Melatonin Biological Basis of Its Function in Health and Disease 2006 PDF

MEDICAL INTELLIGENCE UNIT
PANDI-PERUMAL • CARDINALI
S.R. Pandi-Perumal and Daniel P. Cardinali
Melatonin:
Biological Basis of Its Function
in Health and Disease
MIU
Biological Basis of Its Function in Health and Disease
Melatonin:
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 EUREKAH.COM

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U.S.A. U.S.A.
MELATONIN:
BIOLOGICAL BASIS OF ITS FUNCTION
IN HEALTH AND DISEASE
Medical Intelligence Unit

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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--physiopathol-
ogy. 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: pandi@downstate.edu
Chapters 24, 25
Daniel P. Cardinali, M.D., Ph.D.

Departamento de Fisiolgia
Facultad de Medicina
Universidad de Buenos Aires
Email: cardinal@mail.retina.ar
Chapter 19
CONTRIBUTORS
Aurelio Amodio Keith M. Bagnall
Institute of Medical Pediatrics University of Alberta
Neonatal Intensive Care Unit Edmonton, Alberta, Canada
University of Messina Email: kbagnall@med.ualberta.ca
Messina, Italy Chapter 4
Chapter 16
Ales Balik
Vladimir N. Anisimov Institute of Physiology
Department of Carcinogenesis Academy of Sciences of the Czech
and Oncogerontology Republic
N.N. Petrov Research Institute Prague, Czech Republic
of Oncology Email: abalik@biomed.cas.cz
St. Petersburg, Russia Chapter 8
Email: anisimov@demogr.mpg.de
Chapter 5 Ignacio Barberi
Institute of Medical Pediatrics
Fernando Antón-Tay Neonatal Intensive Care Unit
Departamento de Biología University of Messina
de la Reproducción Messina, Italy
División de Ciencias Biológicas Chapter 16
y de la Salud
Universidad Autónoma Mteropolitana- Christian Bartsch
Iztapalapa Institute of Physiological Chemistry
México, D.F., México University of Tübingen
Chapter 23 Tübingen, Germany
Email: christian.bartsch@uni-tuebingen.de
Chapter 7
Hella Bartsch Bruno Claustrat
Institute of Physiological Chemistry Service de Radioanalyse
University of Tübingen Hôpital Neuro-Cardiologique and
Tübingen, Germany Insitut Fédératif de Neurosciences
Chapter 7 Lyon, Cedex, France
Email: bruno.claustrat@chu-lyon.fr
Gloria Benítez-King Chapter 20
Departamento Neurofarmacologia
Subdirección de Investigaciones Clínicas Rodolfo A. Cutrera
Instituto Nacional de Psiquiatria Ramón Departamento de Fisiología
de la Fuente Muñiz Facultad de Medicinia
México, D.F., México Universidad de Buenos Aires
Email: bekin@imp.edu.mx Buenos Aires, Argentina
Chapter 23 Chapter 19
Verónica Boggio Salvatore Cuzzocrea

Departamento de Fisiología Institute of Pharmacology
Facultad de Medicinia School of Medicine
Universidad de Buenos Aires University of Messina
Buenos Aires, Argentina Torre Biologica—Policlinico
Chapter 19 Universitario Via C. Valeria
Messina, Italy
Jimo Borjigin Email: salvatore.cuzzocrea@unime.it
Department of Embryology Chapter 11
Carnegie Institution of Washington
Baltimore, Maryland, U.S.A. Ariana Daniel
Email: borjigin@ciwemb.edu University of Alberta
Chapter 17 Edmonton, Alberta, Canada
Chapter 4
Jocelyne Brun
Service de Radioanalyse Drew Dawson
Hôpital Neuro-Cardiologique Centre for Sleep Research
Lyon, Cedex, France University of South Australia
Chapter 20 South Australia, Australia
Chapter 9
Guy Chazot
Service de Neurologie Christina Demianczuk
Hôpital Neuro-Cardiologique University of Alberta
Lyon, Cedex, France Edmonton, Alberta, Canada
Christophe Chiquet Jie Deng

Institut Fédératif de Neurosciences Department of Embryology
INSERM, Sérvice d’Ophtalmologie Carnegie Institution of Washington
Hôpital Edouard Herriot Baltimore, Maryland, U.S.A.
Lyon, Cedex, France Chapter 17
Chapter 20
Ana I. Esquifino Michal Karasek
Departamento de Bioquímica Department of Electron Microscopy
y Biología Molecular III Medical University of Lodz
Facultad de Medicina Lodz, Poland
Universidad Complutense Email: karasek@csk.am.lodz.pl
Madrid, Spain Chapter 10
Chapter 19
David García Department of Thyroidology
Departamento Neurofarmacologia Institute of Endocrinology
Subdirección de Investigaciones Clínicas Medical University of Lodz
Instituto Nacional de Psiquiatria Ramón Lodz, Poland
de la Fuente Muñiz Email: mkarbownik@hotmail.com
México, D.F., México Chapter 21
Chapter 23
Jessie Kautz
Marie-Laure Garidou University of Alberta
UMR-CNRS Edmonton, Alberta, Canada
Neurobiologie des Rythmes Chapter 4
Strasbourg, France
Chapter 1 Leonid Kayumov
Department of Psychiatry
Saul S. Gilbert University of Toronto
Centre for Sleep Research University Health Network
University of South Australia Toronto Western Hospital
South Australia, Australia Toronto, Ontario, Canada
Chapter 9 Email: lkayumov@uhnres.utoronto.ca
Chapter 26
Eloisa Gitto
Institute of Medical Pediatrics Tatiana V. Kvetnaia
Neonatal Intensive Care Unit Department of Cell Biology
University of Messina and Pathology
Messina, Italy St. Petersburg Institute of Bioregulation
Chapter 16 and Gerontology of the Russian
Academy of Medical Sciences
Raed Hawa St. Petersburg, Russia
Department of Psychiatry Chapter 13
University of Toronto
University Health Network Igor M. Kvetnoy
Toronto Western Hospital Department of Cell Biology
Toronto, Ontario, Canada and Pathology
Chapter 26 St. Petersburg Institute of Bioregulation
and Gerontology of the Russian
Academy of Medical Sciences
St. Petersburg, Russia
Email: kvetnoy@gerontology.ru
Chapter 13
Leon C. Lack Carlos Mautalen
School of Psychology Sección Osteopatías Médicas
Flinders University Hospital de Clínicas
Adelaide, South Australia, Australia “José de San Martín”
Email: leon.lack@flinders.edu.au Facultad de Medicina
Chapter 15 Universidad de Buenos Aires
Marta G. Ladizesky Chapter 19
Sección Osteopatías Médicas
Hospital de Clínicas Juan C. Mayo
“José de San Martín” Department of Cellular
Facultad de Medicina and Structural Biology
Universidad de Buenos Aires University of Texas Health Science
Buenos Aires, Argentina Center
Chapter 19 San Antonio, Texas, U.S.A.
Chapter 2
Andrzej Lewinski
Department of Thyroidology Marc Moreau
Institute of Endocrinology University of Alberta
Medical University of Lodz Alberta, Canada
Lodz, Poland Chapter 4
Email: alewin@csk.am.lodz.pl
Chapter 3 Yuji Okatani
Department of Clinical Nursing Science
Alan Lowe Kochi Medical School
Department of Psychiatry Nankoku, Kochi, Japan
University of Toronto Email: okataniy@med.kochi-ms.ac.jp
University Health Network Chapter 18
Toronto Western Hospital
Toronto, Ontario, Canada Paul Pévet
Chapter 26 UMR-CNRS
Neurobiologie des Rythmes
Kurt Lushington Strasbourg, France
Centre for Sleep Research Email: pevet@neurochem.u-strasbg.fr
School of Psychology Chapter 1
University of South Australia
South Australia, Australia Arcady A. Putilov
Email: kurt.lushington@unisa.edu.au Institute for Medical
Chapter 9 and Biological Cybernetics
Siberian Branch
James Mahood RAMS
University of Alberta Novosibirsk, Russia
Edmonton, Alberta, Canada Email: putilov@cyber.ma.nsc.ru
Talib Rajwani Ewa Sewerynek
University of Alberta Institute of Endocrinology
Edmonton, Alberta, Canada Medical University of Lodz
Chapter 4 Lodz, Poland
Email: ewa@tyreo.am.lodz.pl
Gerardo Ramírez-Rodríguez Chapter 6
Departamento Neurofarmacologia
Subdirección de Investigaciones Clínicas Colin M. Shapiro
Instituto Nacional de Psiquiatria Ramón Department of Psychiatry
de la Fuente Muñiz University of Toronto
México, D.F., México University Health Network
Chapter 23 Toronto Western Hospital
Toronto, Ontario, Canada
V. James Raso Email: colin.shapiro@uhn.on.ca
University of Alberta Chapter 26
Edmonton, Alberta, Canada
Chapter 4 Valérie Simmoneaux
UMR-CNRS
Russel J. Reiter Neurobiologie des Rythmes
Department of Cellular Strasbourg, France
and Structural Biology Email:
University of Texas Health Science simonneaux@neurochem.u-strasbg.fr
Center Chapter 1
San Antonio, Texas, U.S.A.
Email: reiter@uthscsa.edu Natalia S. Sinitskaya
Chapters 2, 16, 18 Department of Cell Biology
and Pathology
Christophe Ribelayga St. Petersburg Institute of Bioregulation
UMR-CNRS and Gerontology of the Russian
Neurobiologie des Rythmes Academy of Medical Sciences
Strasbourg, France St. Petersburg, Russia
Galena S. Russkikh Marcel G. Smits

Institute for Medical Department of Neurology
and Biological Cybernetics and Sleep-Wake Disorders
Siberian Branch Hospital “De Gelderse Vallei”
RAMS Ede, The Netherlands
Novosibirsk, Russia Chapter 25
Chapter 24
Stanko S. Stojilkovic
Rosa M. Sainz ERRB, NICHD
Department of Cellular National Institutes of Health
and Structural Biology Bethesda, Maryland, U.S.A.
University of Texas Health Science Email: stankos@mail.nih.gov
Center Chapter 8
San Antonio, Texas, U.S.A.
Chapter 2
Dun-Xian Tan Helen R. Wright
Department of Cellular School of Psychology
and Structural Biology Flinders University
University of Texas Health Science Adelaide, South Australia, Australia
Center Email: helen.wright@flinders.edu.au
San Antonio, Texas, U.S.A. Chapter 15
Chapter 2
Hana Zemkova
Cameron J. van den Heuvel Institute of Physiology
Centre for Sleep Research Academy of Sciences of the Czech
University of South Australia Republic
South Australia, Australia Prague, Czech Republic
Email: cameron.vdh@unisa.edu.au Email: zemkova@biomed.cas.cz
Chapter 9 Chapter 8
Akihiko Wakatsuki Irina V. Zhdanova

Department of Obstetrics Department of Anatomy
and Gynecology and Neurobiology
Kochi Medical School Boston University School of Medicine
Nankoku, Kochi, Japan Boston, Massachusetts, U.S.A.
Chapter 18 Email: zhdanova@cajal-1.bu.edu
Chapter 14
Xaioping Wang
University of Alberta Nava Zisapel
Edmonton, Alberta, Canada Department of Neurobiochemistry
Chapter 4 The George S. Wise Faculty
of Life Sciences
Allan F. Wiechmann Tel Aviv University
Department of Cell Biology Tel Aviv, Israel
University of Oklahoma Health Sciences Email: navazis@post.tau.ac.il
Center Chapter 12
Oklahoma City, Oklahoma, U.S.A.
Email: allan-wiechmann@ouhsc.edu
Chapter 22
Janet Wilson
University of Alberta
Edmonton, Alberta, Canada
Chapter 4
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 pro-
duction of this volume.
The editors wish to express their sincere appreciation and owe endless
gratitude to all the contributors for their scholarly contributions that facili-
tated 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 cre-
ativity 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 in-
debted 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 gratifi-
cation from the opportunity afforded to us in the rapidly growing area of
Neuroendocrinology. Because the field is a dynamic one, this book is in-
tended 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 pro-
duction have been intensively investigated, those responsible for the seasonal variations in me-
latonin 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 hor-
mone 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 sea-
sons 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 control-
ling 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.
2 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 spe-
cies differences, particularly among the rodents (Fig. 1, see ref. 3 for review). In rat, lengthen-
ing 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 de-
cline 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 in-
creases 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 endo-
crine 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 im-
portantly, 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 pho-
toperiodic (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 reli-
able) 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 trans-
mission 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 ac-
tivity (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 activi-
ties.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 regula-
tion 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 melato-
nin 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 oxy-
tocin), lateral hypothalamus (hypocretin), habenula nucleus (substance P), thalamic
intergeniculate nucleus (NPY) and raphe nuclei (serotonin). Additionally, the neurons of ptery-
gopalatine (vasoactive intestinal peptide, VIP), trigeminal (pituitary activating adenylate cycle
peptide, PACAP; substance P, calcitonin gene-related peptide) and otic ganglia (PACAP) in-
nervate the pineal gland. Most of these peptides have been found to regulate melatonin synthe-
sis 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 regu-
lation of melatonin synthesis have been investigated in detail, it is not known which transmit-
ters drive the seasonal regulation of melatonin synthesis. Because the hypothalamic clock inte-
grates 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,
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= neu-
ropeptide Y; OT= oxytocin; PACAP= pituitary adenylate cyclase activating peptide; PVN= paraventricular
nuclei of the hypothalamus; SCG= superior cervical ganglia; SCN= suprachiasmatic nuclei of the hypo-
thalamus; 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 differ-
ences 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 synthe-
sis. Among the other pineal transmitters, NPY displays marked seasonal variations in the Euro-
pean hamster.37 Interestingly, the marked increase in NPY immunoreactivity observed in win-
ter 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 varia-
tion 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 ac-
cording to the photoperiod in rat.42,43
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 hy-
droxylated 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
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) fol-

