**Molecular Biochemistry and Pharmacological Mechanisms of Action of Melatonin and Its Indole Derivatives**

**Chapter 1**

**Provisional chapter**

**Introductory Chapter: Melatonin, the Integrative**

**Introductory Chapter: Melatonin, the Integrative** 

DOI: 10.5772/intechopen.81071

In the healthcare scientific environment, nowadays, researchers are inspired by endogenous springs of molecules that can be reinterpreted, better understood, or completely reconsidered in their function and ability to sustain the human organism in maintaining its homeostasis [1]. Melatonin is such a tremendous molecule acting in the center of the integrative molecular mechanisms of the body, based on interlinkages of the regulatory systems: neural, endocrine,

The endogenous indole system represented by biomolecules with indole structure such as tryptophan, serotonin, and, above all, melatonin conducts the integration mechanisms of the organisms in the great informational variety of the environment. Melatonin is responsible for coordinating and synchronizing the expression of the most important physiological effects of the biological rhythm, imposes an order of the biochemical systems functionality and, glob-

The indole ring is considered by scientists as a "privileged" biological structure [4, 5], due to its outstanding ability to form organic active compounds with different affinities for endogenous receptors, mainly for G protein-coupled receptors [6]. The indole structure is widely found at all levels of the biological systems as an important component of the biomolecules and natural products, such as the alkaloids from ergot, essential tryptophan amino acid, serotonin, neuromediator, and melatonin, the main hormone secreted by the pineal gland. As a consequence of its biological effects, the indole nucleus is present in the structure of many marketed medicines [7–10] or dietary supplements [11–13], as well as in the prototypes of

immune, and genetic, all embodying the uniqueness of human architecture [1, 2].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Molecule within the Human Architecture**

**Molecule within the Human Architecture**

Cristina Manuela Drăgoi and

Cristina Manuela Drăgoi and

http://dx.doi.org/10.5772/intechopen.81071

ally, depicts the molecular logic of living [2, 3].

some drugs that are currently under development.

Additional information is available at the end of the chapter

Alina Crenguța NicolaeAdditional information is available at the end of the chapter

Alina Crenguța Nicolae

**1. Introduction**

#### **Introductory Chapter: Melatonin, the Integrative Molecule within the Human Architecture Introductory Chapter: Melatonin, the Integrative Molecule within the Human Architecture**

DOI: 10.5772/intechopen.81071

Cristina Manuela Drăgoi and Alina Crenguța Nicolae Cristina Manuela Drăgoi and

Additional information is available at the end of the chapter Alina Crenguța NicolaeAdditional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81071

### **1. Introduction**

In the healthcare scientific environment, nowadays, researchers are inspired by endogenous springs of molecules that can be reinterpreted, better understood, or completely reconsidered in their function and ability to sustain the human organism in maintaining its homeostasis [1].

Melatonin is such a tremendous molecule acting in the center of the integrative molecular mechanisms of the body, based on interlinkages of the regulatory systems: neural, endocrine, immune, and genetic, all embodying the uniqueness of human architecture [1, 2].

The endogenous indole system represented by biomolecules with indole structure such as tryptophan, serotonin, and, above all, melatonin conducts the integration mechanisms of the organisms in the great informational variety of the environment. Melatonin is responsible for coordinating and synchronizing the expression of the most important physiological effects of the biological rhythm, imposes an order of the biochemical systems functionality and, globally, depicts the molecular logic of living [2, 3].

The indole ring is considered by scientists as a "privileged" biological structure [4, 5], due to its outstanding ability to form organic active compounds with different affinities for endogenous receptors, mainly for G protein-coupled receptors [6]. The indole structure is widely found at all levels of the biological systems as an important component of the biomolecules and natural products, such as the alkaloids from ergot, essential tryptophan amino acid, serotonin, neuromediator, and melatonin, the main hormone secreted by the pineal gland. As a consequence of its biological effects, the indole nucleus is present in the structure of many marketed medicines [7–10] or dietary supplements [11–13], as well as in the prototypes of some drugs that are currently under development.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

As a constitutive element of proteins, the essential indole amino acid, tryptophan, has the most pronounced hydrophobic character of all the amino acids and forms a specific hydrophobic environment that contributes to the stabilization of the endogenous protein structure, special characteristics regarding membrane fluidity and transmembrane potential [14, 15]. Also, tryptophan is one of the most important indolic endogenous precursors, being involved in the biosynthesis of all endogenous compounds with indole structure: the neurotransmitter serotonin, the pineal hormone melatonin, the neuromodulator and neurotransmitter tryptamine, 5-hydroxytryptophan, and 5-hydroxyindoleacetic acid, as also in the activity of some specific enzymes, cytochrome c peroxidase. Tryptophan depletion is part of the cytotoxic process and antiproliferative cellular mechanism mediated by γ-interferon. Low serum tryptophan concentrations are clinically correlated with the appearance of some pathological infectious, autoimmune, and, not the last, malignant processes [16–18].

This molecule was studied under very different circumstances, from interactions with DNA, in association with other therapeutic agents [39], using different animal models [40–45] and cell lines [46–48]. The current scientific interest focuses on revealing melatonin actions on major physiological process, as pregnancy or aging [49, 50], determining its modulatory abilities on different stages of fetus evolution, on healthy aging mechanisms, and on preventing neurodegeneration, melatonin receptors being highly expressed at the placenta level, the BBB mainly by P-glycoprotein overexpression, mediating the mother-fetus interchanges and

Introductory Chapter: Melatonin, the Integrative Molecule within the Human Architecture

http://dx.doi.org/10.5772/intechopen.81071

5

Melatonin also exerts different effects on the glucose metabolism, considering various targets: it stimulates glucose uptake in muscle cells by phosphorylation of insulin receptor substrate-1 through MT2 signaling, MT2 receptors are expressed in hepatocytes, and melatonin therapy

The cardiovascular system physiological, pathophysiological, and molecular endogenous mechanisms are highly influenced by diurnal variations, circadian imbalances affecting gene and protein expression, cardiac remodeling, and promoting ischemia/reperfusion damage [55–64]. Desynchronizations are frequently registered in patients with hypertension, diabetes

Another important research field for melatonin and its derivatives is identifying predictive biomarkers meant to provide extensive control upon pathologic progression and therapy suc-

Melatonin is the integrative molecule in the in vivo milieu of every living cell, mediating the integration complex mechanisms of the individual entity into the environment, synchronizes its cyclic processes, and depicts the circadian distribution of physiological and behavioral

Faculty of Pharmacy, "Carol Davila" University of Medicine and Pharmacy, Bucharest,

[1] Duarte CD, Barreiro EJ, Fraga CAM. Privileged structures: A useful concept for the rational design of new lead drug candidates. Mini-Reviews in Medicinal Chemistry.

cess, markers that are as minimal invasive as possible and readily available [67–72].

restricting the xenobiotic way to the fragile developing organism [51–53].

elevates glucose release from the liver [54].

processes.

Romania

**References**

2007;**7**:1108-1119

**Author details**

mellitus, obesity, and metabolic syndrome [65, 66].

Cristina Manuela Drăgoi\* and Alina Crenguța Nicolae

\*Address all correspondence to: manuela.dragoi@gmail.com

Tryptophan is the precursor of serotonin, a neurotransmitter with indole structure, with vast biological effects. Emergence of imbalances in the serotoninergic metabolism determines the etiology and pathological neuropsychiatric and systemic disorders, including the development of serotonin-secreting tumors [19–22]. Thus, a more complete overview of tryptophan and serotonin biochemistry and the precise relationships and interactions of these molecules with other endogenous constituents or structures may contribute to the therapeutic understanding and solving many psychiatric, autoimmune, and neoplastic disorders [23–25].

In particular, melatonin is an indole neurohormone synthesized mainly in the pineal gland, during the night, being also known as the darkness hormone. Melatonin is not exclusively synthesized by the pineal gland; the retina, the skin, and the gastrointestinal tract are only a few other tissues that produce high amounts of melatonin [26].

The direct precursor of melatonin is the serotonin, naturally synthesized in pinealocytes from L-tryptophan. The regulation system of the melatoninergic synthesis is complex, using central and autonomous pathways, so that there are many pathophysiologic situations where the melatonin secretion is deficient. The alteration of the melatoninergic circadian profile [27] is associated with the susceptibility, development, and evolution of a variety of pathologies, the highest incidence of cancer being registered in shift workers, which have a detrimental day-night alternation [28].

On the other hand, small fluctuations in the steady-state levels of the reactive oxygen and nitrogen species concentrations may play a key role in the intracellular signaling, uncontrolled increases of these highly reactive molecules leading to chain reactions mediated by free radicals, which destroy, without discrimination, proteins, lipids, and DNA, resulting, ultimately, in cell death and being the primary or secondary cause of a wide range of diseases [29–35].

Melatonin was closely analyzed, under all biochemical aspects, considering its antioxidant mechanisms, intrinsic or modulatory at the level of antioxidant enzymes or in connected supplementary scavenging processes, and revealing a unique molecular antioxidant cascade. Its effects were interpreted in conjunction with other endogenous structures or assessed in controlled release formulations, aimed to enhance antioxidant processes and endogenous indole modulatory actions [36–38].

This molecule was studied under very different circumstances, from interactions with DNA, in association with other therapeutic agents [39], using different animal models [40–45] and cell lines [46–48]. The current scientific interest focuses on revealing melatonin actions on major physiological process, as pregnancy or aging [49, 50], determining its modulatory abilities on different stages of fetus evolution, on healthy aging mechanisms, and on preventing neurodegeneration, melatonin receptors being highly expressed at the placenta level, the BBB mainly by P-glycoprotein overexpression, mediating the mother-fetus interchanges and restricting the xenobiotic way to the fragile developing organism [51–53].

Melatonin also exerts different effects on the glucose metabolism, considering various targets: it stimulates glucose uptake in muscle cells by phosphorylation of insulin receptor substrate-1 through MT2 signaling, MT2 receptors are expressed in hepatocytes, and melatonin therapy elevates glucose release from the liver [54].

The cardiovascular system physiological, pathophysiological, and molecular endogenous mechanisms are highly influenced by diurnal variations, circadian imbalances affecting gene and protein expression, cardiac remodeling, and promoting ischemia/reperfusion damage [55–64]. Desynchronizations are frequently registered in patients with hypertension, diabetes mellitus, obesity, and metabolic syndrome [65, 66].

Another important research field for melatonin and its derivatives is identifying predictive biomarkers meant to provide extensive control upon pathologic progression and therapy success, markers that are as minimal invasive as possible and readily available [67–72].

Melatonin is the integrative molecule in the in vivo milieu of every living cell, mediating the integration complex mechanisms of the individual entity into the environment, synchronizes its cyclic processes, and depicts the circadian distribution of physiological and behavioral processes.

### **Author details**

As a constitutive element of proteins, the essential indole amino acid, tryptophan, has the most pronounced hydrophobic character of all the amino acids and forms a specific hydrophobic environment that contributes to the stabilization of the endogenous protein structure, special characteristics regarding membrane fluidity and transmembrane potential [14, 15]. Also, tryptophan is one of the most important indolic endogenous precursors, being involved in the biosynthesis of all endogenous compounds with indole structure: the neurotransmitter serotonin, the pineal hormone melatonin, the neuromodulator and neurotransmitter tryptamine, 5-hydroxytryptophan, and 5-hydroxyindoleacetic acid, as also in the activity of some specific enzymes, cytochrome c peroxidase. Tryptophan depletion is part of the cytotoxic process and antiproliferative cellular mechanism mediated by γ-interferon. Low serum tryptophan concentrations are clinically correlated with the appearance of some pathological

Tryptophan is the precursor of serotonin, a neurotransmitter with indole structure, with vast biological effects. Emergence of imbalances in the serotoninergic metabolism determines the etiology and pathological neuropsychiatric and systemic disorders, including the development of serotonin-secreting tumors [19–22]. Thus, a more complete overview of tryptophan and serotonin biochemistry and the precise relationships and interactions of these molecules with other endogenous constituents or structures may contribute to the therapeutic understanding and solving many psychiatric, autoimmune, and neoplastic disorders [23–25].

In particular, melatonin is an indole neurohormone synthesized mainly in the pineal gland, during the night, being also known as the darkness hormone. Melatonin is not exclusively synthesized by the pineal gland; the retina, the skin, and the gastrointestinal tract are only a

The direct precursor of melatonin is the serotonin, naturally synthesized in pinealocytes from L-tryptophan. The regulation system of the melatoninergic synthesis is complex, using central and autonomous pathways, so that there are many pathophysiologic situations where the melatonin secretion is deficient. The alteration of the melatoninergic circadian profile [27] is associated with the susceptibility, development, and evolution of a variety of pathologies, the highest incidence of cancer being registered in shift workers, which have a detrimental

On the other hand, small fluctuations in the steady-state levels of the reactive oxygen and nitrogen species concentrations may play a key role in the intracellular signaling, uncontrolled increases of these highly reactive molecules leading to chain reactions mediated by free radicals, which destroy, without discrimination, proteins, lipids, and DNA, resulting, ultimately, in cell death and being the primary or secondary cause of a wide range of diseases [29–35].

Melatonin was closely analyzed, under all biochemical aspects, considering its antioxidant mechanisms, intrinsic or modulatory at the level of antioxidant enzymes or in connected supplementary scavenging processes, and revealing a unique molecular antioxidant cascade. Its effects were interpreted in conjunction with other endogenous structures or assessed in controlled release formulations, aimed to enhance antioxidant processes and endogenous

infectious, autoimmune, and, not the last, malignant processes [16–18].

4 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

few other tissues that produce high amounts of melatonin [26].

day-night alternation [28].

indole modulatory actions [36–38].

Cristina Manuela Drăgoi\* and Alina Crenguța Nicolae

\*Address all correspondence to: manuela.dragoi@gmail.com

Faculty of Pharmacy, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania

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Introductory Chapter: Melatonin, the Integrative Molecule within the Human Architecture

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[57] Diaconu CC, Dragoi CM, Bratu OG, et al. New approaches and perspectives for the pharmacological treatment of arterial hypertension. Farmácia. 2018;**66**(3):408-415

[58] Wiggins-Dohlvik K, Han MS, Stagg HW, et al. Melatonin inhibits thermal injury-induced hyperpermeability in microvascular endothelial cells. Journal of Trauma and Acute

[59] Khera AV, Kathiresan S. Is coronary atherosclerosis one disease or many? Setting realistic

[60] Lochner A, Huisamen B, Nduhirabandi F. Cardioprotective effect of melatonin against ischaemia/reperfusion damage. Frontiers in Bioscience (Elite Edition). 2013;**5**:305-315 [61] Diaconu CC, Stănescu AMA, et al. Hyperkalemia and cardiovascular diseases: New

[62] Yang Y, Sun Y, Wei Y, et al. A review of melatonin as a suitable antioxidant against myocardial ischemia-reperfusion injury and clinical heart diseases. Journal of Pineal

[63] Yip HK, Chang YC, Wallace CG, et al. Melatonin treatment improves adipose-derived mesenchymal stem cell therapy for acute lung ischemia-reperfusion injury. Journal of

[64] Dominguez-Rodriguez A, Abreu-Gonzalez P, Reiter RJ. Melatonin for cardioprotection in ST elevation myocardial infarction: Are we ready for the challenge? Heart. 2017;

[65] Drăgoi CM, Nicolae AC, Grigore C, Dinu-Pîrvu CE, Arsene AL. Characteristics of glucose homeostasis and lipidic profile in a hamster metabolic syndrome model, after the co-administration of melatonin and irbesartan in a multiparticulate pharmaceutical formulation. In: Serafinceanu C, Negoiţă O, Elian V, editors. 2nd International Conference on Interdisciplinary Management of Diabetes Mellitus and its Complications—

[66] Mohamad MA, Mitrea N, Nicolae AC, Constantinescu MZ, Drăgoi CM, Arsene AL, et al. The dynamics of adiponectin and leptin on metabolic syndrome patients and age

[67] Vlăsceanu AM, Petraru C, Baconi D, Ghica M, Arsene A, Popa L, et al. Quantitative relationships of urinary cotinine levels in smoking diabetic patients. Farmácia. 2015;**63**(3):

[68] Barbu CG, Arsene AL, Florea S, Albu A, Sirbu A, Martin S, Nicolae AC, Burcea-Dragomiroiu GT, Popa DE, Velescu BS, Dumitrescu IB, Mitrea N, Draganescu D, Lupuliasa D, Spandidos DA, Tsatsakis AM, Dragoi CM, Fica S. Cardiovascular risk assessment in osteoporotic patients using osteoprotegerin as a reliable predictive biochemical marker. Molecular

Medicine Reports. 2017;**16**(5):6059-6067. https://doi.org/10.3892/mmr.2017.7376

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matched healthy subjects. Farmácia. 2014;**62**(3):532-545

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10 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

Research. 2013;**57**:357-366

**103**:647-648

349-356

Pineal Research. 2013;**54**:207-221


**Chapter 2**

**Provisional chapter**

**How Can Molecular Pharmacology Help Understand**

**How Can Molecular Pharmacology Help Understand** 

Melatonin actions are so numerous that a naive reader might become suspicious at such wonders. In a systematic way, we would like to summarize the various approaches that led to what is scientifically sounded in terms of molecular pharmacology: where and how melatonin is acting as a molecule, what can be its action as an antioxidant per se, and its side effects at a molecular level not as a drug or in vivo. Finally, the nature of the relationship between melatonin and mitochondria should be decrypted as well. The road we took from 1987 up to now, and particularly after 1995, will be mentioned with special considerations to the receptors from various species and our goals beyond that; the synthesis and catabolism of melatonin and their link to other enzymes; the discovery

target; and the search for agonists that occulted part of the potential discovery of true and potent antagonists, a situation quite unique among the G-protein-coupled receptors.

Melatonin is a neurohormone synthesized by the pineal gland at night. The longer the night, the higher the concentration of melatonin in the blood. Even if new information is modulating this basic principle, this rhythmicity has been the basis of many published observations linking melatonin to many physiological features of animals, including human. This comprises the daily changes in light and the way the body understands the successions of dark and clear

binding sites, and what's left to understand on this particularly interesting

, MT2

, catabolism

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.79524

**the Multiple Actions of Melatonin: 20 Years of**

**the Multiple Actions of Melatonin: 20 Years of** 

**Research and Trends**

**Research and Trends**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Keywords:** melatonin, molecular pharmacology, MT<sup>1</sup>

http://dx.doi.org/10.5772/intechopen.79524

Jean A. Boutin

Jean A. Boutin

**Abstract**

of the *MT3*

**1. Introduction**

#### **How Can Molecular Pharmacology Help Understand the Multiple Actions of Melatonin: 20 Years of Research and Trends How Can Molecular Pharmacology Help Understand the Multiple Actions of Melatonin: 20 Years of Research and Trends**

DOI: 10.5772/intechopen.79524

Jean A. Boutin Jean A. Boutin

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79524

#### **Abstract**

Melatonin actions are so numerous that a naive reader might become suspicious at such wonders. In a systematic way, we would like to summarize the various approaches that led to what is scientifically sounded in terms of molecular pharmacology: where and how melatonin is acting as a molecule, what can be its action as an antioxidant per se, and its side effects at a molecular level not as a drug or in vivo. Finally, the nature of the relationship between melatonin and mitochondria should be decrypted as well. The road we took from 1987 up to now, and particularly after 1995, will be mentioned with special considerations to the receptors from various species and our goals beyond that; the synthesis and catabolism of melatonin and their link to other enzymes; the discovery of the *MT3* binding sites, and what's left to understand on this particularly interesting target; and the search for agonists that occulted part of the potential discovery of true and potent antagonists, a situation quite unique among the G-protein-coupled receptors.

**Keywords:** melatonin, molecular pharmacology, MT<sup>1</sup> , MT2 , catabolism

#### **1. Introduction**

Melatonin is a neurohormone synthesized by the pineal gland at night. The longer the night, the higher the concentration of melatonin in the blood. Even if new information is modulating this basic principle, this rhythmicity has been the basis of many published observations linking melatonin to many physiological features of animals, including human. This comprises the daily changes in light and the way the body understands the successions of dark and clear

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

periods but also the circannual rhythmicity, during which animals prepare for the harsher winter period during which access to food is more difficult. By "measuring" the length of melatonin synthesis periods (directly proportional to the length of the nights), animals start to modify their physiology in order to prepare the time to come: accumulation of fat (fat storing) for some, food storing for others, and preparing the bodies to reproduction for all animals at the best period to avoid the exposure of the newborns to cold and difficult conditions. Apparently, humans have lost this advanced capacity in our ever lit-up society.

How antioxidant this molecule is and why? What makes it so special? Some of these points

Molecular Pharmacology and Melatonin: 20 Yeras of Research and Trends

http://dx.doi.org/10.5772/intechopen.79524

15

Beyond these biochemical features, melatonin has two unique features: it is very soluble but seems to travel freely through biological membranes, and its possible toxicity is extremely low (although some human cases of undesirable effects were reported), permitting scientists to give on many models very large amounts of the compound in cellulo and in vivo without apparent associated major toxicity. It was thus obvious that in many cases, the activity at those "pharmacological" dosages led to surprisingly numerous therapeutic properties of this molecule. Furthermore, the discovery that melatonin had, in cellulo and in vivo, antioxidant properties added to the multiple possibilities of use of melatonin, leading to this apparent

Melatonin is mostly synthesized starting from tryptophan in the pineal gland by a series of enzymes, the limiting one being arylalkylamine *N*-acetyltransferase (AANAT) also known as the timezyme [6]. Many studies have been performed on this enzyme and its requirements in terms of substrate specificity and inhibitor research, in particular in human [7, 8]. Over the years, several groups hypothesized and reported the possibility that melatonin was also synthesized in mitochondria (see also below, Section 6.4), suggesting that the antioxidant properties of the molecule would confer a strong resistance of mitochondria to the generation of ROS, a common feature of those subcellular organelles. Indeed, if the dogma was, in the 1950s and later on, that melatonin was mainly synthesized in the pineal gland, a fact that was clearly confirmed by surgical removing of the gland would lead to a major reduction of circulating melatonin and to the loss of some of the circadian and circannual rhythms; several papers co-indicated that such local synthesis that does exist should be taken into consideration (see, for instance, Stehle et al. [9]). What is more troubling is the recent precise description of melatonin synthesis in mitochondria, at least in brain-derived organelles [10] as well as the

against the finding that melatonin is synthesized in mitochondria, even if that was recently restricted to brain-derived mitochondria [11]. But melatonin is also known to "travel" freely inside membranes. Thus in order to stay inside mitochondria, it should be sequestered inside them in order to prevent the damages of ROS production—a common and key feature of the respiratory chain—thanks to the antioxidant properties of melatonin (see below, Section 6.1.2,

No revolution, nevertheless, occurred in the way melatonin is synthesized. It comes from several steps. All those enzymes have been cloned and studied, including from human origin. The particular case of AANAT catalyzing the limiting step of the synthesis has been at the source of the seminal work of David Klein's laboratory (see, for instance [6, 7]). This enzyme is destroyed during the day and active only at night. The way it is regulated is different according to species, but it seems a formidable waste of energy to handle it that way (synthesis and immediate destruction for "nothing") [12, 13], strongly suggesting by the way this pathway is regulated that it is of major

). Intuitively, many previous strong knowledge would go

are reviewed in the present chapter based on two decades of research in this area.

paramount of therapeutic properties.

presence of a functional GPCR (MT1

for further discussion).

**2. Melatonin synthesis**

New evidences recently caught our attention and challenge our ways of understanding the melatonin actions and pathways, whether because it seems that light can be "seen" by the body without the relay of melatonin or because one finds that melatonin is synthesized in mitochondria in all parts of the brain, as opposed to the pivotal and ancient statement: melatonin is synthesized by the pineal gland only. Nowadays, one can also see many publications claiming that melatonin helps cure cancer as well as so many other diseases (see a non-exhaustive selection of such actions since 2015 in **Table 1**). All these information should be treated with respect, integrated inside our decade-old knowledge and carefully evaluated as a contribution to a bigger picture. The basics on melatonin can be found in some recent reviews [1–5]. The present chapter concentrates on the molecular pharmacology of melatonin. This small molecule, derived from tryptophan, has a limited number of recognized targets. It is synthesized and catabolized by a limited and well-known number of enzymes that have been described in the past (see **Figure 1** for a simplistic summary). The core of the discussion seems always to be the same: how this molecule can be active on so many pathological events?

