res Cell Motil 2002; 23:223-234. 37. Cereijido M, Ehrenfeld J, Meza I et al. Structural and functional membrane polarity in cultured monolayers of MDCK cells. J Membr Biol 1980; 52:147-159. 38. Contreras RG, Avila G, Gutiérrez C et al. Repolarization of Na+-K+ pumps during establishment of epithelial momolayers. Am J Physiol 1989; 257:C896-C905. 39. Balda MS, González-Mariscal, Contreras RG et al. Assembly and sealing of tight junctions: Possible participation of G-proteins, phospholipase C, protein kinase C and calmodulin. J Membr Biol 1991; 122:193-202. 40. Lowndes JM, Hokin-Neaverson M, Bertics PJ. Kinetics of phosphorylation of Na+/K+-ATPase by protein kinase C. Biochim Biophys Acta 1990; 1052:143-151. 41. Efendiev R, Bertorello AM, Pressley TA et al. Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na+/K+-ATPase molecules to the plasma membrane. Biochemistry 2000; 39:9884-9892. 42. Ramírez G, y Benítez-King G. Melatonin causes protein kinase C down regulation in MDCK cells. Neuroimmunomodulation 1999; 6:447. 43. Benítez-King G, Ramírez G. Protein Kinase C/Calmodulin an intracellular signalling system involved in actin microfilament rearrangements elicited by melatonin. Neuroimmunomodulation 1999; 6:448. 44. García D, Hernández ME, Ramírez-Rodríguez G et al. Melatonin elicited phosphorylation of MDCK cytoskeletal cortical ring proteins [abstract]. Melatonin and Biological Rhythms Symposium Adelaide Australia 2001. 45. Slater Sj, Seiz JL, Stagliano BA et al. Interaction of protein kinase C isozymes with Rho GTPases. Biochemistry 2000; 40:4437-4445. 46. Hall A, Nobes CD. Rho GTPases: Molecular switches that control the organization and dynamics of the actin cytoskeleton. Philos Trans R Soc Lond B Biol Sci 2000; 355:965-970. 47. Richardson BA, Studier EH, Stallone JN et al. Effects of melatonin on water metabolism and renal function in male Syrian hamster (Mesocrisetus auratus). J Pineal Res 1992; 13:49-59. 48. Koopman MG, Koomen GC, Krediet RT. Circadian rhythm of glomerular filtration rate in normal individuals. Clin Sci Lond 1989; 77:105-111.

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CHAPTER 24

Melatonin in Winter Depression Arcady A. Putilov, Galena S. Russkikh and S.R. Pandi-Perumal

Abstracts

I

n many animals, the day length response is mediated by circadian rhythm of melatonin (MLT). Although humans are not generally considered to be photoperiodic, the exposure to bright light was shown to be necessary to suppress MLT secretion and to shift the circadian phase. Moreover, seasonal variations in many human functions has been widely acknowledged. Among them seasonal affective disorder (SAD) is probably the most well known. The therapeutic effect of bright light treatment (BLT) on SAD was predicted theoretically and thereafter demonstrated in numerous trials. Association of winter type of SAD with reduced day length and therapeutic response to BLT may suggest that either photoperiodic time measurement or delayed circadian phase or both play a role in etiology of this disease. However, there were many investigations that argued against the involvement of MLT in SAD pathophysiology and antidepressant response to BLT and only few, but more recent investigations have drawn renewed interest to the MLT hypotheses. Some of the results on SAD pathophysiology raise a question about multi-component nature of biological dysfunction in SAD. Such chronobiological mechanisms as phase resetting and daytime measurement could be primarily responsible for those symptoms of SAD that are closely related to circadian and metabolic dysfunctions in winter depressives, while the disturbances in arousal and mood may be closer associated with other (i.e., nonchronobiological) mechanisms. In particular, the findings on BLT effects in SAD suggest that any simple pathophysiological model of SAD is not adequate and that modification of MLT rhythm might not be necessary for favorite therapeutic response to BLT in the majority of winter depressives. In general, despite several demonstrations that the postulated by a theory changes in MLT rhythm are associated with the antidepressant action of BLT and/or change in season, several findings must be considered to lend no support for or even arguing against the involvement of day length and phase responses in SAD pathogenesis and the mechanism by which LT works. Thus, the question of importance of MLT and circadian phase for manifestation of SAD symptoms and effective BLT is still open.

Winter Depression Although humans are not generally considered to be photoperiodic, seasonal variations in many human functions has been widely acknowledged. Among them seasonal course in affective disorder is probably the most well known and most intriguing example. Approximately 10% of affective disorders have a season-depended course1 and more than 10% of Siberian population reported to have dramatic seasonal variations in mood and behavior.2-4 The seasonal affective disorder (SAD) occurs in it’s most common form—winter depression—during the winter and remits in the spring and summer. It differs from nonseasonal depression not only in its seasonal variation, but also in the presence of such depressive symptoms as fatigue, social withdrawal, oversleeping, overeating, carbohydrate craving and weight

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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gain.5 These symptoms are considered atypical, in contrast with insomnia, early morning wakening, poor appetite and agitation that are often seen in nonseasonal depression. In the light of the numerous findings of triggering and termination of affective illness by environmental factors Wehr et al (1988)6 speculated that these conditions could, at least partly, be the disorders of systems that mediate the organism’s adaptation to changes in physical environment. The SAD symptoms may remit after just one week of 2-4 hour daily exposure to bright light (2500 lux or more).5 Despite extensive experimental investigations, the pathophysiological mechanisms of winter depression and it’s response to bright light remains unknown. The majority of the proposed explanations link them with the circadian rhythms. It was noted that most of these hypotheses are not mutually exclusive (see Refs. 7-10).

Day Length Measurement

SAD5 and antidepressant effect of bright light on depressive symptoms11,12 were predicted on the bases of the observations that, in animals, the day length response is mediated by circadian rhythm of melatonin (MLT) (i.e., Ref. 13) and that, in humans, the exposure to bright light is necessary to suppress MLT secretion.14 The theory assumes that the suprachiasmatic nucleus (SCN) may play a role as a mediator of the response of mood to seasonal variation in day length and that this response may be mirrored in seasonal change of MLT profile. The theoretically predicted ability of bright light treatment (BLT) to reverse the symptoms of SAD was demonstrated in numerous investigations (see for review Refs. 15-20). However, the reports of the therapeutic efficacy of midday light21 were considered to lend no support for the involvement of day length response in SAD pathogenesis and in the mechanism by which BLT works. The efficacy of BLT was found to be rather high at various time of day, including hours when MLT is not secreted by the pineal gland (i.e., Refs. 22-26). In particular, the midday BLT still reverses effectively the symptoms of winter depression.21,27-30 Thus, those experimental investigations that were made soon after the discovering of winter depression and BLT argued against the involvement of photoperiodic time measurement in SAD etiology and therapeutic response to bright light. MLT administered orally in the morning and early evening did not completely reverse the effects of BLT, although it did reproduce the atypical depressive symptoms of SAD (social withdrawal, hyperphagia, appetite and weight increase, carbohydrate craving, hypersomnia, fatigability and reverse diurnal variations).31 In another study,32 MLT given to SAD patients either in the morning or in the evening has neither positive nor negative effect on depressive symptoms, while bright light has the positive effect. The experiments with two beta-adrenergic blockers, which suppress MLT secretion, led to the discrepant results. In the early study, a long acting agent, atenolol, did not reproduce the antidepressant effect of light in most SAD subjects treated in the afternoon, although several subjects responded very well.33 In contrast to earlier published findings, in more recently reported experiment34 an antidepressant response was demonstrated in SAD patients treated in early morning with a short acting beta-adrenergic blocker, propranolol. The subsequent study35 provides additional support for the efficacy of timed beta blockade. Some recent investigations of healthy subjects have drawn renewed interest to the photo periodical hypothesis of SAD. Wehr et al (1993, 1995a)36,37 have shown that the duration of MLT secretion in healthy humans responds to changes in photoperiod in ways that resemble the responses seen in animals. Spiegel et al (1998)38 have demonstrated that the duration of MLT secretion may be shorten or lengthen by sleep curtailment or extension, respectively. Hashimoto et al (1997)39 have found that midday exposure to bright light may change the duration of time of MLT excretion due to advance of phase of the secretion onset. However, the seasonal changes in the circadian patterns of MLT and some other hormones have not been detected in a sample of winter SAD studied by Avery et al (1997).40 The negative

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results were presented by Wehr et al (1995b)41 in their early report. However, the most recent report based on the investigation of winter and summer patterns of MLT secretion in a bigger sample (55 patients with SAD and 55 age- and sex-matched controls) Wehr et al (2000)42 demonstrated that the winter depressives rather than healthy controls respond to the lengthening of day by shortening of the duration of MLT secretion. Thus, the original assumption that the timing of winter depression reminds the mechanism of photoperiodic response in mammals is not rejected yet completely. However, there is yet no solid evidence that the mood response to bright light and other antidepressants in SAD is mediated by normalization of pattern of MLT secretion.