lowed 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 in-
crease 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;
(3) PKA-induced phosphorylation of AA-NAT protects the enzyme from proteasome proteoly-
sis. 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 activ-
ity 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 re-
lease 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 depo-
larization with a resulting release of glutamate, from pineal microvesicles, which in turn inhib-
its 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 inter-
mediate 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 con-
stantly 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 respon-
sive 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 se-
cretion. Although the major (and most reproductive) seasonal modification of the environ-
mental 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 ham-
ster,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.
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 sea-
sonal (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 activ-
ity 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 limit-
ing 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
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) com-
pared 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 dura-
tion 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 mela-
tonin 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 neu-
rotransmitters 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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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 ma-
jor 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 es-
sential molecules thereby compromising their function and leading to cellular death. This re-
view 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. Regard-
less 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 and Ischemia-Reperfusion Injury 13
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 func-
tion.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
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 mea-
surements 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
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 de-
crease 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 radi-
cal (•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 re-
ferred to as “spectacular” protection of cardiac function and morphology. Given that free radi-
cals 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 cowork-
ers23 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 reac-
tants 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 re-
ported 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
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 protec-
tive 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 oc-
curred 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 sug-
gested 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 pro-
duction 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 melato-
nin 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 cer-
tainly 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 devas-
tating 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 mela-
tonin against the resulting damage. Pinealectomy, which results in a relative melatonin defi-
ciency, 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
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 vol-
ume 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 re-
duction 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 administra-
tion (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 spectros-
copy 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 neuro-
logical 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.
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
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 is-
chemia, 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 re-
duced 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 hippoc-
ampus 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 sig-
nificant neurological deficits measured on both day 1 and day 7. When melatonin was con-
tinually 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 obvi-
ously 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
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 contrib-
ute 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 inter-
rupted 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 gas-
trointestinal 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,
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 mu-
cosal 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 me-
senteric 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 adminis-
tration 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 height-
ened 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 destruc-
tion 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 edema-
tous 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 prop-
erties and immunomodulatory actions.
As with other organs, the urinary bladder is functionally and morphologically compro-
mised 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 bio-
chemical 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
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 morphologi-
cal 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 conse-
quence 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 tis-
sue 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 physiologi-
cal concentrations, i.e., pharmacological levels, of antioxidants is always necessary under con-
ditions of massive induced oxidation stress. Indeed, the reason oxidative damage occurs in
these situations is that all physiological antioxidants combined are obviously incapable of cop-
ing 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 func-
tions far outstrip those implied by the term hormone which are, by definitiaon, receptor medi-
ated. 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
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
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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 thy-
roid gland. Accordingly, it has been documented that oxidative stress accompanies certain thy-
roid disturbances or diseases. Up-to-date literature, although not abundant, indicates that me-
latonin 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 rela-
tionship 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 me-
latonin 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 and the Thyroid Gland 27
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 hypophysecto-
mized 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 squir-
rels (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 ad-
enosine kinase. Additionally, we examined the effect of indoleamines on cyclic AMP genera-
tion in rat thyroids in vitro.
Thymidine kinase (TK: thymidine 5'-phosphotransferase, EC 2.7.1.21) is an enzyme re-
sponsible for catalyzing the phosphorylation of thymidine, functioning as a part of the pyrimi-
dine 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 monophos-
phate (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 ana-
logues 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
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 vari-
ous 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 pretreat-
ment 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.
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, de-
pending on animal species, as well as on applied melatonin dose and administration protocol.
Melatonin, employed in late afternoon s.c. injections, decreased circulating thyroid hor-
mone 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 anti-
oxidant 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. Hydro-
gen 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 concentra-
tions). 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
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 concen-
tration 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) produc-
tion 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 re-
duce 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 addi-
tional 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 concentra-
tions 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 mol-
ecules.37,51,56 As regards the protective effects of melatonin against oxidative stress in the thy-
roid 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 hyperthyroid-
ism in human subjects,57 other investigators have found increased nocturnal melatonin con-
centrations 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
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 signifi-
cantly 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 in-
duced 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 melato-
nin in rats.66
Concluding Remarks
On the basis of our early results, a reciprocal relationship between the pineal and the thy-
roid 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 thy-
roid 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 exclu-
sively, 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 ques-
tions still remain to be answered. These are the following:
1. to what extent is the relationship in question a direct one?
2. are there any intermediate substances or factors involved in this regulation?
3. are there any cells in the body capable of producing both thyroid hormones and melatonin?
4. is there a local (paracrine) regulation of thyroid hormone synthesis and of thyrocyte prolif-
eration by melatonin in the gland in question?
5. does melatonin participate in the regulation of the peripheral metabolism of thyroid hor-
mones (i.e., monodeiodination processes)?
6. does melatonin regulate the activity of type II T4-5'-monodeiodinase in pinealocytes?
7. does melatonin control the expression of certain thyroid gland-related genes, e.g., NIS,
TPO, thyroglobulin, pendrin, TSH, TSH receptor (TSHR), etc.?
8. 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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The Role of Melatonin in the Development of Scoliosis 35
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 charac-
teristics 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 pro-
duced 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, nev-
ertheless, 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 mela-
tonin 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 meth-
ods 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

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 particu-
larly 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, treat-
ment may include ‘bracing’ but if the curve continues to progress then extensive surgery be-
comes 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 charac-
teristics associated with a hormonal problem but when the involvement of the hormone mela-
tonin 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 ap-
proach 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 hid-
den and their effects significantly reduced within the normal values from the other scoliosis
patients. The average results from the total group of AIS patients will therefore not be signifi-
cantly 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 appro-
priate 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 prob-
lems 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 pa-
tients, 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 de-
signs should therefore be avoided and the results from such experiments viewed with consider-
able 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 im-
proved evaluation system might provide clear identification of some of the symptoms mani-
fested 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.
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 underly-
ing 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% (consis-
tently).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
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 suscep-
tible 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 even-
tual 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 accompany-
ing 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 find-
ing the solution to this phenomenon. Pinealectomy in young rats and hamsters does not ap-
pear 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 ques-
tionable.
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 se-
rum 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 re-
main 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 produc-
tion 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. Conse-
quently, all measurements are expressed as average values from a group of chickens since longi-
tudinal 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
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 signifi-
cantly 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 chick-
ens that developed scoliosis are not randomly scattered along the continuum of serum melato-
nin 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 thresh-
old 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 scolio-
sis. 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 injec-
tion 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 contradic-
tory 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 an-
other 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 melato-
nin 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 an-
other 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 mela-
tonin 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 (submit-
ted) 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
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 exten-
sive 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 envi-
ronmental conditions. Apparently, the chickens would provide their own ‘darkness’ by hud-
dling together and burying their heads under their wings to achieve a dark environment. Nev-
ertheless, 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 tryp-
tophan 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 mela-
tonin 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 pro-
duction 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 pa-
tients with non-progressive curves. If these experiments can be replicated and the results con-
firmed, 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
affects the shape of the G-proteins which then bind to and activate adenylate cyclase. Alterna-
tively, melatonin can bind intracellularly to cytosolic calmodulin which may then affect cal-
cium signalling by interacting with target enzymes such as adenylate cyclase and phosphodi-
esterase 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 pa-
tients 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 hor-
mone 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 soma-
tostatin 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, an-
other 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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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.
Effect of Melatonin on Life Span and Longevity 45
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 con-
cerning 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 neuroen-
docrine function.5,6 As age advances, the nocturnal production of melatonin decreases in ani-
mals 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 me-
latonin. 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 influ-
ence 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 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 maintain-
ing 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 supple-
mentation 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 mela-
tonin 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
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 supplemen-
tation with melatonin initiated during middle age did not appear to affect age-associated le-
sions 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 lympho-
mas 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 consump-
tion 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 pro-
nounced 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.
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 ac-
celerated 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 re-
ceived 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. Leu-
kemia 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 localiza-
tion. 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. Au-
thors believe that melatonin antagonist ML-23 induces chronic deprivation of melatonin re-
ceptors 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. Treat-
ment 1 pups had access to water, whereas treatment 2 and 3 pups had access to water contain-
ing 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.
Table 1. Summary of experiments on the effect of melatonin on life span and spontaneous tumor incidence in mice
Age at Effects of Melatonin on:

Nos. of Age at Start of the End of Mean Tumor
Strain Sex Mice C/M Treatment, mo Treatment with Melatonin Observation Life Span Incidence References
Balb/c Female 26/12 15 10 mg/l in night drinking water ND +18% No data Pierpaoli and
Regelson, 199414
Balb/c Male 50/50 18 10 mg/l in night drinking water ND Shift to right No data Mocchegiani
of the survival et al, 199817
curve
C57BL/6 Male & 25/45 1.5 2.5 mg/mouse s.c. 22 mo -13% Increases Romanenko,
female twice a wk x 5 mo 198324
Effect of Melatonin on Life Span and Longevity
C57BL/6 Male & 29/57 1.5 2.5 mg/mouse s.c. 22 mo - 20.6% Increases Romanenko,
female twice a wk x 2.5 mo 198524
CC57Br Male & 26/57 1.5 2.5 mg/mouse s.c. 22 mo -12% Increases Romanenko,
female twice a wk x 2.5 mo 198525
C57BL/6J Male 10/10 19 10 mg/l in night drinking water ND + 20% No data Pierpaoli and
Maestroni, 198712
C57BL/6 Male 20/15 19 10 mg/l in night drinking water ND +17% No data Pierpaoli et al,
199110
C57BL/6 Male 1:20/20 18 11 ppm (68 mkg/kg) 1: 24 mo; No effect 1: No effect; Lipman et al,
2:7/13 with lab chow ad libitum 2: 50% survival 2: Increases; 199820
3:38/30 3: died < 2 y 3: No effect
CBA Female 50/50 6 20 mg/l in night drinking water ND +5% Increases Anisimov et al,
200121
C3H Male 20/20 1 2.5 mg/kg/day in night 23 mo + 20% No data Oxenkrug et al,
Female 20/20 8 drinking water 27 mo No effect 200119
C3H/He Female 14/15 12 10 mg/l in night drinking ND No effect Increases Pierpaoli et al,
water 199110
C3H/Jax Female 16/39 3 wk 25-50 mkg/mouse/d 12 mo No data Decreases Subramanian and
with drinking water Kothari, 199127
49
Table continued on next page

50
Table 1. Continued
Age at Effects of Melatonin on:

Nos. of Age at Start of the End of Mean Tumor
Strain Sex Mice C/M Treatment, mo Treatment with Melatonin Observation Life Span Incidence References
HER-2 / Female 30/27/22 2 2.5 mg/kg/day in night ND M1: No effect M1: No effect; Anisimov et al,
neu drinking water 5 d/w monthly M2: -13% M2: Decreases 200228
or constantly
NOD Female 25/30 1 4 mg/kg s.c. at 4:30 PM, 50 wk C:32% No data Conti and
5 times a wk, 4-38 wk survivors Maestroni, 199818
M:90%
survivors
NOD Female 29/17 1 10 mg/l in night drinking water, 50 wk + 17% No data Conti and
5 times a wk, 4-38 wk Maestroni, 199818
NZB Female 10/10 4 10 mg/l in night drinking water 20 mo Survivors: No data Pierpaoli et al,
C:0; M: 40% 199110
NZB/W Female 15/15/15 8 2-3.5 mg/kg s.c.daily at 34 wk Survivors: No data Lenz et al,
8-10 hrs (M1) or at 17-19 hrs C: 20%; 199516
(M2) x 9 mo M1: 60%;
M2: 60%
SAMP-1 Female 20/20 3 20 mg/l in night drinking water ND No effect No effect Rosenfeld,
200223
SAMR-1 Female 10/12 3 20 mg/l in night drinking water ND -11% No effect Rosenfeld,
200223
SHR Female 50/50/50 3 2 or 20 mg/l in night drinking ND No effect; M1: ↓ Anisimov et al,
water M1, M2: 1,9-fold 2003
+3 mo. MLS M2: No effect
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.
Melatonin: Biological Basis of Its Function in Health and Disease
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 influ-
ence 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 melato-
nin 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 corre-
sponding 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 longev-
ity 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 circa-
dian 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. Au-
thors suggest that geroprotector effect of melatonin in worms depend on its capacity to scav-
enge free radicals in cells.
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 mem-
branes, 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 prod-
ucts, 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 mu-
tations 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 nor-
mal 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 anoma-
lies 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 Melato-
nin 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
As oxidative mutagens we used DMH, bleomycin (with S9-mix) and mitomycin C (with-
out 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 acti-
vation. 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 no-
ticed 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 modu-
late 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 concentra-
tions (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 oxida-
tive 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 melato-
nin 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 medi-
ated 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 mela-
tonin to mice with drinking water (15 mg/l) during 40 days also attenuated apoptotic thy-
mocyte 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 experi-
mental models. Treatment with melatonin increases production of antibodies to sheep erythro-
cytes 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 endo-
crine 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
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 hor-
mone (GnRH)-secreting neurons of hypothalamus, Roy and Belsham84 have shown that mela-
tonin 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 Tran-
scription 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 coinci-
dent 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 develop-
ment 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 expres-
sion 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 melato-
nin are the genes controlling the cell cycle, cell/organism defense, protein expression and trans-
port. 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 de-
tected.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,
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 signifi-
cantly. 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 impor-
tant 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 be-
lieved that melatonin may influence tumor growth by interfering with calcium binding and
blocking the formation of the MAPs/calmodulin and tubulin/calmodulin complexes to pre-
vent 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 guide-
lines 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 experi-
ments 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 conclu-
sion about it safety. There are data on suppressive effect of melatonin on development of spon-
taneous 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 ap-
propriate 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 in-
hibitor 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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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 popu-
lation. The evidence obtained during the last 10 years suggests that melatonin exerts certain
effects upon the cardiovascular system. The presence of vascular melatoninergic receptors (bind-
ing sites) has been demonstrated; these receptors are functionally associated with either vaso-
constrictor 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 coro-
nary 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 mela-
tonin suppresses the formation of cholesterol, reduces LDL accumulation in serum and modi-
fies 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 cardiovas-
cular system and melatonin.
It is well-known that serum melatonin concentrations and urinary levels of its main me-
tabolite, 6-sulphatoxymelatonin, decrease in the course of ageing.1 In elderly subjects, the inci-
dence 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 sea-
sonal 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