**Figure 1.** A simplistic summary of melatonin-related proteins. Melatonin is synthesized from tryptophan by a series of enzyme the the limiting step of which is catalyzed by Arylalkylamine N-acetyl transferase (green box); melatonin is excreted mainly unchanged from mammalian bodies or conjugated either by UDP-glucuronosyltranserases or by sulfotranferases, but it is also catabolized by indoleamine-2,3-dioxygenase, myeloperoxidase or cytochrome c (yellow box). At the molecular level, the targets of melatonin are mainly: Its two melatonin receptors, MT1 and MT2 , QR2 (formerly known as *MT3* ). Furthermore, putative targets might exist such as nuclear receptors and particularly Nrf2 that might explain some of the anti-oxidant capacities of melatonin (red box).

How antioxidant this molecule is and why? What makes it so special? Some of these points are reviewed in the present chapter based on two decades of research in this area.

Beyond these biochemical features, melatonin has two unique features: it is very soluble but seems to travel freely through biological membranes, and its possible toxicity is extremely low (although some human cases of undesirable effects were reported), permitting scientists to give on many models very large amounts of the compound in cellulo and in vivo without apparent associated major toxicity. It was thus obvious that in many cases, the activity at those "pharmacological" dosages led to surprisingly numerous therapeutic properties of this molecule. Furthermore, the discovery that melatonin had, in cellulo and in vivo, antioxidant properties added to the multiple possibilities of use of melatonin, leading to this apparent paramount of therapeutic properties.

### **2. Melatonin synthesis**

periods but also the circannual rhythmicity, during which animals prepare for the harsher winter period during which access to food is more difficult. By "measuring" the length of melatonin synthesis periods (directly proportional to the length of the nights), animals start to modify their physiology in order to prepare the time to come: accumulation of fat (fat storing) for some, food storing for others, and preparing the bodies to reproduction for all animals at the best period to avoid the exposure of the newborns to cold and difficult conditions.

New evidences recently caught our attention and challenge our ways of understanding the melatonin actions and pathways, whether because it seems that light can be "seen" by the body without the relay of melatonin or because one finds that melatonin is synthesized in mitochondria in all parts of the brain, as opposed to the pivotal and ancient statement: melatonin is synthesized by the pineal gland only. Nowadays, one can also see many publications claiming that melatonin helps cure cancer as well as so many other diseases (see a non-exhaustive selection of such actions since 2015 in **Table 1**). All these information should be treated with respect, integrated inside our decade-old knowledge and carefully evaluated as a contribution to a bigger picture. The basics on melatonin can be found in some recent reviews [1–5]. The present chapter concentrates on the molecular pharmacology of melatonin. This small molecule, derived from tryptophan, has a limited number of recognized targets. It is synthesized and catabolized by a limited and well-known number of enzymes that have been described in the past (see **Figure 1** for a simplistic summary). The core of the discussion seems always to be the same: how this molecule can be active on so many pathological events?

**Figure 1.** A simplistic summary of melatonin-related proteins. Melatonin is synthesized from tryptophan by a series of enzyme the the limiting step of which is catalyzed by Arylalkylamine N-acetyl transferase (green box); melatonin is excreted mainly unchanged from mammalian bodies or conjugated either by UDP-glucuronosyltranserases or by sulfotranferases, but it is also catabolized by indoleamine-2,3-dioxygenase, myeloperoxidase or cytochrome c (yellow

). Furthermore, putative targets might exist such as nuclear receptors and particularly Nrf2 that

and MT2

, QR2

box). At the molecular level, the targets of melatonin are mainly: Its two melatonin receptors, MT1

might explain some of the anti-oxidant capacities of melatonin (red box).

(formerly known as *MT3*

Apparently, humans have lost this advanced capacity in our ever lit-up society.

14 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

Melatonin is mostly synthesized starting from tryptophan in the pineal gland by a series of enzymes, the limiting one being arylalkylamine *N*-acetyltransferase (AANAT) also known as the timezyme [6]. Many studies have been performed on this enzyme and its requirements in terms of substrate specificity and inhibitor research, in particular in human [7, 8]. Over the years, several groups hypothesized and reported the possibility that melatonin was also synthesized in mitochondria (see also below, Section 6.4), suggesting that the antioxidant properties of the molecule would confer a strong resistance of mitochondria to the generation of ROS, a common feature of those subcellular organelles. Indeed, if the dogma was, in the 1950s and later on, that melatonin was mainly synthesized in the pineal gland, a fact that was clearly confirmed by surgical removing of the gland would lead to a major reduction of circulating melatonin and to the loss of some of the circadian and circannual rhythms; several papers co-indicated that such local synthesis that does exist should be taken into consideration (see, for instance, Stehle et al. [9]). What is more troubling is the recent precise description of melatonin synthesis in mitochondria, at least in brain-derived organelles [10] as well as the presence of a functional GPCR (MT1 ). Intuitively, many previous strong knowledge would go against the finding that melatonin is synthesized in mitochondria, even if that was recently restricted to brain-derived mitochondria [11]. But melatonin is also known to "travel" freely inside membranes. Thus in order to stay inside mitochondria, it should be sequestered inside them in order to prevent the damages of ROS production—a common and key feature of the respiratory chain—thanks to the antioxidant properties of melatonin (see below, Section 6.1.2, for further discussion).

No revolution, nevertheless, occurred in the way melatonin is synthesized. It comes from several steps. All those enzymes have been cloned and studied, including from human origin. The particular case of AANAT catalyzing the limiting step of the synthesis has been at the source of the seminal work of David Klein's laboratory (see, for instance [6, 7]). This enzyme is destroyed during the day and active only at night. The way it is regulated is different according to species, but it seems a formidable waste of energy to handle it that way (synthesis and immediate destruction for "nothing") [12, 13], strongly suggesting by the way this pathway is regulated that it is of major importance for physiology. The enzyme was cloned in our laboratory, and a thorough study of its substrate and co-substrate specificities was reported [8]. It was attempting, at one stage, to try to find specific inhibitors of the enzyme, in order to better understand in in situ situations the roles of melatonin at various locations. Several publications including ours reported those efforts [14–18]. If one particular point should be stressed, it is the elegant ways analogues of an intermediary state of the substrate/co-substrate complex permitted to turn molecules into powerful inhibitors, although overall fragile ones [19], as well as the way that the incorporation of an exotic amino acid in place of a serine permitted to stabilize the enzyme, rendered insensitive to proteolysis [20–22].

Nevertheless, the repartition of the receptors in various organs, and particularly throughout the brain, has been nicely reviewed by Ng et al. [43] with some precisions of their respective functions: these seem to be as follows—improvement of neurogenesis (hippocampus with

ease. In terms of melatonin receptor actions, those are the strongest information available. It seems clear, for example, that most of the protection offered by melatonin in multiple patho-

It would be very complicated to be exhaustive in terms of characterization of those binding sites, as the data is scattered throughout the literature. What is "easy" is that we and others using the binding assay developed around Vakkuri et al.'s 2-[<sup>125</sup>I] iodomelatonin [44] for

In the next sections, three aspects must be covered: the binding, heterodimerization, and

was not sufficient to gain robust information on the receptors. It is only recently that heavily

various states of the receptors and their behavior in that context [46]. Historically the binding studies were largely facilitated by the use of the super-agonist, 2-[125I]-iodomelatonin [44]. Not only this compound is easy to synthesize, but its sensitivity counteracted the very high affinity of melatonin for its receptors, as well as the paucity of the expression of these receptors in relevant tissues. Almost all the laboratories involved in melatonin research have been using this radiotracer. It must be pointed out, though, that attempts to use alternative ligands have been reported, mainly in the spirit of using specific ligands of one or the other of mela-


Functionality of seven-transmembrane domain receptors is a complex science, providing daily new data. The number of coupling pathways at receptors in general is important, and more are discovered often. An excellent review has been published very recently [48], and the contribution of the same authors to the IUPHAR compendium on melatonin receptor functionality [45] should be considered as reference documents to understand the various pathways, at least as of today.

Nevertheless relatively few publications address the functionality of ligands in a global way. Indeed, if some functional data has been produced around a series of chemical analogues completing the classical binding displacement approach, rather few address the global and

H]melatonin became available. This radiotracer permitted to better understand the

laboratory species and from humans, but basic data can be found in Jockers et al. [45].

would also confer a protection against Huntington dis-

Molecular Pharmacology and Melatonin: 20 Yeras of Research and Trends

http://dx.doi.org/10.5772/intechopen.79524

and MT2

H]melatonin, but the specific activity of the tracer

and/or MT<sup>2</sup>

receptor); MT1

17

receptors from several key

have been reported, so far.

. There are cases

a memory maintenance via the inhibition of long-term potentiation by MT<sup>2</sup>

logical situations (as summarized in **Table 1**) are not mediated by its receptors.

establishing the basic molecular pharmacology of the MT1

**3.2. Binding, functionality, and heterodimerization**

tonin receptors [47]. Unfortunately, only ligands specific of MT*<sup>2</sup>*

"standardized" characterizations of a series of ligands on MT1

would regulate the REM sleep; MT1

structure of the melatonin receptors.

labeled [<sup>3</sup>

MT<sup>1</sup>

Initially, several reports were done using [<sup>3</sup>

at the receptors with strong stability in vivo.

#### **3. Melatonin receptor molecular pharmacology**

#### **3.1. Melatonin receptors**

To somewhat summarize the situation, there were two GPCRs (MT1 and MT2 ) found throughout the animal kingdom, an elusive binding site (*MT3* ) that turned out to be an enzyme quinone reductase 2 (QR2) [23] (see below, Section 6.3), another receptor (Mel1c) that was present in fishes, birds, and reptilians and that evolved to a GPR50 in mammals [24], with the curious property to have lost the recognition of melatonin, with a single exception (in platypus [25]), and finally the elusive nuclear receptovr, first described by Becker-André et al. [26] and then retracted [27]. Several other research papers [28] pointed at nuclear receptor(s) to explain the circadian rhythm of some key metabolism enzymes that could logically be dependent on the circadian rhythm of melatonin (see discussion in Jan et al. [29]).

What are the most characterized in the melatonin field, besides the multiple functions of the molecule itself (see below), are the binding characteristics at its receptors. The receptors were first discovered and cloned/characterized by Reppert's group [30] from both hamster and human. This première was followed by a series of cloning, including the discovery that hamster had only one functional melatonin receptor [31], to the contrary of most of the mammals that possess two (MT<sup>1</sup> and MT2 ): human, rat, mice, sheep (although it was long believed to possess also a single receptor form [32]), etc. Cloning was also reported for other species, including birds and fishes [33, 34] and probably insects [35]. This led to the possibility to establish the binding pharmacology of those receptors in several laboratory species—mouse [36], rat [37], and sheep [32]—as well as in human [38]. For years, then, our goal has been to synthesize analogues of melatonin and use them to better understand the MT1 and MT2 roles, as well as to be able to somehow modulate them. It is not the place, here, to review the chemistry that has been explored around melatonin, but recent reviews could be looked at: Mor et al. [39], Garrat and Tsotinis [40], Rivara et al. [41], and Zlotos et al. [42]. The field would have benefit from a quest of specific and stable in vivo binders, particularly antagonists, permitting to explore and understand the limit of the melatonin actions, at least through these particular targets.

Incidentally, one must point out that there are still no antibodies against the receptors. We and many others tried over the last decades to produce such tools with a general negative endpoint. This, of course, has been an obstacle to a better understanding of those receptors. Nevertheless, the repartition of the receptors in various organs, and particularly throughout the brain, has been nicely reviewed by Ng et al. [43] with some precisions of their respective functions: these seem to be as follows—improvement of neurogenesis (hippocampus with a memory maintenance via the inhibition of long-term potentiation by MT<sup>2</sup> receptor); MT1 would regulate the REM sleep; MT1 would also confer a protection against Huntington disease. In terms of melatonin receptor actions, those are the strongest information available. It seems clear, for example, that most of the protection offered by melatonin in multiple pathological situations (as summarized in **Table 1**) are not mediated by its receptors.

It would be very complicated to be exhaustive in terms of characterization of those binding sites, as the data is scattered throughout the literature. What is "easy" is that we and others using the binding assay developed around Vakkuri et al.'s 2-[<sup>125</sup>I] iodomelatonin [44] for establishing the basic molecular pharmacology of the MT1 and MT2 receptors from several key laboratory species and from humans, but basic data can be found in Jockers et al. [45].

In the next sections, three aspects must be covered: the binding, heterodimerization, and structure of the melatonin receptors.

#### **3.2. Binding, functionality, and heterodimerization**

importance for physiology. The enzyme was cloned in our laboratory, and a thorough study of its substrate and co-substrate specificities was reported [8]. It was attempting, at one stage, to try to find specific inhibitors of the enzyme, in order to better understand in in situ situations the roles of melatonin at various locations. Several publications including ours reported those efforts [14–18]. If one particular point should be stressed, it is the elegant ways analogues of an intermediary state of the substrate/co-substrate complex permitted to turn molecules into powerful inhibitors, although overall fragile ones [19], as well as the way that the incorporation of an exotic amino acid in place of a serine permitted to stabilize the enzyme, rendered insensitive to proteolysis [20–22].

quinone reductase 2 (QR2) [23] (see below, Section 6.3), another receptor (Mel1c) that was present in fishes, birds, and reptilians and that evolved to a GPR50 in mammals [24], with the curious property to have lost the recognition of melatonin, with a single exception (in platypus [25]), and finally the elusive nuclear receptovr, first described by Becker-André et al. [26] and then retracted [27]. Several other research papers [28] pointed at nuclear receptor(s) to explain the circadian rhythm of some key metabolism enzymes that could logically be

What are the most characterized in the melatonin field, besides the multiple functions of the molecule itself (see below), are the binding characteristics at its receptors. The receptors were first discovered and cloned/characterized by Reppert's group [30] from both hamster and human. This première was followed by a series of cloning, including the discovery that hamster had only one functional melatonin receptor [31], to the contrary of most of the mam-

to possess also a single receptor form [32]), etc. Cloning was also reported for other species, including birds and fishes [33, 34] and probably insects [35]. This led to the possibility to establish the binding pharmacology of those receptors in several laboratory species—mouse [36], rat [37], and sheep [32]—as well as in human [38]. For years, then, our goal has been to

as well as to be able to somehow modulate them. It is not the place, here, to review the chemistry that has been explored around melatonin, but recent reviews could be looked at: Mor et al. [39], Garrat and Tsotinis [40], Rivara et al. [41], and Zlotos et al. [42]. The field would have benefit from a quest of specific and stable in vivo binders, particularly antagonists, permitting to explore and understand the limit of the melatonin actions, at least through

Incidentally, one must point out that there are still no antibodies against the receptors. We and many others tried over the last decades to produce such tools with a general negative endpoint. This, of course, has been an obstacle to a better understanding of those receptors.

dependent on the circadian rhythm of melatonin (see discussion in Jan et al. [29]).

synthesize analogues of melatonin and use them to better understand the MT1

and MT2

): human, rat, mice, sheep (although it was long believed

) that turned out to be an enzyme—

) found through-

and MT2

roles,

**3. Melatonin receptor molecular pharmacology**

16 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

out the animal kingdom, an elusive binding site (*MT3*

To somewhat summarize the situation, there were two GPCRs (MT1

and MT2

**3.1. Melatonin receptors**

mals that possess two (MT<sup>1</sup>

these particular targets.

Initially, several reports were done using [<sup>3</sup> H]melatonin, but the specific activity of the tracer was not sufficient to gain robust information on the receptors. It is only recently that heavily labeled [<sup>3</sup> H]melatonin became available. This radiotracer permitted to better understand the various states of the receptors and their behavior in that context [46]. Historically the binding studies were largely facilitated by the use of the super-agonist, 2-[125I]-iodomelatonin [44]. Not only this compound is easy to synthesize, but its sensitivity counteracted the very high affinity of melatonin for its receptors, as well as the paucity of the expression of these receptors in relevant tissues. Almost all the laboratories involved in melatonin research have been using this radiotracer. It must be pointed out, though, that attempts to use alternative ligands have been reported, mainly in the spirit of using specific ligands of one or the other of melatonin receptors [47]. Unfortunately, only ligands specific of MT*<sup>2</sup>* have been reported, so far. MT<sup>1</sup> -specific binders have been elusive, despite the wide variety of melatonin analogues that have been synthesized. As stated elsewhere in the present essay, the main focus of chemistry research in this melatonin domain over the last decades was to find alternative ligand agonists at the receptors with strong stability in vivo.

Functionality of seven-transmembrane domain receptors is a complex science, providing daily new data. The number of coupling pathways at receptors in general is important, and more are discovered often. An excellent review has been published very recently [48], and the contribution of the same authors to the IUPHAR compendium on melatonin receptor functionality [45] should be considered as reference documents to understand the various pathways, at least as of today.

Nevertheless relatively few publications address the functionality of ligands in a global way. Indeed, if some functional data has been produced around a series of chemical analogues completing the classical binding displacement approach, rather few address the global and "standardized" characterizations of a series of ligands on MT1 and/or MT<sup>2</sup> . There are cases where given compounds were characterized as partial agonists that turned out to be inverse agonists instead [49], leading to a yet another level of complexity of melatonin receptor pharmacology. We recently embarked in such a task, by screening the agonist/antagonist behavior of a series of compounds (Legros & Boutin, in preparation) after assessing the various methods [50]. We also extended these coupling measurements to a small series of potential antagonists specific of MT<sup>2</sup> [51], to conclude that the compounds were not antagonists but rather partial agonists. As stated and described by Kenakin [52], one should further dig the concept of biased ligands. Indeed, it seems clear, now, that some at least of the downstream pathways of melatonin receptors are elicited by some agonist ligands while not by other ones. This concept has been a bias of the approach to melatonin research. Indeed, while seeking for tools to understand these pathways and their implications in various pathological models, we never had access to real, stable, and potent antagonists, despite past claims for such compounds [53, 54]. Even when large-scale screening campaigns were attempted [55], the poor affinity (compared to the already existing compounds: low μM affinity in the best cases *versus* low nM already available ones) of those newly discovered compounds was not in favor of trying to develop series of chemicals around those hits. A similar situation occurred when we attempted to find peptide ligands at melatonin receptors [56].

interesting routes toward the role of this orphan receptor as well as its implication in cancer development. Obviously many control experiments should be run in these explorations, as it would be attempting to conclude that any receptor can dimerize—and thus regulate the signaling pathway of—any other receptor (see Damian et al. [71] for a counter example, among

Molecular Pharmacology and Melatonin: 20 Yeras of Research and Trends

http://dx.doi.org/10.5772/intechopen.79524

Attempts to crystallize GPCRs have been done for years with various successes. Beside several reports of models of the receptors [72–74], that turned to be more or less disappointing because they poorly brought new information—somewhat as expected. Thus, several lines

several years of technical challenges: expression, stabilization, purification, and functionality measurement [75, 76]. We embarked several years ago in an approach that attempted to be original: as a first step in this purification/crystallization of melatonin receptor(s) project, we cloned melatonin receptors from as many and as various species as possible. Many such melatonin receptors have been reported in the literature, such as various sheep strains, buffalo, fishes, and even coral, many of which has been deposited in GenBank but not described in a publication. The rationale behind this Noah-ark type of research was to systematically use melatonin receptors from those variously evolved species (birds, reptilians, mammals from various environments, some harsh insects, etc.) that have in common their capacity to recognize melatonin—by definition. We aimed at comparing their thermal stability once they were stably expressed in CHO cells. We would choose the most resistant one and use that as a model in the process of purification/reconstitution established previously by Logez et al. in our laboratory [75, 76]. Despite a few success [25], we terminated the program for resource

Melatonin catabolism has been described and discussed in-depth by Hardeland throughout the living kingdom [77]. In short, the main route of melatonin elimination (from the body) is not catabolism but rather conjugation and excretion via the urine. Thus there are three ways to consider: (i) the unchanged melatonin that one can find in urine; (ii) the conjugates, mostly glucuronides and sulfates; and (iii) the catabolism itself. Catabolism means that the molecule is transformed into something quite different from the original molecule. For example, in melatonin case, several reports demonstrated the opening of the indol ring. This opening possibly occurred through cytochrome c [78] or through 1,2-dioxygenase [79]. This was of importance because not too many compounds bear a formyl moiety such as the one produced during the cleavage of the indol cycle by either of these enzymes. This catabolism process would generate several products including N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK) [80]. The same paper, though, pointed out the absence of such metabolite(s) in human urine, strongly suggesting that the main catabolic

route of melatonin would rather be through conjugation, even after oxidative stress.

receptor, after

19

of strategies were further explored. One of them led to a pure, functional MT1

some others).

**3.3. Purification/structure**

limitation reasons.

**4. Melatonin catabolism**

After the first evidences that crystallogenesis of membrane proteins would be a challenge, we thought we would continue to search for ligands with a trial-and-error approach as we did for years, without the help of the visualization of the compounds in the protein as it became "mundane" these last years concerning co-crystallizations of compounds in their soluble protein targets. By multiplying the number of ligands in attempts to better describe the topography of the melatonin-binding site, even using mutagenesis [57–60], we also multiplied the assays on the functionality of the receptors, because we more and more discovered the ways the receptors were transferring their message to the cell. The simplistic view that a handful of such pathways between the receptors and the inner cell existed became obsolete. The by-product of such variety was that we found biased ligands that activated one but not the other(s) signaling pathways downstream melatonin receptor, as it is briefly discussed elsewhere.

Then, another new aspect came up: receptors can dimerize, a feature that was known for quite some time (see Rodbell [61], and see seminal review by Bockaert and Pin, [62]). Even though it was often believed to be an artifact of the purification attempts, the reality of such structures in situ was evidenced when one realized and demonstrated heterodimerisation between various types of such receptors: heterodimers between isoforms of a receptor, GABA [63], heterodimers between two unrelated GPCRs or even between GPCR, and another type of protein [64]. A paramount of examples were published, and their studies were made possible using the BRET technology [65]. In brief, engineering two receptors to make each of them fused with a carefully chosen fluorescent protein leads to a system in which the excitation of one of them results in the emission of fluorescent in the region exciting the other one. This cannot occur if the proteins are not physically in a very close vicinity of one another. Melatonin receptors have been also shown to be able to dimerize with serotonin 5HT2c receptor [66], as well as between MT<sup>1</sup> and MT2 [67] or between MT<sup>1</sup> and GPR50 [68], the melatonin-related receptor (evolved from Mel1c [24] and that has lost its property to bind melatonin [69]). More recently, the heterodimerisation of GPR50 and the transforming growth factor-β receptor [70] potentially open interesting routes toward the role of this orphan receptor as well as its implication in cancer development. Obviously many control experiments should be run in these explorations, as it would be attempting to conclude that any receptor can dimerize—and thus regulate the signaling pathway of—any other receptor (see Damian et al. [71] for a counter example, among some others).

#### **3.3. Purification/structure**

where given compounds were characterized as partial agonists that turned out to be inverse agonists instead [49], leading to a yet another level of complexity of melatonin receptor pharmacology. We recently embarked in such a task, by screening the agonist/antagonist behavior of a series of compounds (Legros & Boutin, in preparation) after assessing the various methods [50]. We also extended these coupling measurements to a small series of potential

rather partial agonists. As stated and described by Kenakin [52], one should further dig the concept of biased ligands. Indeed, it seems clear, now, that some at least of the downstream pathways of melatonin receptors are elicited by some agonist ligands while not by other ones. This concept has been a bias of the approach to melatonin research. Indeed, while seeking for tools to understand these pathways and their implications in various pathological models, we never had access to real, stable, and potent antagonists, despite past claims for such compounds [53, 54]. Even when large-scale screening campaigns were attempted [55], the poor affinity (compared to the already existing compounds: low μM affinity in the best cases *versus* low nM already available ones) of those newly discovered compounds was not in favor of trying to develop series of chemicals around those hits. A similar situation occurred when we

After the first evidences that crystallogenesis of membrane proteins would be a challenge, we thought we would continue to search for ligands with a trial-and-error approach as we did for years, without the help of the visualization of the compounds in the protein as it became "mundane" these last years concerning co-crystallizations of compounds in their soluble protein targets. By multiplying the number of ligands in attempts to better describe the topography of the melatonin-binding site, even using mutagenesis [57–60], we also multiplied the assays on the functionality of the receptors, because we more and more discovered the ways the receptors were transferring their message to the cell. The simplistic view that a handful of such pathways between the receptors and the inner cell existed became obsolete. The by-product of such variety was that we found biased ligands that activated one but not the other(s) signaling

Then, another new aspect came up: receptors can dimerize, a feature that was known for quite some time (see Rodbell [61], and see seminal review by Bockaert and Pin, [62]). Even though it was often believed to be an artifact of the purification attempts, the reality of such structures in situ was evidenced when one realized and demonstrated heterodimerisation between various types of such receptors: heterodimers between isoforms of a receptor, GABA [63], heterodimers between two unrelated GPCRs or even between GPCR, and another type of protein [64]. A paramount of examples were published, and their studies were made possible using the BRET technology [65]. In brief, engineering two receptors to make each of them fused with a carefully chosen fluorescent protein leads to a system in which the excitation of one of them results in the emission of fluorescent in the region exciting the other one. This cannot occur if the proteins are not physically in a very close vicinity of one another. Melatonin receptors have been also shown to be able to dimerize with serotonin 5HT2c receptor [66], as well as between

from Mel1c [24] and that has lost its property to bind melatonin [69]). More recently, the heterodimerisation of GPR50 and the transforming growth factor-β receptor [70] potentially open

and GPR50 [68], the melatonin-related receptor (evolved

pathways downstream melatonin receptor, as it is briefly discussed elsewhere.

attempted to find peptide ligands at melatonin receptors [56].