Daytime MLT Levels The idea of involvement of MLT rhythm in SAD was not supported by most research on the effect of antidepressant treatments on MLT levels. In particular, it was noted that the antidepressants reduce depressive symptoms irrespective of their effect on MLT levels that could either increase (tricyclics and fluvoxamine) or decrease (fluoxetine).43 The results of several clinical studies let us assume that daytime secretion of MLT may be enhanced SAD patients. In a case study of Prasko (1992)44 the abnormally high MLT levels were found in a patient with SAD in the morning and afternoon hours and early morning BLT resulted in lowering and phase advancing of MLT rhythm. Among 10 patients with SAD and 10 studied by Jacobsen et al (1987a)27 one patient hyper secreted MLT in daytime and in this patient, a rise in plasma MLT levels was found after oral administration of 5-hydroxytryptophan. Dietary L-tryptophan, the precursor of 5-hydroxytryptophan, was as effective as light in patients with SAD.45 In a case study of Levitt et al (1991)46 a favorable response to tryptophan treatment was associated with increased nighttime levels of a MLT metabolite excretion and a higher than expected daytime levels were found during placebo treatment. Day/Night differences in urine excretion of MLT were found to be low in the Siberian SAD patients and the signs of normalization of the excretion pattern were noted after remission of symptoms following by BLT, change in season and flight to a south region.47-49 The further research showed that in winter MLT levels in serum in daytime were significantly higher in patients with SAD compared to controls. This difference disappeared after BLT and in summer.50,51 The study of the seasonal changes in diurnal pattern of serum MLT in an Alaskan population (64 degree North) demonstrated the elevated daytime levels in winter and some correlations with SAD-like symptoms.52 By contrast, the abnormalities in the daytime MLT levels of winter depressives were not reported by other groups (i.e., Refs. 21,53-55). The elevated MLT levels in daytime in serum and urine may have extra pineal origin and further experimental studies are required to understand a possible role of daytime MLT in manifestation of typical and atypical symptoms in SAD patients (see Ref. 56).

Circadian Phase Association of winter form of SAD with reduced day length and its beneficial response to BLT may suggest that this disease relates not only to changing in duration of MLT secretion, but also to changing in circadian phase of MLT and other rhythms. When the early experiments did not confirm the assumption that the extension of day length is critical for the antidepressant effect of bright light, another chronobiological response to light—correcting of abnormally phased circadian rhythms—was proposed and intensively tested as a possible mechanism of BLT for SAD. According to the phase-shift hypothesis,57,58 endogenous circadian rhythms in most SAD patients are abnormally phase-delayed with respect to real time or sleep time and BLT in the morning can correct this phase disturbance by advance shift of circadian pacemaker. Hypersomnia and morning fatigue have been proposed as markers of delayed circadian rhythms.59-61 In a number of investigations, the symptom of hypersomnia was found to be a

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predictor of clinical response to BLT in SAD.62-68 Besides, the significant correlation between difficulty awaking and severity of depression was reported by Avery et al (1994).69 However, in some SAD populations the symptom of hypersomnia was not found to be very common and hypersomnic SAD subjects responded well to nighttime LT, although this treatment does not produce the advance of their circadian phase.61 Avery et al (1997)40 assumed that a phase delay of circadian rhythms relative to sleep may explain why SAD subjects experience hypersomnia.40 When, under conditions of internal desynchronization, the temperature minimum is phase-delayed relative to sleep onset, the sleep duration is relatively long.70,71 In the researches of masked temperature rhythms in SAD subjects, neither baseline delays of phase, nor advances caused by BLT were observed (Skwerer et al, 1990).49,72-74 However, in the constant routine studies, both pretreatment phase delay and advance shift following morning BLT was reported for main markers of circadian phase position: the rhythms of body temperature, cortisol and MLT.40,75 In another study using constant routine protocol,10,29 the temperature rhythm showed the tendency to delay in winter and certain parameters of the rhythm were advanced by midday light. The results of SAD studies designed to test the pretreatment MLT phase and its shift following LT are inconsistent. A phase delay of the MLT rhythm in depressed SAD patients was noted in a number of investigations.54,76-78 However, there were also many reports of a normal phase.29,53,55,79-81 Similarly, a phase advance of the MLT rhythm following LT was evidenced by several,54,55,57,58,75,77,82 but not all groups.29,53,78-81 In general, the necessity of pretreatment phase delay of MLT rhythm and its phase advance by morning light for clinical response might be questioned. The phase shift model for the mechanism of SAD and BLT remains a controversial hypothesis.

Timing of Light Treatment The question about the optimal timing of BLT for SAD is of both theoretical and practical importance. The midday treatment has been proposed as a test for not only the hypothesis of normalization of the timing of melatonin secretion by extension of day length with morning plus evening BLT,5,12 but also for the hypothesis of correction of abnormally delayed circadian rhythms with early morning BLT.57,58 The meta-analysis of data from different research centers shows that light is therapeutic at most times of day. However, the efficacy of early morning LT seems to be somewhat higher compared to the efficacy of evening LT and the efficacy of these treatments seems to be higher than the efficacy of treatments scheduled in between (see i.e., review of Ref. 61). It was noted that evening BLT does not potentiate the following response to morning BLT, whereas morning light inhibits the following response to evening light.17,83 Rosenthal and Wehr (1992)8 suggested a hypothetical circadian variation in sensitivity to the antidepressant responses to light. Morning LT may phase advance the hypothetical circadian variation in sensitivity to the antide pressant responses to light in such a way that subsequently administered evening light may fall upon an inert portion of the response curve. The earlier findings of superiority of morning BLT over evening BLT was recently replicated by several groups.84-86 However, no differences in the antidepressant response were found in most studies using a parallel design with random assignment to time of a.m. and p.m. treatment.24,25,50,78,87 The afternoon treatment with MLT in physiological dose was proposed to be an effective antidepressant, because such a treatment has to produce a circadian phase advance. Indeed, the afternoon administration has been shown to improve winter depression.88 In another study,89 the afternoon MLT treatment prevented relapse after total sleep deprivation, although no difference between placebo and MLT conditions was found. The typical for mammal’s phase-response curve (PRC) suggests that only weak, if any, therapeutic response to bright light in the middle of the day, because such a treatment fails to extend

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day length or reset the abnormally phased circadian rhythms. However, the phase-shifting effect of daytime bright light was reported in healthy subjects.90 Therefore, it could be assumed that human circadian pacemaker keeps the sensitivity to light throughout subjective day and midday BLT could advance the circadian phase. However, the findings of considerable improvement caused by day-by-day variable BLT23-25 may be considered as evidence against the phase-shift hypothesis. Besides, the causal link between the antidepressant response and phase shifts in winter depression was not supported satisfactorily (i.e., Refs. 55,67,80,82,91,92). Although the positive correlation between the advance phase shift and antidepressant response was detected in several studies,4,58,87,93 even negative relations between advance shift of a circadian rhythm and clinical response were reported.40,72 Lewy et al (2000)94 found the statistically significant correlation between the decrease in depression ratings and the amount of phase advance when phase advances greater or equal to 1.5 hours were excluded from the analysis. They and other researches of SAD do not recommend early morning BLT for patients with early morning awakenings and crushing evening tiredness. Figure 2 illustrates that pretreatment phase of MLT rhythm could predict the differential response to morning and afternoon BLT. The data suggest that early morning decay and early evening rise of MLT predict worse response to morning BLT, whereas the late morning decay and late evening rise predicted worse response to afternoon BLT.4,95

Sensitivity to Light

Several groups4,55,58,68,96 noted that patients with SAD did not differ much from controls on baseline MLT phase position (which might be no significantly delayed), but more pronounced differences from controls were found for the extent of advance shift of circadian phase of MLT rhythm following morning BLT. It has to be noted that the symptoms of hypersomnia and late awakening may be a sign of both delay phase shift and long duration of MLT excretion. Since the recent showed the close link between time of MLT secretion and sleep onset observations,97-99 the using of the onset of MLT secretion as a marker of circadian phase position57,59,100 may lead to underestimation of circadian phase differences between winter depressives and healthy subjects.4,89 It is not excluded that the more robust phase difference between patients and controls exist on the offset of MLT secretion. Since several reports37,101-103 suggest that the MLT secretion in patients with SAD is more sensitive to light than in normal subjects, the increased sensitivity to light rather than the initial phase delay may explain the phase shifts in SAD patients. However, Partonen et al (1997)104 Did not find the difference between patients with SAD and controls in the suppression of MLT by evening bright light. Thompson et al (1997)55 suggested that there may be instability of circadian rhythms in SAD mediated by a high-amplitude PRC, rather than a fixed phase abnormality.55 Another explanation suggest that bright light does not shift the phase of circadian pacemaker, but shift only phase of the overt rhythms by simple strengthening of pacemaker’s effluence on them.105 The relationship between suppression of MLT and amelioration of SAD symptoms was reported by Kjellman et al (1993).106 Patients were exposed to bright light at 2200-2300 and lowered or unchanged MLT levels at 2300 compared to the levels at 2200 were considered as a normal reaction. Such a reaction was found in 22 of 25 responders to morning BLT and in only 3 of 8 nonresponders. We suggested that even when BLT does not cause the phase shift (this effect could be a direct and noncircadian on its nature), MLT suppression could still enhance the therapeutic action of light.26 Taking together the findings on the effects of BLT on circadian phase position in winter depression provides little evidence for the phase shifting nature of SAD and BLT, although phase shifts may play a certain role in the manifestation of several depressive symptoms in winter and their reduction following BLT.

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Multi-Component Physiological Response to Light Thus, after more than 16 year of SAD research, there is no simple chronophysiological model of mechanism of SAD and BLT that would be considered as fully supported by the collected facts. Most hypotheses of SAD pathology are not mutually exclusive. Even being a relatively homogeneous group of patients in symptomatology, winter depressives are not necessarily homogeneous in the etiology of their illness.9 Many facts let us suggest that not one but several physiological mechanisms are involved in regulating mood of SAD patients. Although in general, the epidemiological studies support the idea of rather close link between seasonal changes in photoperiod and symptoms of SAD, the significant differences between annual phases of main depressive symptoms were also has been noted.4 The seasonal responses of MLT phase and duration of MLT secretion may play a certain role in manifestation of several specific neurovegetative symptoms of SAD. However, the annual photoperiodic cycle seem not to be the only important determinant for seasonal variations in psychic symptoms. The findings of the BLT studies suggesting the multi-component nature of the physiological response to bright light.87 The changes of photoperiod and temperature may independently trigger the changes in several systems of physiological regulation and MLT rhythm may be more or less directly involved in the mechanisms underlying the effects of BLT on different groups of symptoms. This could explain the contradicting results of investigations on SAD Chronophysiology. Most experiments were designed to test only one physiological abnormality, while the therapeutic action of bright light could be mediated not only this, but also several other physiological effects (such as change of duration of MLT secretion, shift of circadian phase, increase of nonREMS pressure, enhancement of metabolic rate and sympatho-adrenal activity, etc.). Each of these effects could not be necessarily observed in the vast majority of patients.