Cardiovascular Effects of Melatonin 61
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 be-
tween 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 demon-
strated that blood pressure falls during the night. Nicolau et al8 suggested that cardiac mortal-
ity 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 demon-
strate 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 car-
diovascular diseases, compared to circadian and seasonal variations in melatonin concentrations,
are not very well documented. Data obtained in animals indicate that the cardiovascular re-
sponse 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 recep-
tor 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, to-
gether with their functional associations with vasoconstrictor or vasodilatory effects of melato-
nin. 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, de-
rived from healthy heart donors, has been described.22 It has been suggested that MT2 melato-
nin receptors, expressed in vascular smooth muscles, mediate vasodilation, in contrast to vascu-
lar 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 ob-
served during acute myocardial infarction.26 Urinary 6-sulphatoxymelatonin excretion was sig-
nificantly 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 coro-
nary 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 dur-
ing 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
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 pa-
tients 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 dis-
eases, 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 spe-
cific inhibition of beta1-receptors. Nathan et al34 have demonstrated a dose-dependent rela-
tionship 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 disor-
ders 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 pa-
tients, 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 hypercholester-
olemia in rats (Fig. 2). The results of that study confirm that melatonin participates in the
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 af-
fected 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 activ-
ity 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 prod-
ucts 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 melato-
nin treatment reduced LDL susceptibility to oxidative modification in normolipidemic
post-menopausal women. Thus, the oxidised form of LDL-cholesterol (ox-LDL) plays a prin-
cipal 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 sup-
pressed 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 ap-
pears 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 hyperten-
sive rats, which either demonstrate a lower content of endogenous antioxidants or a greater
sensitivity to free radicals of the vascular tissue.
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 postmeno-
pausal 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 sug-
gested that, because a normal night-time decline of blood pressure protects women from car-
diovascular 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 car-
diovascular risk in postmenopausal women. Doolen et al56 attempted at determining whether
oestrogen modulates the function of vascular melatonin receptors. They have found that estra-
diol 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 vasocon-
striction, while MT2 receptors mediated melatonin-induced vasodilatation.57
Weekley45 found that melatonin relaxed the smooth muscles, lining the rat aorta. The vas-
cular endothelium may contribute to the regulation of vascular smooth muscle tone by pro-
ducing 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 hy-
drogen 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 modu-
lates 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 path-
ways 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 ef-
fect 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 intra-
cellular 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 contractil-
ity 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 mela-
tonin has been demonstrated during the last 10 years of studies.72-78 The results of epidemio-
logical 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 sug-
gested that physiological melatonin concentrations are important to reduce the
ischemia-reperfusion arrhythmias, myocyte damage and mortality, while pharmacological con-
centrations 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 neutro-
phil inhibition in myocardial ischemia-reperfusion.83 Melatonin reduced the damage induced
by chemical hypoxia and reoxygenation in rat cardiomyocytes. 84 Melatonin alone or in
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 cardiomyopa-
thy, 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 sup-
presses 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 hu-
mans 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 myo-
cardial infarction is associated with a nocturnal melatonin deficit in serum and increased oxi-
dative 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 hy-
pertensive96 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
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 me-
latonin. 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. interference with arterial response to catecholamines,
2. reduction of norepinephrine efflux from perivascular nerves,
3. decrease of noradrenergic activity,
4. suppression of prostaglandin production,
5. influence on nitric oxide production,
6. via specific receptors.
Additionally, melatonin may reduce blood pressure via the following mechanisms:
1. by a direct effect on the hypothalamus;
2. as an antioxidant which lowers blood pressure;
3. by decreasing the levels of catecholamines, or
4. 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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Pineal Gland and Cancer—An Epigenetic Approach to the Control of Malignancy 71
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 sum-
marized 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 develop-
ment 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 sub-
stitutional 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 mecha-
nisms against the malignant process improving survival. This means that melatonin via indi-
rect 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 melato-
nin 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 spe-
cifically 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.

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 mecha-
nisms 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 mani-
festing 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 per-
formed important experimental studies.13 A central finding was that surgical removal of the
pineal gland (pinealectomy) stimulated both primary tumor growth and formation of me-
tastases 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-divi-
sion of normal tissues. The question arose how this general anti-proliferative effect of the pi-
neal 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 melato-
nin 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 circa-
dian 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 melato-
nin was administered in most subsequent experimental and clinical studies in the late after-
noon or in the evening to achieve optimal tumor-inhibitory effects.
A considerable number of investigations have dealt with the effect of melatonin on experi-
mental mammary cancers in female Sprague-Dawley rats induced by 7,12-
dimethylbenz-[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 affect-
ing 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 hy-
droxylate melatonin to yield 6-hydroxymelatonin32,33 and DMBA leading to the forma-
tion of 3,4-dihydrodiol-1,2-epoxy derivative, the ultimal carcinogen.29,34 It can be assumed
furthermore that metabolically activated intermediary free radical forms of DMBA are scav-
enged 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 pre-
dominantly 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 ex-
amples are the behavior of transplantable prostatic or mammary cancers. The growth of experi-
mental prostatic cancers is effectively blocked by melatonin only if they are androgen-depen-
dent 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 in-
bred rats44,45 were found to be inhibited by melatonin only at an early and slow-growing pas-
sage 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 poten-
tially important actions even on general development of multi-cellular organisms.55,56
Experiments with spontaneous endometrial carcinomas in BDII/Han rats57 illustrate com-
plex 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 at-
tained 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 in-
clude immune-mediated processes leading to tumor rejection. Melatonin possesses well-docu-
mented 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 mela-
tonin on in vivo experimental tumor growth may thus be understood as a process involving both
neuro-immunoendocrine as well as metabolic mechanisms.
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 hor-
mone 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 there-
fore 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. Accord-
ing 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 inacti-
vates 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 biop-
sies.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 cellu-
lar signals.
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 per-
formed 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 re-
fractory 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 hor-
mone 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 observa-
tion 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%) com-
pared to controls;100 patients suffering from glioblastoma melatonin given with brain irradia-
tion significantly elevated one-year survival (+37%) compared to irradiation alone;101 in pa-
tients with heavily pretreated metastatic breast cancer melatonin co-administered with tamoxifen
highly significantly extended one-year survival (+39%) compared to antiestrogen alone;102 pa-
tients 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 signifi-
cantly increased one-year survival (+25%) compared to irradiation alone.104
The greatest number of patients treated by Lissoni with melatonin received the pineal hor-
mone 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 treat-
ment 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 indi-
rectly 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 col-
leagues are encouraging and justify a further systematic verification under double-blind pla-
cebo 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.
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 tu-
mors) 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 depen-
dency 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 de-
tect 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 pa-
tients 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
melatonin was depressed by 28% compared to patients with small BPH but patients with T2-
and 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 con-
cluded 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 noctur-
nal 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 dif-
ferent 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 melato-
nin. 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 con-
trols. 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 be-
ing 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.
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 concen-
trations 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 bio-
synthesis 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 obser-
vation was made in female BDII/Han rats during the development of spontaneous endome-
trial adenocarcinomas.139 F344 Fischer rats with an early tumor passage (derived from the
above-mentioned DMBA-induced mammary tumors) showed an elevated melatonin produc-
tion 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-de-
rived biopterin and of γ-interferon in plasma).142 In contrast to that, rats with tumors of simi-
lar 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 accompa-
nied 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 ob-
served 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 deriva-
tives 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 malig-
nant process. Such hopes, however, do not appear to be justified on the basis of the
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 meta-
static 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 cen-
tral 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 develop-
ment 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 devel-
opment 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 malig-
nancy, 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 depres-
sion 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 determi-
nations of nocturnal urinary 6-sulfatoxymelatonin (being a reliable estimate of nocturnal me-
latonin production in cancer patients121) may be included in the monitoring program of onco-
logical 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 carci-
noma124 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.
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 oscilla-
tion of the SCN and thereby the secretion of pineal melatonin is synchronized to environmen-
tal 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 in-
creasing depression of circulating melatonin in cancer patients and tumor-bearing animals in
the presence of localized primary tumors129 together with temporal neuroendocrine distur-
bances147,148 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 suppres-
sion 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 geo-
graphic patterns connected with seasonally modulated circadian rhythms of melatonin at dif-
ferent 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 suppres-
sion of nocturnal melatonin secretion. This so-called “light pollution” may not only have ex-
tinguished previous seasonal endocrine and behavioural patterns including reproduction in
humans161 but could also contribute to a higher risk for the development of hormone-depen-
dent 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 melato-
nin164-166 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 hypoth-
esized 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 can-
cer 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 evi-
dence that patients after pinealectomy due to the presence of a pineal tumor exhibit an in-
creased 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 patho-
physiological 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 ani-
mals 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 cir-
culating melatonin could decisively modulate the development of endometrial cancer in
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 hu-
man 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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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 cal-
cium and electrophysiological recordings revealed that reduction in gonadotropin release re-
sults 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 re-
lease 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 cal-
cium 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 func-
tions, including GnRH release, are preserved, and are critically important in synchronizing the
external photoperiods and reproductive functions through still not well characterized mecha-
nisms.
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 pro-
cess that reflects on activity of numerous pathways. The activity of melatonin-producing en-
zyme 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 partici-
pates 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

Expression and Signal Transduction Pathways of Melatonin Receptors in Pituitary 89
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, de-
pending 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 melato-
nin feedback action and are responsible for the phase-shifting effects on the circadian rhythms.4-6
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 ex-
pression of MT2 subtype is more localized,6,13 Other forms or states of the mammalian mela-
tonin 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 in-
hibiting 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 activa-
tion 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 ac-
companied with facilitation of phospholipase C activity, an enzyme that produces two intrac-
ellular messengers, inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG).24,25 Activa-
tion 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 tem-
perature 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 inhibi-
tion 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 exog-
enous 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 mim-
icked 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 repro-
duction.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
Melatonin probably influences reproduction at three levels: the hypothalamic GnRH neu-
rons, 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 immortal-
ized 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 ex-
pressed 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 concentra-
tion 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 path-
ways.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 antago-
nists for these two receptors limits investigations on their expression and function. For ex-
ample, 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 chick-
ens, 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 con-
taining 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
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 melato-
nin 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 iden-
tity 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 pitu-
itary.67 Our recent RT-PCR analysis, however, suggested that both subtypes of melatonin re-
ceptor mRNAs are also expressed in anterior pituitary from neonatal rats, but that the expres-
sion 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 ob-
served 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 path-
ways.
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 hy-
pothalamic 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 neu-
ronal 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
Figure 2. Typical patterns of calcium signal-

ing 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 concen-
tration range, the baseline oscillations in 0.1
to 10 nM concentration range, and the
biphasic oscillatory and non-oscillatory re-
sponses 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 cal-
cium 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 partici-
pate in modulating voltage-dependent Ca2+ influx and Ca2+ release from IP3-sensitive intracel-
lular 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;
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 con-

trolled by GnRH concentration, followed by baseline oscillations, which frequency is con-
trolled 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 fre-
quently 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
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 characteris-
tics 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+ re-
lease 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
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 pitu-
itary cells.96 Thus, the expression and functional coupling of ryanodine-sensitive channels in
neonatal gonadotrophs probably coincides with expression and function of melatonin recep-
tors 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 phos-
phorylation 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
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 ex-
tracellular 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
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 pro-
vides an effective mechanism for amplification of signals from VGCC through Ry store to IP3 store (indi-
cated 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+ re-
lease 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+ oscil-
lations 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
shown to inhibit the GnRH-induced increase in DAG,40 suggesting that it may inhibit phos-
pholipase C activity. However, this is an unlikely explanation, because potentiation rather than
inhibition of phospholipase C has been found in cells transiently expressing melatonin recep-
tor, 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 be-
tween the cAMP and phosphoinositide signaling pathways is well documented. For example,
in hepatocytes the frequency of calcium oscillations triggered by hormones linked to IP3 pro-
duction 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 ef-
fects 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) con-
centrations, 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 melato-
nin 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 prob-
ably 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 endog-
enous 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 re-
ceptors 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.
In contrast to the physiological relevance of transient expression of melatonin receptors in

gonadotrophs, the mechanism by which the developmental down-regulation of melatonin re-
ceptor 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 ex-
ample 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 melato-
nin 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 Distribu-
tion and appearance of specific transcription factors that precede terminal differentiation of pitu-
itary cell types thus may also parallel developmental disappearance of melatonin receptor.
Perspectives
Although the importance of melatonin in control of reproductive functions is well estab-
lished, 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 pitu-
itary pars distalis cells appears to be related to the control of hormonal status during the devel-
opment 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 devel-
opments, 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, specifi-
cally 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 secre-
tion. The actions of melatonin in gonadotrophs are better characterized. Activation of melato-
nin 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 path-
ways 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 sub-
units 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
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 ani-
mals. 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. Further-
more, 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 mamma-
lian 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 lutein-
izing hormone and follicle-stimulating hormone.112 Whether or not these two GnRH recep-
tors 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 mecha-
nism 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 ther-
moregulation and sleep, we will examine the interaction between melatonin, sleep and ther-
moregulation.
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 mela-
tonin 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).