18 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

[51], to conclude that the compounds were not antagonists but

antagonists specific of MT<sup>2</sup>

MT<sup>1</sup>

and MT2

[67] or between MT<sup>1</sup>

Attempts to crystallize GPCRs have been done for years with various successes. Beside several reports of models of the receptors [72–74], that turned to be more or less disappointing because they poorly brought new information—somewhat as expected. Thus, several lines of strategies were further explored. One of them led to a pure, functional MT1 receptor, after several years of technical challenges: expression, stabilization, purification, and functionality measurement [75, 76]. We embarked several years ago in an approach that attempted to be original: as a first step in this purification/crystallization of melatonin receptor(s) project, we cloned melatonin receptors from as many and as various species as possible. Many such melatonin receptors have been reported in the literature, such as various sheep strains, buffalo, fishes, and even coral, many of which has been deposited in GenBank but not described in a publication. The rationale behind this Noah-ark type of research was to systematically use melatonin receptors from those variously evolved species (birds, reptilians, mammals from various environments, some harsh insects, etc.) that have in common their capacity to recognize melatonin—by definition. We aimed at comparing their thermal stability once they were stably expressed in CHO cells. We would choose the most resistant one and use that as a model in the process of purification/reconstitution established previously by Logez et al. in our laboratory [75, 76]. Despite a few success [25], we terminated the program for resource limitation reasons.

### **4. Melatonin catabolism**

Melatonin catabolism has been described and discussed in-depth by Hardeland throughout the living kingdom [77]. In short, the main route of melatonin elimination (from the body) is not catabolism but rather conjugation and excretion via the urine. Thus there are three ways to consider: (i) the unchanged melatonin that one can find in urine; (ii) the conjugates, mostly glucuronides and sulfates; and (iii) the catabolism itself. Catabolism means that the molecule is transformed into something quite different from the original molecule. For example, in melatonin case, several reports demonstrated the opening of the indol ring. This opening possibly occurred through cytochrome c [78] or through 1,2-dioxygenase [79]. This was of importance because not too many compounds bear a formyl moiety such as the one produced during the cleavage of the indol cycle by either of these enzymes. This catabolism process would generate several products including N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK) [80]. The same paper, though, pointed out the absence of such metabolite(s) in human urine, strongly suggesting that the main catabolic route of melatonin would rather be through conjugation, even after oxidative stress.

### **5. The melatonin paradox**

The field suffered from two paradoxes: safety and high affinity to natively poorly expressed receptors. First, melatonin is a very safe molecule, as far as we know; there is no report of human toxicity for overdose, and in mice the lethal dose is superior to 800 mg/kg [81]. Nevertheless, the French Agency for Food, Environmental and Occupational Health & Safety (www.anses.fr) emitted a report—in French—pointing at several cases of toxicity linked to melatonin consumption. Although they were ~100 cases in France reported during a 30-year period survey (i.e., a relatively modest number of cases, some of which have not been univocally linked to melatonin intake), the literature on clinical trials of melatonin is large enough to consider melatonin as reasonably safe [82], with the usual cases of deliberate overdose. In any case, it is not unusual to find reports in the literature where the dosage of melatonin in vivo or in cellulo is important. It was reported at several occasions—even if it probably depends on the cell type—cells treated with 1 mM of melatonin without apparent cell toxicity and even, in some cases, with beneficial effects [83–87]. Why is it a flaw? Because one can treat almost anything with this compound, at almost whatever dosage, and observe something, including relevant benefits for the situation (see **Table 1** and further examples in Boutin [88]). Furthermore, melatonin has a friendly behavior in terms of pharmacokinetics. When compared to another multi-card compound, resveratrol, it seems that unlike it, melatonin circulates in the blood after oral consumption at a fairly high concentration, while only 1 to 2% of resveratrol was found at the peak after ingestion of 25 mg/kg of resveratrol [89].

synthesizes melatonin. Nevertheless, a report [91] shows that, at least in the European hamster, the circannual rhythm could be maintained even after pinealectomy, thanks to light action in an accelerated photoperiodic regime, demonstrating the hypothalamic integration of the photoperiodic signal even in pinealectomized animals and, thus, in the absence of pineal

Molecular Pharmacology and Melatonin: 20 Yeras of Research and Trends

http://dx.doi.org/10.5772/intechopen.79524

21

Melatonin circadian rhythm can be measured in the blood from healthy volunteers and is clearly linked to the successions of days and nights. The timezyme (AANAT) is the master key of this process: active during dark periods and inactive during day (as the enzyme is

Melatonin exerts protective actions far beyond mammals, as several reports showed the role of melatonin in protecting yeast [92], bacteria [93], zebrafish [94], and plants [95] from various types of insult. For a discussion of melatonin throughout evolution, see also Tan et al. [96]. For decades, melatonin has been described as a compound able to fight almost any pathological situations. Tekbas et al. [97] even seriously considered melatonin as an antibiotic and Anderson et al. as an anti-Ebola virus agent [98]. A sample of those numerous actions can be found in Boutin [88], up to 2015. **Table 1** of the present essay is the follow-up of that particular list of beneficial properties. Many of those properties of melatonin seem to be linked to the capacity of the compound to limit reactive oxygen species (ROS) actions. ROS have been first documented as an "infamous" group of highly reactive molecules responsible for oxidative stress. In an enlightening review, Roy and coworkers [99] defined the field of reactive oxygen species, by starting to recall that ROS are also regulating signaling pathways in physiological situations. They also emphasized the fact that treatments with so-called antioxidants failed to show efficacy or/and positive effects in the prevention of diseases or health complications coming from oxidative stress. Nevertheless, it seems that according to a common belief, melatonin falls outside that particular category and is the ultimate scavenger/antioxidant molecule that has multiple capacities to prevent almost

It is sometimes complicated to find common sense in such a plethora of actions. **Table 1** lists some of these many actions, as published between 2015 and today. There is no way to be able to understand why melatonin has been reported for so many years in so many pathological situations. And the purpose of the present essay is not to do so. It is rather to make a compendium of those actions and to let the community know that such papers exist and that the

It is attempting, though, to make a rapid survey of those publications and to conclude that the common factor is the production of ROS. Then, we can hypothesize that most of these beneficial actions were due to a capacity of melatonin to induce antioxidant enzymatic defenses. To conclude on this working hypothesis, one will have to identify the nuclear receptor mediating this property. Nuclear factor erythroid 2-related factor 2 (Nrf2) might be a good candidate,

reason why melatonin is so ubiquitously active remains a mystery.

but a wishful thinking is certainly not a proof of fact.

gland synthesis of melatonin.

*6.1.1. Foreword*

any diseases.

destroyed by the light-induced proteasome).

Finally, as stated elsewhere in the present essay, the affinity of melatonin for its receptors is in the low nanomolar range [45]. Many strict analogues have been synthesized with similar high affinities for the receptors. Thus, it has been complicated and sometimes impossible to start new chemical programs ad initio, or at least starting from molecules issued from high-throughput screening campaigns, for example, from which hits are rather in the high micromolar range. Therefore, new compounds with unexpected structures have been slowly emerged in the field. As a representative example, D600 (hydroxyl-verapamil) is one of the few compounds showing strong specificity for MT1 [90]. No chemical program to date has been published to explore this observation and to deliver a specific ligand at MT1 receptor with some pharmacological properties (and specificity) rendering it amenable to in cellulo or in vivo experiments.

### **6. Melatonin actions**

#### **6.1. Overall**

Melatonin is the core master of rhythms. This part of the story is beyond any doubt. It translates the succession of days (light) and night (darkness) to the body. In the absence of light, the pineal gland (and more particularly the AANAT, the limiting step of melatonin biosynthesis) synthesizes melatonin. Nevertheless, a report [91] shows that, at least in the European hamster, the circannual rhythm could be maintained even after pinealectomy, thanks to light action in an accelerated photoperiodic regime, demonstrating the hypothalamic integration of the photoperiodic signal even in pinealectomized animals and, thus, in the absence of pineal gland synthesis of melatonin.

Melatonin circadian rhythm can be measured in the blood from healthy volunteers and is clearly linked to the successions of days and nights. The timezyme (AANAT) is the master key of this process: active during dark periods and inactive during day (as the enzyme is destroyed by the light-induced proteasome).

#### *6.1.1. Foreword*

**5. The melatonin paradox**

20 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

of 25 mg/kg of resveratrol [89].

in cellulo or in vivo experiments.

**6. Melatonin actions**

**6.1. Overall**

of the few compounds showing strong specificity for MT1

The field suffered from two paradoxes: safety and high affinity to natively poorly expressed receptors. First, melatonin is a very safe molecule, as far as we know; there is no report of human toxicity for overdose, and in mice the lethal dose is superior to 800 mg/kg [81]. Nevertheless, the French Agency for Food, Environmental and Occupational Health & Safety (www.anses.fr) emitted a report—in French—pointing at several cases of toxicity linked to melatonin consumption. Although they were ~100 cases in France reported during a 30-year period survey (i.e., a relatively modest number of cases, some of which have not been univocally linked to melatonin intake), the literature on clinical trials of melatonin is large enough to consider melatonin as reasonably safe [82], with the usual cases of deliberate overdose. In any case, it is not unusual to find reports in the literature where the dosage of melatonin in vivo or in cellulo is important. It was reported at several occasions—even if it probably depends on the cell type—cells treated with 1 mM of melatonin without apparent cell toxicity and even, in some cases, with beneficial effects [83–87]. Why is it a flaw? Because one can treat almost anything with this compound, at almost whatever dosage, and observe something, including relevant benefits for the situation (see **Table 1** and further examples in Boutin [88]). Furthermore, melatonin has a friendly behavior in terms of pharmacokinetics. When compared to another multi-card compound, resveratrol, it seems that unlike it, melatonin circulates in the blood after oral consumption at a fairly high concentration, while only 1 to 2% of resveratrol was found at the peak after ingestion

Finally, as stated elsewhere in the present essay, the affinity of melatonin for its receptors is in the low nanomolar range [45]. Many strict analogues have been synthesized with similar high affinities for the receptors. Thus, it has been complicated and sometimes impossible to start new chemical programs ad initio, or at least starting from molecules issued from high-throughput screening campaigns, for example, from which hits are rather in the high micromolar range. Therefore, new compounds with unexpected structures have been slowly emerged in the field. As a representative example, D600 (hydroxyl-verapamil) is one

date has been published to explore this observation and to deliver a specific ligand at MT1 receptor with some pharmacological properties (and specificity) rendering it amenable to

Melatonin is the core master of rhythms. This part of the story is beyond any doubt. It translates the succession of days (light) and night (darkness) to the body. In the absence of light, the pineal gland (and more particularly the AANAT, the limiting step of melatonin biosynthesis)

[90]. No chemical program to

Melatonin exerts protective actions far beyond mammals, as several reports showed the role of melatonin in protecting yeast [92], bacteria [93], zebrafish [94], and plants [95] from various types of insult. For a discussion of melatonin throughout evolution, see also Tan et al. [96]. For decades, melatonin has been described as a compound able to fight almost any pathological situations. Tekbas et al. [97] even seriously considered melatonin as an antibiotic and Anderson et al. as an anti-Ebola virus agent [98]. A sample of those numerous actions can be found in Boutin [88], up to 2015. **Table 1** of the present essay is the follow-up of that particular list of beneficial properties. Many of those properties of melatonin seem to be linked to the capacity of the compound to limit reactive oxygen species (ROS) actions. ROS have been first documented as an "infamous" group of highly reactive molecules responsible for oxidative stress. In an enlightening review, Roy and coworkers [99] defined the field of reactive oxygen species, by starting to recall that ROS are also regulating signaling pathways in physiological situations. They also emphasized the fact that treatments with so-called antioxidants failed to show efficacy or/and positive effects in the prevention of diseases or health complications coming from oxidative stress. Nevertheless, it seems that according to a common belief, melatonin falls outside that particular category and is the ultimate scavenger/antioxidant molecule that has multiple capacities to prevent almost any diseases.

It is sometimes complicated to find common sense in such a plethora of actions. **Table 1** lists some of these many actions, as published between 2015 and today. There is no way to be able to understand why melatonin has been reported for so many years in so many pathological situations. And the purpose of the present essay is not to do so. It is rather to make a compendium of those actions and to let the community know that such papers exist and that the reason why melatonin is so ubiquitously active remains a mystery.

It is attempting, though, to make a rapid survey of those publications and to conclude that the common factor is the production of ROS. Then, we can hypothesize that most of these beneficial actions were due to a capacity of melatonin to induce antioxidant enzymatic defenses. To conclude on this working hypothesis, one will have to identify the nuclear receptor mediating this property. Nuclear factor erythroid 2-related factor 2 (Nrf2) might be a good candidate, but a wishful thinking is certainly not a proof of fact.


**Authors Date Protection against Targets Amount Species Ref**

Hermoso et al. 2016 Steatosis Liver 10 mg/kg Rat [129]

Hu et al. 2017 BBB damage BBB 15 mg/kg Rat [131]

Karaer et al. 2015 Radiation damage Inner ear 5 mg/kg Rat [135] Karimfar et al. 2015 Cryopreservation stress Sperm 0.001–1 mM Human [136]

Li et al. 2016 Cadmium-induced toxicity Testicles 1 mg Mouse [142] Li et al. 2015 Maturation defect Oocyte 0.001–1 μM Pig [143] Lopez et al. 2017 MPTP-toxicity Brain 10 mg/kg Mouse [144] Lv et al. 2018 Cr(VI) toxicity Testicles 25 mg/kg Mouse [145] Ma et al. 2018 Oxidative injury Heart 100 μM Rat [146]

neurons

cells


Heart mitochondria

Kidney 15 mg/kg/day Rat [125]

Molecular Pharmacology and Melatonin: 20 Yeras of Research and Trends

Organs 10 mg/kg Mouse [127]

Kidneys 5–20 mg/kg Mouse [128]

Liver 2 mg/kg Rat [130]

Brain 10 mg/kg Mouse [132]

Retina 10 mg/kg/day Rat [133]

Liver / Mouse [134]

Organs 1 mg/kg Rat [137]

/ 10 mg/kg Rat [138]

Liver 5 mg/kg/day Rat [140]

Ovaries 20 mg/kg/day Mouse [147]

Kidney 20 mg/kg/day Rat [149]

10 mg/kg/day Rat [139]

10 mg/kg/day Rat [148]

1 μM Goat [126]

http://dx.doi.org/10.5772/intechopen.79524

23

Ghaznavi et al. 2016 Gentamicin-induced

Goc et al. 2017 Sodium nitroprusside

Hsu et al. 2017 Trauma-induced

Ji et al. 2017 Sepsis-associated

Jiang et al. 2016 Diabetic-induced

Jin et al. 2016 Non-alcoholic fatty liver

Khaksar et al. 2017 Fluoxetine-induced tissue

Khalil et al. 2015 Zonisamide-induced

Lebda et al. 2018 Thioacetamide-induced

Ma et al. 2017 Tripterygium glycosides

Mehrzadi et al. 2016 Gentamicin-induced

Lee et al. 2016 H2

Ghosh et al. 2017 Copper ascorbate-induced

Goudarzi et al. 2017 Cyclophophamide-induced

toxicity

damage

toxicity

stress

hemorrhage

encephalopathy

inflammation

disease

injury

toxicity

Koc et al. 2016 Apoptosis Olfactive

fibrosis

O2

toxicity

toxicity

Ma et al. 2015 Adriamicyn-toxicity Breast cancer


**Authors Date Protection against Targets Amount Species Ref** Abdel-Moneim et al. 2015 *Naja naja* venom toxicity — 10 mg/kg Rat [100]

Al-Olayan et al. 2015 Aluminum-induced injury Neurons 10 mg/kg Rat [102] Al-Rasheed et al. 2016 CCl4-induced toxicity Liver 20 mg/kg Rat [103] Amin et al. 2015 Diabetes-induced apoptosis Heart 10 mg/kg Rat [104]

Barberino et al. 2017 Cisplatin-induceddamage Ovaries 5–20 mg/kg Mouse [107] Berkiks et al. 2017 Ccognitive disorders Brain 5 mg/kg Rat [108] Cao et al. 2017 Subarachnoid hemorrhage Brain 150 mg/kg Rat [109] Cebi et al. 2018 Radioiodine treatment Testicles 12 mg/kg/day Rat [110]

Chen et al. 2017 Neuropathic pain / ??? Rat [114]

Das et al. 2017 Mitochondrial dysfunction Hepatocytes 10–20 mg/kg/day Mouse [116]

Dos Santos et al. 2018 Lupus nephritis injury Kidney 10 mg/kg/day Mouse [119]

Ewida et al. 2016 Metabolic syndroma Kidney 5 mg/kg Rat [121]

organ

cells

/ 10 mg/kg Rat [101]

Heart 20–50 mg/kg Rat [105]

Kidney 10 mg/kg Rat [106]

Kidney 90 mg Rat [111]

Pancreas 0.5–2 mM Rat [113]

Liver 10 mg/kg Rat [115]

Heart 100 μM Rat [117]

Brain 10 mg/kg Mouse [118]

Muscle 2.5–5 mg/kg Rat [122]

Intestine 45 mg/day Rat [123]

Mitochondria 5–50 mg/kg Rat [124]

120 mg/kg Rat [112]

4 mg/L Carp [120]

Allagui et al. 2015 Aluminum-induced

Asghari et al. 2017 Aluminum phosphide

Banaei et al. 2016 Ischemia–reperfusion

Chang et al. 2016 Ischemia–reperfusion

Chen et al. 2016 Endoplasmic reticulum

Czechowska et al. 2015 Thioacetamide-induced

Ding et al. 2018 Post-traumtic cardiac

Favero et al. 2017 Fibromyalgia-related

Fernandez-Gil et al. 2017 Radiotherapy-iondued

Galley et al. 2017 Paclitaxel-induced

Ding et al. 2015 Traumatic injury-induced

toxicity

22 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

toxicity

injury

injury

stress

fibrosis

function

apoptosis

Drag-Kozak et al. 2018 Cadmium-induced toxicity Reproductive

alterations

toxicity

dysfunction

Chang et al. 2015 Ischemia/reperfusion injury Adipose stem


**Authors Date Protection against Targets Amount Species Ref**

Uygur et al. 2016 As-induced apoptosis Testicles 25 mg/kg/day Rat [179]

Wang et al. 2018 Intracerebral Hemorrahge Brain ??? Rat [182]

Xue et al. 2017 Kainic-induced cell death Brain 20 mg/kg Mouse [184] Yang et al. 2018 Subarachnoid hemorrhage Brain 0.1–10 μM Mouse [185]

Yu et al. 2015 Ischemia/reperfusion injury Heart 10 mg/kg/day Rat [190] Yu et al. 2015 Ischemia/reperfusion injury Heart 20 mg/kg/day Rat [191]

Zhang et al. 2017 Diabetic cardiomyopathy Heart 20 mg/kg/day Mouse [195] Zhang et al. 2017 Arsenic-induced injury Liver 5–20 mg/kg Rat [196]

Zhao et al. 2017 NaF-induced injury Embryos 50–100 μM? Mouse [198] Zhou et al. 2017 Ischemia/reperfusion injry Heart 20 mg/kg Mouse [199]

Kobylinska et al. 2017 Lead-induced cell death Tobacco cells 200 nM Plant [201] Wang et al. 2017 Drought stress Arabidopsis ??? Plant [202]

endothelium

Testicles 10 mg/kg/day Rat [178]

http://dx.doi.org/10.5772/intechopen.79524

25

Molecular Pharmacology and Melatonin: 20 Yeras of Research and Trends

Heart 50 μM Rat [180]

Brain 30 μg/kg/day Rat [181]

Blood samples 10 mg/kg Rat [183]

Blood samples 3 mg/day Human [183]

Macrophages 50–100 mg/kg Mouse [186]

Kidney 10 mg/kg Rat [187]

Heart 10 mg/kg Rat [188]

Oocytes ??? Pig [189]

Thyroids 0.001–10 mM Pig [192]

Heart 20 mg/kg/day Mouse [193]

Oocytes 30 mg/kg Mouse [194]

Brain 50 μM Rat [197]

10 μM Rat [200]

Torabi et al. 2017 Cyclophosphamide-

Vazan et al. 2015 Epinephrine-induced

Vinod et al. 2016 Aging-induced NO rhythm loss

Wang et al. 2016 Smoke-induiced vascular

Wang et al. 2016 Smoke-induiced vascular

Yi et al. 2017 Stress-induced

Yildirim et al. 2016 Ureteral obstruction-

Yu et al. 2018 Ischemia–reperfusion

Yu et al. 2018 MEHP-induced meiosis

Zasada et al. 2015 Nitrobenzene-induced

Zhai et al. 2017 Pathological cardiac

Zhang et al. 2017 Bisphenol A-induced

Zhang et al. 2016 β-amyloid-induced

**Plants**

induced toxicity

injury

injury

injury

inflammation

induced injury

injury

defect

peroxidation

hypertrophy

toxicity

damages

Zhu et al. 2018 Oxidative stress Heart


**Authors Date Protection against Targets Amount Species Ref** Mirhoseini et al. 2017 Torsion/detorsion model Testicles 25 μg/kg Rat [150]

Naseri et al. 2017 Irradiation-induced toxicity Brain 100 mg/kg Rat [154]

O'Neal-Moffitt et al. 2015 Alzheimer neiuropathology / Ad libitum??? Mouse [156]

Ozsoy et al. 2016 Mitochondrial dysfunction Liver 10 mg/kg Rat [159] Ozsoy et al. 2015 6-hydroxydopamine stress Neurons 10 mg/kg Rat [160]

Pang et al. 2016 Frozen–thawed cycles Sperm 0.01–1 mM Bovine [162] Patino et al. 2016 o2 & Glucose deprivation Brain slices 10–30 μM Rat [163]

Rajput et al. 2017 Alcohol-induced stress Brain 20 mg/kg Mouse [165]

Sarihan et al. 2015 TCDD-induced injury Heart 5 mg/kg/day Rat [167] Scheuer et al. 2016 UVR-induced erythrema Skin 0.5–12.5% Human [168]

Shao et al. 2015 LPS-induced mastitis Breast 5–20 mg/kg Mouse [171]

Shokrzadeh et al. 2015 Cyclophosphamide toxicity Lung 2.5–20 mg/kg Mouse [173] Sinha et al. 2018 Hypoxy/Ischemy Brain 10 mg/kg Mouse [174] Tanabe et al. 2015 Oxidative stress ??? 100 μg/kg Mouse [175]

Tas et al. 2015 Ischemia/reperfusion injury Intestine 50 mg/kg Rat [177]

nigra

Heart mitochondria

Liver 10 mg/kg Rat [151]

Pineal gland 10 mg/kg/day Rat [153]

Brain 10–30 mg/kg Mouse [155]

Mouth 45 mg/kg/day Rat [157]

Testicles 10 mg/kg Rat [158]

/ 10–100 mg/kg Rat [161]

Pancreas 50 mg/kg Rat [166]

Estomac 10 mg/kg Rat [169]

Colon 2.5 mg/kg/day Rat [170]

Brain 5–20 mg/kg Rat [172]

Aorta 10 mg/kg/day Rat [176]

10–30 mg/kg Rat [164]

0.125–4 μM Goat [152]

Montasser et al. 2017 Methotrexate-induced

Mukherjee et al. 2015 Isoproterenol-induced

Naskar et al. 2015 MPTP-induced

Ortiz et al. 2015 Radiation-induced

Othman et al. 2016 Bisphenopl A-induced

Pal et al. 2016 Stress-induced behavior

Sadek and Khattab 2017 Arginine-induced

Shahrokhi et al. 2016 Ischmia/reperfusion-

Shang et al. 2016 Colitis-induced neuron

Shokri et al. 2015 Pilocarpine-induced

Tang et al. 2017 Abdominal aortic

Munoz et al. 2017 Cumene peroxide-induced

toxicity

24 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

damage

stress

Parkinsonism

mucositis

toxicity

changes

Paul et al. 2018 Oxidative stress Substancia

pancreatis

oxidative stress

damage

epilesy

aneurysm


*6.1.2. Melatonin as an antioxidant molecule*

Yang et al. 2017 Glucocorticoid-induced

Yu et al. 2017 Ischemia–reperfusion

Zhou et al. 2018 rotenone-induced cell

impairment

injury

Zhao et al. 2018 Ab-induced neurotoxicity Primary

death

Zhu et al. 2015 Myocardial infarction Adipose stem

**Table 1.** Some of the actions of melatonin observed in various pathophysiological situations.

can melatonin induce those antioxidant defenses?