Conclusion In conclusion, we are still far from understanding of the mechanisms underlying the seasonal variations in affective disorders. A question about the role of photoperiodic time measurement and circadian phase shifts in the pathophysiology of SAD and antidepressant effect of BLT remains to be open. Several recent investigations of MLT profile provide some indirect evidence for involvement of the SCN in pathogenesis of SAD and response to BLT. It is likely that in subsets of patients with SAD the changes in an individual profile of MLT secretion could be linked with the beneficial effects of BLT and spontaneous remission in spring and summer. These changes may include either shortening of duration of MLT secretion or phase-advance of MLT rhythm or both. Nevertheless, if the SCN and/or MLT rhythmicity are involved in the physiological response to BLT, this does not necessary imply that they are the cause of the improvement in symptoms and therefore the chronobiological hypotheses of SAD etiology must be regarded as being still controversial.

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CHAPTER 25

Delayed Sleep Phase Syndrome: A Melatonin Onset Disorder Marcel G. Smits and S.R. Pandi-Perumal

Summary

D

elayed sleep phase syndrome (DSPS) is a common but little reported cause of severe insomnia. Characteristic symptoms of this poorly defined circadian rhythm disorder are sleep onset insomnia and trouble to awake at conventional hours. Generally DSPS patients feel more alert at night than in the morning. Many suffer from hypersomnia and fatigue during the day. The prevalence of depression and personality disorders in DSPS is high. Delayed melatonin onset plays a key role in the pathophysiology of this circadian rhythm disorder. The possibility to assess melatonin onset relatively easy in saliva resulted in the finding that melatonin onset is delayed in several patients with poorly understood disorders such as chronic fatigue syndrome, chronic whiplash syndrome and children with idiopathic chronic sleep onset insomnia. Exogenous melatonin is probably the best treatment for DSPS. The time of administration of this chronobiotic drug determines probably the success rate of this treatment considerably. Five hours before endogenous melatonin onset seems to advance sleep-wake rhythm most. Other possibilities to treat DSPS are bright light (> 3000 lux), and chronotherapy. Patients with DSPS need clear, solid timecues to entrain their circadian rhythm at the desired 24-hour rhythm.

Introduction

The delayed sleep phase syndrome (DSPS) was first described by Weitzman et al in 1981.1 They found that about 7% of a large population (450 patients), seen for a primary insomniac complaint, suffered from a circadian sleep wake rhythm disorder. In the following years the clinical picture of DSPS became known more detailed. New diagnostic tools made it possible to assess DSPS more accurately. Furthermore, several successful treatments became available. Delayed melatonin onset offered to play a key-role in the pathophysiology of DSPS. Consequently clinicians discovered other, hitherto not well understood disorders, such as chronic whiplash syndrome, chronic fatigue and idiopathic insomnia in children, characterised by a delayed melatonin onset. This chapter summarises the current knowledge of DSPS, particularly its clinical significance.

Clinical Aspects of DSPS The characteristic combination of symptoms, described by the discoverers of DSPS included: (1) chronic inability to fall asleep at a desired clock time; (2) when not on a strict schedule, the patients have a normal sleep pattern and after a sleep of normal length awaken spontaneously and feel refreshed; and (3) a long history of unsuccessful attempts to treat the 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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problem. Neither gender-related nor psychiatric disorder dependencies were noted. The pioneer publication led to the recognition of the existence of circadian rhythm sleep disorders (CRSD) other than DSPS.2 To the criteria of Weizman et al Joseph-Vanderpool et al3 added1 disrupted work or social functioning due to sleep patterns,2 Repeated unsuccessful attempts to sleep and wake at earlier times, and3 poor morning alertness. In 1990 the Diagnostic Classification Steering Committee of the American Sleep disorders Association listed as minimal criteria for the diagnosis of CRSD delayed sleep for at least a month or excessive sleepiness with sleep log evidence of delayed sleep. When the patient is freed of a conflicting schedule, the delayed sleep is of normal length and quality. In addition, 24-hour polysomnography or temperature monitoring should be consistent with this history, and the patient should have no other insomnia disorder.4 Later several studies have been reported to strengthen the definition of DSPS. Reviewing all articles on DSPS with primary data on DSPS published through 1993 Regestein et al5 concluded that DSPS involves undesirably late bedtimes and arising times, early night insomnia, and poor morning alertness but lack of insomnia on vacations. In his own group of 33 DSPS patients mean bedtime and arising time were 4:00 a.m. and 10:38 a.m. respectively. In 1999 Dagan6 presented as far as now the largest survey of DSPS patients, He described 322 patients suffering from circadian rhythm sleep disorders (CRSD). The diagnosis of CRSD was made according to the criteria of the ICSD4 in addition to meeting more severe criteria, namely, that the individual was unable to go to sleep before 2:00 a.m. and extreme difficulty waking up before 10:00 a.m. for at least a year prior to assessment. A total of 83.5% were of the DSPS type; 89.6% reported that the onset of the CRSD occurred in early childhood or adolescence; CRSD exhibited no gender differences. 47% reported subjective sensitivity to light, while only 19.6% of the control group did so (p<0.02). Night eating was reported by 52% in comparison to only 10.7% of the controls (p<0.00005). 28% complained of suffering from gastrointestinal tract problems, such as irritable bowel syndrome and inflammatory bowel diseases, as opposed to only 7.1% of the controls (p<0.005). Of the CRSD patients, 83% were unmarried, as opposed to 44.4% of the controls (p<0.00005). There are some indications that DSPS can cause headache and migraine variants.7 The high sensitivity to light suggests that light supersensitivity could be involved in the pathophysiology of DSPS. The use of dark sunglasses to avoid bright light could have an impact on the synchronisation of the circadian time structure and pacemaker of these types of patients The underlying problem in CRSD is that the patient cannot sleep when sleep is desired, needed, or expected. Sleep episodes occur at inappropriate times, and as a result, wake periods occur at undesired times. Therefore, the patient complains of insomnia or excessive sleepiness, when forced to comply with a normal societal sleep-wake routine.6 Late-night eating is not surprising when one sleeps during the day. Individuals with DSPS may suffer not only from the a deviation of the sleep-wake cycle, but also from disturbances in other circadian rhythms, such as body temperature, melatonin, growth hormone and cortisol secretion and shift in the times of feeling hungry, which leads to their having “breakfast” at noon, “lunch” in the evening and “dinner” in the middle of the night.6

Epidemiology

DSPS is the most frequently occurring CRSD. Dagan6 found that 83.5% of 322 CRSD patients were of the DSPS type. Weitzman et al1 reported that DSPS patients are younger than the general insomniac population. The prevalence of DSPS in adolescence is more than 7%.8,9 In middle aged adults a prevalence of 0.7% is found.10 The reason for this decrease of the prevalence with age is unknown.

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Comorbidity

Weitzman et al1 reported that DSPS patients as a group did not have a specific psychiatric disorder. Regestein et al5 however reported that in their group of 33 DSPS patients 25 patients were, or had been depressed. In the CRSD patients described by Dagan6 (83.5% suffered from DSPS), learning disorders (attention deficit disorder, attention deficit hyperactivity disorder or dyslexia) occurred in 19.3% and personality disorders in 22.4%. According to Dagan these disturbances are related to or are an outcome of CRSD. A young child who fails to attain sufficient sleep at night is unlikely to be alert during the school day and is likely to have difficulty keeping up with other children. Frequently, parents, teachers, doctors, psychologists, and even the patients themselves, believe that the biological sleep-wake problem and accompanying dysfunction at school or work stems from motivational or psychological problems, a belief tat the patients tend to adopt themselves over the years. Subjection of the DSPS persons to such accusations and attitudes by loved ones, teachers, and supervisors from early childhood or adolescence onward adds further psychological distress to the practical difficulties of coping with life. Indeed, this may contribute to the high prevalence of personality disorders among DSPS patients and to the difficulties they experience in marrying. The high prevalence of personality disorders in CRSD patients has also been found by Dagan.11 It is well known that sleep disorders are a common symptom and complication of numerous psychopathologies, such as depression, anxiety, post-traumatic stress disorder (PTSD), and so on. However no existing psychopathology is characterised by a sleep disorder of the circadian type. According to Regestein,5 DSPS has been linked occasionally with psychopathology. Treatment response was poor in DSPS patients who manifested significant psychopathology12,13 found in 14 of 22 dsps patinets psychometric signs of depression. Since some patients with DSPS are psychometrically normal (regestein 6) dsps does not necessarely cause depression.

Onset of DSPS

The ICSD,4 states that for DSPS , adolescence is the most common age of onset. However, Dagan6 reported that the onset of CRSD occurred in early childhood in 64.3% and the beginning of the puberty in 25.3% of CRSD patients (83.5% were of the DSPS type). In 10.4% CRSD age of onset was during adulthood. When CRSD develops during adulthood Dagan suggests that a head trauma or even minor brain injury is its trigger.6 There is increasing evidence that a delayed sleep phase syndrome might develop following a whiplash injury.14-17 Thus in cases of brain concussion syndrome or in chronic whiplash syndrome, in which patients exhibit symptoms of a disturbed sleep-wake pattern, the possibility of DSPS must be considered. Some patients developed a chronic fatigue syndrome-like clinical picture, with late melatonin onset, following a viral infection.18 Consequently it is suggested that next to a trauma, also a (viral ?) encephalitis might induce DSPS.18 After travelling in the eastern direction by plane, passing a number of time zones, a temporary delayed sleep phase syndrome arises, causing jetlag. The same happens, when a daytime shift is assumed after several nightshifts, causing shift maladaptation syndrome. It is reasonable to suppose that frequently occurring jet lag and or frequently occurring shift maladaptation syndrome are risk factors for developing DSPS.