The Role of Thermoregulation in the Soporific Effects of Melatonin 107
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/hyp-
notic for the treatment of insomnia has since proved disappointing (e.g., ref. 4). Further, de-
spite 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 fol-
lowing 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
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 tempera-
ture 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 Further-
more, 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 in-
creased 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 circa-
dian 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 maxi-
mum.
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 melato-
nin blocked the temperature increase associated with photic suppression of nocturnal melato-
nin.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 demon-
strated 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 perspec-
tive, 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
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 tempera-
ture 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 stron-
gest 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 melato-
nin 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 con-
cluded 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 exam-
ined 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 at-
tenuated. 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 melato-
nin administration.
However, the apparent variability in the soporific and thermoregulatory effects of exog-
enous 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.
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 tempera-
tures 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 adeny-
late cyclase through a cell surface receptor. An early autoradiographic study localized putative
melatonin receptors predominantly in the median eminence (implicated in seasonal/reproduc-
tive regulation) and the SCN.72 Soon after, two distinct melatonin binding sites were identi-
fied; 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 pitu-
itary and preoptic area in the fetal brain (e.g., refs 75, 76). In decreasing order of abundance in
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 melato-
nin 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 melato-
nin 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 demon-
strating 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 signifi-
cant decrease in circulating noradrenaline levels in humans following the daytime administra-
tion 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 vasodi-
latation 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 consis-
tent 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 vascu-
lature 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 endog-
enous 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 inhib-
iting 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
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 (adminis-
tered 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 effi-
cacy 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 tempera-
ture 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 hypothala-
mus, basal forebrain, and the dorsal raphe nuclei. When taken together, these findings pro-
vided 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 pro-
vided a specific neurophysiological mechanism through which an increase in peripheral skin
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 administra-
tion 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 neu-
roanatomical findings are taken together with the peripheral effects of melatonin on tempera-
ture, 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 melato-
nin 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 indi-
rectly 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 vasodi-
lator would be an oversimplification. Given the direct action of melatonin on the thermoregu-
latory system, it would be parsimonious to consider that the thermoregulatory effects of mela-
tonin 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 infor-
mation 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 thermoregu-
latory 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 ca-
pacity or an elevated nocturnal core temperature.
Figure 2. Schematic illustration of the interaction between melatonin, sleep and the thermoregulatory and
circadian systems.
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Am J Physiol 1997; 272(4 Pt 2):H1609-14.
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 me-
latonin may result in lowered sleep efficacy very often associated with advancing age. Dimin-
ished 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 radi-
cals 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 cen-
tury.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

The Role of Melatonin in Human Aging and Age-Related Diseases 119
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 dis-
ease), 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. There-
fore, 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 therapeu-
tic 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 (oscilla-
tor, 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 be-
tween 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
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 mela-
tonin 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
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 gen-
eral 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 func-
tioning (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 indi-
viduals 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 me-
latonin 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 char-
acterized 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 adapta-
tion 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 corre-
lates 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 asso-
ciation with circadian timing system.
On the other hand, Rodenbeck and Hajak presented hypothesis that the diminished mela-
tonin secretion in insomniacs seems to represent the result of very slowly developing neuroen-
docrine 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 de-
crease 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, numer-
ous 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 dis-
eases.37-39
Recently, Pierpaoli and Lesnikov suggested that the pineal, due to its influence on the func-
tion 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 formu-
lated 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 demon-
strated.45 Although the question whether the balance between the amount of free radical for-
mation and the activity of the antioxidative defense might influence the aging process in gen-
eral 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
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 me-
latonin concentrations were demonstrated in postmenopausal women in comparison with pre-
menopausal individuals.49 Age-related decline in nocturnal melatonin secretion in postmeno-
pausal 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 post-
menopausal women on hormone replacement therapy (HRT) but not in untreated postmeno-
pausal 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 dem-
onstrated 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 amy-
loid-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 over-
come 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 stud-
ies 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 implica-
tions of these findings is that the loss of this antioxidant may contribute to the incidence or
severity of some age-associated diseases.
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 impor-
tance, 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 hor-
monal replacement therapy in women or suggested supplementation with dehydroepiandrosterone.
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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. Reac-
tive oxygen species can modulate a wide range of toxic oxidative reactions. These include initia-
tion of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inac-
tivation 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) syn-
thetase (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 experi-
mental 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

edited by S.R. Pandi-Perumal and Daniel Cardinali. ©2006 Eurekah.com.
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 glucocorti-
coids, 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 pro-
duction 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 super-
oxide). 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 ex-
press iNOS.11 Small amounts of peroxynitrite are produced under basal physiological condi-
tions, 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 prob-
able 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 ad-
ducts 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 condi-
tions is expanding. Although there are a number of experimental difficulties related to delinea-
tion of the actual role of peroxynitrite in acute and chronic inflammatory conditions, theoreti-
cal 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
Role of Endogenous and Exogenous Melatonin in Inflammation 129
of superoxide with superoxide dismutase.9 Although chemical considerations favor the pro-
duction 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 over-
production of oxygenderived free radicals. The formation of nitrotyrosine staining as an indi-
cation of “increased nitrosative stress,” and peroxynitrite formation has recently been demon-
strated 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 inflam-
mation. 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 interac-
tions 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 nitra-
tion 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 aconi-
tase, 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 actu-
ally 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 vari-
ous forms of shock23,24 and can exacerbate ischemia/reperfusion injury in some25 but not other26
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 pro-
nounced nitrotyrosine staining was present, suggesting the presence of more, biologically ac-
tive peroxynitrite in the alveolar macrophage and in the airway epithelial cells. The more pro-
nounced 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 in-
creases and leukocyte migration occurs, there is an involvement of several mediators including
neutrophil-derived free radicals, such as hydrogen peroxide, superoxide and hydroxyl radi-
cal.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 conse-
quent 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 phar-
macological 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 su-
peroxide dismutase scavenger compound36 against the suppression of mitochondrial respira-
tion, 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 pro-
cess, 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 in-
jury 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 demonstra-
tion 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 deple-
tion 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 scav-
enger 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.
In vitro studies in macrophages and other cell types have established that endogenous anti-
oxidants (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, re-
cent 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 re-
sponse 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 inflamma-
tion.
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, histo-
logical 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 inflam-
mation 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 demonstra-
tion 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 indica-
tor 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 sub-
stantial 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 activa-
tion50,51 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 Further-
more, substantial evidence exists to support the fact that PARS activation is important in in-
flammation.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.
In addition to the reduction of ROS production and PARS activation, melatonin also re-
duced 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 examina-
tion.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 produc-
tion,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 syn-
thase 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. Bio-
chemical 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 inflamma-
tion, 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 contrib-
ute 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
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.
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 poly-
morphonuclear 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 pharmaco-
logical 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 melato-
nin may interfere with the inflammatory process (Fig. 1): (1) melatonin inhibits NO produc-
tion, 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 tran-
scriptional 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 inhib-
ited 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 sec-
ondarily 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- forma-
tion. (3) In addition to O2-. melatonin also scavenges other radical oxygen species including.
OH. (4) Melatonin additionally scavenges ONOO-. (5). Finally, melatonin reduces the re-
cruitment 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 en-
dothelial 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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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 andro-
gen 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 ex-
clusion of AR in the cells, an effect that may explain the attenuation of AR activity. The path-
way 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 stud-
ies 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 sensitiv-
ity of target cells to androgens, which may be of special importance in androgen related dis-
eases 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, dif-
ferentiation and maintenance of male reproductive function, support sexually dimorphic func-
tions of non-reproductive functions and enhance prostate cancer growth.4 Besides the induction

Heterologous Modulation of Androgen Receptor Nucleo-Cytoplasmic Shuttling 139
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 evi-
dence indicate that the hormone may directly regulate prostate cell growth in vivo under con-
ditions 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 carci-
noma 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 de-
creases 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 con-
centrations 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 medi-
ated 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 tran-
scriptional 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 induc-
tion 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 treat-
ment (10 nM, 24 h) resulted in 7 fold induction of the reporter gene activity (Fig. 1). Melato-
nin (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 expres-
sion 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 promi-
nent at lower concentrations of androgen.18,19 This suggests that the two agents have contra-
dicting effects on an unidentified factor in the AR cascade, leading to diminution of melatonin
response at high androgen concentrations.
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. Immunore-
active 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 (unpub-
lished). 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 me-
latonin 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 experi-
ments 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 accu-
mulation 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 sus-
tained 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
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 pros-

tate 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 pre-
treatment with melatonin did not impair androgen binding capacity of the AR.19 The possibil-
ity 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.
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 ap-
peared 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
Leptomycin B completely blocked the melatonin-mediated nuclear exclusion of the AR indi-

cating 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 ex-
pression.
The intracellular signaling pathways mediating the nuclear exclusion of the androgen re-
ceptor (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 meta-
static 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 sec-
ond line treatments.27,28
It should be noted that in androgen-independent prostate carcinoma cells in vivo expres-
sion 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 abla-
tion 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 acti-
vated by endogenous hormones other than DHT. In addition, ligand independent mecha-
nisms 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
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 pro-
moter 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 re-
gardless 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 prolifera-
tion 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
A recent clinical study has shown that melatonin restores the sensitivity of metastatic pros-
tate 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 pre-
sumably 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 reac-
tions.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 spe-
cific 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.
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16. Gilad E, Laufer M, Matzkin H et al. Melatonin receptors in PC3 human prostate tumor cells. J
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17. Rimler A, Lupowitz Z, Zisapel N. Differential regulation by melatonin of cell growth and andro-
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18. Rimler A, Culig Z, Levy-Rimler G et al. Melatonin elicits nuclear exclusion of the human andro-
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19. Rimler A, Culig Z, Lupowitz Z et al. Nuclear exclusion of the androgen receptor by melatonin. J
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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 quan-
titatively 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 ac-
tion of aromatic amino acid decarboxylase (AAAD), Serotonin is converted into
N-acetylserotonin by the arylalkylamine-N-acetyltransferase (NAT) enzyme and then into me-
latonin 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 circa-
dian clock, situated in the hypothalamic suprachiasmatic nucleus (SCN)12 via norepinephrine
release from pineal sympathetic nerve endings. Norepinephrine as well as other pineal trans-
mitters (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

Extrapineal Melatonin: Location and Role in Pathological Processes 149
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 pro-
cesses 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 concentra-
tion 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 bio-
genic amines. Different types of cells widely dispersed throughout the organism have a com-
mon 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 Decar-
boxylation” 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 im-
munohistochemistry 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, neuropep-
tide 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 rhyth-
mic 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 com-
partments of melatonin synthesis.
The presence and rhythmical production melatonin in pineal gland have been well investi-
gated. As soon as highly sensitive techniques of analysis and identification became available,
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 seroto-
nin) was demonstrated.6,34,35,51,56,83,84,93 There are presented the evidences of melatonin syn-
thesis 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. Experi-
mental 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 mela-
tonin 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
Thus, the integrated application of methods of biological testing, thin-layer chromatogra-

phy, 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 distri-
bution 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 indi-
cate 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 me-
latonin 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 sub-
stances, acting within the nervous system as neurotransmitters and neurohormones; and, lo-
cally 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 patho-
logical processes in the organism. To ascertain of the morphologic fundamentals of the hor-
monal function in non-endocrine cells would enable to understand better the intercellular
mechanisms of the adaptation and compensation of the functional disorders appearing perma-
nently 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
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 estab-
lishment a normal sleep-wake cycle in children with neurologic syndromes, mental retarda-
tion, 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 dem-
onstrated 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 cyto-
static therapy, autoimmune and gastrointestinal diseases, pharmacological and toxicological influ-
ence, 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 approxi-
mately 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 num-
ber 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 ani-
mals 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 melato-
nin 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 tryp-
tophan 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
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 leuko-
cytes 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 gas-
trointestinal tract.95 Receptors for melatonin and enzymes involved in its synthesis from tryp-
tophan were also detected in all gastrointestinal tract tissues. Maximum amounts of this hor-
mone 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 gastrointesti-
nal 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 me-
latonin 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 func-
tions of gastrointestinal tract, microcirculation, and mucosal cell proliferation.
In vitro and in vivo experiments with animals showed that melatonin inhibits motor activ-
ity 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 synthe-
sis 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 dinitroben-
zene, 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
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 de-
creased incidence of ulceration, increased blood flow in the stomach wall, and normaliza-
tion 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 improve-
ment 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. Experi-
ments in animals showed that pinealectomy stimulates proliferative activity of cells in vari-
ous 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 neu-
rocrine 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 ex-
periments 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 mecha-
nism 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 stimula-
tion 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 sub-
mucosal 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 distur-
bances 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 func-
tion of controlling cell proliferation rates (especially those “out of step” with the biological
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 modu-
lation 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 re-
mains 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 me-
latonin 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 hista-
mine 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 prolif-
erative 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 anti-
gen), 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 deter-
mination 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 hu-
man 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
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 mecha-
nism of melatonin role in regulating proliferative activity of tumor cells remains unclear. Tak-
ing 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 mela-
tonin 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 stron-
ger.
The second variant. A decrease of urinary aMT6s excretion parallels a reduced secretion of
melatonin from cellular sources into blood. Melatonin binding in the tumor increases under
such conditions and via paracrine mechanisms results in suppression of tumor cell proliferation.
As compared with the first variant the second one is more favourable for the prognosis of the
patient. Hence it follows that the maintenance of urinary aMT6s within normal limits or
above in cancer patients could be regarded as an unfavourable sign for prognosis which may
gives evidence for defects within endogenous adaptative mechanisms.
Collectively, the data discussed above open promising perspectives for the elaboration of
new approaches for improvement of antitumor therapy, using the drugs which could change
the level of biologically active substances into tumours, particularly melatonin.
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CHAPTER 14
Sleep and Melatonin in Diurnal Species