*6.1.3. Melatonin as a ligand of Nrf2?*

Forman et al. in two seminal papers explained that the notion of hydroxyl radical scavengers is an extreme case of wishful thinking [230, 231]. Later on, he and his coworkers clearly showed that a unique molecule could not be a scavenger of superoxides, hydrogen peroxides, or other hydroperoxides or hydroxyl radicals. Indeed, all chemicals inside a cell react chemically with radical species, that is, proteins, lipids, nucleic acids, etc. Thus, because all organic compounds react with radicals with rate constants approaching the diffusion limitation, no compound can be better than the sum of the others to scavenge those ROS [231]. This can apply to melatonin. Like many other chemicals, whether indol-based or not, this compound, even at large concentrations, cannot be, per se, a scavenger. Therefore claims that melatonin is a super scavenger, with many advantages over other similar naturally occurring compounds, must be taken with extreme caution, despite several in-depth reviews, such as the one by Galano et al. [232]. Even the use of "direct" detection methods of radicals (to prove this hypothesis) should be handled with much caution [233]. Nevertheless, melatonin sustains antioxidant properties (see Rodriguez et al. [234] for review). Indeed, it can increase the expression of antioxidant enzymes (see, e.g., Mahrzadi et al. [149] and references therein). Melatonin can also act as a potent antiapoptotic agent in many cells [235], maybe through an antioxidant type of activity, as a relationship between ROS and apoptosis and autophagy has been well documented. How

**Authors Date Protection against Targets Amount Species Ref** Xue et al. 2017 Kainic-induced cell death N2a cells 50–100 μM Mouse [184] Yang et al. 2017 Iron overload senescence MSCstem cells 10 nM–100 μM Mouse [224]

> Isolated knee joints

neurons

cells

1 μM Mouse [225]

http://dx.doi.org/10.5772/intechopen.79524

27

0.1–100 μM Mouse [227]

5 μM Rat [229]

H9c2 10 μM Rat [226]

Molecular Pharmacology and Melatonin: 20 Yeras of Research and Trends

SH-SY5Y cells 50–500 μM Human [228]

At the time (2003) Rodriguez et al. wrote their review [234] on antioxidant capacities of melatonin, Nrf2 was not really an identified and recognized partner in this process. The relationship between melatonin actions and the role of nuclear factor erythroid 2-related factor 2 (Nrf2) has been reported more than 50 times in the literature these last years, starting around 2009 [236].


**Table 1.** Some of the actions of melatonin observed in various pathophysiological situations.

#### *6.1.2. Melatonin as an antioxidant molecule*

**Authors Date Protection against Targets Amount Species Ref** Xu et al. 2016 Thermotolerance Tomato plants 10 μM Plant [203] Zheng et al. 2017 Salt-stress Plant cells — Plant [204]

Baburina et al. 2017 Aging Mitochondria 7 mg/kg/day Rat [205]

Charao et al. 2015 Paraquat-induced toxicity A549 cells 10 μg/mL Human [207]

Fu et al. 2017 Chloranil-induced toxicity PC12 cells 25–200 μM Mouse [209] Gurer-Orhan et al. 2016 b-amyloid-induced damage Cells 10–100 μM Hamster [210] Han et al. 2017 Obesity-associated stress Oocytes 30 mg/kg/day Mouse [211] Janjetovic et al. 2017 UVB-induced damage Melanocytes 50 μM Human [212]

Liu et al. 2015 Hypoxia-induced N2a cells 5 μg/mL Mouse [214]

Mehrzadi et al. 2017 H2O2-induced toxicity MSC 10 nM–1 mM Human [85]

Pang et al. 2017 Early apoptosis Oocytes 1 nM Bovine [218]

Song et al. 2015 LPS-induced inflammation Stem cells 100 nM Mouse [220]

2015 Methamphetamine-toxicity C6 cells 1–100 nM Rat [213]

cells

myotubes

Hep3B

ARPE-19 cells 200 μM Human [206]

L02 cells 1 μM Human [208]

Podocytes 0.1–1 mM Mouse [83]

Oocytes 1 nM–1 mM Pig [84]

Trophoblasts 1 mM Human [86]

Adipocytes 100 μM Human [221]

SHSY-5Y cells 10 μM Human [222]

SH-SY5Y cells 0.01–1 μM Human [223]

1 mM Rat [87]


Cardiomycyte cell line

1.5–6 mg/mL Rat [215]

10 nM Mouse [216]

10 nM Human [217]

**Cells**

Jumnongprakhon

Sagrillo-Fagundes

Sanchez-Bretano et al. 2017 H2

et al.

et al.

Bardak et al. 2017 2-ethylpyridine-induced

Chen et al. 2015 Bile acid-induced oxidative

Ji et al. 2016 Angiotensin-II-indued

Miao et al. 2018 benzo(a)pyrene meiotic

Tan et al. 2016 Oxidative stress-induced

Waseem et al. 2017 Oxaliplatin-induced

Wongprayoon et al. 2017 Methamphetamine-

Xie et al. 2015 Hypoxia-induced

stress

26 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

stress

injury

Lu et al. 2015 LPS-induced hypertrophy Myocardial

Maarman et al. 2017 Uric acid-induced toxicity C2C12

failure

Ozerkan et al. 2015 CCl4-induced cytotoxicity HepG2 &

O2

cell death

toxicity

induced stress

hypertrophy

2016 Hypoxia-reoxygenation toxicity

Forman et al. in two seminal papers explained that the notion of hydroxyl radical scavengers is an extreme case of wishful thinking [230, 231]. Later on, he and his coworkers clearly showed that a unique molecule could not be a scavenger of superoxides, hydrogen peroxides, or other hydroperoxides or hydroxyl radicals. Indeed, all chemicals inside a cell react chemically with radical species, that is, proteins, lipids, nucleic acids, etc. Thus, because all organic compounds react with radicals with rate constants approaching the diffusion limitation, no compound can be better than the sum of the others to scavenge those ROS [231]. This can apply to melatonin. Like many other chemicals, whether indol-based or not, this compound, even at large concentrations, cannot be, per se, a scavenger. Therefore claims that melatonin is a super scavenger, with many advantages over other similar naturally occurring compounds, must be taken with extreme caution, despite several in-depth reviews, such as the one by Galano et al. [232]. Even the use of "direct" detection methods of radicals (to prove this hypothesis) should be handled with much caution [233]. Nevertheless, melatonin sustains antioxidant properties (see Rodriguez et al. [234] for review). Indeed, it can increase the expression of antioxidant enzymes (see, e.g., Mahrzadi et al. [149] and references therein). Melatonin can also act as a potent antiapoptotic agent in many cells [235], maybe through an antioxidant type of activity, as a relationship between ROS and apoptosis and autophagy has been well documented. How can melatonin induce those antioxidant defenses?

#### *6.1.3. Melatonin as a ligand of Nrf2?*

At the time (2003) Rodriguez et al. wrote their review [234] on antioxidant capacities of melatonin, Nrf2 was not really an identified and recognized partner in this process. The relationship between melatonin actions and the role of nuclear factor erythroid 2-related factor 2 (Nrf2) has been reported more than 50 times in the literature these last years, starting around 2009 [236]. Nrf2 is a key factor in the induction of antioxidant protein defenses of the cell. It binds to a region called EpRE—also known as ARE [230]. This transcription factor (belonging to the huge family of Cap'n'collar transcription factors) is neutralized in cellulo by another factor, Kelch-like ECH-associated protein 1 (Keap1). The heterodimer is directed to the proteasome where the proteins are destroyed. Upon some conditions, including pharmacological ones (for instance, sprout-derived chemicals [237]), the dimer is open, and the free Nrf2 migrates to the nucleus of the cell where it associates with the EpRE region. This translates by the induction of several key proteins of the antioxidant cellular armada, such as heme oxygenase 1, quinone reductase 1, glutathione S-transferase π1, etc., but also enzymes from the phase 2 drug metabolism, such as UDP glucuronosyltransferases. There is a large literature indicating that melatonin induces Nrf2 expression and/or its separation with its corepressor, Keap1 (about 50 publications reported at least the induction of Nrf2 by melatonin). Furthermore, it has been shown several times that upon melatonin treatment, the cytosolic Nrf2 migrates to the nucleus where it can exert its inductive function. One question remains unanswered, though; it is the possibility that Nrf2 was the elusive nuclear factor described at several occasions [26]? Unfortunately, the tridimensional structure of Nrf2 and/or of its complex with Keap1 has not been reported. It seems that Nrf2 has no a priori structure and is only adopting define 3D shape either once linked to Keap1 (a complex that is then directed to the proteasome) or when in complex with a ligand. Much more need to be done to understand this relationship that might enlighten part of the observation of **Table 1**.

**6.3. Through QR2**

with very fast exchange) baptized *MT3*

we discovered that QR2 was indeed *MT3*

**6.4. Through mitochondria**

binding, signing the presence of MT1

tion for melatonin exerting its antioxidant capacities [88].

As stated previously, it was rapidly discovered that two melatonin-binding sites were GPCR in mammals and an extra one, Mel1c, in reptilians and birds. The group of Dubocovich also pointed at a binding site, ML2 [248, 249], with rather unconventional properties (particularly

ticular receptor, after having obtained similar results for the pharmacological description of this particular "receptor" [250]. We had the chance to identify it by using a series of inverse

which affinity chromatography succeeded. The binding site was an enzyme with a peculiar story, quinone reductase 2 (QR2 a.k.a. NQO2) [23]. The activity of this enzyme was first described in the early 1960s as a reductase using unconventional donors as co-substrates, such as *N*-benzyl, *N*-methyl, or *N*-ribosyl dihydronicotinamides, and Talalay's group established that the enzyme was the enzyme once described by Liao et al. [251]. Interestingly, they clearly established the nature of the enzyme and particularly its incapacity to recognize NADH or NAD(P)H as co-substrates, as well as its sensitivity to some chemical, in an orthogonal way to QR1. For instance, QR2 is insensitive to the reference QR1 inhibitor, dicoumarol. When

KO cell lines [252], KO mouse strain [253] and various tools that would help to understand the potential role of this enzyme (see Vella et al. [254] and references there in). Although the enzyme was identified during a pure melatonin-related program, it turned out to have nothing in common, a priori, with the melatoninergic systems. Indeed, while able to bind melatonin with a rather strong affinity—in the nM range—QR2 is only poorly inhibited by melatonin, in the 50 μM range, suggesting that melatonin regulation was not a player in the QR2 game. Indeed, as often in the drug metabolism area, enzymes from both phases I and II, such as cytochrome P450, UGTs, or glutathione S-transferases, are often enzymes with enough plasticity in their catalytic sites in order to accommodate xenobiotics that are, by definition, molecules of various chemical structures issued from the environment at large.

Nevertheless, I suggested that QR2 inhibition at high dose of melatonin could be an explana-

Incidentally, a couple of papers reported not only the synthesis of melatonin in mitochondria but also the presence in these organelles—at least those isolated from the brain—of a measurable

reported for genes encoding for these GPCRs, it seems possible to hypothesize that those binding sites were a leftover from the brain preparation of mitochondria, a possibility reinforced by the difficulty of preparing "pure" mitochondria from these lipid- and membrane-rich organs. Beyond these hypothetical technical considerations lays also the fact that our laboratory had experienced "very" often cells with no binding activity, suggesting that mitochondria would express melatonin receptors only in melatonin receptor-rich organs—such as the brain—an indirect suggestion that the presence of those receptors in these organelles might be a "simple" signature of a difficult

pharmacology techniques, comprising an analogue of a specific *MT3*

. In 1999 we embarked in an attempt to clone this par-

Molecular Pharmacology and Melatonin: 20 Yeras of Research and Trends

http://dx.doi.org/10.5772/intechopen.79524

, we had to reinforce this observation by generating

receptors. Again, as long as the mitochondrial DNA is not

ligand, MCA-NAT, on

29

#### **6.2. Through MT1 /MT<sup>2</sup>**

The specificity of actions linked to the binding of melatonin to one of its receptors, MT1 and MT2 , is still a matter of debate. Indeed, a thorough survey of its action is not possible in vivo in wildtype animals, because we are still lacking reliable and isoform-specific antagonists (see discussion in Jockers et al. [45]). It is possible, though, to study the role of one or the other of the receptors using either natural KO animals [such as the Siberian hamster, but not the European hamster (Gautier & Boutin [281])] or, alternatively, MT1 or MT2 (or both) KO animals, which have been engineered [238–240], but results are slow to be issued [241–243] (see also discussion in Jockers et al. [45]). Nevertheless, general conclusions can be drawn from accumulated data, as reviewed by the same authors [45]. It is difficult, as of today, without drowning in the 3970 available reviews on melatonin, to clearly segregate between the subtype roles. Among the clearest facts, mice lacking MT1 receptors exhibit higher mean blood glucose levels than wildtype mice [244]. Those KO animals tend to be more glucose intolerant and insulin resistant than their wildtype counterparts. Through many different parameters, both MT1 and MT2 receptors seem to have a role in the phase shift of circadian rhythms, as demonstrated by several lines of indications, including knockout animals, the use of specific MT<sup>2</sup> antagonists (luzindole, 4P-PDOT), as well as ex vivo experiments. Melatonin can activate an immune response. Remarkably, that was proposed as early as 1926 by Berman. This activity seems to depend on the MT1 receptor [245], but opposite claims have also been published [246]. Liu et al. showed that it was MT<sup>2</sup> that was the receptor implicated in axogenesis and the formation of functional synapses [247].

Nevertheless, it seems to me improbable that even only some of the actions in **Table 1** were through the binding of melatonin onto its receptors.

#### **6.3. Through QR2**

Nrf2 is a key factor in the induction of antioxidant protein defenses of the cell. It binds to a region called EpRE—also known as ARE [230]. This transcription factor (belonging to the huge family of Cap'n'collar transcription factors) is neutralized in cellulo by another factor, Kelch-like ECH-associated protein 1 (Keap1). The heterodimer is directed to the proteasome where the proteins are destroyed. Upon some conditions, including pharmacological ones (for instance, sprout-derived chemicals [237]), the dimer is open, and the free Nrf2 migrates to the nucleus of the cell where it associates with the EpRE region. This translates by the induction of several key proteins of the antioxidant cellular armada, such as heme oxygenase 1, quinone reductase 1, glutathione S-transferase π1, etc., but also enzymes from the phase 2 drug metabolism, such as UDP glucuronosyltransferases. There is a large literature indicating that melatonin induces Nrf2 expression and/or its separation with its corepressor, Keap1 (about 50 publications reported at least the induction of Nrf2 by melatonin). Furthermore, it has been shown several times that upon melatonin treatment, the cytosolic Nrf2 migrates to the nucleus where it can exert its inductive function. One question remains unanswered, though; it is the possibility that Nrf2 was the elusive nuclear factor described at several occasions [26]? Unfortunately, the tridimensional structure of Nrf2 and/or of its complex with Keap1 has not been reported. It seems that Nrf2 has no a priori structure and is only adopting define 3D shape either once linked to Keap1 (a complex that is then directed to the proteasome) or when in complex with a ligand. Much more need to be done to understand this relationship that might enlighten part of the observation of **Table 1**.

The specificity of actions linked to the binding of melatonin to one of its receptors, MT1

, is still a matter of debate. Indeed, a thorough survey of its action is not possible in vivo in wildtype animals, because we are still lacking reliable and isoform-specific antagonists (see discussion in Jockers et al. [45]). It is possible, though, to study the role of one or the other of the receptors using either natural KO animals [such as the Siberian hamster, but not the European

have been engineered [238–240], but results are slow to be issued [241–243] (see also discussion in Jockers et al. [45]). Nevertheless, general conclusions can be drawn from accumulated data, as reviewed by the same authors [45]. It is difficult, as of today, without drowning in the 3970 available reviews on melatonin, to clearly segregate between the subtype roles. Among the clear-

mice [244]. Those KO animals tend to be more glucose intolerant and insulin resistant than their

to have a role in the phase shift of circadian rhythms, as demonstrated by several lines of indica-

well as ex vivo experiments. Melatonin can activate an immune response. Remarkably, that was

Nevertheless, it seems to me improbable that even only some of the actions in **Table 1** were

or MT2

receptors exhibit higher mean blood glucose levels than wildtype

and

(or both) KO animals, which

receptors seem

receptor [245],

that was

and MT2

antagonists (luzindole, 4P-PDOT), as

**6.2. Through MT1**

est facts, mice lacking MT1

MT2

**/MT<sup>2</sup>**

28 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

hamster (Gautier & Boutin [281])] or, alternatively, MT1

tions, including knockout animals, the use of specific MT<sup>2</sup>

through the binding of melatonin onto its receptors.

wildtype counterparts. Through many different parameters, both MT1

proposed as early as 1926 by Berman. This activity seems to depend on the MT1

but opposite claims have also been published [246]. Liu et al. showed that it was MT<sup>2</sup>

the receptor implicated in axogenesis and the formation of functional synapses [247].

As stated previously, it was rapidly discovered that two melatonin-binding sites were GPCR in mammals and an extra one, Mel1c, in reptilians and birds. The group of Dubocovich also pointed at a binding site, ML2 [248, 249], with rather unconventional properties (particularly with very fast exchange) baptized *MT3* . In 1999 we embarked in an attempt to clone this particular receptor, after having obtained similar results for the pharmacological description of this particular "receptor" [250]. We had the chance to identify it by using a series of inverse pharmacology techniques, comprising an analogue of a specific *MT3* ligand, MCA-NAT, on which affinity chromatography succeeded. The binding site was an enzyme with a peculiar story, quinone reductase 2 (QR2 a.k.a. NQO2) [23]. The activity of this enzyme was first described in the early 1960s as a reductase using unconventional donors as co-substrates, such as *N*-benzyl, *N*-methyl, or *N*-ribosyl dihydronicotinamides, and Talalay's group established that the enzyme was the enzyme once described by Liao et al. [251]. Interestingly, they clearly established the nature of the enzyme and particularly its incapacity to recognize NADH or NAD(P)H as co-substrates, as well as its sensitivity to some chemical, in an orthogonal way to QR1. For instance, QR2 is insensitive to the reference QR1 inhibitor, dicoumarol. When we discovered that QR2 was indeed *MT3* , we had to reinforce this observation by generating KO cell lines [252], KO mouse strain [253] and various tools that would help to understand the potential role of this enzyme (see Vella et al. [254] and references there in). Although the enzyme was identified during a pure melatonin-related program, it turned out to have nothing in common, a priori, with the melatoninergic systems. Indeed, while able to bind melatonin with a rather strong affinity—in the nM range—QR2 is only poorly inhibited by melatonin, in the 50 μM range, suggesting that melatonin regulation was not a player in the QR2 game. Indeed, as often in the drug metabolism area, enzymes from both phases I and II, such as cytochrome P450, UGTs, or glutathione S-transferases, are often enzymes with enough plasticity in their catalytic sites in order to accommodate xenobiotics that are, by definition, molecules of various chemical structures issued from the environment at large.

Nevertheless, I suggested that QR2 inhibition at high dose of melatonin could be an explanation for melatonin exerting its antioxidant capacities [88].

#### **6.4. Through mitochondria**

Incidentally, a couple of papers reported not only the synthesis of melatonin in mitochondria but also the presence in these organelles—at least those isolated from the brain—of a measurable binding, signing the presence of MT1 receptors. Again, as long as the mitochondrial DNA is not reported for genes encoding for these GPCRs, it seems possible to hypothesize that those binding sites were a leftover from the brain preparation of mitochondria, a possibility reinforced by the difficulty of preparing "pure" mitochondria from these lipid- and membrane-rich organs. Beyond these hypothetical technical considerations lays also the fact that our laboratory had experienced "very" often cells with no binding activity, suggesting that mitochondria would express melatonin receptors only in melatonin receptor-rich organs—such as the brain—an indirect suggestion that the presence of those receptors in these organelles might be a "simple" signature of a difficult separation between all the kinds of membranes present in a neuronal cell. There were several reports over the last decade showing a protective effect of melatonin onto mitochondria functions (see **Table 2**). Then several reviews suggested that melatonin was synthesized by mitochondria (see, for instance, Manchester et al., 2015 [255], Reiter et al., 2017 [256] and 2018 [257]). Particularly interesting is the fact that Cellular and Molecular Life Science published a special issue in 2017 (volume 74, issue 21) dealing with melatonin and mitochondria, emphasizing the interest of the community for these observations and their consequences. A reason for this hypothesis was given: mitochondria, like chloroplasts in plants, evolved from bacteria. Because originally cyanobacteria were subjected to heavy exposition to toxic free radicals, they evolved in keeping melatonin as an antioxidant, scavenging these radicals and thus preserving their integrity. Because this happened about 3 billion years ago, melatonin has been selected to protect and defend those microorganisms.

Of course, when bacteria colonized eukaryotic cells, the trait was maintained throughout evolution, including in mammals. Thus, no matter how high or low the blood melatonin concentration is, this particular intra-mitochondria concentration remains constant (not depending on the circadian rhythm), protecting mitochondria from the never ending production of free radicals that is the signature of sane mitochondria. An impressive series of publications were issued in these last few years (see **Table 2**) dealing with situations where toxicity was prevented by melatonin. This can be further extended to the protection afforded by mitochondria-synthesized melatonin to oocytes [278]. Finally, one can also add the observation that mitochondria melatonin protects plants from drought episodes [202]. Particularly interesting was the last one in which Suofu et al. [10] demonstrated the presence of the main melatonin synthesis enzymes, arylalkylamine *N*-acetyltransferase (AANAT) and acetylserotonin *O*-methyltransferase (HIOMT), in mitochondria matrix, as well as the high concentration of melatonin inside those mitochondria matrix. Furthermore, they showed the presence of MT1 receptor and the actual coupling of this receptor, turning this observation into a major progress in the domain, as rare are the receptors signaling in the mitochondria. This observation was challenged by Ahluwalia et al. [279] (replied by Suofu et al. [11]) that was able to show the presence of the melatonin receptors in muscle fibers, but not in mitochondria thereof. It is clear that this breakthrough information will be better understood after the observation will be confirmed independently. Of course, questions remain in the skeptical reader mind: if the melatonin system evolved from bacteria over several billion years, then the genetic material should have evolved together with it, meaning that the mitochondrial DNA should encode for MT1 , AANAT, and ASMT, which does not seem to be the case. This observation would also lead to an extra complexity involving the protein importation system (TOM, Tim, etc.) and the *ad hoc* addressing sequence(s) onto those proteins, all of which have not been seen so far. Furthermore, the discovery and description of an inward transport of melatonin in mitochondria [280] are not fitting an in situ synthesis. For those of us who have been working with subcellular organelles, it is very hard to assess the purity of those organelles because of the continuum that exists between all the membranes from cells. One should also add to this the particular complexity of the brain tissue that is by essence very lipid-rich, leading to an extra difficulty in preparing pure membranes or pure subcellular organelles. Nevertheless, the several evidences on the melatonin actions at the level of mitochondria cannot be doubted and change our view of its role and of the role of MT<sup>1</sup> , as MT2 seems to be absent from the organelle.