Familial Traits

According to the ICDS4 no familial pattern is known for DSPS. However, in the CRSD patients described by Dagan6 (83.5% suffered from DSPS), a familial trait existed in 44% of patients. After the discovery of clock genes in animals, clock-genes are studied in humans with CRSD. Toh et al found a mutation of the Per2 clock gen in familial advanced sleep phase

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syndrome.19 Ebisawa et al20 reported a mutation of the Per 3 gene in familial DSPS. However, another study could not confirm this finding.21

DSPS versus Owls

According to Dagan,2 DSPS patients differ from night chronotyped (“owls”) in regard to the rigidity of their maladjusted circadian timekeeping. “While owls” prefer late evening hours, they are flexible and can adjust, if required, to the demands of the environmental setting. DSPS patients seem to be unable to change their “circadian clock” by means of motivation or education

Treatment The first attempts to treat DSPS with strictly behavioural interventions took the logical step of advancing bedtimes across several days, using ranging from 5-30 minutes, every 1 or 2 days. All such approaches, in which an advance of sleep time has been attempted, hav proven largely unsuccessful (Campbell et al, Sleep Med Rev 1999; 179-200). In a similar procedure, called chronotherapy, bedtimes and rising times were systematically delayed over a period of days untill the desired bedtime was achieved. Once achieved, strict adherance to the new sleep-wake cycle was thought to be critical to maintaining a positve outcome.1,22 In practice chronotherapy is demanding and compliance is low. In the mid-1980s timed bright light exposure was demonstrated to effectively reset the human endogenous pacemaker. Therefore bright light (>2500 lux) treatment was used in the treatment of DSPS. The time of administration is considered to be very important. Phase advances are induced when light exposure is scheduled after the minimum of core body temperature. Phase delays are induced when light exposure is scheduled before the minimum of the core body tempera7ture. In practice activity during the day, movements during the night and many other factors prevent good assessment of the minimum of the core body temperature. The (salivary) melatonin rhythm is contrary to the 24-hour core body temperature. The maximum of the 24-hour melatonin curve corresponds with the minimum of the 24-h core body temperature curve. So nowadays salivary melatonin curve can be used to determine the best time of exposure to bright light . The duration of the exposure to bright light differs in studies on bright light in DSPS. Usually treatment during at least 30 minutes is recommended. Also concerning the amount of brightness (lux) needed, no consensus exists. Usually at least 2500 lux is applied. Bright light treatment can be performed with special lamps and with special glasses.23 Vitamin B12 has been reported to benefit patients with DSPS in an open treatment study.24 However this finding has not been confirmed in a placebo-controlled study. Regestein et al5 reported that in their group of 33 dsps patients 17 patients showed little treatment response with sleep hygiene improving measures, bright light, chronotherapy, Vitamin B12, benzodiazepines and trazolam. Since the discovery that exogenous administration of the pineal hormone melatonin can reset the biological clock, several investigators have used melatonin to treat DSPS patients. Dahlitz25 was the first who reported a placebo controlled study showing melatonin to be effective in DSPS patients. His findings were confirmed in 3 other placebo-controlled studies.26-28 The time of melatonin administration remains a matter of discussion. Dahlitz administered the melatonin at 22:00 hour. N several open label studies melatonin is administerd 2 hours before the desired bed time. Just as the influence of bright light at the human endogenous pacemaker depends upon the time of administration, also the time ad which melatonin is administered, determines its effects. Lewy29 found that melatonin, administered 5 hours before endogenous melatonin onset, advances circadian rhythms most. When melatonin is administered 10 hours after melatonin onset, circadian rhythms are delayed most. Therefore melatonin was administered 5 hours

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before melatonin onset in 2 placebo controlled trials in DSPS patients.26,27 Consequently we recommend tis time of administration of the melatonin.30 In most studies on melatonin doses of 3-5 mg were used. Reliable dose-finding studies have not yet been reported. Two placebo-controlled studies have shown the efficacy of melatonin on children with idiopathic chronic sleep onset insomnia, which is probably identical to childhood onset DSPS.31,32 Melatonin does not only improve sleep, but also quality of life, both in adults33 and in children.31 Probably melatonin is the best treatment for DSPS, if necessary combined with light therapy in the morning. Patients should also adhere a strict sleep-wake schedule, using strong time cues, next to this treatment.

Pathophysiology

Weitzman et al1 hypothesised in their pioneer publication that DSPS is a disorder of the circadian sleep-wake rhythm in which the “advance” portion of the phase response curve is small. There are several possible reasons for the inability of the DSPS patient to accomplish phase advances to earlier hours. First, the endogenous circadian period (i.e., internal cycle in the absence of external timing information) that regulates the sleep/wake cycle may be particularly long. The length of the endogenous circadian period normally changes over the lifespan and is longer during early life in most species.5 Longer endogenous circadian periods may underly the preference of youth for later bedtimes. Some may have endogenous cycles that are simply too long to make a large readjustment needed to conform to a 24-hour day at an appropriate timing. For example, an individual whose endogenous cycle is 25.5 hours may be able, after exposure to a 24-hour light/dark cycle, to shorten the internal cycle to 24 hours. However, the person may be unable to shorten the internal cycle any more, which would be temporarily needed to advance the sleep hours to early clock times. Another reason a patient may not be able to resist the tendency to lengthen de internal day is exposure to insufficient bits of external timing information, often termed “time cues.” The most important time cue is the length of the day/night cycle. A paucity of timing information could also occur because of too few social contacts or consistent arising times. University life , for example, may involve too few obligations to induce normal sleep hours.34 A paucity of time cues could also occur if day light becomes too dimmed if there is insufficient exposure to outdoor light, if sunglasses are excessively used or if the patient is blind.

Drug Induced Delayed Sleep Phase Syndrome Two reports describe a strong association between Haldol and the development of DSPS. Wirz-Justice et al describe a patient with chronic schizophrenia, treated with haloperidol, and showing signs of CRSD. Changing medication to clozapine improved the sleep-wake pattern evidently. The antidepressant Fluvoxamine also has been described to provoke DSPS.2

Diagnosis Sleep-wake rhythm disorders can be assessed by sleep diaries. Several techniques can help to objectivate the subjective reports.

Wrist Actigraphy Actigraphic monitoring measures movements in periods of usually 30 seconds. The motion-sensing device at the size of a matchbox, attached to the nondominant wrist, is worn 24 h. a day for 7 consecutive days. Dagan recommends the activity monitoring as far as

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possible, under “free-running-like conditions,” that is, when patients are on vacation from work or school and while relieved of all constraints on their sleep-wake cycle.2 Actigraphic data can be analysed by means of a computerised algorithm for sleep onset and offset, among other endpoints derived by such monitoring.35

Polysomnography Polysomnography, preferably by ambulatory techniques, assesses sleep onset, offset and sleep architecture. Sleep architecture, polygraphically recorded at the patient’s usual late bedtimes has reported as normal), deeper or of heterogeneous patterns.5 Endogenous melatonin assesses the timing of the biological clock (see further). Especially melatonin onset, which can be measured relatively easy in saliva helps to diagnose circadian rhythm disorders and consequently sleep-wake rhythm disorders.

Biological Clock The biological clocks of almost all human beings have a natural (“free running”) endogenous circadian cycle that is longer than 24 hours.5 This cycle induces progressively later bedtimes in the absence of temporal cues and constraints. Everyday the cycle must be reset or “entrained” to a shorter period in order to keep the circadian rhythm with its sleep and wake phases in time with the 24-hour period of earth and society. DSPS patients find this correctional shift of the sleep phase back to earlier hours for work-days particularly difficult. Therefore, they find their daily sleep schedules gradually shifting toward progressively later times in the 24-hour day. Weitzman used the analogy of living on a one-way street and being unable to back up oven one or two houses. 5 This analogy suggested the “delaying” treatment “Chronotherapy”, explained earlier. The endogenous melatonin rhythm plays a key-role in the synchronisation of the biological clock. The process of production and release of the pineal hormone melatonin (summarized in Fig. 1) is controlled by the endogenous biological clock, which is located in the suprachiasmatic nucleus (SCN) of the hypothalamus.36 Suprachiasmatic projections regulate the pineal gland (PG) and run dorsal to the paraventricular nucleus of the hypothalamus and innervate paraventricular cells37,38 that project through the medial forebrain bundle to intermediolateral cell column of the spinal cord.39 These nerve projections stimulate preganglionic cells that innervate the superior cervical ganglia (SCG). These ganglia are of primary importance to the sympathetic innervation of the pineal gland40 and mediate all known biochemical and physiological functions of the pineal gland. Postganglionic noradrenergic cells in the SCG project to the pineal gland via the inferior carotid nerve and the coronary nerve.42 A late Dim Light Melatonin Onset can be due to defects of clock genes,41,42 enzymes involved in the melatonin synthesis,26 or due to dysfunctioning neural connections between the retina and pineal gland.14 Dysfunction could be induced by damage of these connections, caused by trauma in brain or neck or by encephalitis. During the day usually there is hardly any melatonin production by the pineal gland. Between 19:30 and 21:30 endogenous melatonin production starts to rise. A peak is reached at about 2:00. The basal value is reached in the morning. Melatonin, administered 5 hours before endogenous melatonin onset, or bright light administered during the last part of the melatonin curve, when melatonin secretion is decreasing, advances the endogenous melatonin rhythm. Melatonin, administered 10 hours after melatonin onset, or bright light, administered during the first half of the melatonin curve, when melatonin secretion is increasing, delays the melatonin curve (Fig. 2). As sleep-wake rhythm (as well as 24-hour temperature rhythm and cortisol rhythm) is linked to the melatonin rhythm, appropriate administration of melatonin and bright light can be used to shift the sleep-wake rhythm.