Irina V. Zhdanova
Abstract
M
elatonin secretion, occurring at night in both diurnal and nocturnal species, provides an
important circadian signal for initiating different types of behavior. In diurnal
vertebrates, e.g., humans, macaques, zebrafish, this pineal hormone also has a pro-
nounced 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 mecha-
nisms 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 physiologi-
cal 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 con-
verted to serotonin (5HT) by a two-step process, catalyzed by the enzymes tryptophan hy-
droxylase 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 expo-
sure. 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

Sleep and Melatonin in Diurnal Species 163
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 associ-
ates 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 sleepi-
ness, 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, mela-
tonin 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 ampli-
tude 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 pro-
duce 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 treat-
ment 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 dura-
tion 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
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 physi-
ological 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 synchro-
nized 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 melato-
nin 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 sig-
nificant 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 manipu-
lation feasible. To have all these features in one laboratory animal species is problematic, how-
ever 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, impor-
tantly, 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 impor-
tant 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
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) diaz-
epam, 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 transpar-
ency 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 pharma-
cological treatments. Most importantly, an enormous accumulated experience in conducting
large-scale genetic screens and multiple mutant zebrafish phenotypes available,20 with the ge-
netic 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 under-
standing of melatonin’s action in various target cells. These receptors inhibit cAMP accumula-
tion 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 re-
main 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 depen-
dency, 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 concen-
trations (Fig. 3B,C). These differences between the effects of melatonin and those of the com-
monly 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
Figure 3. Melatonin and conventional sedatives promote rest behavior in larval zebrafish. Melatonin,
diazepam and sodium pentobarbital (barbital) significantly and dose-dependently reduced zebrafish loco-
motor 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 docu-
mented in multiple brain areas, including those involved in sleep regulation, e.g., hypothala-
mus 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 melato-
nin might result from an overall reduction in alertness or may entirely depend on the periph-
eral 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.
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 in-
duce 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 mela-
tonin treatment does not appear to either significantly affect normal nighttime sleep or in-
crease morning-after sleepiness. The effects of both physiological and pharmacological doses of
melatonin are not accompanied by any dramatic changes in electrophysiological sleep architec-
ture, i.e., temporal distribution and duration of different sleep stages. Similarly, we have re-
ported 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 seda-
tion, 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 Interest-
ingly, 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 develop-
ment 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 mecha-
nisms 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 treat-
ment could be of substantial assistance to those suffering from insomnia of different origin,
including age-related sleep disturbances or circadian rhythm sleep disorders.
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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hours before habitual bedtime, on sleep in normal young humans. Sleep 1996; 19(5):423-431.
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hours before habitual bedtime, on sleep in normal young humans. Sleep 1996; 19(5):423-431.
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 circa-
dian 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 im-
paired 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.

The Effect of Different Wavelengths of Light in Changing the Phase of the Melatonin Circadian Rhythm 171
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 expo-
sure 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, bed-
times 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.
time will be decreased. This will inevitably lead to excessive daytime sleepiness, fatigue, moodi-
ness 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 awak-
ening 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 symp-
toms 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 stud-
ies35,36 suggest that evening bright light before and after arriving at the destination of a west-
ward flight and conversely, morning bright light for an eastward flight should benefit the trav-
eller 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 physi-
cal 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 perfor-
mance, 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
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 Sea-
sonal 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 carbo-
hydrate 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 hypoth-
esis, 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 substanti-
ated.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 circa-
dian rhythm a light stimulus needs to be presented before the endogenous core body tempera-
ture 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 (in-
door) 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 differen-
tial 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 wave-
lengths 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 num-
ber 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
identified as the most potent wavelength region for regulating the human pineal gland. There-
fore, 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 differ-
ential 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 mecha-
nism 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 melato-
nin 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 under-
lying 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 be-
hind 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 re-
ceives 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 loca-
tion 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
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 sup-
pressing 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 sug-
gested 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 com-
pared 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 sup-
pressed 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
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 pace-
maker. At the level of the circadian clock, animal researchers have proposed that the mamma-
lian 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 circa-
dian times, melatonin phase delays were, on average, greater than phase advances on that first
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).
Figure 3C. Mean melatonin concentration for the blue LED (470 nm) group showing melatonin suppres-
sion 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). Simi-
larly, 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
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 wave-
length 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 sys-
tem. 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 Possibili-
ties 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 hu-
mans.70 This has been more recently supported by Rea and colleagues.106 However, after in-
ducing 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 in-
duced 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 How-
ever, this has tentatively been disputed by a further study which took into account neuroana-
tomical 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
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, there-
fore, are of potential clinical benefit.
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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 sub-
jected 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, endothe-
lium, 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 acti-
vate 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 Oxi-
dative 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 polysac-
charides 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 generat-
ing 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 erythro-
cyte 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 mem-
branes are rich in polyunsaturated fatty acids and because the human newborn, especially if

Clinical Utility of the Antioxidant Melatonin in the Newborn 185
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 mecha-
nisms, 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 coun-
tries. 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. pH in umbilical arterial cord blood < 7.00;
2. apgar score < 4 more than 5 minutes;
3. multiple organ failure, and
4. 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, nei-
ther 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 com-
mencing 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 chan-
nels, 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 radi-
cals, 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
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 deple-
tion 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 develop-
ment 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 dis-
turb 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 surfac-
tant 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 differ-
ence was not statistically significant. There exist, however, several potential causes of intracellu-
lar 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, cata-
lase, and glutathione peroxidase) or nonenzymatic (vitamins C, E, β-carotene, and allopu-
rinol).
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 natu-
rally 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; exog-
enously administered dietary antioxidants in high doses (vitamin C and β-carotene in particu-
lar) 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 interac-
tion 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 chro-
matography with electrochemical detection (HPLC-EC) and electron spin resonance spectros-
copy (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.
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 effi-
cient ˙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 in-
jury. 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 ONOO-
was 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 ancil-
lary in vitro study, this group also reported that melatonin prevented DNA strand breaks nor-
mally 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 pro-
duction.
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
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 addi-
tional 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 new-
borns 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). Mela-
tonin 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 adminis-
tration in the 74 RDS newborns enrolled in this study and also show that a significant incre-
ment 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 du-
ration 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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Diurnal 5-HT Production and Melatonin Formation 193
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 decarboxy-
lase (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 com-
parison, the regulation of TPH and its contribution in melatonin production is less well under-
stood. 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 sig-
naling 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 stimu-
late NAT transcription and increase NAT protein stability, which leads to increased metabo-

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, electrophore-
sed, 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.
Diurnal 5-HT Production and Melatonin Formation 195
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, acti-
vated alpha-adrenoceptor increases 5-HT secretion.1 Both types of adrenergic signaling effec-
tively 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 signal-
ing 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 regu-
lation. 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 adenos-
ine 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 pro-
tein kinase A. J Neurochem 1995; 65:882-888.
9. Aloyo VJ, Walker RF. Alpha-adrenergic control of serotonin release from rat pineal glands. Neu-
roendocrinology 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.
CHAPTER 18
Melatonin and Mitochondrial Respiration

Yuji Okatani, Akihiko Wakatsuki and Russel J. Reiter
Abstract
I
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 mito-
chondrial 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 melato-
nin 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 discov-
ery 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 ac-
tions 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 evi-
dence: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 and Mitochondrial Respiration 197
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 sep-
sis.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 Oxi-
dative 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 func-
tion is the efficiency of electron movement through the electron transport chain and its cou-
pling 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, corre-
sponding 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 mito-
chondria 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 mitochon-
dria.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 me-
tabolism. Recently, Martin and Acuna-Castroviejo and colleagues18 demonstrated that melato-
nin (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 Melato-
nin also prevented the decreases in the activities of these respiratory chain complexes induced
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 mito-
chondria from rat brain and liver. Oxidative stress induced by tert-butyl-hydroperoxide re-
duced 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 pathogen-
esis 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 metabo-
lize 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. Oxy-
gen 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 respira-
tory activity (Fig. 1).13 The ADP/O was calculated as the ratio of the added ADP concentra-
tion to the consumption of oxygen during State 3 respiration. Uncoupled respiration was in-
duced 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). Mito-
chondrial lipid peroxidation was elevated and GPx activity was decreased during ischemia/
reperfusion; these changes were counteracted in rats treated with melatonin (Table 1). Addi-
tionally, electron microscopic observation demonstrated that mitochondria from rats that un-
derwent 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.
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.
Table 1. Functional characteristics of rat liver mitochondria during ischemia and

subsequent reperfusion
Melatonin +
Ischemia/ Ischemia/
Characteristic Control Ischemia Reperfusion Reperfusion
RCI 4.13 ± 0.15 (11) 1.22 ± 0.04 (11)** 1.55 ± 0.18 (11) 2.55 ± 0.26 (11)††
ADP/0 2.55 ± 0.06 (11) 1.44 ± 0.19 (11)** 1.71 ± 0.07 (11) 2.14 ± 0.09 (11)††
State 3 24.76 ± 0.99 (11) 11.14 ± 1.13 (11)** 12.89 ± 0.91 (11) 18.29 ± 1.98 (11)†
respiration
State 4 6.24 ± 0.16 (11) 8.95 ± 0.25 (11)* 7.83 ± 0.42 (11) 6.86 ± 0.29 (11)
respiration
DNP uncoupled 21.05 ± 0.49 (11) 10.54 ± 0.63 (11)** 9.70 ± 0.94 (11) 14.45 ± 1.88 (11)†
respiration
TEARS 1.481 ± 0.07 (11) 1.52 ± 0.07 (11) 2.10 ± 0.13 (11)** 1.66 ± 0.08 (11)†
GPx activity 5.65 ± 0.36 (11) 4.86 ± 0.44 (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 de-
rived from its stimulatory effects on GPx and SOD. PTP opening is triggered by the associa-
tion of calcium overload with an inducer, such as oxidative stress or high phosphate concentra-
tion, conditions encountered during ischemia/reperfusion. The opening of this pore leads to
the destruction of the mitochondrial membrane potential and mitochondrial swelling, result-
ing in mitochondrial uncoupling and inhibition of ATP synthesis. Although the mitochon-
drial 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.
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 ex-
perimental 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 predomi-
nance 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 mito-
chondrial dysfunction in the processes of aging.7,8 Since the mitochondria are the site of utili-
zation of the bulk of the inspired oxygen, they are also the site of abundant free radical genera-
tion.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 mitochon-
drial 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.
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 mela-
tonin. 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.
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 investi-
gated 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.
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 sig-
nificantly increased RCI, State 3 respiration, DNP-dependent uncoupled respiration, and com-
plex 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 demon-
strated 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 reduc-
tion 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 trans-
port 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 func-
tion 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
Figure 8. Schematic diagram of the hypothesized melatonin involvement in mitochondrial electron trans-
port system and oxidative phosphorylation. Melatonin easily crosses biological membranes to reach the
mitochondrial matrix. Melatonin can scavenge oxygen free radicals directly. Additionally, melatonin’s an-
tioxidant 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 mitochon-
dria, 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 mitochon-
drial 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 oxida-
tively 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 anti-
oxidant 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 un-
known. 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 me-
latonin may be another indirect way by which melatonin limits molecular destruction of essen-
tial molecules by reactive oxygen species.
Another possible mechanism for the protective effects of melatonin on mitochondrial func-
tion may involve the stabilization of mitochondrial membranes. Melatonin reportedly stabi-
lizes cell membrane fluidity, thereby preserving the dysfunction of these structures.47,48 Mela-
tonin 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 mecha-
nism 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 reduc-
ing 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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Melatonin and Bone Physiology 209
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. Treat-
ment 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 re-
sorption. Additionally, melatonin could impair osteopenia by improving slow sleep and restor-
ing growth hormone secretion. The feasibility of the use of melatonin in therapy for augment-
ing 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 os-
teoblasts 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 in-
volved 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 treat-
ment 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,

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 mela-
tonin, 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 produc-
tion without modifying alkaline phosphatase or osteocalcin.21
Several osteoblast-derived local factors control osteoclast activity. One of them is the osteo-
clast 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 phos-
phatase 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 indicat-
ing 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, mela-
tonin stimulates several antioxidative enzymes that increase its efficiency as an antioxidant.
Melatonin protects macromolecules in all compartments of the cell making them more resis-
tant 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. Experimen-
tal 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 heteroge-
neous 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 modu-
lating 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
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 biochemi-
cal 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 re-
sorption) 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. Uri-
nary deoxypyridinoline increased significantly after ovariectomy an effect prevented by mela-
tonin 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 ovariec-
tomy 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 remodel-
ing 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 col-
lagen concentrations, as well as the urinary excretion of calcium and hydroxyproline were mea-
sured. The study demonstrated that pinealectomy augmented bone resorption while melato-
nin 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 re-
duced bone resorption parameters without affecting bone formation. Therefore, although me-
latonin increases the proliferation, differentiation, and bone nodule formation activity of os-
teoblasts 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 skel-
eton 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
melatonin levels), providing that appropriate levels of circulating estradiol were present. More-
over, 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 mani-
fested.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 fragil-
ity, 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.
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latonin 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 dy-
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27. Reiter RJ, Tan DX, Allegra M. Melatonin: Reducing molecular pathology and dysfunction due to
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28. Reiter RJ, Tan DX, Qi W et al. Pharmacology and physiology of melatonin in the reduction of
29. Roth JA, Kim BG, Lin WL et al. Melatonin promotes osteoblast differentiation and bone forma-
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30. Sack RL, Lewy AJ, Erb DL et al. Human melatonin production decreases with age. J Pineal Res
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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 con-
sumption 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 re-
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mans. Psychoneuroendocrinology 1979; 4:351-362.
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 melato-
nin (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 sug-
gested that a periodic central disturbance of hypothalamic activity could account for the peri-
odicity 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 mecha-
nisms. Migraine patients are sensitive to light, even at the interictal period, and display photo-
phobia. 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