**7. Future paths?**

**Table 2.** Melatonin protects mitochondria against various stresses.

Trying to summarize the literature on subjects like melatonin is obviously impossible. One will give his/her view on some of the points that are the most attractive to him/her. It was thus vain to attempt to solve issues with such an essay on this neurohormone. The future will tell if melatonin is an exceptional molecule with many capacities. What is clear, as of today, is that melatonin has been described on a plethora of situations with beneficial endpoints. If melatonin is an antioxidant—but the concept behind this word is different from one author

**Protection against Authors Year Reference** Doxorubicin Xu and Ashraf 2002 [258] Oxidative stress Jou et al. 2004 [259] NO synthase induced dysfunction Escames et al. 2006 [260] Apoptosis Han et al. 2006 [261] Ischemia-Reperfusion Petrosillo et al. 2006 [262] Apoptosis Luchetti et al. 2007 [263] Oxidative stress Jou et al. 2007 [264] UV exposition Fischer et al. 2008 [265] Aging Petrosillo et al. 2008 [266] Oxidative stress Hibaoui et al. 2009 [267] Permeability transition Jou et al. 2010 [268] Permeability transition Jou et al. 2011 [269] Bisphenol A Anjum et al. 2011 [270] CCl4 Chechshevik et al. 2012 [271] Isoproterenol Mukherjee et al. 2012 [272] Ischemia-Reperfusion Yang et al. 2013 [273] UV exposition Canonico et al. 2013 [274] Demyelination induced stress Kashani et al. 2014 [275] Cd Guo et al. 2014 [276] Isoproterenol Mukherjee et al. 2015 [272] Ischemic-Stroke Yang et al. 2015 [277] Lipid toxicity Ozsoy et al. 2016 [159] Aging Baburina et al. 2017 [205] Lipid toxicity Das et al. 2017 [116] Paclitaxel Galley et al. 2017 [124] Copper Ghosh et al. 2017 [126]

Molecular Pharmacology and Melatonin: 20 Yeras of Research and Trends

http://dx.doi.org/10.5772/intechopen.79524

31


**Table 2.** Melatonin protects mitochondria against various stresses.

### **7. Future paths?**

separation between all the kinds of membranes present in a neuronal cell. There were several reports over the last decade showing a protective effect of melatonin onto mitochondria functions (see **Table 2**). Then several reviews suggested that melatonin was synthesized by mitochondria (see, for instance, Manchester et al., 2015 [255], Reiter et al., 2017 [256] and 2018 [257]). Particularly interesting is the fact that Cellular and Molecular Life Science published a special issue in 2017 (volume 74, issue 21) dealing with melatonin and mitochondria, emphasizing the interest of the community for these observations and their consequences. A reason for this hypothesis was given: mitochondria, like chloroplasts in plants, evolved from bacteria. Because originally cyanobacteria were subjected to heavy exposition to toxic free radicals, they evolved in keeping melatonin as an antioxidant, scavenging these radicals and thus preserving their integrity. Because this happened about 3 billion years ago, melatonin has been selected to protect and defend those microorganisms.

30 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

Of course, when bacteria colonized eukaryotic cells, the trait was maintained throughout evolution, including in mammals. Thus, no matter how high or low the blood melatonin concentration is, this particular intra-mitochondria concentration remains constant (not depending on the circadian rhythm), protecting mitochondria from the never ending production of free radicals that is the signature of sane mitochondria. An impressive series of publications were issued in these last few years (see **Table 2**) dealing with situations where toxicity was prevented by melatonin. This can be further extended to the protection afforded by mitochondria-synthesized melatonin to oocytes [278]. Finally, one can also add the observation that mitochondria melatonin protects plants from drought episodes [202]. Particularly interesting was the last one in which Suofu et al. [10] demonstrated the presence of the main melatonin synthesis enzymes, arylalkylamine *N*-acetyltransferase (AANAT) and acetylserotonin *O*-methyltransferase (HIOMT), in mitochondria matrix, as well as the high concentration of melatonin inside those

coupling of this receptor, turning this observation into a major progress in the domain, as rare are the receptors signaling in the mitochondria. This observation was challenged by Ahluwalia et al. [279] (replied by Suofu et al. [11]) that was able to show the presence of the melatonin receptors in muscle fibers, but not in mitochondria thereof. It is clear that this breakthrough information will be better understood after the observation will be confirmed independently. Of course, questions remain in the skeptical reader mind: if the melatonin system evolved from bacteria over several billion years, then the genetic material should have evolved together with

does not seem to be the case. This observation would also lead to an extra complexity involving the protein importation system (TOM, Tim, etc.) and the *ad hoc* addressing sequence(s) onto those proteins, all of which have not been seen so far. Furthermore, the discovery and description of an inward transport of melatonin in mitochondria [280] are not fitting an in situ synthesis. For those of us who have been working with subcellular organelles, it is very hard to assess the purity of those organelles because of the continuum that exists between all the membranes from cells. One should also add to this the particular complexity of the brain tissue that is by essence very lipid-rich, leading to an extra difficulty in preparing pure membranes or pure subcellular organelles. Nevertheless, the several evidences on the melatonin actions at the level of mitochondria cannot be doubted and change our view of its role and of the role of

receptor and the actual

, AANAT, and ASMT, which

mitochondria matrix. Furthermore, they showed the presence of MT1

it, meaning that the mitochondrial DNA should encode for MT1

seems to be absent from the organelle.

MT<sup>1</sup>

, as MT2

Trying to summarize the literature on subjects like melatonin is obviously impossible. One will give his/her view on some of the points that are the most attractive to him/her. It was thus vain to attempt to solve issues with such an essay on this neurohormone. The future will tell if melatonin is an exceptional molecule with many capacities. What is clear, as of today, is that melatonin has been described on a plethora of situations with beneficial endpoints. If melatonin is an antioxidant—but the concept behind this word is different from one author to another—it is not as a scavenger of radical oxygen species, but most probably through its capacity to induce cellular defenses against oxidative stress. Melatonin has different known targets; two, MT1 and MT2 , are well described, but these receptors bring more unexpected novelties over the years, an enzyme—QR2—the study of which could be part of an explanation for the antioxidant properties of melatonin, and, finally, a pathway, linked to Nrf2 that seems to be another part of the explanation for these properties. There are many routes still to explore to understand what is behind this molecule, and the spectacular associated with it should be concealed and mastered until beyond (and despite) our hopes; facts will be revealed.

synthesis. Proceedings of the National Academy of Sciences. 2001;**98**:8083-8088. DOI:

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**Chapter 3**

**Provisional chapter**

**An Overview of Melatonin as an Antioxidant Molecule:**

Melatonin is an endogenous hormone derived from tryptophan that is mainly released from the pineal gland in the dark. Melatonin regulates many biological functions such as sleep, circadian rhythm, immunity, and reproduction. Melatonin has a free radical scavenger, anti-inflammatory, and antioxidant effects. It scavenges reactive oxygen and nitrogen species and increases antioxidant defenses, thus it prevents tissue damage and blocks transcriptional factors of pro-inflammatory cytokines. Due to its small size and amphiphilic nature, it increases the efficacy of mitochondrial electron transport chain and reduces electron leakage. Melatonin prevents degenerative changes in the central nervous system in models of Alzheimer's and Parkinson's disease and reduces free radical damage to DNA which may lead to cancer and many other situations. Consequently, melatonin has beneficial effects including stimulation of antioxidant enzymes, inhibition of lipid peroxidation,

**Keywords:** melatonin, antioxidant, free radical, oxidative stress, anti-inflammatory,

Melatonin, N-acetyl-5-methoxytryptamine, which was first isolated from bovine pineal glands [1], is an endogenous neurohormone derived from tryptophan [2]. Melatonin controls various physiologic processes, including circadian rhythms, mood regulation, anxiety, sleep, appetite, immune responses, and cardiac functions [3]. The sleep–wake cycle is the most overt circadian rhythm [4]. More or less sleep shows negative effects on biological and physiological processes including alterations in metabolic, endocrine, and immune pathways that lead to health problems

**An Overview of Melatonin as an Antioxidant** 

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.79421

**A Biochemical Approach**

Aysun Hacışevki and Burcu Baba

Aysun Hacışevki and Burcu Baba

http://dx.doi.org/10.5772/intechopen.79421

**Abstract**

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

and so it contributes to protection from oxidative damages.

neurohormone, tryptophan, disease

**Molecule: A Biochemical Approach**


#### **An Overview of Melatonin as an Antioxidant Molecule: A Biochemical Approach An Overview of Melatonin as an Antioxidant Molecule: A Biochemical Approach**

DOI: 10.5772/intechopen.79421

Aysun Hacışevki and Burcu Baba Aysun Hacışevki and Burcu Baba

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79421

#### **Abstract**

.

angiogenesis and cardio/gastroprotection. Proceedings of the National Academy of Sciences of the United States of America. 2018;**115**:E1942-E1943. DOI: 10.1073/

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pnas.1722131115

3927-3940. DOI: 10.1007/s00018-017-2616-8

58 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

International Journal of Medical Sciences. 2018. [In press]

Melatonin is an endogenous hormone derived from tryptophan that is mainly released from the pineal gland in the dark. Melatonin regulates many biological functions such as sleep, circadian rhythm, immunity, and reproduction. Melatonin has a free radical scavenger, anti-inflammatory, and antioxidant effects. It scavenges reactive oxygen and nitrogen species and increases antioxidant defenses, thus it prevents tissue damage and blocks transcriptional factors of pro-inflammatory cytokines. Due to its small size and amphiphilic nature, it increases the efficacy of mitochondrial electron transport chain and reduces electron leakage. Melatonin prevents degenerative changes in the central nervous system in models of Alzheimer's and Parkinson's disease and reduces free radical damage to DNA which may lead to cancer and many other situations. Consequently, melatonin has beneficial effects including stimulation of antioxidant enzymes, inhibition of lipid peroxidation, and so it contributes to protection from oxidative damages.

**Keywords:** melatonin, antioxidant, free radical, oxidative stress, anti-inflammatory, neurohormone, tryptophan, disease

#### **1. Introduction**

Melatonin, N-acetyl-5-methoxytryptamine, which was first isolated from bovine pineal glands [1], is an endogenous neurohormone derived from tryptophan [2]. Melatonin controls various physiologic processes, including circadian rhythms, mood regulation, anxiety, sleep, appetite, immune responses, and cardiac functions [3]. The sleep–wake cycle is the most overt circadian rhythm [4]. More or less sleep shows negative effects on biological and physiological processes including alterations in metabolic, endocrine, and immune pathways that lead to health problems

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

involving obesity, diabetes, hypertension, and respiratory diseases [4–6]. Timing of melatonin secretion is closely associated with the timing of sleep propensity, and it also coincides with decreases in core body temperature, alertness, and performance [7]. Melatonin regulates memory formation by directly affecting hippocampal neurons. There are antinociceptive, antidepressant, anxiolytic, antineophobic, and locomotor activity regulating effects of melatonin [3, 8]. Melatonin plays important roles in neurogenesis, neuroprotection, maintenance of oxidant/antioxidant balance, modulation of cardiovascular and/or immune system, and diabetes control. It exerts a direct antioxidant effect on tissues/organs and antiapoptotic effects on cells [9]. Other actions of melatonin include inhibition of dopamine release in the hypothalamus and retina, involvement in the aging process and pubertal development, blood pressure control, and free radical scavenging [7]. Melatonin dysfunction may contribute to many divergent diseases, such as neurodegenerative diseases, circadian and mood disorders, insomnia, type 2 diabetes, and pain [3]. Low levels of melatonin have been shown in Parkinson's disease (PD), Alzheimer's disease (AD), insomnia, epilepsy, ischemic injury, and neuropsychiatric disorders; in addition, roles for melatonin in the development of cataracts, aging, and retinitis have also been reported [10]. Melatonin has been utilized in several countries for circadian rhythm disorders, sleep disturbances, jet lag, and sleep– wake cycle disturbances in blind people and shift workers [7, 11, 12].

called hydroxyindole-O-methyltransferase or HIOMT). The last step is the rate-limiting step

An Overview of Melatonin as an Antioxidant Molecule: A Biochemical Approach

http://dx.doi.org/10.5772/intechopen.79421

61

Melatonin synthesis depends on intact beta-adrenergic receptor function. Norepinephrine activates the N-acetyltransferase, and beta-receptor blockers depress melatonin secretion [29]. Both AA-NAT and ASMT activities are controlled by noradrenergic and neuropeptidergic projections to the pineal gland. The pineal gland receives input from postganglionic fibers, leading to the release of norepinephrine. Norepinephrine induces its α1/β-adrenoceptors that activate adenylate cyclase-cAMP system. Thus, intracellular levels of the second messengers include cAMP, Ca2+, phosphatidylinositol, diacetylglycerol, and protein kinase C increase. These mes-

The pineal gland is located outside the blood brain barrier, and loses it connections with the central nervous system, having sympathetic innervation as its main source. This may explain for the pineal gland ability to have a large uptake of tryptophan leading to a high melatonin production and secretion in response to darkness [18]. Once synthesized, melatonin is quickly released into the systemic circulation to reach central and peripheral target tissues. The effects of melatonin depend on the localization and types of melatonin receptors [15]. Melatonin activates two high-affinity G-protein-coupled receptors, termed MT1 and MT2. The MT1 and MT2 lead to an inhibition of the adenylate cyclase in target cells and regulate a variety of cellular and physiological processes including neuronal firing, arterial vasoconstriction, cell proliferation, immune responses, and reproductive and metabolic functions [8, 16, 27, 31–33]. MT1 and MT2 receptors are 350 and 362 amino acids long, located on chromosome 4q35.1 and chromosome 11q21-q22, respectively. MT1 receptors are expressed in the brain, cardiovascular system, immune system, testes, ovary, skin, liver, kidney, adrenal cortex, placenta, breast, retina, pancreas, and spleen. MT2 has been found in the immune system, brain, retina, pituitary, blood vessels, testes, kidney, gastrointestinal tract, mammary glands, adipose tissue, and the skin [27, 31, 32]. The MT3 receptor has a low affinity, unlike MT1 and MT2; it is not coupled to G proteins; it has a nanomolar affinity for melatonin, and it is not sensitive

**Figure 1.** Biosynthesis of melatonin from tryptophan (TPH, tryptophan-5-hydroxylase; AADC, L-aromatic amino acid

decarboxylase; AA-NAT, arylalkylamine N-acetyltransferase; HIOMT, hydroxyindole-O-methyltransferase).

sengers induce the expression and activity of AA-NAT and HIOMT [7, 14, 15, 18, 30].

in the biosynthesis of melatonin (**Figure 1**) [18, 20, 25–28].

Melatonin is secreted primarily by the pineal gland in response to darkness [2, 13, 14]. It was later found to be also present or synthesized in extrapineal sites such as retina, Harderian gland, lymphocytes, gastrointestinal tract, bone marrow cells, platelets and skin [13, 15–17]. The neurohormone melatonin is not stored in the pineal gland but rather is released into the bloodstream and can penetrate all body tissues [18]. The synthesis of melatonin shows a clear circadian rhythm with low levels during the daytime and its secretory peak at night [19, 20]. The nocturnal synthesis and release of melatonin by the pineal gland are strictly controlled by the suprachiasmatic nucleus (SCN) clock and inhibited by lighting conditions [19, 21]. In humans and other mammals, detection of light drives activity in retinal ganglion cells that project to the SCN in the hypothalamus, causing the release of inhibitory γ-amino butyric acid that suppresses the circuit controlling melatonin synthesis and release [22]. Serum melatonin reaches a peak value (80–150 pg/mL) between midnight and 3 a.m., while its concentration during the day is low (10–20 pg/mL) [23]. Both normal melatonin patterns and the influence of light can vary considerably between individuals, either in terms of personal characteristics or as a consequence of aging or a chronic disease [24]. Serum concentrations of melatonin vary considerably with age, and infants secrete very low levels of melatonin before 3 months of age. Amplitude of the nocturnal peak in melatonin secretion reaches the highest levels between the 4th and 7th year of age [15, 19]. Other factors that alter melatonin levels are nightwork, impaired light–dark cycles, and obesity. Additionally, some nutritional factors could change melatonin production [13].

Melatonin, hormone of darkness, is synthesized from tryptophan, which is an essential amino acid by the pineal gland. The synthesis of melatonin is a multistep process. Firstly, tryptophan is hydroxylated by tryptophan-5-hydroxylase (TPH) to form 5-hydroxytryptophan, which is subsequently decarboxylated to 5-hydroxytryptamine (serotonin) by L-aromatic amino acid decarboxylase (AADC). Serotonin is N-acetylated by arylalkylamine N-acetyltransferase (AA-NAT, also called "Timezyme," is the rate-limiting enzyme for melatonin synthesis), to form N-acetylserotonin, which is converted to N-acetyl-5 methoxytryptamine (melatonin) by N-acetylserotonin-O-methyltransferase (ASMT, also called hydroxyindole-O-methyltransferase or HIOMT). The last step is the rate-limiting step in the biosynthesis of melatonin (**Figure 1**) [18, 20, 25–28].

involving obesity, diabetes, hypertension, and respiratory diseases [4–6]. Timing of melatonin secretion is closely associated with the timing of sleep propensity, and it also coincides with decreases in core body temperature, alertness, and performance [7]. Melatonin regulates memory formation by directly affecting hippocampal neurons. There are antinociceptive, antidepressant, anxiolytic, antineophobic, and locomotor activity regulating effects of melatonin [3, 8]. Melatonin plays important roles in neurogenesis, neuroprotection, maintenance of oxidant/antioxidant balance, modulation of cardiovascular and/or immune system, and diabetes control. It exerts a direct antioxidant effect on tissues/organs and antiapoptotic effects on cells [9]. Other actions of melatonin include inhibition of dopamine release in the hypothalamus and retina, involvement in the aging process and pubertal development, blood pressure control, and free radical scavenging [7]. Melatonin dysfunction may contribute to many divergent diseases, such as neurodegenerative diseases, circadian and mood disorders, insomnia, type 2 diabetes, and pain [3]. Low levels of melatonin have been shown in Parkinson's disease (PD), Alzheimer's disease (AD), insomnia, epilepsy, ischemic injury, and neuropsychiatric disorders; in addition, roles for melatonin in the development of cataracts, aging, and retinitis have also been reported [10]. Melatonin has been utilized in several countries for circadian rhythm disorders, sleep disturbances, jet lag, and sleep–

Melatonin is secreted primarily by the pineal gland in response to darkness [2, 13, 14]. It was later found to be also present or synthesized in extrapineal sites such as retina, Harderian gland, lymphocytes, gastrointestinal tract, bone marrow cells, platelets and skin [13, 15–17]. The neurohormone melatonin is not stored in the pineal gland but rather is released into the bloodstream and can penetrate all body tissues [18]. The synthesis of melatonin shows a clear circadian rhythm with low levels during the daytime and its secretory peak at night [19, 20]. The nocturnal synthesis and release of melatonin by the pineal gland are strictly controlled by the suprachiasmatic nucleus (SCN) clock and inhibited by lighting conditions [19, 21]. In humans and other mammals, detection of light drives activity in retinal ganglion cells that project to the SCN in the hypothalamus, causing the release of inhibitory γ-amino butyric acid that suppresses the circuit controlling melatonin synthesis and release [22]. Serum melatonin reaches a peak value (80–150 pg/mL) between midnight and 3 a.m., while its concentration during the day is low (10–20 pg/mL) [23]. Both normal melatonin patterns and the influence of light can vary considerably between individuals, either in terms of personal characteristics or as a consequence of aging or a chronic disease [24]. Serum concentrations of melatonin vary considerably with age, and infants secrete very low levels of melatonin before 3 months of age. Amplitude of the nocturnal peak in melatonin secretion reaches the highest levels between the 4th and 7th year of age [15, 19]. Other factors that alter melatonin levels are nightwork, impaired light–dark cycles, and obesity. Additionally, some nutritional factors could change melatonin production [13].

Melatonin, hormone of darkness, is synthesized from tryptophan, which is an essential amino acid by the pineal gland. The synthesis of melatonin is a multistep process. Firstly, tryptophan is hydroxylated by tryptophan-5-hydroxylase (TPH) to form 5-hydroxytryptophan, which is subsequently decarboxylated to 5-hydroxytryptamine (serotonin) by L-aromatic amino acid decarboxylase (AADC). Serotonin is N-acetylated by arylalkylamine N-acetyltransferase (AA-NAT, also called "Timezyme," is the rate-limiting enzyme for melatonin synthesis), to form N-acetylserotonin, which is converted to N-acetyl-5 methoxytryptamine (melatonin) by N-acetylserotonin-O-methyltransferase (ASMT, also

wake cycle disturbances in blind people and shift workers [7, 11, 12].

60 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

Melatonin synthesis depends on intact beta-adrenergic receptor function. Norepinephrine activates the N-acetyltransferase, and beta-receptor blockers depress melatonin secretion [29]. Both AA-NAT and ASMT activities are controlled by noradrenergic and neuropeptidergic projections to the pineal gland. The pineal gland receives input from postganglionic fibers, leading to the release of norepinephrine. Norepinephrine induces its α1/β-adrenoceptors that activate adenylate cyclase-cAMP system. Thus, intracellular levels of the second messengers include cAMP, Ca2+, phosphatidylinositol, diacetylglycerol, and protein kinase C increase. These messengers induce the expression and activity of AA-NAT and HIOMT [7, 14, 15, 18, 30].

The pineal gland is located outside the blood brain barrier, and loses it connections with the central nervous system, having sympathetic innervation as its main source. This may explain for the pineal gland ability to have a large uptake of tryptophan leading to a high melatonin production and secretion in response to darkness [18]. Once synthesized, melatonin is quickly released into the systemic circulation to reach central and peripheral target tissues. The effects of melatonin depend on the localization and types of melatonin receptors [15]. Melatonin activates two high-affinity G-protein-coupled receptors, termed MT1 and MT2. The MT1 and MT2 lead to an inhibition of the adenylate cyclase in target cells and regulate a variety of cellular and physiological processes including neuronal firing, arterial vasoconstriction, cell proliferation, immune responses, and reproductive and metabolic functions [8, 16, 27, 31–33]. MT1 and MT2 receptors are 350 and 362 amino acids long, located on chromosome 4q35.1 and chromosome 11q21-q22, respectively. MT1 receptors are expressed in the brain, cardiovascular system, immune system, testes, ovary, skin, liver, kidney, adrenal cortex, placenta, breast, retina, pancreas, and spleen. MT2 has been found in the immune system, brain, retina, pituitary, blood vessels, testes, kidney, gastrointestinal tract, mammary glands, adipose tissue, and the skin [27, 31, 32]. The MT3 receptor has a low affinity, unlike MT1 and MT2; it is not coupled to G proteins; it has a nanomolar affinity for melatonin, and it is not sensitive

**Figure 1.** Biosynthesis of melatonin from tryptophan (TPH, tryptophan-5-hydroxylase; AADC, L-aromatic amino acid decarboxylase; AA-NAT, arylalkylamine N-acetyltransferase; HIOMT, hydroxyindole-O-methyltransferase).

to Na+2, Mg+2, and Ca+2. The MT3 is equivalent to enzyme quinone reductase II [27]. The relationship between multiple physiological function of melatonin and this enzyme is possibly involved in the regulation of cellular redox status, although the exact role of this relationship remains unclear [16, 34]. Melatonin appears to be a natural ligand for the retinoid-related orphan nuclear hormone receptor family (RZR/ROR). RZR/RORα is expressed in a variety of organs, whereas RZRβ is specific for the brain and retina [35]. In addition, melatonin interacts with intracellular proteins such as calmodulin, calreticulin, or tubulin and antagonizes the binding of Ca2+ to calmodulin [7]. ROR/RZR has been proposed to work in coordination with the plasma membrane receptors MT1/MT2 to regulate gene expression. The low-affinity interaction between melatonin and calmodulin may be involved in its antioxidant action as well as other signaling processes [15, 16]. The membrane receptors have been defined in the central nervous system and in peripheral organs, such as liver, gastrointestinal tract, skin, kidney, heart, and adipose and lymphoid tissues in many mammalians [33]. Melatonin also acts through nonreceptor-mediated mechanisms, for example, serving as a scavenger for reactive oxygen species (ROS) and reactive nitrogen species (RNS) [27]. Melatonin and its metabolites have potent antioxidative and radioprotective properties [36]. Melatonin has been proven to be an efficient oxidant scavenger of a variety of radical and nonradical reactants [37].

as a result of normal intracellular metabolism in mitochondria and peroxisomes, as well as from diverse cytosolic enzyme systems such as lipoxygenases, NADPH oxidase, and cytochrome P450. Furthermore, various external agents including ionizing radiation, ultraviolet light, environmental toxins, inflammatory, and cytokines can trigger ROS production [16, 44].