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Figure 1. Physiology of melatonin secretion. The 24 hour rhitmicity of melatonin is controlled by the Supra Chiasmatic Nucleus. Postganglionic retinal nerve fibers transport information about light-dark to the Supra Chiasmatic Nucleus, and through preganglionic fibres to the Superior Cervical Ganglion. Postganglionic fibres transport this information further to the Pineal Gland. The pineal gland produces melatonin. Light inhibits the secretion of melatonin. Reprinted with permission from reference 18.

Figure 2. Schematic representation of the endogenous melatonin curve. Melatonin, administered 5 hours before melatonin onset or bright light, administered during the second part of the curve, when melatonin secretion is decreasing, advances the melatonin curve.

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Practice Points When patients complain of sleep difficulties the doctor should ask some additional clinical questions about their sleep-wake habits. If DSPS is suspected, Dagan2 suggests asking the following questions: 1. Hunger times: The patients should be questioned about his/her preferable eating hours whether she/he eats or is hungry during the night, and whether she/he eats early in the morning. 2. Hours of alertness: DSPS patients, even when they wake up early and should thus become more and more tired as the day passes, will paradoxically become more alert as evening approaches. 3. Heredity: patients should be asked about close family members with the same characteristics. 4. Functional difficulties: DSPS patients ofthe have trouble functioning in everyday life. The hallmark of their problem is a severe difficulty to wake up in the morning. 5. Rigidity of the biological rhythm: DSPS patients have very rigid biological clocks. Therefore, it is extremely difficult for them to adjust to environmental demands, eve for a very limited time. They should be asked about their sleep-wake habits during vacation time 6. Head injury: patients displaying symptoms of DSPS should be inquired about prior head and neck injuries (even minor). 7. Drug side-effect: It is advised for psychiatrists treating patients with psychotropic drugs to take into consideration DSPS as a possible side effect of this group of drugs.

Conclusion DSPS is a circadian rhythm disorder, characterised by delayed circadian rhythms including sleep-wake rhythm, 24-hours temperature rhythm and cortisol rhythm. The clinical picture is poorly defined. DSPS was initially described in adults, but the disorder often begins in childhood and is relatively common among adolescents. DSPS is unfamiliar to many doctors. Therefor many patients had for years been wrongly diagnosed by neurologists, paediatricians, and psychiatrists as psychophysiological insomniacs and therefore were unsuccessfully treated with sleeping pills. The combination of the early onset of CRSD, ease of diagnosis, high frequency of misdiagnosis and erroneous treatment, potentially harmful psychological and adjustment consequences, and availability of promising treatments all indicate the importance of greater awareness of this disorder on the part of paediatricians, family doctors, psychiatrists, neurologists, as well as psychologists and teachers. Several clinical signs and symptoms strongly suggest DSPS. Assessment of (salivary) endogenous melatonin onset may strongly help the diagnosis and the treatment of DSPS. The diagnosis of clock-gene defects might be a promising diagnostic tool in the near future. Treatment with melatonin seems to be the best therapeutic strategy, often combined with sleep hygiene improving measures and bright light. Appropriate timing of melatonin and bright light is essential for a good therapeutic effect. Studies to determine the optimal dose of melatonin treatment as was as optimal duration and intensity of bright light treatment are urgently needed, as well as studies to establish long term effects of melatonin treatment

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6. Dagan Y, Eisenstein M. Circadian rhythm sleep disorders: Toward a more precise definition and diagnosis. Chronobiol Int 1999; 16(2):213-22. 7. Nagtegaal JE, Smits MG, Swart AC et al. Melatonin-responsive headache in delayed sleep phase syndrome: Preliminary observations. Headache 1998; 38(4):303-7. 8. Regestein QR, Pavlova M. Treatment of delayed sleep phase syndrome. Gen Hosp Psychiat 1995; 17:335-45. 9. Pelayo RP, Thorpy MJ, Glovinsky P. Prevalence of delayed sleep phase syndrome among adolescents. J Sleep Res 1988; 17:392. 10. Ando K, Kripke DF, Ancoli-Israel S. Estimated prevalence of delayed and advanced sleep phase syndromes. J Sleep Res 1995;24:509. 11. Dagan Y, Sela H, Omer H et al. High prevalence of personality disorders among circadian rhythm sleep disorders (CRSD) patients. J Psychosom Res 1996; 41(4):357-63. 12. Billiard M, Touchon JCBBA. Delayed Sleep Phase Syndrome: Subjective and objective data, chronotherapy and follow up. Sleep Res 1993; 22:172. Sleep Res 1993; 22:172. 13. Thorpy MJ, Korman E, Spielman AJ et al. Delayed sleep phase syndrome in adolescents. J Adolesc Health Care 1988; 9(1):22-7. 14. Nagtegaal JE, Kerkhof GA, Smits MG et al. Traumatic brain injury-associated delayed sleep phase syndrome. Funct Neurol 1997; 12(6):345-8. 15. Smits MG, Nagtegaal JE. Post-traumatic delayed sleep phase syndrome. Neurology 2000; 55(6):902-3. 16. Wieringen Sv, Jansen T, Smits MG et al. Melatonin for chronic whiplash syndrome with delayed melatonin onset. Randomised, placebo-controlled trial. Clin Drug Invest 2001; 21(12):813-20. 17. Patten SB, Lauderdale WM. Delayed sleep phase disorder after traumatic brain injury. J Am Acad Child Adolesc Psychiatry 1992; 31(1):100-2. 18. Smits MG, Rooy Rv, Nagtegaal JE. Influence of melatonin on quality of life in patients with chronic fatigue and late melatonin onset. JCFS 2002; 10(3/4):25-36. 19. Toh KL, Jones CR, He Y et al. An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome. Science 2001; 291(5506):1040-3. 20. Ebisawa T, Uchiyama M, Kajimura N et al. Association of structural polymorphisms in the human period3 gene with delayed sleep phase syndrome. EMBO Rep 2001; 2(4):342-6. 21. Robilliard DL, Archer SN, Arendt J et al. The 3111 Clock gene polymorphism is not associated with sleep and circadian rhythmicity in phenotypically characterized human subjects. J Sleep Res 2002; 11(4):305-12. 22. Czeisler CA, Richardson GS, Coleman RM et al. Chronotherapy: Resetting the circadian clocks of patients with delayed sleep phase insomnia. Sleep 1981; 4(1):1-21. 23. Cole RJ, Smith JS, Alcala YC et al. Bright-light mask treatment of delayed sleep phase syndrome. J Biol Rhythms 2002; 17(1):89-101. 24. Okawa M, Mishima K, Nanami T et al. Vitamin B12 treatment for sleep-wake rhythm disorders. Sleep 1990; 13(1):15-23. 25. Dahlitz M, Alvarez B, Vignau J et al. Delayed sleep phase syndrome response to melatonin. Lancet 1991; 337(8750):1121-4. 26. Nagtegaal JE, Kerkhof GA, Smits MG et al. Delayed sleep phase syndrome: A placebo-controlled cross-over study on the effects of melatonin administered five hours before the individual dim light melatonin onset. J Sleep Res 1998; 7(2):135-43. 27. Smits MG, Laurant M, Nagtegaal JE et al. Influence of melatonin on vigilance and cognitive functions in delayed sleep phase syndrome. Chronobiol Int 1997; 14(Suppl 1):159. 28. Kayumov L, Brown G, Jindal R et al. A randomized, double-blind, placebo-controlled crossover study of the effect of exogenous melatonin on delayed sleep phase syndrome. Psychosom Med 2001; 63(1):40-8. 29. Lewy AJ, Ahmed S, Jackson JM et al. Melatonin shifts human circadian rhythms according to a phase-response curve. Chronobiol Int 1992; 9(5):380-92. 30. Smits MG, Nagtegaal JE. Melatonin for cluster headache. Cephalalgia 2002; 22(8):695. 31. Smits MG, Stel H van, Heijden K van et al. Melatonin improves health status and sleep in children with idiopathic sleep onset insomnia. A randomized placebo-controlled study. 2003; In press. 32. Smits MG, Nagtegaal JE, van der Heijden J et al. Melatonin for chronic sleep onset insomnia in children: A randomized placebo-controlled trial. J Child Neurol 2001; 16(2):86-92. 33. Nagtegaal JE, Laurant M, Kerkhof GA et al. Melatonin improves quality of life in patients with delayed sleep phase syndrome. J Psychosom Res 2000; 48:45-50. 34. Wirz-Justice A, Pringle C. The nonentrained life of a young gentleman at Oxford. Sleep 1987; 10(1):57-61.

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35. Sadeh A. Evaluating night wakings in sleep-disturbed infants: A methodological study of parental reports and actigraphy. Sleep 1996; 19(10):757-62. 36. Arendt J. Biochemistry of the pineal gland. In: Arendt J, ed. Melatonin and the mammalian pineal gland. Cambridge: University Press, 1995:27-63. 37. Klein DC, Smoot R, Weller JL et al. Lesions of the paraventricular nucleus area of the hypothalamus disrupt the suprachiasmatic leads to spinal cord circuit in the melatonin rhythm generating system. Brain Res Bull 1983; 10(5):647-52. 38. Hastings MH, Herbert J. Neurotoxic lesions of the paraventriculo-spinal projection block the nocturnal rise in pineal melatonin synthesis in the Syrian hamster. Neurosci Lett 1986; 69(1):1-6. 39. Swanson LW, Kuypers HG. A direct projection from the ventromedial nucleus and retrochiasmatic area of the hypothalamus to the medulla and spinal cord of the rat. Neurosci Lett 1980; 17(3):307-12. 40. Ariens-Kappers J. The development, topographical relations and innervation of the epiphysis cerebri in the albino rat. Z Zellforsch 1960; 52:163-215. 41. Horst van der TJ, Muijtjens M, Kobayashi K et al. Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature 1999; 398:627-30. 42. Kay A. PAS, present and future:clues to the origins of circadian clocks. Science 1997; 276:753-4.