Melatonin, Light and Migraine 215
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 relation-
ship 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, tem-
perature, 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 ana-
tomical or pathophysiological data reported in humans.9 Results have been obtained in ani-
mals, 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 originat-
ing 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 explana-
tion for the hemicranial distribution of migraine. With regard to the parasympathetic innerva-
tion, 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 vasomo-
tor 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 ob-
served.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).
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 mela-
tonin 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 aggra-
vated 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 ten-
dency 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 path-
ways.28 Electrophysiological investigations suggest that it occurs at the cortical level. The ob-
served 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 ad-
dition, 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 mi-
graine. 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 hypofunc-
tion 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 ca-
rotid 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
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, espe-
cially 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 compat-
ible 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 in-
volved both in long-term phase-adjustments and rapid changes of light-induced pineal me-
tabolism.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, head-
ache 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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CHAPTER 21
Melatonin in Protection against Oxidative

Damage Caused by Potential Carcinogens
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 stud-
ies, 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 con-
ditions; they are, however, effectively counteracted by natural defense mechanisms.1 Any inter-
nal 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 conse-
quence, 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, oxida-
tive 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 mecha-
nisms.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 metabo-
lites of the damage to macromolecules and, in this way, protect against cancer.1

Melatonin in Protection against Oxidative Damage Caused by Potential Carcinogens 221
Exogenous or endogenous factors are classified as carcinogens on the basis of epidemiologi-

cal 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 superox-
ide dismutase (SOD), γ-glutamylcysteine synthetase, glutathione peroxidase (GSH-Px), glu-
tathione 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 reac-
tion 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 reac-
tion—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 antioxi-
dants—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 effec-
tively 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
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 admin-
istration of melatonin to organisms decreases organ susceptibility to oxidative stress after tis-
sues 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
Dose of Melatonin
Species/Organ/ Which Reduced or
Tissue/Cellular Effect of Prevented the Effect
Compartment Carcinogen/Dose Carcinogen of Carcinogen Ref.
Male Sprague No treatment ↑ MDA+4-HDA 15 mg/kg b.w., 26

Dawley rats; 2x daily, 8 days
hepatic homogenates
exposed to FeSO4
(15 µM) + H2O2
(0.1 mM)
Male Sprague Ferrous citrate, ↑ MDA 60 µg infused 27
Dawley rats; 3.4 mM of iron ↓glutathione into substantia nigra
substantia nigra infused into or 10 mg/kg b.w.,
substantia nigra systemically, 7 days
Male Sprague Ferric nitrilotriacetate ↑ nuclear 25 mg/kg b.w., 30 min 32
Dawley rats; kidney (Fe-NTA), 15 mg 8oxodGuo before Fe-NTA
Fe/kg b.w. ↑ MDA+4-HDA 50 mg/kg b.w., 30 min
before Fe-NTA
Male Syrian hamsters Cadmium chloride ↑ MDA+4-HDA 6 x 15 mg/kg b.w. 39
Brain, heart, (CdCl2), 1 mg/kg 6 x 5 mg/kg b.w.
lung b.w. 1 x 15 mg/kg b.w.
kidney ↑ MDA+4-HDA 1 x 15 mg/kg b.w.
Male Wistar rats; Potassium bromate ↑ nuclear 10 mg/kg b.w., 41
kidney (KBrO3), 80 mg/kg 8oxodGuo every 6 h for 24 h
b.w. before KBrO3 inj.
Male Sprague Ionizing radiation ↑ nuclear 4 x 50 mg/kg b.w., 47
Dawley rats; liver (IR), 800 cGy 8oxodGuo every 30 min before
↓ microsomal MF exposure to IR
Male Syrian hamsters 17β-estradiol (E2), ↑ nuclear 15 mg/kg b.w., 0.5 h 51
Kidney collected 5 h 75 mg/kg b.w. 8oxodGuo before and 2 h after
after E2 treatment E2 treatment
Liver ↑ nuclear
collected 3 h after 8oxodGuo
E2 treatment
Male Syrian hamsters 17β-estradiol (E2), ↑ nuclear 15 mg/kg b.w., 52
Kidney collected 5 h 75 mg/kg b.w. 8oxodGuo 2 h and 0.5 h
after E2 treatment before and 2 h
and 4 h after
E2 treatment
Table 1. Continued
Dose of Melatonin
Species/Organ/ Which Reduced or
Tissue/Cellular Effect of Prevented the Effect
Compartment Carcinogen/Dose Carcinogen of Carcinogen Ref.
Male Sprague Dawley δ-Aminolevulinic **10 mg/kg b.w., 55

rats acid (ALA), 3x daily (15 days) 56
Liver, kidney, 40 mg/kg ↑ nuclear 8oxodGuo 57
lung, spleen b.w. every second
Liver, kidney day for 15 days ↓ microsomal and
mitochondrial MF
Hepatic microsomal ↑ MDA+4-HDA
membranes, lung
spleen, blood serum
Male Sprague Phenylhydrazine 15 mg/kg b.w., 26
Dawley rats (PHZ) 2 x daily, 8 days
Spleen, blood serum 15 mg/kg b.w., ↑ MDA+4-HDA
Liver 7 days ↓ microsomal MF
Male Sprague Safrole, ↑ DNA adducts 0.2 mg/kg or 61
Dawley rats; liver 300 mg/kg b.w. 0.4 mg/kg b.w.,
15 min before, 3 h
and 20 h after
safrole inj.
Male Sprague Dawley Safrole, 100 mg/kg ↑ DNA adducts 4 x 0.15 mg/kg b.w., 62
rats; liver from b.w. s.c., every 2 h, first
pinealectomized dose 15 min
animals before safrole
↑ DNA adducts Physiological
concentration of
melatonin due to
its higher night
secretion by the
pineal gland
Male Sprague Dawley 2-Nitropropane ↑ MDA+4-HDA 2.5, 5.0, 10.0 mg/kg 65
rats; liver, lung, (2-NP), 4 mmol/ b.w., 0.5 h before
kidney kg b.w. 2-NP treatment
Male Wistar rats; Phosphine (PH3), ↑ nuclear 10 mg/kg b.w., 69
Brain and liver 2 mg/kg b.w. 8oxodGuo 30 min before PH3
Brain, lung and liver ↑ MDA+4-HDA injection
Female mice, liver 7,12-dimethylbenz ↓ GSH-Px, SOD, 4.2 mg/kg b.w., 71
(a)anthracene ↓ CAT 21 days,
(DMBA), 20 mg/kg simultaneously
b.w., 21 days with DMBA
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).
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 in-
creased 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 pro-
duction 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 accu-
mulates 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 dam-
age 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 car-
cinogen in animal models.40 An increased level of 8oxodGuo in rat kidney, due to bromium
treatment, was effectively reduced by melatonin (Table 1).41
Mercury
Mercury—a heavy metal—reveals numerous toxic effects to organisms.42 It is likely in-
volved in the pathomechanism of Alzheimer’s disease. Its cytotoxic effects in SHSY5Y neuro-
blastoma 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 Numer-
ous 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
Species/Tissue/ Carcinogen/ Effect of Reduced or Prevented
Cell/Compartment Concentration Carcinogen the Effect of Carcinogen Ref.
Rat liver Ferrous sulfate ↑ MDA+4-HDA 0.4, 0.8, 1.2 22

homogenates (FeSO4) (15 µM) + and 1.6 mM
H2O2 (0.1 mM)
Monkey liver Ferrous sulfate ↑ MDA+4-HDA 0.4, 0.5, 0.6, 23
homogenates (FeSO4) (15 µM) + 0.8 and 1.0 mM
H2O2 (0.1 mM)
Hamster testis Ferrous sulfate ↑ MDA+4-HDA 1.0, 2.0, 2.5, 24
homogenates (FeSO4) (30 µM) + 5.0 mM
H2O2 (0.1 mM)
Male Sprague Ferrous citrate, ↑ MDA 0.5, 1.0, 2.0, 27
Dawley rats, 1.0 µM 3.0 and 4.0 mM
cortical
homogenates
Male Sprague Ferric nitrilotriacetate ↑ MDA+4-HDA 0.5, 1.0, 2.0, 32
Dawley rats, (Fe-NTA) (50 µM) 4.0 mM
kidney
homogenates
Male Sprague Ferric chloride ↓ MF 0.3, 1.0, 3.0 mM 28
Dawley rats FeCl3 ↑ MDA+4-HDA 29
Hepatic (0.2 mM)+ ADP 30
microsomes (1.7 mM)+ NADPH
(0.2 mM)

Table 2. Continued
Concentration of
Melatonin Which
Species/Tissue/ Carcinogen/ Effect of Reduced or Prevented
Cell/Compartment Concentration Carcinogen the Effect of Carcinogen Ref.
Wistar rat Potassium dichromate 34

hepatocytes [K2Cr2O7; Cr6+]
2.5 µM ↑ DNA single- 0.5, 1.0 mM
strand breaks
0.5 µM ↑ LDH leakage 0.5, 1.0 mM
from cells
0.5 µM ↑ MDA 0.5, 1.0 mM
0.5 µM ↓ Vit. C, E 1.0 mM
0.5 µM ↓ CAT 0.5 mM
Purified calf Chromium chloride ↑ 8oxodGuo 0.25, 0.5, 1.0, 35
thymus DNA (CrCl3) [Cr(III)] 2.5, 5.0, 10.0 µM 36
(0.5 mM) + H2O2
(0.5 mM)
Purified calf Chromium chloride ↑ 8oxodGuo 0.05, 0.1, 0.5, 37
thymus DNA (CrCl3) (Cr3+) 1.0, 10.0 µM
(0.5 mM) + H2O2
(0.5 mM)
Purified monkey Chromium chloride ↑ 8oxodGuo 10.0 µM 24
liver DNA (CrCl3) (Cr3+)
(0.5 mM) + H2O2
(0.5 mM)
SHSY5Y Mercury chloride ↓ glutathione 1.0 µM (30-min- 43
neuroblastoma (HgCl2) (180 nM) preincubation)
cells
SHSY5Y Cobalt chloride ↓ glutathione 1.0 µM (12-hour 45
neuroblastoma (CoCl2) (0.3 mM) preincubation)
cells
Human skin X-ray irradiation, ↑ MDA 0.1 mM 48
fibroblasts 8 Gy
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 car-
cinogenesis; this animal model shares numerous mechanistic features with estrogen-related
tumors in human females, making its use appropriate for investigating the mechanisms of
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 concen-
tration is found in patients suffering from inherited or acquired porphyrias—acute intermit-
tent 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 accumu-
lation 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 carcino-
genesis. In several studies, ALA has been shown to induce oxidative damage to macromol-
ecules, 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 pre-
vented 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 tox-
ins 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 physi-
ological concentrations, protected—in dose dependent manner—against safrole-caused DNA
damage.61,62 Safrole, injected at night, when the blood concentration of melatonin is physi-
ologically 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 physi-
ological concentrations, prevented the oxidative damage, exerted by the carcinogen, when used
in pharmacological concentrations.
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 impor-
tant 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 hydro-
carbons 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 pre-
vented 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 develop-
ment, 6-sulphatoxymelatonin excretion is abolished,72 constitutes an additional piece of evi-
dence for melatonin treatment under conditions of increased exposure to carcinogens—to
prevent cancer initiation.
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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 per-
haps to modulate neuronal activity in response to signals from the photoreceptors. The retinal
photoreceptors themselves express melatonin receptors and are therefore target cells for mela-
tonin 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 ph-
agocytosis of photoreceptor outer segment disks as part of a renewal process, modulate neu-
rotransmitter 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 experimen-
tally 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 in-
volved in dark adaptation. The sites of action of retinally-synthesized melatonin are the photo-
receptor 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 com-
pelling 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 benefi-
cial 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.