An Overview of Melatonin as an Antioxidant Molecule: A Biochemical Approach

water or highly toxic hydroxyl radical [16]. Although hydroxyl radical formation can occur in several ways, by far the most important mechanism in vivo is likely to be the transition metalcatalyzed decomposition of superoxide anion and hydrogen peroxide [46]. Hydroxyl radicals are generated from hydrogen peroxide during cellular oxygen metabolism via the Fenton and Haber-Weiss reactions (**Figure 2**) [47], in the presence of free iron or copper ions [48]. The OH•

O2

Alkoxyl radicals that are formed from the reduction of peroxides, are less reactive than OH• and significantly more reactive than ROO radicals, provided that R is the same in both species. Therefore, they are suggested to be ideal candidates to evaluate the efficiency of antioxidants and also the reactivity of any species reacting with ROS. As regards RNS, the chemical

In healthy organisms, there is a delicate balance between the production and the removal of free radicals, which guarantees that they remain in low/moderate concentrations. Under such conditions, free radicals have beneficial effects [41]. ROS and RNS play important roles in regulation of a wide variety of physiology functions like gene expression, cellular growth, differentiation, modulation of chemical reactions, and induction of transcription factors such as nuclear factor-kappa B (NF-кB) and activator protein-1 (AP-1) and activation of signal transduction pathways. They also participate in blood pressure control, are mediators in the biosynthesis of prostaglandins, function in embryonic development, and act as signaling molecules within the individual cell and among cells during their life span [44–46]. The harmful and useful effects of ROS/RNS are associated with their concentrations, the cell type and the subcellular compartments that are produced, and their timing of production [16]. An imbalance between excessive ROS and RNS generation and rate of their elimination by the antioxidant capacity leads to oxidative stress [49, 50]. It has been shown that oxidative stress is involved in over 100 diseases, as their cause or consequence [51]. Oxidative stress results in macromolecular damage and is implicated in various disease states such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging [52]. The cellular dysfunctions caused by excessive ROS and/or RNS might produce loss of energy metabolism, altered cell signaling

•− during

63

•− is quickly converted

is converted into

•− forming per-

O2

http://dx.doi.org/10.5772/intechopen.79421

interacts with transition metals (Fe2+, Cu1+,

is a mild oxidant, and its reactivity is between

Mitochondria are the major source of ROS and RNS production [45]. Generation of O<sup>2</sup>

enzymatically by superoxide dismutases (SODs). After that, H<sup>2</sup>

etc.) [16, 40, 41]. It can also be produced by ultraviolet and ionizing radiations [41].

reactivity and direct toxicity of NO• are quite low. However, it reacts with O<sup>2</sup>

[41, 42, 44].

oxidative phosphorylation takes place mainly in the mitochondria. O<sup>2</sup>

is formed during the Fenton reaction when H<sup>2</sup>

oxynitrite, which is a powerful oxidant. NO<sup>2</sup>

those of NO• and ONOO−

**Figure 2.** Fenton and Haber-Weiss reactions.

to H<sup>2</sup> O2

In the circulation, melatonin is partially bound to albumin and can also bind to hemoglobin [38]. Melatonin metabolism is a rapid process, and its half-life in humans varies between 10 and 60 min following exogenous administration. It is deactivated mostly by the liver and excreted in the urine [13, 26]. There are three major pathways of melatonin degradation: (1) the classical hepatic degradation pathway that generates 6-hydroxymelatonin, (2) the alternative indolic pathway that produces 5-methoxyindole acetic acid (5-MIAA) or 5-methoxytryptophol (5-MTOL), and (3) the kynurenic pathway that produces the main brain metabolites of melatonin, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), and its deformylated product N1-acetyl-5-methoxykynuramine (AMK). These metabolites are highly remarkable and are generated enzymatically, pseudoenzymatically, by free radical, and via photochemical processes. Recently, it was reported that AFMK and AMK detoxify reactive species and preserve tissues from damage by reactive intermediates [39]. This chapter summarizes effects of melatonin and its metabolites as antioxidants and their clinical significance in several diseases.

### **2. Free radicals**

Free radicals are atoms or molecules that containing one or more unpaired electrons in the external orbitals of the molecules, usually unstable and highly reactive. The free radical chemical reactivity is directly associated with the damage that they can inflict to biological molecules. In biology system, oxygen-derived radicals and nitrogen-derived radicals are two types of free radicals. Oxygen-derived radicals, such as superoxide (O<sup>2</sup> •−), hydroxyl radicals (OH•), alkoxyl radicals (RO•), as well as nonradicals such as hydrogen peroxide (H<sup>2</sup> O2 ), ozone and hypochlorous acid, are defined as reactive oxygen species (ROS). ROS are produced during the oxygen metabolism. Nitrogen-derived radicals and nonradicals, such as nitrogen dioxide (NO<sup>2</sup> ), nitric oxide radicals (NO•), and peroxynitrite (ONOO), are known as reactive nitrogen species (RNS) which are derived from nitric oxide and superoxide by inducible nitric oxide synthase (iNOS) and NADPH oxidase, respectively [16, 40–44]. Oxidants are produced as a result of normal intracellular metabolism in mitochondria and peroxisomes, as well as from diverse cytosolic enzyme systems such as lipoxygenases, NADPH oxidase, and cytochrome P450. Furthermore, various external agents including ionizing radiation, ultraviolet light, environmental toxins, inflammatory, and cytokines can trigger ROS production [16, 44]. Mitochondria are the major source of ROS and RNS production [45]. Generation of O<sup>2</sup> •− during oxidative phosphorylation takes place mainly in the mitochondria. O<sup>2</sup> •− is quickly converted to H<sup>2</sup> O2 enzymatically by superoxide dismutases (SODs). After that, H<sup>2</sup> O2 is converted into water or highly toxic hydroxyl radical [16]. Although hydroxyl radical formation can occur in several ways, by far the most important mechanism in vivo is likely to be the transition metalcatalyzed decomposition of superoxide anion and hydrogen peroxide [46]. Hydroxyl radicals are generated from hydrogen peroxide during cellular oxygen metabolism via the Fenton and Haber-Weiss reactions (**Figure 2**) [47], in the presence of free iron or copper ions [48]. The OH• is formed during the Fenton reaction when H<sup>2</sup> O2 interacts with transition metals (Fe2+, Cu1+, etc.) [16, 40, 41]. It can also be produced by ultraviolet and ionizing radiations [41].

Alkoxyl radicals that are formed from the reduction of peroxides, are less reactive than OH• and significantly more reactive than ROO radicals, provided that R is the same in both species. Therefore, they are suggested to be ideal candidates to evaluate the efficiency of antioxidants and also the reactivity of any species reacting with ROS. As regards RNS, the chemical reactivity and direct toxicity of NO• are quite low. However, it reacts with O<sup>2</sup> •− forming peroxynitrite, which is a powerful oxidant. NO<sup>2</sup> is a mild oxidant, and its reactivity is between those of NO• and ONOO− [41, 42, 44].

In healthy organisms, there is a delicate balance between the production and the removal of free radicals, which guarantees that they remain in low/moderate concentrations. Under such conditions, free radicals have beneficial effects [41]. ROS and RNS play important roles in regulation of a wide variety of physiology functions like gene expression, cellular growth, differentiation, modulation of chemical reactions, and induction of transcription factors such as nuclear factor-kappa B (NF-кB) and activator protein-1 (AP-1) and activation of signal transduction pathways. They also participate in blood pressure control, are mediators in the biosynthesis of prostaglandins, function in embryonic development, and act as signaling molecules within the individual cell and among cells during their life span [44–46]. The harmful and useful effects of ROS/RNS are associated with their concentrations, the cell type and the subcellular compartments that are produced, and their timing of production [16]. An imbalance between excessive ROS and RNS generation and rate of their elimination by the antioxidant capacity leads to oxidative stress [49, 50]. It has been shown that oxidative stress is involved in over 100 diseases, as their cause or consequence [51]. Oxidative stress results in macromolecular damage and is implicated in various disease states such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging [52]. The cellular dysfunctions caused by excessive ROS and/or RNS might produce loss of energy metabolism, altered cell signaling

**Figure 2.** Fenton and Haber-Weiss reactions.

to Na+2, Mg+2, and Ca+2. The MT3 is equivalent to enzyme quinone reductase II [27]. The relationship between multiple physiological function of melatonin and this enzyme is possibly involved in the regulation of cellular redox status, although the exact role of this relationship remains unclear [16, 34]. Melatonin appears to be a natural ligand for the retinoid-related orphan nuclear hormone receptor family (RZR/ROR). RZR/RORα is expressed in a variety of organs, whereas RZRβ is specific for the brain and retina [35]. In addition, melatonin interacts with intracellular proteins such as calmodulin, calreticulin, or tubulin and antagonizes the binding of Ca2+ to calmodulin [7]. ROR/RZR has been proposed to work in coordination with the plasma membrane receptors MT1/MT2 to regulate gene expression. The low-affinity interaction between melatonin and calmodulin may be involved in its antioxidant action as well as other signaling processes [15, 16]. The membrane receptors have been defined in the central nervous system and in peripheral organs, such as liver, gastrointestinal tract, skin, kidney, heart, and adipose and lymphoid tissues in many mammalians [33]. Melatonin also acts through nonreceptor-mediated mechanisms, for example, serving as a scavenger for reactive oxygen species (ROS) and reactive nitrogen species (RNS) [27]. Melatonin and its metabolites have potent antioxidative and radioprotective properties [36]. Melatonin has been proven to

62 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

be an efficient oxidant scavenger of a variety of radical and nonradical reactants [37].

**2. Free radicals**

dioxide (NO<sup>2</sup>

In the circulation, melatonin is partially bound to albumin and can also bind to hemoglobin [38]. Melatonin metabolism is a rapid process, and its half-life in humans varies between 10 and 60 min following exogenous administration. It is deactivated mostly by the liver and excreted in the urine [13, 26]. There are three major pathways of melatonin degradation: (1) the classical hepatic degradation pathway that generates 6-hydroxymelatonin, (2) the alternative indolic pathway that produces 5-methoxyindole acetic acid (5-MIAA) or 5-methoxytryptophol (5-MTOL), and (3) the kynurenic pathway that produces the main brain metabolites of melatonin, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), and its deformylated product N1-acetyl-5-methoxykynuramine (AMK). These metabolites are highly remarkable and are generated enzymatically, pseudoenzymatically, by free radical, and via photochemical processes. Recently, it was reported that AFMK and AMK detoxify reactive species and preserve tissues from damage by reactive intermediates [39]. This chapter summarizes effects of melatonin and its metabolites as antioxidants and their clinical significance in several diseases.

Free radicals are atoms or molecules that containing one or more unpaired electrons in the external orbitals of the molecules, usually unstable and highly reactive. The free radical chemical reactivity is directly associated with the damage that they can inflict to biological molecules. In biology system, oxygen-derived radicals and nitrogen-derived radicals are two

and hypochlorous acid, are defined as reactive oxygen species (ROS). ROS are produced during the oxygen metabolism. Nitrogen-derived radicals and nonradicals, such as nitrogen

nitrogen species (RNS) which are derived from nitric oxide and superoxide by inducible nitric oxide synthase (iNOS) and NADPH oxidase, respectively [16, 40–44]. Oxidants are produced

), nitric oxide radicals (NO•), and peroxynitrite (ONOO), are known as reactive

•−), hydroxyl radicals

O2

), ozone

types of free radicals. Oxygen-derived radicals, such as superoxide (O<sup>2</sup>

(OH•), alkoxyl radicals (RO•), as well as nonradicals such as hydrogen peroxide (H<sup>2</sup>

and cell cycle, gene mutations, and impaired cellular transport mechanisms. The oxidative stress promotes decreased biological activities, immune activation, and inflammation [50]. It seems that both high levels of ROS (oxidative stress) and excessively low levels of ROS (reductive stress) are deleterious and apparently play a causative role in the pathologies caused by malfunctioning processes related to the dramatic change of redox environment [53].

fluidity and damage membrane proteins [60, 61]. Vitamin E and Vitamin C are the most frequently used antioxidant vitamins [62] that are thought to have a protective effect by either reducing or preventing oxidative damage [63]. Vitamin E belongs to the group of fat-soluble vitamins existing in eight different forms. The methylation pattern of the chroman ring determines the classification as α, β, γ, and δ tocopherols. These compounds have antioxidant properties. Vitamin E scavenges peroxyl radicals and hence acts to break the chain reaction of lipid peroxidation [64]. Besides its antioxidant role, vitamin E might also have a structural role in stabilizing membranes [46, 65, 66]. Vitamin C, which is readily water soluble, is an important antioxidant and thus works in aqueous environments of the body [46, 57, 67]. As an antioxidant, ascorbate is an efficient scavenger, or reducing antioxidant, capable of donating its electrons to ROS and eliminating them [44]. Loss of one electron generates the ascorbyl radical intermediate, and loss of two electrons generates dehydroascorbate (DHA, which can also be formed via dismutation of the ascorbyl radical) [61, 68]. It makes ascorbate a powerful important antioxidant [44]. Vitamin C serves as a co-antioxidant with vitamin E to regenerate α-tocopherol from α-tocopherol radicals in membranes and lipoproteins and protect protein thiol group against oxidation by increasing intracellular levels of GSH [46, 61, 69]. Vitamin C can also neutralize ROS (e.g., hydrogen peroxide) [46, 70]. Recently, toxicity of ascorbic acid

An Overview of Melatonin as an Antioxidant Molecule: A Biochemical Approach

http://dx.doi.org/10.5772/intechopen.79421

65

An efficient antioxidant should not only be ubiquitous but should also be present in adequate amounts in cells and easily reacts with a wide variety of free radicals which have short halflife due to high reactivity. A good antioxidant has the ability to cross physiologic barriers and to be quickly transported into the cells. Thus, it must be available to all cells. It is also important for an antioxidant to be available. Antioxidants should be available when needed. They should be easily acquired through the diet or produced in situ. Antioxidants should be suitable for regeneration. The reaction between an antioxidant and a free radical yields an oxidized form of the antioxidant which has less scavenging activity than the original compound. Therefore, many antioxidants have physiologically reducing mechanisms, or its oxidized forms can still efficiently react with new free radicals. An ideal antioxidant should be conserved by the kidneys. Otherwise, large urinary losses would occur and the half-life will be short. An important aspect to consider for evaluating the suitability of a compound as an antioxidant is its toxicity. It should be nontoxic prior to and after the free radical scavenging process takes place. In addition, it is also important to be aware of possible interactions with

Melatonin is a potent direct scavenger of free radicals. Unlike most of other radical scavengers, it is a multifunctional antioxidant. Melatonin can easily pass through cell membranes because of its high lipophilicity and hydrophilicity [73]. Melatonin is also widespread within cells. Its concentrations in human serum and cerebrospinal fluid vary widely. Melatonin is endogenously generated, and it is ingested in the food as it is widely available in fruits and vegetables. Hence, melatonin is produced internally and is also ingested in the diet. Only small amounts of melatonin are excreted into the urine in its unchanged form. It has minimal toxicity. Numerous in vivo studies on animals involving massive doses of melatonin have shown that acute and chronic toxicity of melatonin is extremely low [41, 74]. Unlike most small molecule biological antioxidants such as ascorbic acid, α-tocopherol, lipoic acid, etc., melatonin does not undergo redox cycling and, thus, does not promote oxidation. Melatonin

has also been attributed to its autoxidation [45].

any drug that may be concurrently consumed [41, 71, 72].

### **3. Antioxidants**

Based on the oxidative stress related to free radical theory, the antioxidants are the first line of choice to take care of the stress [45]. Antioxidants act as free radical scavengers and can prevent oxidative reactions that lead to various diseases [54]. The antioxidant defense system includes endogenous (enzymatic and nonenzymatic) and exogenous (dietary) antioxidants that interact in establishing redox homeostasis in the body [49]. Endogenous antioxidants, which are products of the body's metabolism, may be enzymatic or nonenzymatic compounds localized generally in the cytoplasm and diverse cell organelles [45, 49]. In eukaryotics, various antioxidant enzymes, for instance, SOD, catalase (CAT), and some peroxidases, transform ROS into more stable molecules (e.g., water and O<sup>2</sup> ) via complex cascade of reactions [45]. One of the most effective intracellular enzymatic antioxidants is SOD. In humans, there are three forms of SOD: cytosolic CuZn-SOD, mitochondrial Mn-SOD, and extracellular SOD. SOD catalyzes the dismutation of O2 •− to H<sup>2</sup> O2 , decreasing the amount of O2 •− and thereby lowering the formation of ONOO− [44, 50]. Other important enzymatic antioxidants include CAT, glutathione peroxidase (GPx), glutathione reductase (GR), and peroxiredoxins (Prxs). These enzymes neutralize hydrogen peroxide, yielding water (CAT, GPx) and oxygen molecule (CAT) [45, 49]. CAT which is found in the peroxisomes and cytoplasm [55] presents a molecule of ferric ion at its active site and converts two molecules of H<sup>2</sup> O2 into one molecule each of water and diatomic oxygen [37]. Glutathione peroxidase can be found in many subcellular compartments including the mitochondria and nucleus depending on the family member [55]. Selenium, as a selenocysteine, is a component of the active site of GPx [37, 55, 56]. GPx uses reduced glutathione (GSH) as a substrate to transfer electrons to H<sup>2</sup> O2 (and other peroxides), thereby converting it into two molecules of water [37]. When hydrogen peroxide is metabolized by glutathione peroxidase, reduced glutathione is oxidized to glutathione disulfide (GSSG) which is converted back to GSH by the enzyme GR [57–59].

Small molecular nonenzymic antioxidants (e.g., GSH, NADPH, thioredoxin, vitamin E (α-tocopherol), vitamin C (ascorbic acid), and trace metals, such as selenium) also function as direct scavengers of ROS [45]. In particular, glutathione plays a central role in defense against oxidative stress [54]. The antioxidant properties of GSH which is a tripeptide, γ-l-glutamyll-cysteinyl-glycine, depend on the presence of a peptide bond between the amino group of cysteine and the alpha-carboxyl group, which provide an excellent protection against aminopeptidases, and the expression of the thiol group which derive from the cysteine residue. Complexation of metal ions, participation in the oxidation reactions, and formation of thiol radicals and disulfides are the most important functions of thiol groups in the biological systems [49]. Maintaining or reestablishment of redox homeostasis are ensured by endogenous and exogenous antioxidants that act synergistically [49, 60], such as during the regeneration of vitamin E by GSH or vitamin C to prevent lipid peroxidation, which can affect membrane fluidity and damage membrane proteins [60, 61]. Vitamin E and Vitamin C are the most frequently used antioxidant vitamins [62] that are thought to have a protective effect by either reducing or preventing oxidative damage [63]. Vitamin E belongs to the group of fat-soluble vitamins existing in eight different forms. The methylation pattern of the chroman ring determines the classification as α, β, γ, and δ tocopherols. These compounds have antioxidant properties. Vitamin E scavenges peroxyl radicals and hence acts to break the chain reaction of lipid peroxidation [64]. Besides its antioxidant role, vitamin E might also have a structural role in stabilizing membranes [46, 65, 66]. Vitamin C, which is readily water soluble, is an important antioxidant and thus works in aqueous environments of the body [46, 57, 67]. As an antioxidant, ascorbate is an efficient scavenger, or reducing antioxidant, capable of donating its electrons to ROS and eliminating them [44]. Loss of one electron generates the ascorbyl radical intermediate, and loss of two electrons generates dehydroascorbate (DHA, which can also be formed via dismutation of the ascorbyl radical) [61, 68]. It makes ascorbate a powerful important antioxidant [44]. Vitamin C serves as a co-antioxidant with vitamin E to regenerate α-tocopherol from α-tocopherol radicals in membranes and lipoproteins and protect protein thiol group against oxidation by increasing intracellular levels of GSH [46, 61, 69]. Vitamin C can also neutralize ROS (e.g., hydrogen peroxide) [46, 70]. Recently, toxicity of ascorbic acid has also been attributed to its autoxidation [45].

and cell cycle, gene mutations, and impaired cellular transport mechanisms. The oxidative stress promotes decreased biological activities, immune activation, and inflammation [50]. It seems that both high levels of ROS (oxidative stress) and excessively low levels of ROS (reductive stress) are deleterious and apparently play a causative role in the pathologies caused by

Based on the oxidative stress related to free radical theory, the antioxidants are the first line of choice to take care of the stress [45]. Antioxidants act as free radical scavengers and can prevent oxidative reactions that lead to various diseases [54]. The antioxidant defense system includes endogenous (enzymatic and nonenzymatic) and exogenous (dietary) antioxidants that interact in establishing redox homeostasis in the body [49]. Endogenous antioxidants, which are products of the body's metabolism, may be enzymatic or nonenzymatic compounds localized generally in the cytoplasm and diverse cell organelles [45, 49]. In eukaryotics, various antioxidant enzymes, for instance, SOD, catalase (CAT), and some peroxidases, transform ROS into more stable

intracellular enzymatic antioxidants is SOD. In humans, there are three forms of SOD: cytosolic CuZn-SOD, mitochondrial Mn-SOD, and extracellular SOD. SOD catalyzes the dismutation of

50]. Other important enzymatic antioxidants include CAT, glutathione peroxidase (GPx), glutathione reductase (GR), and peroxiredoxins (Prxs). These enzymes neutralize hydrogen peroxide, yielding water (CAT, GPx) and oxygen molecule (CAT) [45, 49]. CAT which is found in the peroxisomes and cytoplasm [55] presents a molecule of ferric ion at its active site and converts two

dase can be found in many subcellular compartments including the mitochondria and nucleus depending on the family member [55]. Selenium, as a selenocysteine, is a component of the active site of GPx [37, 55, 56]. GPx uses reduced glutathione (GSH) as a substrate to transfer electrons

hydrogen peroxide is metabolized by glutathione peroxidase, reduced glutathione is oxidized to

Small molecular nonenzymic antioxidants (e.g., GSH, NADPH, thioredoxin, vitamin E (α-tocopherol), vitamin C (ascorbic acid), and trace metals, such as selenium) also function as direct scavengers of ROS [45]. In particular, glutathione plays a central role in defense against oxidative stress [54]. The antioxidant properties of GSH which is a tripeptide, γ-l-glutamyll-cysteinyl-glycine, depend on the presence of a peptide bond between the amino group of cysteine and the alpha-carboxyl group, which provide an excellent protection against aminopeptidases, and the expression of the thiol group which derive from the cysteine residue. Complexation of metal ions, participation in the oxidation reactions, and formation of thiol radicals and disulfides are the most important functions of thiol groups in the biological systems [49]. Maintaining or reestablishment of redox homeostasis are ensured by endogenous and exogenous antioxidants that act synergistically [49, 60], such as during the regeneration of vitamin E by GSH or vitamin C to prevent lipid peroxidation, which can affect membrane

glutathione disulfide (GSSG) which is converted back to GSH by the enzyme GR [57–59].

(and other peroxides), thereby converting it into two molecules of water [37]. When

) via complex cascade of reactions [45]. One of the most effective

into one molecule each of water and diatomic oxygen [37]. Glutathione peroxi-

•− and thereby lowering the formation of ONOO−

[44,

malfunctioning processes related to the dramatic change of redox environment [53].

**3. Antioxidants**

molecules (e.g., water and O<sup>2</sup>

O2

, decreasing the amount of O2

64 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

O2 •− to H<sup>2</sup> O2

to H<sup>2</sup> O2

molecules of H<sup>2</sup>

An efficient antioxidant should not only be ubiquitous but should also be present in adequate amounts in cells and easily reacts with a wide variety of free radicals which have short halflife due to high reactivity. A good antioxidant has the ability to cross physiologic barriers and to be quickly transported into the cells. Thus, it must be available to all cells. It is also important for an antioxidant to be available. Antioxidants should be available when needed. They should be easily acquired through the diet or produced in situ. Antioxidants should be suitable for regeneration. The reaction between an antioxidant and a free radical yields an oxidized form of the antioxidant which has less scavenging activity than the original compound. Therefore, many antioxidants have physiologically reducing mechanisms, or its oxidized forms can still efficiently react with new free radicals. An ideal antioxidant should be conserved by the kidneys. Otherwise, large urinary losses would occur and the half-life will be short. An important aspect to consider for evaluating the suitability of a compound as an antioxidant is its toxicity. It should be nontoxic prior to and after the free radical scavenging process takes place. In addition, it is also important to be aware of possible interactions with any drug that may be concurrently consumed [41, 71, 72].