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CHAPTER 26

Melatonin as an Antidepressant for Treatment of Delayed Sleep Phase Syndrome with Comorbid Depression Leonid Kayumov, Alan Lowe, Raed Hawa and Colin M. Shapiro

A

high prevalence of depressive symptoms in delayed sleep phase syndrome (DSPS) patients is commonly reported.23,41,42,57 Regestein and Monk41 reported that 75% of their patients with DSPS had previous or present severe depression, compared with 16% of nonDSPS chronic insomnia patients and 2% of sleep apnea patients. Weitzman et al63 and Alvarez et al1 found psychopathologic abnormalities in half of adult DSPS patients. Ferber and Boyle23 reported a high prevalence of depression in adolescents with DSPS. Dagan et al17,18 found increased prevalence of personality disorders in DSPS patients and significantly higher scores on the dependent, passive-aggressive, borderline, sadistic, and anti-social subscales of the Personality Disorders Questionnaire as compared with normal sleepers. Some patients with DSPS do not have any particular psychopathology, i.e.,DSPS is not necessarily associated with depression. Moreover Schrader et al48 have explained the reported high prevalence of depression in DSPS as consequence of referral or self-referral bias. DSPS differs from the sleep disturbance associated with most psychiatric disorders where it is common to find that once the psychiatric condition is treated, the sleep problem diminishes. In DSPS the sleep disruption persists despite treatment of depression. Whether DSPS leads to clinical depression directly or vice versa is unknown, however there appears to be a strong relationship between these two clinical entities. Previously psychiatric researchers have hypothesized a phase-advanced change in circadian rhythm disruption leading to the development of depression. In the phase advance hypothesis of affective disorders29,37-39,51 circadian rhythms are thought to be abnormally advanced with respect to sleep-wake cycle. According to this theory sleep in depressed patients resembles sleep in normal subjects whose circadian rhythms of core body temperature and some biochemical parameters are shifted earlier relative to their sleep schedules. Since rapid eye movement (REM) sleep is predominately under control of a circadian process C,6,7 this apparent advance in circadian rhythms’ phases provides a plausible explanation for the short REM sleep latency and temporal redistribution of REM sleep that had been frequently observed in depression.13,31,35,43 Advancing sleep in some of depressed patients has been shown to have an antidepressant effect.62 Sleep deprivation in the second (but not first) half of the night was somewhat antidepressive and transiently helpful in some cases.5,44,47 However, phase advance of circadian rhythms has not been consistently observed in all chronobiological studies of depression.24,28,59,60 The most consistent finding is that the nadir of the circadian rhythm in plasma level of cortisol is abnormally phase advanced in endogenous depression.9,26,49

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Figure 1. CES-D and HDRS-17 scores during a nine-week randomized, double-blind, placebo-controlled crossover study in DSPS patients with comorbid depression.

Internal coincidence theory of depression61 implies an internal phase angle disturbance between sleep and other circadian rhythms. According to this theory sleep induces or exacerbates depression when sleep coincides with a circadian phase that is sensitive to these effects of sleep. This postulate can be considered separately from the question whether or not circadian rhythms are phase advanced in depression. Indeed, in DSPS patients with comorbid features of depression, an internal phase angle disturbance may exist because sleep is not as delayed as the other circadian rhythms or conversely the circadian rhythms are not as delayed as sleep-wake cycle. Although early morning awakening is one of the characteristic biologic markers of depression, the diurnal mood variation with better mood in the evening might induce delayed bedtimes and delayed rise times. However late sleeping itself may exacerbate or precipitate depression25,55,62 probably due to sleep not being as phase shifted as other circadian rhythms. Theoretically, manipulations correcting the phase angle disturbance between sleep-wake cycle and circadian rhythms should result in treatment of both DSPS and comorbid depression. Bright light has been used successfully to reset the circadian pacemaker and manipulate the phase angle between the core body temperature, melatonin secretion, sleep-wake cycle and the environment. However, light can produce very large phase-shifts if inappropriately administered.15,22,21 In DSPS there is a small daily phase reset required to maintain the entrainment, thus melatonin, which is a less potent zeitgeber compared to light, may be the more appropriate tool. It has been shown that exogenous melatonin affects the phase of underlying biological rhythms as well as the phase of the sleep-wake oscillator.10,20 Because melatonin can cause phase advance or delay, depending on the timing of administration according to a phase response curve,36 it may potentially compromise sleep quality if it is given at an inappropriate circadian time. In our recent randomized, double blind, placebo-controlled study we assessed the effect of exogenous melatonin in 8 patients with established diagnosis of DSPS and comorbid depression. The diagnosis of depression was based on prestudy clinical interviews and high scores on the CES-D and Hamilton Depression scales.30,40 During melatonin treatment the depression scale scores decreased but they increased again when the subjects were placed back onto placebo (Fig. 1). The patients with depressive features treated with melatonin had significantly more TST than when they were on placebo. REM sleep latencies on the baseline, melatonin and placebo limbs of the trial were within the normal range. Interestingly, the patients showed normal distribution of REM sleep on melatonin treatment, i.e., increase of REM sleep duration from the 1st sleep cycle towards the end of the night. On the baseline and placebo

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conditions the duration of REM sleep did not differ statistically in sleep cycles across the night. Another important parameter of REM sleep—REM density—at the baseline condition and on placebo demonstrated a flattened distribution, being in the 4th and 5th sleep cycles not higher than in the 1st cycle. It is opposite to the normal distribution of REM sleep variables.2 The overall REM density in all sleep cycles demonstrated a tendency to be higher on melatonin treatment than on placebo or on baseline limb of the trial. Numerous studies have linked disruptions in the sleep–wake cycle and fundamental circadian dysfunctions to affective illnesses.25,34,37-39,62,61 However the heterogeneity of depression and the masking of endogenous circadian rhythms by sleep-wake patterns, rest-activity cycles, light, ambient temperature and other factors have produced somewhat conflicting results.24,28 It seems simplistic to associate pathophysiological mechanisms of depression with only a phase advance of main circadian rhythms. Moreover Healy and Waterhouse32 and Wirz-Justice66 concluded that there is no primary disruption of the circadian system in depression, but that circadian function may be disturbed as a part of the multiple manifestations of the primary pathophysiology of these disorders. Different types of depression may have different underlying chronobiologic pathology and the so-called internal circadian phase angle disturbance may be due to phase variability,58 phase advance or phase delay of major circadian rhythms. It has been reported that core body temperature and cortisol rhythms during a constant routine are phase-delayed relative to sleep in patients with seasonal affective disorder (SAD).3 The onset of melatonin secretion is also phase-delayed in SAD patients maintained in dim light conditions compared with controls.19,46 It has been widely accepted that the profile of endogenous melatonin secretion is a good measure of the circadian clock.16,36,45 Melatonin also has a potential role as a marker for depression. Numerous studies showed reduced levels of plasma or serum melatonin in depressed patients.4,8,53,64 Other investigations however failed to demonstrate any difference in melatonin levels between depressed and control subjects.54,56 Stewart and Halbreich54 even showed an increase in daytime levels of melatonin in depressed patients. The discrepancies between the reported results can be explained by environmental factors and differences in age and type of depression. On the other hand many investigators showed that antidepressants increase both plasma melatonin levels and 24-hour urinary output of aMT6s in depressed patients.27,33,52 A clue as to different etiologies of DSPS (genotypic orphenotypic) with or without comorbid depression may be inferred from alterations in the circadian profile of endogenous melatonin secretion. Altered rhythm of melatonin secretion in DSPS was recently reported by Shibui et al50 who suggested that not only the delay of the circadian clock but also a functional disturbance of the sleep-wake mechanism underlies DSPS. Our findings suggest a relationship between the magnitude of DSPS symptoms, severity of comorbid depressive features and abnormalities in the circadian pattern of circulating melatonin as judged by the excretion of its major metabolite – aMT6s. The patients with marked comorbid depression and extreme symptoms of DSPS showed an abnormal circadian pattern of melatonin secretion on placebo treatment with the peak during the period between 8:00 h and 15:00 h. This finding suggests that sleep was not as delayed as the other circadian rhythms producing an internal phase angle disturbance. Since we did not define profiles of other major circadian rhythms (core body temperature, secretion of cortisol and thyroid stimulating hormone etc), we were able only indirectly address this issue. The DSPS patients with comorbid depression demonstrated a normalization of the circadian pattern of excreted aMT6s after oral administration of 5 mg of melatonin with the usual nocturnal rise and rapid decline of aMT6s during daytime hours. This may explain why the patients did not report any hangover effects as judged by the subjective assessment of the circadian pattern of sleepiness, fatigue and alertness. It is reasonable to speculate that phase advance in melatonin output and possibly in other circadian rhythms along with advance of sleep-wake cycle produced by exogenous melatonin, resulted in amelioration of symptoms of depression. This is contrary to a surprisingly widespread opinion that melatonin in fact causes or exacerbates depression.12,14,65 Despite frequent citation of this finding in lay and medical literature, we were able to locate only one reference

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directly reporting negative effect of melatonin on depression.11 These authors reported the increased dysphoria in a small number of patients with unipolar and bipolar depression after administration of enormous pharmacological doses of melatonin (ranging from 150 mg to 1600 mg). Zhdanova et al67,68 have shown dramatic differences in pharmacological properties and pharmacokinetics of high and low doses of the hormone. These studies also suggest that the magnitude of melatonin’s phase-shifting and sleep-inducing effects may be significantly influenced by the dosage and the time of its administration. In our study, administration of 5 mg melatonin for four weeks, three to five hours before imposed sleep period from 24:00 h to 8:00 h, significantly advanced sleep onset latency compared to placebo in DSPS patients with comorbid depression. The common polysomnographic markers for depression such as short REM sleep latency and increased duration of REM sleep were not observed in DSPS patients with depressive features. It is noteworthy that in DSPS patients with comorbid depression, the exogenous melatonin had an impact on the circadian profile scores of sleepiness, fatigue, and alertness. Patients were less sleepy, less fatigued and more alert in comparison with when they were taking placebo. Our data derived from both objective and subjective measures, can be interpreted as indicating that melatonin has antidepressant properties. It is also possible that correction of a disrupted circadian rhythm allows mood to improve. The overall findings demonstrate that when appropriately used, melatonin can be beneficial for the treatment of both DSPS and depression.