Influence of Melatonin on the Health and Diseases of the Retina 233
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 synthe-
sized 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 en-
zyme, 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 radioac-
tive 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 pertus-
sis 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
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, respec-
tively. 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 in-
tracellular signaling due to coupling to different effectors, and (4) selective regulation of signal-
ing 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 immuno-
cytochemical 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 dem-
onstrated 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 pharmacologi-
cal 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 melato-
nin 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, investi-
gators 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 sensitiv-
ity 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 pro-
duced 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
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 permis-
sion 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 local-
ization 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
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 photo-
receptor 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 ex-
press 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 horizon-
tal 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 activ-
ity 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 hori-
zontal 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
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 con-
firmed in humans (MT1),30 and Mel1a receptor mRNA was localized to chicken photorecep-
tors.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 photorecep-
tors,10,36,44,54,55 intracrine melatonin signaling in photoreceptors likely contributes substan-
tially 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 photorecep-
tor 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 photore-
ceptor 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 sensi-
tive 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 conse-
quence 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 path-
way, 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 regula-
tion 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 sug-
gest 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.
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 Melato-
nin 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 contrib-
ute to the etiology of age-related macular degeneration.69,70 Melatonin produced by the photo-
receptors 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 chal-
lenged 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
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 melato-
nin 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 spe-
cific 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 sur-
vival.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
whether alterations in normal melatonin levels may contribute to a disruption of normal reti-
nal 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 me-
latonin 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 degenera-
tions that ensue from the death of photoreceptors. We propose that endogenous retinal mela-
tonin 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 pho-
toreceptors to light-induced damage.
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Melatonin Synchronizes Cell Physiology through Cytoskeletal Rearrangements 243
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 cul-
ture 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 organiza-
tion 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 microfila-
ment 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 activ-
ity 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

Moreover, the hormone inhibits calmodulin-dependent enzyme activities such as cAMP phos-
phodiesterase activity in vitro,11 Ca2+ Mg2+-ATPase,12 nitric oxide synthase13 and calmodulin ki-
nase 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 hor-
mone.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 incuba-
tion 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 mela-
tonin 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 addi-
tion, 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 reorga-
nization 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 melano-
phores;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 de-
scribed.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 Melato-
nin is a lipophylic molecule that crosses the plasmatic membrane and it has been found distrib-
uted 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 kid-
ney transporting epithelia since they form epithelial cell monolayers that resemble the interca-
lated 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 cul-
tured 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 capa-
bility 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
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 syn-
chronizes 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 de-
graded 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 mela-
tonin 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 mela-
tonin 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 mono-
layers cultured only with the vehicle.28 By 9 and 12 h after melatonin incubation, dome forma-
tion 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
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 mela-
tonin 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 incuba-
tion 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 corre-
lates 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 com-
ponents regulates the sealing of the tight junctions and the paracellular pathway of ion trans-
port.29 In the presence of cytochalasin B, cytoplasmic and cortical microfilaments are dis-
rupted, 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 path-
way 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
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 Incuba-
tion 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 path-
way, 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. Alto-
gether, these data suggest that melatonin effects on microfilament organization are related to
the melatonin action on vectorial water transport in MDCK cells.
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 incu-
bated 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+ AT-
Pase 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. Further-
more, 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 ob-
served.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 inhibi-
tors 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 in-
volved 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 frac-
tion to the cytosol in an initial period of melatonin incubation, and decreased levels were
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. Fluores-
cent 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 to-
gether 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 adhe-
sion 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 hor-
mone 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
Figure 5. Schematic representation of the mechanisms by which melatonin causes microfilament reorgani-
zation 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 phospho-
rylation 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 synchro-
nized 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
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 ac-
tin 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 fellow-
ship from CONACYT for GRR.
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Melatonin in Winter Depression 253
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 circa-
dian phase. Moreover, seasonal variations in many human functions has been widely acknowl-
edged. 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 mea-
surement 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 patho-
physiology 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 pathophysi-
ology 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 circa-
dian 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 affec-
tive 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 depres-
sion—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 symp-
toms as fatigue, social withdrawal, oversleeping, overeating, carbohydrate craving and weight

gain.5 These symptoms are considered atypical, in contrast with insomnia, early morning wak-
ening, 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 envi-
ronment.
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 pathophysiologi-
cal 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 circa-
dian 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 antide-
pressant response was demonstrated in SAD patients treated in early morning with a short
acting beta-adrenergic blocker, propranolol. The subsequent study35 provides additional sup-
port 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
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 lengthen-
ing 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 pa-
tients 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 sum-
mer.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 under-
stand 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 experi-
ments did not confirm the assumption that the extension of day length is critical for the anti-
depressant effect of bright light, another chronobiological response to light—correcting of
abnormally phased circadian rhythms—was proposed and intensively tested as a possible mecha-
nism 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
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 morn-
ing 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 fol-
lowing 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 contro-
versial 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 adminis-
tered 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 repli-
cated 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 dif-
ference between placebo and MLT conditions was found.
The typical for mammal’s phase-response curve (PRC) suggests that only weak, if any, thera-
peutic response to bright light in the middle of the day, because such a treatment fails to extend
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 im-
provement 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 posi-
tive 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 recom-
mend 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 differ-
ential 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 pro-
nounced 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 ex-
cluded 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.
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 neces-
sarily 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 be-
tween seasonal changes in photoperiod and symptoms of SAD, the significant differences be-
tween 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 an-
nual 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 physiologi-
cal 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 abnormal-
ity, 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 sea-
sonal variations in affective disorders. A question about the role of photoperiodic time mea-
surement 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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A Melatonin Onset Disorder 263
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 find-
ing 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. Conse-
quently 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 signifi-
cance.
Clinical Aspects of DSPS

The characteristic combination of symptoms, described by the discoverers of DSPS in-
cluded: (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

264 Melatonin: Biological Basis of its Function in Health and Disease
problem. Neither gender-related nor psychiatric disorder dependencies were noted. The pio-
neer 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 re-
ported 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 adoles-
cence; 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 irri-
table bowel syndrome and inflammatory bowel diseases, as opposed to only 7.1% of the con-
trols (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 im-
pact 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 popu-
lation. 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 un-
known.
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 disor-
ders (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 them-
selves, 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 atti-
tudes 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. Treat-
ment 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 begin-
ning 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 evi-
dence 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 pa-
tients 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 melato-
nin 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 tempo-
rary 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
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 tem-
perature. 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 tempera-
ture. 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, Vita-
min 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 stud-
ies.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 ef-
fects. 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
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 idio-
pathic 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
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”) endog-
enous circadian cycle that is longer than 24 hours.5 This cycle induces progressively later bed-
times 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 correc-
tional shift of the sleep phase back to earlier hours for work-days particularly difficult. There-
fore, 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 physi-
ological 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 in-
volved 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. Be-
tween 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 melato-
nin 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.
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.
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. There-
fore, 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 child-
hood 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 misdiag-
nosis 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) endog-
enous melatonin onset may strongly help the diagnosis and the treatment of DSPS. The diag-
nosis 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 melato-
nin 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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Melatonin as an Antidepressant 273
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 circa-
dian 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 ef-
fect.62 Sleep deprivation in the second (but not first) half of the night was somewhat antide-
pressive 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

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 exacer-
bates 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 depres-
sion, the diurnal mood variation with better mood in the evening might induce delayed bed-
times and delayed rise times. However late sleeping itself may exacerbate or precipitate depres-
sion25,55,62 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 adminis-
tered.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 appropri-
ate 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 adminis-
tration 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 depres-
sion. 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 pla-
cebo (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 dura-
tion from the 1st sleep cycle towards the end of the night. On the baseline and placebo
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 circa-
dian 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 underly-
ing 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 de-
pression. 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 melato-
nin 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 distur-
bance 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 melato-
nin 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 hor-
mone 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 wide-
spread 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
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 com-
pared 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 appro-
priately 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 Cadmium (Cd) 222, 224
Adenosine kinase (AK) 27, 28 Caenorhabditis elegans 51
Adolescent 35, 270, 273 Calcitonin gene-related peptide (CGRP) 3, 4,
Adolescent idiopathic scoliosis (AIS) 35-38, 215
41, 42 Calcium channel 28, 64, 88, 93, 95, 98
Advanced sleep phase syndrome (ASPS) Calcium oscillations 95, 96, 99
170-172, 180, 265 Calmodulin 42, 54, 55, 64, 243, 244
Age-related disease 118, 119, 122-124 Cancer initiation 220, 221, 228
Aging 45, 48, 52, 60, 87, 118-122, 124, 196, Capsaicin 108
197, 201, 202, 204, 205 Carcinogen 48, 52, 72, 220-228
Alpha-adrenergic signaling 193 Carcinogenesis 28, 52, 55, 155, 220, 221,
Alzheimer’s disease 119, 122, 123, 152, 206, 226-228
225 Cardiovascular system 60, 65
Androgen 73, 74, 91, 138-145 Carrageenan-induced acute inflammation 130
Androgen receptor (AR) 6, 138-145 Catecholamine 66, 149
Antioxidant 12, 13, 15, 16, 20-22, 28-30, Cell proliferation 54, 55, 144, 152-156, 210
52-55, 63-66, 122-124, 127-131, Ceruloplasmin 184
152-154, 184, 186-189, 196-198, Cervical cancer 76
200-202, 205, 206, 209, 210, 212, 220, Chicken 35, 38-42, 91, 110, 152
221, 224, 238, 239 Cholera toxin (CTX) 140, 141
Apoptosis 30, 53, 129, 184, 185, 201, 237 Cholesterol 60-63, 65, 66
APUD 149, 151, 152, 155 Chromium (Cr ) 52, 224, 226
Arvicanthis ansorgei 2 Chronic lung disease (CLD) 186
Chronobiotic 113, 217, 263
B Circadian rhythm 38-41, 45, 51, 61, 71, 72,
76, 79, 80, 88, 89, 91, 92, 106-108, 111,
β-adrenoceptor blocker 62 113, 114, 118-121, 123, 124, 148, 150,
Beta-adrenergic signaling 193 152, 153, 162-164, 166, 167, 170-176,
Beta-carotene 64 178-180, 210, 215, 217, 232-234, 237,
Biological clock 119, 266, 268, 270 239, 250, 253-258, 263-268, 270,
Biological rhythm 88, 152, 154, 243, 270, 273-276
274 Cobalt (Co) 225, 226
Bone alkaline phosphatase 211 Colorectal cancer 77
Bone remodeling 209, 211 Corticotropin-releasing hormone (CRH) 62,
Brain 12, 13, 16-19, 23, 30, 38, 47, 53, 75, 122
90-92, 109-113, 123, 127, 164-167, 184, CREB 6, 7
185, 188, 193, 196-198, 200, 215, 217, Cyclic AMP 27, 144, 233, 236
222-224, 228, 244, 265, 268 Cyclooxygenase-2 (COX-2) 17, 132-134
Breast cancer 54, 72-76, 80, 224 Cytoskeleton 244, 246, 247, 251
Bright light treatment (BLT) 253-258, 266,
270
Bromium 224
D G
7,12-dimethylbenz(a)anthracene (DMBA) 53, γ-interferon 53, 78
55, 72, 73, 78, 79, 223, 228 GABA 3, 7, 111, 112, 165, 217, 234, 236
δ-aminolevulinic acid 223, 227 Gene expression 7, 9, 54, 94, 134, 138, 139,
Delayed sleep phase syndrome (DSPS) 171, 141, 143, 145, 184, 210
174, 263-268, 270, 273-276 Glutamate (Glu) 3, 4, 7, 198, 199, 217
Diabetes 46, 55 Glutathione peroxidase (GPx) 13, 16, 19, 20,
Diffuse neuroendocrine system (DNES) 149, 52, 54, 65, 122, 130, 185, 186, 196, 198,
151 200, 202-204, 221, 223, 239
Diurnal species 111, 163, 164, 167 Glutathione reductase (GRd/GSH-Rd) 13,
DNA 19, 27, 28, 31, 52, 53, 122, 127, 19, 20, 52, 122, 130, 154, 196, 198, 221
129-131, 133, 138, 139, 141, 143, 144, GnRH receptor 88, 92, 93, 99, 101
184, 188, 197, 200-202, 205, 220, 223, Gonadotrophs 88, 91-101
224, 226-228, 243 Gonadotropin-releasing hormone (GnRH)
DNA damage 52, 53, 184, 200-202, 220, 227 54, 88-101
Dose-dependency 162, 165 Growth 26-29, 31, 35, 36, 38, 39, 41, 42, 54,
Drosophila melanogaster 51 55, 71-75, 78-81, 88, 118, 128, 138,
139, 143, 144, 151, 152, 155, 156, 209,
E 210, 212, 264
Growth hormone (GH) 35, 42, 65, 96, 209,
17β-estradiol 74, 222, 226 212, 264
EC cell 150, 151, 155
Electron transport system 196, 197, 204, 205 H
Endometrial cancer 73, 74, 76, 80
Endothelin-1 64 5-hydroxytryptamine 4, 193
Enterochromaffin cells 149, 150, 152 5-hydroxytryptophan (5-HT) 4, 5, 148-151,
Estradiol 64, 65, 73, 74, 209-212, 222, 226, 162, 193, 195, 255
227 Heart 12-16, 19, 22, 36, 54, 55, 60-62,
Ethanol 45, 108, 153, 154 64-66, 153, 222, 224
European hamster 2, 4, 7-9 Hemithyroidectomy 28
Hepatic ischemia/reperfusion 198
F High-density lipoprotein (HDL) 62, 66
Hormone 1, 3, 4, 22, 26-31, 35, 36, 40, 42,
Ferric nitrilotriacetate (Fe-NTA) 222, 224, 45, 46, 51, 54, 60-62, 64, 65, 71-77,
225 79-81, 88, 90, 92, 98-101, 106, 109,
Fetal ischemia 200 110, 121, 122, 124, 138, 139, 141, 143,
Focal adhesion contact 247, 249 148-155, 162-167, 209, 212, 215, 217,
Free radical 12, 14-16, 19, 21, 22, 28-30, 47, 232, 243-247, 249, 251, 254, 264, 266,
51, 52-54, 63, 65, 73, 118, 119, 122, 268, 275, 276
127, 129-132, 134, 152, 154, 184-188, Hormone replacement therapy (HRT) 64,
196-198, 200-202, 204-206, 209, 210, 123
217, 220, 221, 225, 227, 238, 239, 243 Hormone secretion 90, 209
Free radical scavenger 12, 22, 29, 52, 118, Hydrochloric acid 154
119, 122, 152, 187, 196, 200, 201, 206, Hydrogen peroxide (H2O2) 29, 30, 52, 64,
209, 210, 220, 221, 243 127, 130, 131, 185, 187, 188, 196, 197,
Funambulus pennanti 27, 29 205, 221, 222, 224-226, 238
Hydroxyindole-O-methyltransferase
(HIOMT) 4-9, 31, 148, 150, 151, 162,
193, 233
Index 281
Hydroxyl radical 13, 15, 22, 28, 29, 63, 64, Light emitting diode (LED) 174-180
122, 127, 130, 133, 185, 187, 196, 221, Lipid peroxidation 15-17, 19, 21, 22, 30, 52,
238 63, 65, 123, 127, 132, 185, 187-189,
Hypercholesterolemia 60, 62 197, 198, 200, 203, 220-222, 224,
Hypertension 60, 62, 63, 65, 66 226-228
Hypnotic 107, 112, 113, 165, 167 Low-density lipoprotein (LDL) 60, 62, 63,
Hypocretin (HCRT) 3, 4 65, 66
Lung carcinoma 48
I Luteinizing hormone-releasing hormone
(LHRH) 92
Immunocompetence 118, 119, 122 Lymphokine activated killer (LAK) 54
Indian palm squirrel 27 Lymphoma 47, 48, 228
Indoloamine 64
Inflammation 127-134, 150, 186, 188, 189, M
214, 217
Inflammatory bowel disease 132, 264 Macaque 162, 164, 165, 167
Insomnia 107, 113, 121, 122, 164, 167, 171, Macrophage 54, 78, 127, 128, 130-132, 184,
172, 212, 254, 263, 264, 267, 273 185, 197
Intercellular adhesion molecule (ICAM) 21, Macrophage-colony stimulating factor
132, 134 (M-CSF) 54
Interleukin-1 (IL-1) 53, 184 MDCK 243-250
Interleukin-2 (IL-2) 75 Melatonin 1-9, 12-22, 26-31, 35-37, 39-42,
Interleukin-4 (IL-4) 53, 128 45-55, 60-66, 71-81, 88-92, 94, 96-101,
Interleukin-6 (IL-6) 54, 143, 184, 189 106-114, 118-124, 127, 129-134,
Interleukin-12 (IL-12) 54 138-145, 148-156, 162-168, 170-180,
Intermediate filament 244 187-189, 193, 195-206, 209-212,
Ionizing radiation (IR) 28, 55, 152, 155, 222, 214-217, 220-228, 232-240, 243-251,
223, 225, 226 253, 254, 256, 263-270, 274-276
Iron 65, 127, 133, 184, 185, 221, 222, 227 Melatonin 1 (MEL1) 110, 111
Ischemia/reperfusion (I/R) 12-22, 60, 64, Melatonin 2 (MEL2) 110, 111
129, 132, 154, 185, 188, 196-198, 200 Melatonin receptor 41, 48, 53, 61, 63, 64, 73,
74, 88-92, 97-101, 109-111, 133, 139,
J 140, 144, 153, 162, 164-167, 210,
232-237, 243
Jet lag 107, 170, 172, 180, 265 Mercury 225, 226
Microfilament 243, 244, 246-250
K Microtubule 244
Middle cerebral artery occlusion (MCAO)
Karyometry 28 16-18
Kennedy’s disease 138, 143, 145 Migraine 214-217, 264
Mitochondria 13, 22, 52, 54, 94, 127-131,
L 153, 189, 196-201, 203-206, 223, 227
MT1 and MT2 receptor 89-91
Langendorff rat heart model 13 Mutagenesis 52
Larynx cancer 77 Myocardial infarction 16, 61, 65, 66
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
N Pertussis toxin (PTX) 88, 92, 97, 99, 100,