Melatonin is a potent direct scavenger of free radicals. Unlike most of other radical scavengers, it is a multifunctional antioxidant. Melatonin can easily pass through cell membranes because of its high lipophilicity and hydrophilicity [73]. Melatonin is also widespread within cells. Its concentrations in human serum and cerebrospinal fluid vary widely. Melatonin is endogenously generated, and it is ingested in the food as it is widely available in fruits and vegetables. Hence, melatonin is produced internally and is also ingested in the diet. Only small amounts of melatonin are excreted into the urine in its unchanged form. It has minimal toxicity. Numerous in vivo studies on animals involving massive doses of melatonin have shown that acute and chronic toxicity of melatonin is extremely low [41, 74]. Unlike most small molecule biological antioxidants such as ascorbic acid, α-tocopherol, lipoic acid, etc., melatonin does not undergo redox cycling and, thus, does not promote oxidation. Melatonin can be considered a suicidal or terminal antioxidant. It undergoes molecular rearrangement, effectively removing the free electron from the system. Each of these products of rearrangement is also a potent antioxidant in its own right. Furthermore, most of these processes involve more than one ROS per step, so that one melatonin molecule has the capacity to scavenge up to 10 ROS versus the classic antioxidants that scavenge one or less ROS [17, 20, 70, 74]. It has been found that melatonin promotes the repair of oxidized DNA. This is probably due to the melatonin's capability of transforming guanosine radical to guanosine by electron transfer [42]. It was shown that melatonin reduced the formation of 8-hydroxy-2′ deoxyguanosine (8-OH-dG), a damaged DNA product, 60–70 times more effective than some classic antioxidants (ascorbate and α-tocopherol) [75]. Additionally, the relative position of melatonin and its metabolites in the antioxidant "pecking order" (electrochemical potential) may contribute greatly to its utility in biological systems [76]. Melatonin protects lipids, proteins, and nuclear DNA from oxidative damage suggests that its intracellular distribution is wide [17]. Melatonin turned out to be considerably more efficient than the majority of its naturally occurring structural analogs, indicating that the substituents of the indole moiety strongly influenced reactivity and selectivity [77].

halide ions, haloperoxyl radicals are significantly more reactive than the alkylperoxyl radical;

by melatonin [85]. Not only melatonin but also several of its metabolites that are formed when it functions as a direct free radical scavenger, i.e., cyclic 3-hydroxymelatonin (c3OHM), AFMK, AMK, etc., are also radical scavengers [57, 86]. Melatonin and its metabolites work in a "task-division" way, with some of them acting mainly as free radical scavengers, while others act as metal chelating agents and inhibitors of the hydroxyl radical (OH•) production [87]. The sequential scavenging of ROS by melatonin and its metabolites is known as melatonin's antioxidant cascade [16]. The efficiency of AMK for scavenging ROS and preventing protein oxidation has been reported to be higher than that of AFMK. Therefore, it seems that at least in general, their protective activities against oxidative stress follow the order AMK > melato-

Electron donation is the principal mechanism by which melatonin detoxifies the free radicals [17]. While melatonin has the capability of donating one or more electrons to free radicals resulting in their detoxification, the metabolites that are formed during this process, i.e., c3OHM, AFMK, and AMK, also have similar capabilities [90]. After donating an electron to OH•, melatonin becomes a free radical itself, the indolyl radical cation. However, its reactivity is very low, and, therefore, it is not toxic to cells [41]. Oxidation of melatonin by hydroxyl radicals leads to several hydroxylated products which can be explained by interaction of melatonin with two hydroxyl radicals, one acting by hydrogen abstraction and the other by

6-Hydroxymelatonin (6OHM) is the major hepatic metabolite and photodegradation product of melatonin. It is an efficient metabolite for protecting against oxidative damage induced by

ity induced by quinolinic acid. It also lowers Fe(II)-induced neurotoxicity and iron-induced lipid peroxidation. It also inhibits the oxidative damage induced by this metal, UV radiation, thiobarbituric acid, and cyanide. It may be more efficient than melatonin in this capacity. Moreover, it inhibits oxidative stress induced by Cu+2-ascorbate mixtures and OH• production by sequestering Cu+2 ions. 6OHM also protects DNA damage induced by Fenton reagents

It was showed that the main hydroxylated metabolite of melatonin interaction with hypochlorous acid is 2-hydroxymelatonin (2OHM). Subsequently, 2OHM and its keto tautomer, melatonin 2-indolinone, were the oxidative products of melatonin's interaction with oxoferryl hemoglobin or OH• [93]. 4-Hydroxymelatonin (4OHM) is an excellent peroxyl radical scavenger and also a preventing antioxidant by inhibiting Cu(II). This effect would reduce the Cu(I) availability, which is the redox state required for the OH• to be formed, via Fenton-like reactions. 4OHM terminates the oxidant effects of copper-ascorbate mixtures. The key structural feature in the antioxidant activity of 4OHM is the presence of phenolic group, unlike 2OHM which has a relative low antioxidant protection [94]. 4OHM and 2OHM are generated during the UV-induced metabolism of melatonin. Further investigation needs to understand the antioxidant activity of these two compounds, as well as their potential role in protecting biomolecules against oxidative damage [87].

O2

and O2

combining with the reaction partner especially, at the sides C2, C3, C6, and C7 [91].

OO•) was found to be potently trapped

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67

An Overview of Melatonin as an Antioxidant Molecule: A Biochemical Approach

•−, 6OHM can reduce neurotoxic-

accordingly, the trichloromethylperoxyl radical (CCl<sup>3</sup>

UV irradiation. Due to its capability of scavenging <sup>1</sup>

**4.1. Effects of melatonin and its metabolites on reactive oxygen species**

nin > AFMK [88] (**Table 1**).

and UV radiation [84, 92].

### **4. Melatonin and its metabolites as antioxidants**

Melatonin is an indoleamine with two side chains, a 5-methoxy group and 3-amide group. Its molecular weight is 232.2 g/mol [42]. Melatonin has multifunctional activities in addition to its function as a synchronizer of the biological clock and seasonal reproduction [78, 79]. One such activity is its antioxidant capacity. Melatonin and its metabolites were found to have important antioxidant properties owing to their direct and indirect antioxidant actions. Melatonin can easily cross cell membranes [80] and the blood brain barrier [78] and protects various biomolecules against damage caused by free radicals by acting as a direct scavenger to detoxify reactive oxygen and nitrogen species. In addition, melatonin can indirectly reduce oxidative stress by increasing the activities of antioxidative defense systems; stimulating the expression and function of a number of antioxidant enzymes, as well as glutathione, another very important nonenzymatic, low molecular weight antioxidant; interacting synergistically with other antioxidants; and increasing the efficiency of the mitochondrial electron transport chain [40, 78–82]. Also, melatonin has a chelating property which may contribute in reducing metal-induced toxicity [83]. Melatonin was shown to be much more specific than its structural analogs in undergoing reactions, which lead to the termination of the radical reaction chain and in avoiding prooxidant, C- or O-centered intermediates [33, 38]. Moreover, it has been shown that it has an ability to scavenge free radicals, including hydroxyl radicals, hydrogen peroxide, peroxyl radicals, singlet oxygen, nitric oxide, and peroxynitrite. It was demonstrated that melatonin inhibits the activity of NO synthase, beside it's NO and peroxynitrite scavenging activity [84].

Melatonin, an endogenously produced indoleamine, is a highly effective antioxidant and free radical scavenger [82]. Melatonin has been reported to neutralize the most toxic oxidizing agents, hydroxyl radical and the peroxynitrite anion, generated within the cells. Moreover, melatonin reportedly scavenges singlet oxygen (<sup>1</sup> O2 ), superoxide anion radical, hydrogen peroxide, nitric oxide, and hypochlorous acid (HClO) [17]. Due to the electron-deficient nature of halide ions, haloperoxyl radicals are significantly more reactive than the alkylperoxyl radical; accordingly, the trichloromethylperoxyl radical (CCl<sup>3</sup> OO•) was found to be potently trapped by melatonin [85]. Not only melatonin but also several of its metabolites that are formed when it functions as a direct free radical scavenger, i.e., cyclic 3-hydroxymelatonin (c3OHM), AFMK, AMK, etc., are also radical scavengers [57, 86]. Melatonin and its metabolites work in a "task-division" way, with some of them acting mainly as free radical scavengers, while others act as metal chelating agents and inhibitors of the hydroxyl radical (OH•) production [87]. The sequential scavenging of ROS by melatonin and its metabolites is known as melatonin's antioxidant cascade [16]. The efficiency of AMK for scavenging ROS and preventing protein oxidation has been reported to be higher than that of AFMK. Therefore, it seems that at least in general, their protective activities against oxidative stress follow the order AMK > melatonin > AFMK [88] (**Table 1**).

#### **4.1. Effects of melatonin and its metabolites on reactive oxygen species**

can be considered a suicidal or terminal antioxidant. It undergoes molecular rearrangement, effectively removing the free electron from the system. Each of these products of rearrangement is also a potent antioxidant in its own right. Furthermore, most of these processes involve more than one ROS per step, so that one melatonin molecule has the capacity to scavenge up to 10 ROS versus the classic antioxidants that scavenge one or less ROS [17, 20, 70, 74]. It has been found that melatonin promotes the repair of oxidized DNA. This is probably due to the melatonin's capability of transforming guanosine radical to guanosine by electron transfer [42]. It was shown that melatonin reduced the formation of 8-hydroxy-2′ deoxyguanosine (8-OH-dG), a damaged DNA product, 60–70 times more effective than some classic antioxidants (ascorbate and α-tocopherol) [75]. Additionally, the relative position of melatonin and its metabolites in the antioxidant "pecking order" (electrochemical potential) may contribute greatly to its utility in biological systems [76]. Melatonin protects lipids, proteins, and nuclear DNA from oxidative damage suggests that its intracellular distribution is wide [17]. Melatonin turned out to be considerably more efficient than the majority of its naturally occurring structural analogs, indicating that the substituents of the indole moiety

Melatonin is an indoleamine with two side chains, a 5-methoxy group and 3-amide group. Its molecular weight is 232.2 g/mol [42]. Melatonin has multifunctional activities in addition to its function as a synchronizer of the biological clock and seasonal reproduction [78, 79]. One such activity is its antioxidant capacity. Melatonin and its metabolites were found to have important antioxidant properties owing to their direct and indirect antioxidant actions. Melatonin can easily cross cell membranes [80] and the blood brain barrier [78] and protects various biomolecules against damage caused by free radicals by acting as a direct scavenger to detoxify reactive oxygen and nitrogen species. In addition, melatonin can indirectly reduce oxidative stress by increasing the activities of antioxidative defense systems; stimulating the expression and function of a number of antioxidant enzymes, as well as glutathione, another very important nonenzymatic, low molecular weight antioxidant; interacting synergistically with other antioxidants; and increasing the efficiency of the mitochondrial electron transport chain [40, 78–82]. Also, melatonin has a chelating property which may contribute in reducing metal-induced toxicity [83]. Melatonin was shown to be much more specific than its structural analogs in undergoing reactions, which lead to the termination of the radical reaction chain and in avoiding prooxidant, C- or O-centered intermediates [33, 38]. Moreover, it has been shown that it has an ability to scavenge free radicals, including hydroxyl radicals, hydrogen peroxide, peroxyl radicals, singlet oxygen, nitric oxide, and peroxynitrite. It was demonstrated that melatonin inhibits the activity of NO synthase, beside it's NO and peroxynitrite scavenging activity [84]. Melatonin, an endogenously produced indoleamine, is a highly effective antioxidant and free radical scavenger [82]. Melatonin has been reported to neutralize the most toxic oxidizing agents, hydroxyl radical and the peroxynitrite anion, generated within the cells. Moreover,

O2

oxide, nitric oxide, and hypochlorous acid (HClO) [17]. Due to the electron-deficient nature of

), superoxide anion radical, hydrogen per-

strongly influenced reactivity and selectivity [77].

66 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

melatonin reportedly scavenges singlet oxygen (<sup>1</sup>

**4. Melatonin and its metabolites as antioxidants**

Electron donation is the principal mechanism by which melatonin detoxifies the free radicals [17]. While melatonin has the capability of donating one or more electrons to free radicals resulting in their detoxification, the metabolites that are formed during this process, i.e., c3OHM, AFMK, and AMK, also have similar capabilities [90]. After donating an electron to OH•, melatonin becomes a free radical itself, the indolyl radical cation. However, its reactivity is very low, and, therefore, it is not toxic to cells [41]. Oxidation of melatonin by hydroxyl radicals leads to several hydroxylated products which can be explained by interaction of melatonin with two hydroxyl radicals, one acting by hydrogen abstraction and the other by combining with the reaction partner especially, at the sides C2, C3, C6, and C7 [91].

6-Hydroxymelatonin (6OHM) is the major hepatic metabolite and photodegradation product of melatonin. It is an efficient metabolite for protecting against oxidative damage induced by UV irradiation. Due to its capability of scavenging <sup>1</sup> O2 and O2 •−, 6OHM can reduce neurotoxicity induced by quinolinic acid. It also lowers Fe(II)-induced neurotoxicity and iron-induced lipid peroxidation. It also inhibits the oxidative damage induced by this metal, UV radiation, thiobarbituric acid, and cyanide. It may be more efficient than melatonin in this capacity. Moreover, it inhibits oxidative stress induced by Cu+2-ascorbate mixtures and OH• production by sequestering Cu+2 ions. 6OHM also protects DNA damage induced by Fenton reagents and UV radiation [84, 92].

It was showed that the main hydroxylated metabolite of melatonin interaction with hypochlorous acid is 2-hydroxymelatonin (2OHM). Subsequently, 2OHM and its keto tautomer, melatonin 2-indolinone, were the oxidative products of melatonin's interaction with oxoferryl hemoglobin or OH• [93]. 4-Hydroxymelatonin (4OHM) is an excellent peroxyl radical scavenger and also a preventing antioxidant by inhibiting Cu(II). This effect would reduce the Cu(I) availability, which is the redox state required for the OH• to be formed, via Fenton-like reactions. 4OHM terminates the oxidant effects of copper-ascorbate mixtures. The key structural feature in the antioxidant activity of 4OHM is the presence of phenolic group, unlike 2OHM which has a relative low antioxidant protection [94]. 4OHM and 2OHM are generated during the UV-induced metabolism of melatonin. Further investigation needs to understand the antioxidant activity of these two compounds, as well as their potential role in protecting biomolecules against oxidative damage [87].


[10, 16]. AFMK is obviously more stable than many other oxidative metabolites or its secondary product, AMK [39]. AFMK reduces lipid peroxidation and oxidative DNA damage induced by a variety of oxidative stressors under various conditions [16]. It protects neuronal cell from injuries caused by hydrogen peroxide and amyloid-β (Aβ) peptide [85, 88, 93]. It has been suggested that neuroprotection of AFMK against radiation-induced oxidative damage

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69

Lipid peroxidation is a natural metabolic process under normal aerobic conditions, and it is one of the most investigated consequences of ROS action on membrane structure and function [44]. Alterations in the fluidity of membranes result in negative effects on their functions such as signal transduction processes and implicate in aging as well as in diseases [102]. Melatonin is known to be a stabilizer or protector of cell and organelle membranes because of its inhibitory effects on lipid peroxidation. Melatonin and its metabolites scavenge free radicals and thus terminate the initiation and propagation of lipid peroxidation [103]. Although melatonin and its metabolites, AFMK and AMK, are peroxyl radical scavengers, it is indicated that melatonin's ability to resist lipid peroxidation may also involve its metabolite, c3-OHM [104]. For the reaction with the peroxyl radical, c3-OHM was several orders of magnitude faster than melatonin, AFMK and AMK, and it was roughly 100-fold faster than water soluble vitamin E (Trolox) [96, 105]. Melatonin also directly scavenges the alkoxyl radical, a product resulting from the transition metal-catalyzed degradation of lipid peroxides. This is important for the control of lipid peroxidation since the alkoxyl radical can abstract a hydrogen atom from a polyunsaturated fatty acids; the resulting

peroxyl radical can obviously continue the propagation of lipid degradation [104, 106].

Reactive nitrogen species represent another category of potentially destructive substances, which react with melatonin [77]. ONOO• itself is a very damaging species able to react with proteins, lipids, and DNA. Therefore, the reaction between two rather innocuous free radicals produces a much more reactive one [41]. Melatonin readily combines with a superoxide releasing NO, thus preventing the formation of peroxynitrite, a free radical even more harm-

Scavenging of nitric oxide by melatonin in a nitrosation reaction is well documented. Whether this can be regarded as a detoxification reaction keeping NO from forming, the more dangerous peroxynitrite is uncertain because nitrosomelatonin easily decomposes, thereby releasing NO. Melatonin also scavenges peroxynitrite, but it is difficult to discriminate direct reactions with peroxynitrite and with hydroxyl radicals generated by decomposition of peroxynitrous

peroxynitrite [33, 77]. There is evidence for the formation of cyclic 2-hydroxymelatonin, cyclic 3-hydroxymelatonin, and 6-hydroxymelatonin about the reaction of melatonin with ONOO−

formation of 6-hydroxymelatonin required an activated peroxynitrite that can only exist in the absence of bicarbonate [41, 107, 108]. AFMK has the ability to interact with the ABTS

reaction and/or nitrated intermediates occur in the oxidation. In addition, the

adduct (ONOOCO<sup>2</sup>

. Therefore, it was suggested that

• seems to be more important than direct scavenging of

− ) which

in the melato-

.

**4.2. Effects of melatonin and its metabolites on reactive nitrogen species**

ful than NO. It has been described as a direct peroxynitrite scavenger [40].

It was suggested that one electron is transferred from melatonin to ONOO−

acid. The interaction with products from the peroxynitrite-CO<sup>2</sup>

•−) and NO<sup>2</sup>

6-hydroxymelatonin is not generated in the presence of CO<sup>2</sup>

carbonate radicals (CO<sup>3</sup>

nin +ONOO−

to the brain is due to its free radical scavenging function [88].

**Table 1.** Antioxidant effects of melatonin and its metabolites [89].

7-Hydroxymelatonin has been rarely considered, although the calculated activation energy for the respective reaction is as low as that for 6-hydroxylation. 3-Hydroxylation leads to an unusual compound cyclic 3-hydroxymelatonin (c3-OHM) [91]. c3-OHM is an intermediate metabolite of melatonin [16]. c3-OHM effectively scavenges OH•, ABTS•+ (2,20-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) [95], and peroxyl radicals [96] and can also chelate Cu(II), preventing its reduction and the consequent OH• production via Fenton-like reactions [93, 97]. It is demonstrated that c3-OHM inhibits oxidative DNA damage and 8-OHdG lesions, induced by Fenton reagents, under in vitro conditions [98]. Indeed, c3-OHM is considered a footprint molecule, excreted in small amounts in the urine, and evidence of the in vivo scavenging activity of melatonin [41]. c3-OHM also undergoes oxidation resulting in the formation of AFMK [16, 77, 99].

AFMK is one of the metabolites of melatonin and can be formed by both enzymatic or pseudoenzymatic and nonenzymatic metabolic pathways [10, 88]. Pyrole ring cleavage of melatonin by varied enzymes including indoleamine 2,3-dioxygenase (IDO), myeloperoxidase (MPO), and hemoperoxidases, varied pseudoenzymatic catalysts such as oxoferryl hemoglobin and in varied reactions with ROS involving free radicals and singlet oxygen, generates AFMK [39, 88, 100]. Melatonin oxidation by MPO and IDO generally requires O<sup>2</sup> •− that produced in large amounts in inflammatory circumstances [100]. Besides, there are also multiple hydroxylations, which are formed in the peroxidase and peroxidase-like reactions and in the conversion of c3-OHM to AFMK [39]. Nonenzymatically, direct reaction of melatonin with highly reactive oxygen species (e.g., hydroxyl radical and singlet oxygen) formed AFMK [100]. The formation of AFMK by singlet oxygen deserves attention, as this reactive oxygen species is formed under the influence of UV light [101]. In light of these findings, it appears that AFMK is a product common to several interactions of melatonin with oxygen-based reactants [85]. The generation of AMK occurs via deformylation of AFMK [10, 16, 77]. These compounds are also major melatonin metabolites in detoxifying ROS and reducing oxidative stress [10, 16]. AFMK is obviously more stable than many other oxidative metabolites or its secondary product, AMK [39]. AFMK reduces lipid peroxidation and oxidative DNA damage induced by a variety of oxidative stressors under various conditions [16]. It protects neuronal cell from injuries caused by hydrogen peroxide and amyloid-β (Aβ) peptide [85, 88, 93]. It has been suggested that neuroprotection of AFMK against radiation-induced oxidative damage to the brain is due to its free radical scavenging function [88].

Lipid peroxidation is a natural metabolic process under normal aerobic conditions, and it is one of the most investigated consequences of ROS action on membrane structure and function [44]. Alterations in the fluidity of membranes result in negative effects on their functions such as signal transduction processes and implicate in aging as well as in diseases [102]. Melatonin is known to be a stabilizer or protector of cell and organelle membranes because of its inhibitory effects on lipid peroxidation. Melatonin and its metabolites scavenge free radicals and thus terminate the initiation and propagation of lipid peroxidation [103]. Although melatonin and its metabolites, AFMK and AMK, are peroxyl radical scavengers, it is indicated that melatonin's ability to resist lipid peroxidation may also involve its metabolite, c3-OHM [104]. For the reaction with the peroxyl radical, c3-OHM was several orders of magnitude faster than melatonin, AFMK and AMK, and it was roughly 100-fold faster than water soluble vitamin E (Trolox) [96, 105]. Melatonin also directly scavenges the alkoxyl radical, a product resulting from the transition metal-catalyzed degradation of lipid peroxides. This is important for the control of lipid peroxidation since the alkoxyl radical can abstract a hydrogen atom from a polyunsaturated fatty acids; the resulting peroxyl radical can obviously continue the propagation of lipid degradation [104, 106].

#### **4.2. Effects of melatonin and its metabolites on reactive nitrogen species**

7-Hydroxymelatonin has been rarely considered, although the calculated activation energy for the respective reaction is as low as that for 6-hydroxylation. 3-Hydroxylation leads to an unusual compound cyclic 3-hydroxymelatonin (c3-OHM) [91]. c3-OHM is an intermediate metabolite of melatonin [16]. c3-OHM effectively scavenges OH•, ABTS•+ (2,20-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) [95], and peroxyl radicals [96] and can also chelate Cu(II), preventing its reduction and the consequent OH• production via Fenton-like reactions [93, 97]. It is demonstrated that c3-OHM inhibits oxidative DNA damage and 8-OHdG lesions, induced by Fenton reagents, under in vitro conditions [98]. Indeed, c3-OHM is considered a footprint molecule, excreted in small amounts in the urine, and evidence of the in vivo scavenging activity of melatonin [41]. c3-OHM also undergoes oxidation resulting in the formation of AFMK [16, 77, 99]. AFMK is one of the metabolites of melatonin and can be formed by both enzymatic or pseudoenzymatic and nonenzymatic metabolic pathways [10, 88]. Pyrole ring cleavage of melatonin by varied enzymes including indoleamine 2,3-dioxygenase (IDO), myeloperoxidase (MPO), and hemoperoxidases, varied pseudoenzymatic catalysts such as oxoferryl hemoglobin and in varied reactions with ROS involving free radicals and singlet oxygen, generates AFMK

Lipoxygenase

**melatonin**

**Antioxidative enzymes that are stimulated by** 

•− that produced in

[39, 88, 100]. Melatonin oxidation by MPO and IDO generally requires O<sup>2</sup>

**ROS/RNS neutralized by melatonin and its** 

Hydroxyl radical Superoxide dismutase Hydrogen peroxide Glutathione peroxidase

Nitric oxide Glutathione reductase Alkoxyl radical Glutamyl-cysteine ligase

Peroxynitrite Cyclooxygenase Singlet oxygen Heme oxygenase Hydrogen peroxide Nitric oxide synthase

Hypochlorous acid Paraoxonase Others Myeloperoxidase

**Table 1.** Antioxidant effects of melatonin and its metabolites [89].

Superoxide anion radical Catalase

68 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

**metabolites**

large amounts in inflammatory circumstances [100]. Besides, there are also multiple hydroxylations, which are formed in the peroxidase and peroxidase-like reactions and in the conversion of c3-OHM to AFMK [39]. Nonenzymatically, direct reaction of melatonin with highly reactive oxygen species (e.g., hydroxyl radical and singlet oxygen) formed AFMK [100]. The formation of AFMK by singlet oxygen deserves attention, as this reactive oxygen species is formed under the influence of UV light [101]. In light of these findings, it appears that AFMK is a product common to several interactions of melatonin with oxygen-based reactants [85]. The generation of AMK occurs via deformylation of AFMK [10, 16, 77]. These compounds are also major melatonin metabolites in detoxifying ROS and reducing oxidative stress Reactive nitrogen species represent another category of potentially destructive substances, which react with melatonin [77]. ONOO• itself is a very damaging species able to react with proteins, lipids, and DNA. Therefore, the reaction between two rather innocuous free radicals produces a much more reactive one [41]. Melatonin readily combines with a superoxide releasing NO, thus preventing the formation of peroxynitrite, a free radical even more harmful than NO. It has been described as a direct peroxynitrite scavenger [40].