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Index A

C

Acetylcholine (ACh) 3, 4, 7, 153 Adenosine kinase (AK) 27, 28 Adolescent 35, 270, 273 Adolescent idiopathic scoliosis (AIS) 35-38, 41, 42 Advanced sleep phase syndrome (ASPS) 170-172, 180, 265 Age-related disease 118, 119, 122-124 Aging 45, 48, 52, 60, 87, 118-122, 124, 196, 197, 201, 202, 204, 205 Alpha-adrenergic signaling 193 Alzheimer’s disease 119, 122, 123, 152, 206, 225 Androgen 73, 74, 91, 138-145 Androgen receptor (AR) 6, 138-145 Antioxidant 12, 13, 15, 16, 20-22, 28-30, 52-55, 63-66, 122-124, 127-131, 152-154, 184, 186-189, 196-198, 200-202, 205, 206, 209, 210, 212, 220, 221, 224, 238, 239 Apoptosis 30, 53, 129, 184, 185, 201, 237 APUD 149, 151, 152, 155 Arvicanthis ansorgei 2

Cadmium (Cd) 222, 224 Caenorhabditis elegans 51 Calcitonin gene-related peptide (CGRP) 3, 4, 215 Calcium channel 28, 64, 88, 93, 95, 98 Calcium oscillations 95, 96, 99 Calmodulin 42, 54, 55, 64, 243, 244 Cancer initiation 220, 221, 228 Capsaicin 108 Carcinogen 48, 52, 72, 220-228 Carcinogenesis 28, 52, 55, 155, 220, 221, 226-228 Cardiovascular system 60, 65 Carrageenan-induced acute inflammation 130 Catecholamine 66, 149 Cell proliferation 54, 55, 144, 152-156, 210 Ceruloplasmin 184 Cervical cancer 76 Chicken 35, 38-42, 91, 110, 152 Cholera toxin (CTX) 140, 141 Cholesterol 60-63, 65, 66 Chromium (Cr ) 52, 224, 226 Chronic lung disease (CLD) 186 Chronobiotic 113, 217, 263 Circadian rhythm 38-41, 45, 51, 61, 71, 72, 76, 79, 80, 88, 89, 91, 92, 106-108, 111, 113, 114, 118-121, 123, 124, 148, 150, 152, 153, 162-164, 166, 167, 170-176, 178-180, 210, 215, 217, 232-234, 237, 239, 250, 253-258, 263-268, 270, 273-276 Cobalt (Co) 225, 226 Colorectal cancer 77 Corticotropin-releasing hormone (CRH) 62, 122 CREB 6, 7 Cyclic AMP 27, 144, 233, 236 Cyclooxygenase-2 (COX-2) 17, 132-134 Cytoskeleton 244, 246, 247, 251

B β-adrenoceptor blocker 62 Beta-adrenergic signaling 193 Beta-carotene 64 Biological clock 119, 266, 268, 270 Biological rhythm 88, 152, 154, 243, 270, 274 Bone alkaline phosphatase 211 Bone remodeling 209, 211 Brain 12, 13, 16-19, 23, 30, 38, 47, 53, 75, 90-92, 109-113, 123, 127, 164-167, 184, 185, 188, 193, 196-198, 200, 215, 217, 222-224, 228, 244, 265, 268 Breast cancer 54, 72-76, 80, 224 Bright light treatment (BLT) 253-258, 266, 270 Bromium 224

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D

G

7,12-dimethylbenz(a)anthracene (DMBA) 53, 55, 72, 73, 78, 79, 223, 228 δ-aminolevulinic acid 223, 227 Delayed sleep phase syndrome (DSPS) 171, 174, 263-268, 270, 273-276 Diabetes 46, 55 Diffuse neuroendocrine system (DNES) 149, 151 Diurnal species 111, 163, 164, 167 DNA 19, 27, 28, 31, 52, 53, 122, 127, 129-131, 133, 138, 139, 141, 143, 144, 184, 188, 197, 200-202, 205, 220, 223, 224, 226-228, 243 DNA damage 52, 53, 184, 200-202, 220, 227 Dose-dependency 162, 165 Drosophila melanogaster 51

γ-interferon 53, 78 GABA 3, 7, 111, 112, 165, 217, 234, 236 Gene expression 7, 9, 54, 94, 134, 138, 139, 141, 143, 145, 184, 210 Glutamate (Glu) 3, 4, 7, 198, 199, 217 Glutathione peroxidase (GPx) 13, 16, 19, 20, 52, 54, 65, 122, 130, 185, 186, 196, 198, 200, 202-204, 221, 223, 239 Glutathione reductase (GRd/GSH-Rd) 13, 19, 20, 52, 122, 130, 154, 196, 198, 221 GnRH receptor 88, 92, 93, 99, 101 Gonadotrophs 88, 91-101 Gonadotropin-releasing hormone (GnRH) 54, 88-101 Growth 26-29, 31, 35, 36, 38, 39, 41, 42, 54, 55, 71-75, 78-81, 88, 118, 128, 138, 139, 143, 144, 151, 152, 155, 156, 209, 210, 212, 264 Growth hormone (GH) 35, 42, 65, 96, 209, 212, 264

E 17β-estradiol 74, 222, 226 EC cell 150, 151, 155 Electron transport system 196, 197, 204, 205 Endometrial cancer 73, 74, 76, 80 Endothelin-1 64 Enterochromaffin cells 149, 150, 152 Estradiol 64, 65, 73, 74, 209-212, 222, 226, 227 Ethanol 45, 108, 153, 154 European hamster 2, 4, 7-9

F Ferric nitrilotriacetate (Fe-NTA) 222, 224, 225 Fetal ischemia 200 Focal adhesion contact 247, 249 Free radical 12, 14-16, 19, 21, 22, 28-30, 47, 51, 52-54, 63, 65, 73, 118, 119, 122, 127, 129-132, 134, 152, 154, 184-188, 196-198, 200-202, 204-206, 209, 210, 217, 220, 221, 225, 227, 238, 239, 243 Free radical scavenger 12, 22, 29, 52, 118, 119, 122, 152, 187, 196, 200, 201, 206, 209, 210, 220, 221, 243 Funambulus pennanti 27, 29

H 5-hydroxytryptamine 4, 193 5-hydroxytryptophan (5-HT) 4, 5, 148-151, 162, 193, 195, 255 Heart 12-16, 19, 22, 36, 54, 55, 60-62, 64-66, 153, 222, 224 Hemithyroidectomy 28 Hepatic ischemia/reperfusion 198 High-density lipoprotein (HDL) 62, 66 Hormone 1, 3, 4, 22, 26-31, 35, 36, 40, 42, 45, 46, 51, 54, 60-62, 64, 65, 71-77, 79-81, 88, 90, 92, 98-101, 106, 109, 110, 121, 122, 124, 138, 139, 141, 143, 148-155, 162-167, 209, 212, 215, 217, 232, 243-247, 249, 251, 254, 264, 266, 268, 275, 276 Hormone replacement therapy (HRT) 64, 123 Hormone secretion 90, 209 Hydrochloric acid 154 Hydrogen peroxide (H2O2) 29, 30, 52, 64, 127, 130, 131, 185, 187, 188, 196, 197, 205, 221, 222, 224-226, 238 Hydroxyindole-O-methyltransferase (HIOMT) 4-9, 31, 148, 150, 151, 162, 193, 233

281

Index Hydroxyl radical 13, 15, 22, 28, 29, 63, 64, 122, 127, 130, 133, 185, 187, 196, 221, 238 Hypercholesterolemia 60, 62 Hypertension 60, 62, 63, 65, 66 Hypnotic 107, 112, 113, 165, 167 Hypocretin (HCRT) 3, 4

I Immunocompetence 118, 119, 122 Indian palm squirrel 27 Indoloamine 64 Inflammation 127-134, 150, 186, 188, 189, 214, 217 Inflammatory bowel disease 132, 264 Insomnia 107, 113, 121, 122, 164, 167, 171, 172, 212, 254, 263, 264, 267, 273 Intercellular adhesion molecule (ICAM) 21, 132, 134 Interleukin-1 (IL-1) 53, 184 Interleukin-2 (IL-2) 75 Interleukin-4 (IL-4) 53, 128 Interleukin-6 (IL-6) 54, 143, 184, 189 Interleukin-12 (IL-12) 54 Intermediate filament 244 Ionizing radiation (IR) 28, 55, 152, 155, 222, 223, 225, 226 Iron 65, 127, 133, 184, 185, 221, 222, 227 Ischemia/reperfusion (I/R) 12-22, 60, 64, 129, 132, 154, 185, 188, 196-198, 200