140, 141, 233
2-Nitropropane (2-NP) 223, 228 Phases 37, 55, 71, 74, 258, 268, 273
N-acetyltransferase (NAT) 5-9, 31, 51, 78, Phenylhydrazine (PHZ) 223, 227
88, 148, 150, 151, 162, 193, 233, 234 Phosphine (PH3) 223, 228
Neonatal sepsis 186 Phospholipase C (PLC ) 6, 90, 92, 98, 99,
Neurodegenerative disease 119, 123 144, 168
Neuropeptide Y (NPY) 3, 4, 7, 148, 149 Photoperiodic variation 2, 8, 148
Night shift 80, 172, 180 Photoreceptor 150, 162, 173, 179, 217,
Nitric oxide (NO• ) 13, 17, 19, 20, 30, 52, 232-240
63, 64, 66, 127, 128, 133, 188, 196, 221, Photoreceptor cell 150, 232, 234, 235, 237
244 Pineal gland 3-9, 16, 17, 26, 27, 31, 35,
Nitric oxide synthase (NOS) 13, 19, 20, 30, 38-40, 45, 48, 71-73, 79, 80, 88-90, 106,
52, 64, 127-130, 132-134, 196, 221, 244 110, 118, 119, 122, 138, 148-152,
Nitrotyrosine 21, 129-132, 188 162-164, 166, 174, 193, 194, 196, 206,
Non-human primate 164 211, 214, 215, 217, 223, 232, 233, 235,
Norepinephrine (NE) 3, 4, 6, 7, 9, 31, 66, 78, 243, 250, 254, 268, 269
123, 148, 193, 215 Pinealectomy 15-17, 27, 29, 35, 38, 39, 41,
Nuclear factor-κB (NF-κB) 133, 210 42, 45, 46, 54, 63, 72, 73, 80, 148, 150,
152, 154, 205, 211, 227
O Pituitary 3, 4, 27, 62, 88-92, 96, 99-101, 110,
122, 215
Organ transplantation 21 Pituitary adenylate cyclase-activating peptide
Orphan nuclear receptors 243 (PACAP) 3, 4, 7, 215
Osteoclasts 209, 210, 212 Platelet-derived endothelial cell growth factor
Osteoporosis 202, 209, 210, 212 (PD-ECGF) 27
Osteoprotegerin 209, 210 PMN 132, 134, 184
OT 4 Poly (ADP-ribose) synthetase (PARS) 127,
Ovarian cancer 74, 76 129, 131, 132
Oxidative damage 16, 19-22, 26, 30, 52, 63, Polymorphonuclear leucocyte 15, 184
184, 186, 187, 198, 200, 202, 205, Polysomnography 264, 268
220-227 Post-transcriptional mechanism 193
Oxidative phosphorylation 22, 122, 185, 197, Postmenopausal women 63-65, 123
199, 205 Premature ventricular contractions (PVC) 14,
Oxidative stress 16, 21, 22, 26, 28-30, 52, 65, 15
119, 127, 184-186, 188, 189, 198, 200, Prostaglandin 28, 66, 132-134, 151, 154,
201, 204, 205, 217, 220-222, 224, 228 185, 217
Oxytocin 3, 4, 148, 149 Prostaglandin E 154
Prostate cancer 76, 77, 79, 138, 139, 143-145
P Protein kinase A (PKA) 6, 7, 88, 90, 97, 98,
101, 143, 144, 193
Pancreas 12, 21, 148, 149, 153, 155 Protein kinase C (PKC) 6, 54, 74, 94, 96, 98,
Paraventricular nucleus (PVN) 3, 4, 268 138, 143, 144, 243, 244, 248-251
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
Index 283
R T
Rapid eye movement (REM) 164, 212, 258, Temazepam 108
273-276 Thermoregulation 106-109, 111, 113
Reactive C protein 62 Thymidine kinase (TK) 27
Reactive oxygen species (ROS) 28-30, 52, 53, Thyroid 26-31, 46, 77, 138, 148, 149, 151,
127, 130-132, 184, 186, 188, 205, 220, 153, 275
221 Thyroid cancer 28, 77
REM sleep 212, 273-276 Thyroid gland 26-31, 148, 149
Reproduction 1, 80, 88, 90, 91, 99, 100 Thyroid hormone 26, 29-31, 46, 138
Respiratory distress syndrome (RDS) 185, Thyroxine (T4) 26, 28-31
186, 189 Transferrin 184
Retina 39, 91, 92, 149, 150, 163, 176, 179, Transforming growth factor-β 54
180, 193, 232-240, 268 Trigeminovascular system 214, 217
Retinoid family 243 Tryptophan (TRP) 5, 9, 41, 45, 71, 78, 129,
148, 150, 152, 153, 162, 193, 233, 255
S Tryptophan hydroxylase (TPH/TPOH) 5, 7,
162, 193-195, 233
Safrole 52, 223, 227 Tumor necrosis factor-α (TNF-α) 21, 54,
Scoliosis 35-42 132, 186, 189
Seasonal affective disorder (SAD) 152, 153,
170, 173, 253-258, 275 U
Seasonal photoperiod 88
Senescence-accelerated mouse (SAM) 48, 50, Urinary bladder 12, 21
201-203 Use of melatonin 16, 75, 124, 209, 212, 228
Serotonin 3-6, 42, 88, 99, 106, 148-155, 162,
193, 237 V
Serotonin N-acetyltransferase 88, 193
Shift work 121, 170, 172 Vasoactive intestinal peptide (VIP) 3, 4, 7,
Shock 64, 129 215
Siberian hamster 2, 7-9, 90 Vasopressin (VP) 3, 4, 7, 148, 149
Sleep 62, 71, 75, 79, 106-109, 112-114, Ventricular fibrillation (VF) 14, 15
118-124, 152, 162-168, 170-173, 175, Vitamin B12 266
178, 180, 209, 212, 215, 254-257, Vitamin C 15, 64, 187, 221
263-268, 270, 273-276 Vitamin E 30, 63, 64, 184, 187, 188, 198,
Sleep disorder 62, 107, 113, 120, 121, 124, 221, 224, 238
163, 167, 170, 171, 175, 178, 264, 265
Somatostatin (SOM) 4, 42, 148, 149, 151 W
Stomach cancer 77
Stroke 12, 13, 16, 17, 23 Water transport 243, 244, 246-248, 250, 251
Substance P (SP) 3, 4, 148, 149, 151, 215 Wavelength 170, 173-176, 178-180, 216
Superior cervical ganglia (SCG) 3, 4, 31, 215, Winter depression 170, 173, 253-257
268 Wrist actigraphy 267
Superoxide dismutase (SOD) 20, 51, 52, 54,
129, 130, 154, 184, 185-187, 196, 198, Z
200, 221, 223, 228, 238
Suprachiasmatic nucleus (SCN) 3, 4, 60, 61, Zebrafish 162, 164-166, 234
71, 79, 80, 88-92, 110, 111, 113, 119, Zymosan 131, 132
121, 148, 163, 214, 215, 217, 243, 254,
258, 268
Syrian hamster 2, 7, 8, 27-29, 54, 222, 226
MEDICAL INTELLIGENCE UNIT
PANDI-PERUMAL • CARDINALI
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Biological Basis of Its Function in Health and Disease

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Melatonin:
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MEDICAL INTELLIGENCE UNIT

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

S.R. Pandi-Perumal, M.Sc.

Daniel P. Cardinali, M.D., Ph.D.

LANDES BIOSCIENCE EUREKAH.COM

Medical Intelligence Unit

Copyright ©2006 Eurekah.com

Library of Congress Cataloging-in-Publication Data

1. Mechanisms Underlying Seasonal Regulation

2. Oxidative Stress-Mediated Damage during in Vivo

3. Melatonin and the Thyroid Gland ....................................................... 26

4. The Role of Melatonin in the Development of Scoliosis ...................... 35

5. Effect of Melatonin on Life Span and Longevity .................................. 45

6. Cardiovascular Effects of Melatonin ..................................................... 60

7. Pineal Gland and Cancer—An Epigenetic Approach to the Control

8. Expression and Signal Transduction Pathways of Melatonin

11. Role of Endogenous and Exogenous Melatonin in Inflammation ....... 127

12. Heterologous Modulation of Androgen Receptor

14. Sleep and Melatonin in Diurnal Species ............................................. 162

17. Diurnal 5-HT Production and Melatonin Formation ........................ 193

19. Melatonin Use As a Bone-Protecting Substance ................................. 209

21. Melatonin in Protection against Oxidative Damage Caused

22. Influence of Melatonin on the Health

23. Melatonin Synchronizes Cell Physiology through Cytoskeletal

26. Melatonin as an Antidepressant for Treatment of Delayed

Index .................................................................................................. 279

Daniel P. Cardinali, M.D., Ph.D.

Verónica Boggio Salvatore Cuzzocrea

Christophe Chiquet Jie Deng

Galena S. Russkikh Marcel G. Smits

Akihiko Wakatsuki Irina V. Zhdanova

In the last two decades, our understanding of the organization of the

Mechanisms Underlying Seasonal Regulation

Annual Variations of the Melatonin Pattern

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

Nervous and Endocrine Inputs Regulating the Annual Rhythm

Molecular Mechanisms Underlying the Annual Changes

one includes a first step of acetylation by arylalkylamine-N-acetyl transferase (AA-NAT) fol-

Molecular Mechanisms Underlying the Daily Changes

Molecular Mechanisms Underlying the Photoperiodic and Seasonal

Oxidative Stress-Mediated Damage during

Melatonin and Ischemia-Reperfusion Injury

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

Melatonin and Cardiac I/R Injury

Melatonin and Neural I/R Injury

Melatonin and I/R Injury in Other Organs

Melatonin and the Thyroid Gland

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

Melatonin and Thyroid Growth Processes

In another study, hemithyroidectomy increased dThdPase activity in the remaining thyroid

Melatonin and Thyroid Function

Oxidative Stress, the Thyroid Gland and Melatonin

Pineal-Thyroid Relationship in Humans

Thyroid Hormone-Stimulation of Pineal Function

The Role of Melatonin

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

Problems with Studying Scoliosis and Melatonin

The Pinealectomised Chicken Model for the Study of Scoliosis

Serum Melatonin Levels in Humans with Scoliosis

A Proposed Model by Which Low Levels of Serum Melatonin Can

Effect of Melatonin on Life Span

Effect of Melatonin on Longevity in Mice

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