J Jet lag 107, 170, 172, 180, 265

K Karyometry 28 Kennedy’s disease 138, 143, 145

L Langendorff rat heart model 13 Larynx cancer 77 Light 2-4, 7, 26, 27, 31, 39-41, 65, 72, 80, 81, 88, 90, 91, 100, 107, 108, 113, 119, 121, 129, 131, 142, 148-150, 152, 162-164, 166, 170-176, 178-180, 187, 193, 194, 202, 209, 214-217, 232, 234-240, 243, 246, 253-258, 263, 264, 266-270, 274, 275

Light emitting diode (LED) 174-180 Lipid peroxidation 15-17, 19, 21, 22, 30, 52, 63, 65, 123, 127, 132, 185, 187-189, 197, 198, 200, 203, 220-222, 224, 226-228 Low-density lipoprotein (LDL) 60, 62, 63, 65, 66 Lung carcinoma 48 Luteinizing hormone-releasing hormone (LHRH) 92 Lymphokine activated killer (LAK) 54 Lymphoma 47, 48, 228

M Macaque 162, 164, 165, 167 Macrophage 54, 78, 127, 128, 130-132, 184, 185, 197 Macrophage-colony stimulating factor (M-CSF) 54 MDCK 243-250 Melatonin 1-9, 12-22, 26-31, 35-37, 39-42, 45-55, 60-66, 71-81, 88-92, 94, 96-101, 106-114, 118-124, 127, 129-134, 138-145, 148-156, 162-168, 170-180, 187-189, 193, 195-206, 209-212, 214-217, 220-228, 232-240, 243-251, 253, 254, 256, 263-270, 274-276 Melatonin 1 (MEL1) 110, 111 Melatonin 2 (MEL2) 110, 111 Melatonin receptor 41, 48, 53, 61, 63, 64, 73, 74, 88-92, 97-101, 109-111, 133, 139, 140, 144, 153, 162, 164-167, 210, 232-237, 243 Mercury 225, 226 Microfilament 243, 244, 246-250 Microtubule 244 Middle cerebral artery occlusion (MCAO) 16-18 Migraine 214-217, 264 Mitochondria 13, 22, 52, 54, 94, 127-131, 153, 189, 196-201, 203-206, 223, 227 MT1 and MT2 receptor 89-91 Mutagenesis 52 Myocardial infarction 16, 61, 65, 66

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N 2-Nitropropane (2-NP) 223, 228 N-acetyltransferase (NAT) 5-9, 31, 51, 78, 88, 148, 150, 151, 162, 193, 233, 234 Neonatal sepsis 186 Neurodegenerative disease 119, 123 Neuropeptide Y (NPY) 3, 4, 7, 148, 149 Night shift 80, 172, 180 Nitric oxide (NO• ) 13, 17, 19, 20, 30, 52, 63, 64, 66, 127, 128, 133, 188, 196, 221, 244 Nitric oxide synthase (NOS) 13, 19, 20, 30, 52, 64, 127-130, 132-134, 196, 221, 244 Nitrotyrosine 21, 129-132, 188 Non-human primate 164 Norepinephrine (NE) 3, 4, 6, 7, 9, 31, 66, 78, 123, 148, 193, 215 Nuclear factor-κB (NF-κB) 133, 210

O Organ transplantation 21 Orphan nuclear receptors 243 Osteoclasts 209, 210, 212 Osteoporosis 202, 209, 210, 212 Osteoprotegerin 209, 210 OT 4 Ovarian cancer 74, 76 Oxidative damage 16, 19-22, 26, 30, 52, 63, 184, 186, 187, 198, 200, 202, 205, 220-227 Oxidative phosphorylation 22, 122, 185, 197, 199, 205 Oxidative stress 16, 21, 22, 26, 28-30, 52, 65, 119, 127, 184-186, 188, 189, 198, 200, 201, 204, 205, 217, 220-222, 224, 228 Oxytocin 3, 4, 148, 149

P Pancreas 12, 21, 148, 149, 153, 155 Paraventricular nucleus (PVN) 3, 4, 268 Parkinson’s disease 119, 122, 123, 152, 206 Pepsin 154 Peptide histidine isoleucine (PHI) 215 Perinatal asphyxia 185, 188 Peroxynitrite 13, 21, 64, 127-133, 188, 196, 221

Pertussis toxin (PTX) 88, 92, 97, 99, 100, 140, 141, 233 Phases 37, 55, 71, 74, 258, 268, 273 Phenylhydrazine (PHZ) 223, 227 Phosphine (PH3) 223, 228 Phospholipase C (PLC ) 6, 90, 92, 98, 99, 144, 168 Photoperiodic variation 2, 8, 148 Photoreceptor 150, 162, 173, 179, 217, 232-240 Photoreceptor cell 150, 232, 234, 235, 237 Pineal gland 3-9, 16, 17, 26, 27, 31, 35, 38-40, 45, 48, 71-73, 79, 80, 88-90, 106, 110, 118, 119, 122, 138, 148-152, 162-164, 166, 174, 193, 194, 196, 206, 211, 214, 215, 217, 223, 232, 233, 235, 243, 250, 254, 268, 269 Pinealectomy 15-17, 27, 29, 35, 38, 39, 41, 42, 45, 46, 54, 63, 72, 73, 80, 148, 150, 152, 154, 205, 211, 227 Pituitary 3, 4, 27, 62, 88-92, 96, 99-101, 110, 122, 215 Pituitary adenylate cyclase-activating peptide (PACAP) 3, 4, 7, 215 Platelet-derived endothelial cell growth factor (PD-ECGF) 27 PMN 132, 134, 184 Poly (ADP-ribose) synthetase (PARS) 127, 129, 131, 132 Polymorphonuclear leucocyte 15, 184 Polysomnography 264, 268 Post-transcriptional mechanism 193 Postmenopausal women 63-65, 123 Premature ventricular contractions (PVC) 14, 15 Prostaglandin 28, 66, 132-134, 151, 154, 185, 217 Prostaglandin E 154 Prostate cancer 76, 77, 79, 138, 139, 143-145 Protein kinase A (PKA) 6, 7, 88, 90, 97, 98, 101, 143, 144, 193 Protein kinase C (PKC) 6, 54, 74, 94, 96, 98, 138, 143, 144, 243, 244, 248-251

283

Index

R

T

Rapid eye movement (REM) 164, 212, 258, 273-276 Reactive C protein 62 Reactive oxygen species (ROS) 28-30, 52, 53, 127, 130-132, 184, 186, 188, 205, 220, 221 REM sleep 212, 273-276 Reproduction 1, 80, 88, 90, 91, 99, 100 Respiratory distress syndrome (RDS) 185, 186, 189 Retina 39, 91, 92, 149, 150, 163, 176, 179, 180, 193, 232-240, 268 Retinoid family 243

Temazepam 108 Thermoregulation 106-109, 111, 113 Thymidine kinase (TK) 27 Thyroid 26-31, 46, 77, 138, 148, 149, 151, 153, 275 Thyroid cancer 28, 77 Thyroid gland 26-31, 148, 149 Thyroid hormone 26, 29-31, 46, 138 Thyroxine (T4) 26, 28-31 Transferrin 184 Transforming growth factor-β 54 Trigeminovascular system 214, 217 Tryptophan (TRP) 5, 9, 41, 45, 71, 78, 129, 148, 150, 152, 153, 162, 193, 233, 255 Tryptophan hydroxylase (TPH/TPOH) 5, 7, 162, 193-195, 233 Tumor necrosis factor-α (TNF-α) 21, 54, 132, 186, 189

S Safrole 52, 223, 227 Scoliosis 35-42 Seasonal affective disorder (SAD) 152, 153, 170, 173, 253-258, 275 Seasonal photoperiod 88 Senescence-accelerated mouse (SAM) 48, 50, 201-203 Serotonin 3-6, 42, 88, 99, 106, 148-155, 162, 193, 237 Serotonin N-acetyltransferase 88, 193 Shift work 121, 170, 172 Shock 64, 129 Siberian hamster 2, 7-9, 90 Sleep 62, 71, 75, 79, 106-109, 112-114, 118-124, 152, 162-168, 170-173, 175, 178, 180, 209, 212, 215, 254-257, 263-268, 270, 273-276 Sleep disorder 62, 107, 113, 120, 121, 124, 163, 167, 170, 171, 175, 178, 264, 265 Somatostatin (SOM) 4, 42, 148, 149, 151 Stomach cancer 77 Stroke 12, 13, 16, 17, 23 Substance P (SP) 3, 4, 148, 149, 151, 215 Superior cervical ganglia (SCG) 3, 4, 31, 215, 268 Superoxide dismutase (SOD) 20, 51, 52, 54, 129, 130, 154, 184, 185-187, 196, 198, 200, 221, 223, 228, 238 Suprachiasmatic nucleus (SCN) 3, 4, 60, 61, 71, 79, 80, 88-92, 110, 111, 113, 119, 121, 148, 163, 214, 215, 217, 243, 254, 258, 268 Syrian hamster 2, 7, 8, 27-29, 54, 222, 226

U Urinary bladder 12, 21 Use of melatonin 16, 75, 124, 209, 212, 228

V Vasoactive intestinal peptide (VIP) 3, 4, 7, 215 Vasopressin (VP) 3, 4, 7, 148, 149 Ventricular fibrillation (VF) 14, 15 Vitamin B12 266 Vitamin C 15, 64, 187, 221 Vitamin E 30, 63, 64, 184, 187, 188, 198, 221, 224, 238

W Water transport 243, 244, 246-248, 250, 251 Wavelength 170, 173-176, 178-180, 216 Winter depression 170, 173, 253-257 Wrist actigraphy 267

Z Zebrafish 162, 164-166, 234 Zymosan 131, 132

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