Scavenging of nitric oxide by melatonin in a nitrosation reaction is well documented. Whether this can be regarded as a detoxification reaction keeping NO from forming, the more dangerous peroxynitrite is uncertain because nitrosomelatonin easily decomposes, thereby releasing NO. Melatonin also scavenges peroxynitrite, but it is difficult to discriminate direct reactions with peroxynitrite and with hydroxyl radicals generated by decomposition of peroxynitrous acid. The interaction with products from the peroxynitrite-CO<sup>2</sup> adduct (ONOOCO<sup>2</sup> − ) which carbonate radicals (CO<sup>3</sup> •−) and NO<sup>2</sup> • seems to be more important than direct scavenging of peroxynitrite [33, 77]. There is evidence for the formation of cyclic 2-hydroxymelatonin, cyclic 3-hydroxymelatonin, and 6-hydroxymelatonin about the reaction of melatonin with ONOO− . It was suggested that one electron is transferred from melatonin to ONOO− in the melatonin +ONOO− reaction and/or nitrated intermediates occur in the oxidation. In addition, the 6-hydroxymelatonin is not generated in the presence of CO<sup>2</sup> . Therefore, it was suggested that formation of 6-hydroxymelatonin required an activated peroxynitrite that can only exist in the absence of bicarbonate [41, 107, 108]. AFMK has the ability to interact with the ABTS cation radical as well as with ROS/RNS to form AMK. When AMK interacts with the ABTS cation radical or with ONOO− , it forms products that may also be ROS and RNS scavengers [59]. AMK was described as better a NO scavenger than melatonin or AFMK [88]. AMK effectively inhibits neuronal nitric oxide synthase activity and reduces intracellular NO levels [93].

free radical and/or toxic reactant generation is alleviated [90, 114]. In addition, AFMK and AMK also have the ability to downregulate prooxidative and pro-inflammatory enzymes including iNOS [102] and cyclooxygenase-2 (COX-2) and to carry out free radical avoidance functions [93].

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Mitochondria are critical in the control of metabolism and responsible for orchestrating cellular energy production. Therefore, they are central to the maintenance of life and the gatekeepers of cell death [116]. The production of energy in the form of ATP is crucial to optimal cell function, including aiding in repairing any cellular damage that has occurred and in improving survivability of the cell, of the tissue, and of the organism [90]. Up to 95% of the ATP produced in aerobic cells is a result of mitochondrial oxidative phosphorylation [59]. The ETC which is coupled to oxidative phosphorylation [59] is a system of oxidoreductase protein complexes (complexes I, II, III, and IV) [85]. Deficiencies in the ETC can result in the leakage of electrons which thereafter generate free radicals and other toxic reactants which leads to molecular damage in mitochondria; this damage culminates in and promotes what are referred to as mitochondria-related diseases [85]. Mitochondria are the primary source of free radicals [44, 45]. Increased free radical generation, enhanced mitochondrial iNOS activity, enhanced NO production, decreased respiratory complex activity, impaired electron transport system, and opening of mitochondrial permeability transition pores have all been

Melatonin has important actions at the level of mitochondria [85]. Melatonin exhibits remarkable functional versatility to protect the morphological and functional aspects of the cell membrane scavenging free radicals, enhancing the activity of the antioxidant enzymes, and optimizing the transfer of electrons through the ETC in the inner mitochondrial membrane [118]. Melatonin increases the efficiency of the ETC and thus reduces electron leakage and free radical generation [38, 75, 105] that is a consequence of the respiratory process by stimulating complex I and complex IV of the mitochondrial respiratory chain that are involved in oxidative phosphorylation [38, 58, 59, 118]. By directly detoxifying ROS/RNS, melatonin enhances ATP production via maintaining high levels of mitochondrial GSH, protects mitochondrial proteins and DNA from oxidative damage, and improves ETC activity [16, 90, 118]. Moreover, AMK, like its precursor melatonin, promotes mitochondrial complex I activity to elevate ATP production by lowering electron leak-

**4.4. Effects of melatonin and its metabolites on the mitochondria**

suggested as factors responsible for impaired mitochondrial function [117].

age and inhibiting the opening of the mitochondrial permeability transition pore [93].

Heavy metals are known to cause oxidative deterioration of biomolecules by initiating free radical-mediated chain reaction resulting in lipid peroxidation, protein oxidation, and oxidation of nucleic acid like DNA and RNA [119]. The ability of antioxidants to chelate and deactivate transition metals prevents such metals from participating in the initiation of lipid peroxidation and oxidative stress through metal-catalyzed reaction [120]. Chemical mean of inhibiting metal-induced oxidation is chelation. This particular process is directly involved in the OH•-inactivating ligand (OIL) behavior of antioxidants. There are two different ways of action in the protection exerted by OIL species against OH•-induced oxidative damage: (i) inhibiting the reduction of metal ions; thus, their reduced forms are not available for Fentonlike reactions or (ii) deactivating OH• after being produced by Fenton-like reactions [87].

**4.5. Effects of melatonin and its metabolites on transition metals**

#### **4.3. Effects of melatonin and its metabolites on antioxidant enzymes**

Cells are protected against oxidative stress by an interacting network of antioxidant enzymes [70]. Antioxidative enzymes provide a major defense mechanism against free radical damage either by metabolizing them to less reactive species or to nontoxic by-products [85]. The activities of antioxidative enzymes depend on the duration and severity of oxidative stress. Under prolonged oxidative stress conditions, free radicals directly damage the antioxidant enzymes or reduce enzyme activities [90, 109]. Besides its ability to directly neutralize a number of free radicals and reactive oxygen and nitrogen species, melatonin stimulates several antioxidative enzymes which increase its efficiency as an antioxidant [58]. The major antioxidative enzymes such as intracellular superoxide dismutases (CuZn-SOD and Mn-SOD), the selenium-containing glutathione peroxidases and catalase, are stimulated by melatonin under basal conditions [43, 75, 110]. Melatonin plays a significant role in maintaining indirect protection versus free radical injury by stimulating gene expression of antioxidant enzymes including those for SOD and GSH-Px [43, 58, 62, 111]. Melatonin affects both antioxidant enzyme activity and cellular mRNA levels for these enzymes under physiological circumstances and during increased oxidative stress, presumably through epigenetic mechanisms. These properties in a single molecule are unique for an antioxidant, and both actions protect against pathologically generated free radicals [43, 62].

The concentration of the intracellular antioxidant, glutathione, is very high in many cells. During high oxidative stress conditions total glutathione levels can be reduced [90]. Melatonin maintains the activities of enzymes that enhance intracellular levels of reduced GSH. The recycling of GSH may well be a major effect of melatonin in reducing oxidative stress. GSH is oxidized to its disulfide, GSSG, which is then quickly reduced back to GSH by GR, an enzyme which has been demonstrated to be stimulated by melatonin. The ability of melatonin to regulate the GSH/ GSSG balance by modulating enzyme activities seems to involve an action of melatonin at a nuclear binding site [85, 112]. The other GSH-metabolizing enzyme, i.e., CAT, also increases its activity in response to melatonin [85]. Furthermore, one of the melatonin actions is stimulation of gamma-glutamylcysteine synthetase that is the rate-limiting enzyme in glutathione production, thus glutathione levels do not drop significantly [36, 43, 58, 75, 77, 85, 86, 90, 110, 112, 113].

There are a number of prooxidative enzymes in multicellular organisms which generate free radicals [90]. Melatonin not only upregulates the expression of genes involved in detoxifying free radicals, but it also suppresses the activity or expression of genes involved in the generation of free radicals [16, 113]. Melatonin inhibits the prooxidative enzyme nitric oxide synthase which generates NO• and lipoxygenase which result in the formation of the superoxide anion [90, 113, 114]. Although NO• is not a strong free radical, when it couples with O2 •−, it forms the peroxynitrite anion which is potently reactive and damaging [90]. Lipoxygenase reaction is another possible source of ROS and other radicals. It catalyzes the hydroperoxidation of polyunsaturated fatty acids [115]. The prooxidative enzymes inhibited by melatonin also include myeloperoxidase and eosinophil peroxidase [110]. As a result,

free radical and/or toxic reactant generation is alleviated [90, 114]. In addition, AFMK and AMK also have the ability to downregulate prooxidative and pro-inflammatory enzymes including iNOS [102] and cyclooxygenase-2 (COX-2) and to carry out free radical avoidance functions [93].

#### **4.4. Effects of melatonin and its metabolites on the mitochondria**

cation radical as well as with ROS/RNS to form AMK. When AMK interacts with the ABTS

[59]. AMK was described as better a NO scavenger than melatonin or AFMK [88]. AMK effectively inhibits neuronal nitric oxide synthase activity and reduces intracellular NO levels [93].

Cells are protected against oxidative stress by an interacting network of antioxidant enzymes [70]. Antioxidative enzymes provide a major defense mechanism against free radical damage either by metabolizing them to less reactive species or to nontoxic by-products [85]. The activities of antioxidative enzymes depend on the duration and severity of oxidative stress. Under prolonged oxidative stress conditions, free radicals directly damage the antioxidant enzymes or reduce enzyme activities [90, 109]. Besides its ability to directly neutralize a number of free radicals and reactive oxygen and nitrogen species, melatonin stimulates several antioxidative enzymes which increase its efficiency as an antioxidant [58]. The major antioxidative enzymes such as intracellular superoxide dismutases (CuZn-SOD and Mn-SOD), the selenium-containing glutathione peroxidases and catalase, are stimulated by melatonin under basal conditions [43, 75, 110]. Melatonin plays a significant role in maintaining indirect protection versus free radical injury by stimulating gene expression of antioxidant enzymes including those for SOD and GSH-Px [43, 58, 62, 111]. Melatonin affects both antioxidant enzyme activity and cellular mRNA levels for these enzymes under physiological circumstances and during increased oxidative stress, presumably through epigenetic mechanisms. These properties in a single molecule are unique for an antioxidant, and both actions protect

The concentration of the intracellular antioxidant, glutathione, is very high in many cells. During high oxidative stress conditions total glutathione levels can be reduced [90]. Melatonin maintains the activities of enzymes that enhance intracellular levels of reduced GSH. The recycling of GSH may well be a major effect of melatonin in reducing oxidative stress. GSH is oxidized to its disulfide, GSSG, which is then quickly reduced back to GSH by GR, an enzyme which has been demonstrated to be stimulated by melatonin. The ability of melatonin to regulate the GSH/ GSSG balance by modulating enzyme activities seems to involve an action of melatonin at a nuclear binding site [85, 112]. The other GSH-metabolizing enzyme, i.e., CAT, also increases its activity in response to melatonin [85]. Furthermore, one of the melatonin actions is stimulation of gamma-glutamylcysteine synthetase that is the rate-limiting enzyme in glutathione production, thus glutathione levels do not drop significantly [36, 43, 58, 75, 77, 85, 86, 90, 110, 112, 113]. There are a number of prooxidative enzymes in multicellular organisms which generate free radicals [90]. Melatonin not only upregulates the expression of genes involved in detoxifying free radicals, but it also suppresses the activity or expression of genes involved in the generation of free radicals [16, 113]. Melatonin inhibits the prooxidative enzyme nitric oxide synthase which generates NO• and lipoxygenase which result in the formation of the superoxide anion [90, 113, 114]. Although NO• is not a strong free radical, when it couples

•−, it forms the peroxynitrite anion which is potently reactive and damaging [90].

Lipoxygenase reaction is another possible source of ROS and other radicals. It catalyzes the hydroperoxidation of polyunsaturated fatty acids [115]. The prooxidative enzymes inhibited by melatonin also include myeloperoxidase and eosinophil peroxidase [110]. As a result,

**4.3. Effects of melatonin and its metabolites on antioxidant enzymes**

against pathologically generated free radicals [43, 62].

with O2

, it forms products that may also be ROS and RNS scavengers

cation radical or with ONOO−

70 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

Mitochondria are critical in the control of metabolism and responsible for orchestrating cellular energy production. Therefore, they are central to the maintenance of life and the gatekeepers of cell death [116]. The production of energy in the form of ATP is crucial to optimal cell function, including aiding in repairing any cellular damage that has occurred and in improving survivability of the cell, of the tissue, and of the organism [90]. Up to 95% of the ATP produced in aerobic cells is a result of mitochondrial oxidative phosphorylation [59]. The ETC which is coupled to oxidative phosphorylation [59] is a system of oxidoreductase protein complexes (complexes I, II, III, and IV) [85]. Deficiencies in the ETC can result in the leakage of electrons which thereafter generate free radicals and other toxic reactants which leads to molecular damage in mitochondria; this damage culminates in and promotes what are referred to as mitochondria-related diseases [85]. Mitochondria are the primary source of free radicals [44, 45]. Increased free radical generation, enhanced mitochondrial iNOS activity, enhanced NO production, decreased respiratory complex activity, impaired electron transport system, and opening of mitochondrial permeability transition pores have all been suggested as factors responsible for impaired mitochondrial function [117].

Melatonin has important actions at the level of mitochondria [85]. Melatonin exhibits remarkable functional versatility to protect the morphological and functional aspects of the cell membrane scavenging free radicals, enhancing the activity of the antioxidant enzymes, and optimizing the transfer of electrons through the ETC in the inner mitochondrial membrane [118]. Melatonin increases the efficiency of the ETC and thus reduces electron leakage and free radical generation [38, 75, 105] that is a consequence of the respiratory process by stimulating complex I and complex IV of the mitochondrial respiratory chain that are involved in oxidative phosphorylation [38, 58, 59, 118]. By directly detoxifying ROS/RNS, melatonin enhances ATP production via maintaining high levels of mitochondrial GSH, protects mitochondrial proteins and DNA from oxidative damage, and improves ETC activity [16, 90, 118]. Moreover, AMK, like its precursor melatonin, promotes mitochondrial complex I activity to elevate ATP production by lowering electron leakage and inhibiting the opening of the mitochondrial permeability transition pore [93].

#### **4.5. Effects of melatonin and its metabolites on transition metals**

Heavy metals are known to cause oxidative deterioration of biomolecules by initiating free radical-mediated chain reaction resulting in lipid peroxidation, protein oxidation, and oxidation of nucleic acid like DNA and RNA [119]. The ability of antioxidants to chelate and deactivate transition metals prevents such metals from participating in the initiation of lipid peroxidation and oxidative stress through metal-catalyzed reaction [120]. Chemical mean of inhibiting metal-induced oxidation is chelation. This particular process is directly involved in the OH•-inactivating ligand (OIL) behavior of antioxidants. There are two different ways of action in the protection exerted by OIL species against OH•-induced oxidative damage: (i) inhibiting the reduction of metal ions; thus, their reduced forms are not available for Fentonlike reactions or (ii) deactivating OH• after being produced by Fenton-like reactions [87].

Melatonin is able to prevent the oxidative actions of metals by neutralizing the produced ROS and capturing such metals to form chelates [83]. It was demonstrated that the interplay of melatonin with metals such as aluminum, cadmium, copper, iron, lead, and zinc depended on concentration. Melatonin chelates both iron(III) and iron(II), which is the form that attends the Fenton reaction. If iron is bound to a protein (e.g., hemoglobin), melatonin restores the highly covalent iron such as oxyferryl (FeIV-O) hemoglobin back to iron(III), thereby reestablishing the biological activity of the protein [89]. It is suggested that, under physiological circumstances, direct chelation mechanism would be the major chelation route for Cu(II). It was demonstrated that melatonin and its metabolites, 3OHM, AFMK, and AMK, fully inhibited the oxidative stress induced by Cu(II)-ascorbate mixtures, via Cu(II) chelation [97]. Melatonin decreases the Cu(II)/H<sup>2</sup> O2 -induced damage to proteins and protects against copper-mediated lipid peroxidation, which led to the suggestion that the antioxidant and neuroprotective effects of melatonin may involve removing toxic metals from the central nervous system [42].

in regulating mitochondrial homeostasis [33, 38]. Mitochondrial dysfunction, i.e., cell energy impairment, apoptosis, and overproduction of ROS, is a final common pathogenic mechanism in aging and in neurodegenerative disease [43, 123]. Melatonin may be possible to treat neurodegenerative disorders by inhibiting mitochondrial cell death pathways. It may easily protect brain mitochondrial membranes from free radical attack, stabilizing them. The ability of melatonin to prevent GSH loss probably reflects its effect on the activities of the GSH redox cycle enzymes [33, 38, 83, 103]. Moreover, several neurological diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, and Wilson's disease (hepatolenticular degeneration) are characterized by an overload of copper and/or other metals. Melatonin and

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73

its metabolites, c3OHM, AFMK, and AMK, have the copper sequestering ability [89].

energetics [17].

Excessive and/or sustained increase in ROS generation plays a pivotal role in the initiation, progression, and clinical consequences of cardiovascular diseases (CVDs) [64, 124]. Clinically, melatonin is being increasingly recognized in the pathophysiology of CVD. Low levels of serum melatonin as well as its urinary metabolite, 6-sulphatoxymelatonin, have been reported in various CVDs including coronary heart disease, angina, congestive heart failure, and myocardial infarcts [125]. Melatonin plays an important role in the regulation of several parameters of the cardiovascular system, including blood pressure, and is considered to be a putative antihypertensive agent [126]. It may have cardio-protective properties via its direct free radical scavenger activity and its indirect antioxidant activity together with its significant anti-inflammatory properties [127, 128]. Mitochondrial respiration, mainly at the level of complex I and complex III, is an important source of ROS generation and hence a potential contributor of cardiac reperfusion injury [129]. Most of the beneficial actions of melatonin at the heart level may depend on its effect on mitochondrial bioenergetics mediated through various mechanisms including general antioxidant actions at the level of ETC dysfunction, electron leakage, and mitochondrial oxidative damage and also through a direct action of melatonin on mitochondrial permeability transition pore opening [127]. It was reported that melatonin protects against mitochondrial dysfunction associated with cardiac ischemia reperfusion, by preventing alterations to several parameters involved in mitochondrial bio-

Melatonin may also exhibit anticancer and protective oncostatic activity through several mechanisms, including inhibition of cancer cell proliferation, decrease in oxidative stress, and increase in immune system activity [130, 131]. Oxidative stress has complex and different effects on each type of cancer development [132]. Oxidation of cellular lipids and proteins can adversely affect several steps of the carcinogenic process through changes in a variety of cell regulatory functions, including signal transduction and gene expression. ROS are postulated to be involved in carcinogenesis process, especially in the stages of initiation and promotion [133]. It appears that the DNA damage is predominantly linked with the initiation process [132]. Free radicals and ROS generated by environmental carcinogens, or by metabolic alterations, cause DNA damage and genetic instability [134]. Furthermore, DNA damage, apoptosis resistance, enhanced proliferation, mutation, COX-2 upregulation, oxidative stress, tumor vascularity, and metastatic potential may be caused by nitric oxide synthase overexpression and increased nitric oxide and other RNS productions [132]. A growing body of evidence implicates melatonin's antioxidant/free radical scavenging actions in the inhibition of cancer development and growth [75]. Melatonin is a powerful scavenger of ROS, such as

### **5. Melatonin and its metabolites as anti-inflammatory agents**

Inflammation is an essential response to tissue injuries induced by physical, chemical, or biological insults [17]. The production of inflammatory cytokines including TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), or IL-6 attenuates by melatonin in numerous experimental models of inflammation [2]. Melatonin has several additional anti-inflammatory effects, which are probably related to a direct interaction with specific binding sites located in lymphocytes and macrophages [103]. Anti-inflammatory activity of melatonin includes inhibition of the activation of COX-2 and iNOS, as well as blocking of the transcriptional factors that triggers proinflammatory cytokine production. These include not only NF-кB but also HIF, Nrf2, cAMP, CREB, STAT, PPARs, and AP-1 [2, 43, 121]. Melatonin may be useful for the treatment of inflammatory disease, as it reduces inflammatory injury by blocking transcription factors and NF-κB, thereby decreasing further ROS formation within cells [43]. In peripheral monocytes, melatonin and, even more, AFMK suppressed TNF-α and IL-8 production and, in macrophages, COX-2 and iNOS expression. Moreover, melatonin was found to be efficiently oxidized to AFMK by macrophages [91]. AMK was reported to downregulate COX-2—but not COX-1—expression in macrophages, an effect shared by its precursors AFMK and melatonin [122].

### **6. The clinical significance of melatonin**

Melatonin plays important roles in neurogenesis, neuroprotection, maintenance of oxidant/ antioxidant balance, and modulation of cardiovascular and/or immune system. It also exerts a direct antioxidant effect on tissues/organs and antiapoptotic effects on cells [9]. Melatonin has been investigated in a wide range of diseases, such as neurodegenerative, cardiovascular, liver, and kidney diseases, cancer, and diabetes [43].

Melatonin is a ubiquitously acting direct free radical scavenger and also an indirect antioxidant. Melatonin and its metabolites are efficient in scavenging ROS and RNS. It plays an effective role in regulating mitochondrial homeostasis [33, 38]. Mitochondrial dysfunction, i.e., cell energy impairment, apoptosis, and overproduction of ROS, is a final common pathogenic mechanism in aging and in neurodegenerative disease [43, 123]. Melatonin may be possible to treat neurodegenerative disorders by inhibiting mitochondrial cell death pathways. It may easily protect brain mitochondrial membranes from free radical attack, stabilizing them. The ability of melatonin to prevent GSH loss probably reflects its effect on the activities of the GSH redox cycle enzymes [33, 38, 83, 103]. Moreover, several neurological diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, and Wilson's disease (hepatolenticular degeneration) are characterized by an overload of copper and/or other metals. Melatonin and its metabolites, c3OHM, AFMK, and AMK, have the copper sequestering ability [89].

Melatonin is able to prevent the oxidative actions of metals by neutralizing the produced ROS and capturing such metals to form chelates [83]. It was demonstrated that the interplay of melatonin with metals such as aluminum, cadmium, copper, iron, lead, and zinc depended on concentration. Melatonin chelates both iron(III) and iron(II), which is the form that attends the Fenton reaction. If iron is bound to a protein (e.g., hemoglobin), melatonin restores the highly covalent iron such as oxyferryl (FeIV-O) hemoglobin back to iron(III), thereby reestablishing the biological activity of the protein [89]. It is suggested that, under physiological circumstances, direct chelation mechanism would be the major chelation route for Cu(II). It was demonstrated that melatonin and its metabolites, 3OHM, AFMK, and AMK, fully inhibited the oxidative stress induced by Cu(II)-ascorbate mixtures, via Cu(II) chelation

O2

**5. Melatonin and its metabolites as anti-inflammatory agents**

macrophages, an effect shared by its precursors AFMK and melatonin [122].

**6. The clinical significance of melatonin**

liver, and kidney diseases, cancer, and diabetes [43].

copper-mediated lipid peroxidation, which led to the suggestion that the antioxidant and neuroprotective effects of melatonin may involve removing toxic metals from the central

Inflammation is an essential response to tissue injuries induced by physical, chemical, or biological insults [17]. The production of inflammatory cytokines including TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), or IL-6 attenuates by melatonin in numerous experimental models of inflammation [2]. Melatonin has several additional anti-inflammatory effects, which are probably related to a direct interaction with specific binding sites located in lymphocytes and macrophages [103]. Anti-inflammatory activity of melatonin includes inhibition of the activation of COX-2 and iNOS, as well as blocking of the transcriptional factors that triggers proinflammatory cytokine production. These include not only NF-кB but also HIF, Nrf2, cAMP, CREB, STAT, PPARs, and AP-1 [2, 43, 121]. Melatonin may be useful for the treatment of inflammatory disease, as it reduces inflammatory injury by blocking transcription factors and NF-κB, thereby decreasing further ROS formation within cells [43]. In peripheral monocytes, melatonin and, even more, AFMK suppressed TNF-α and IL-8 production and, in macrophages, COX-2 and iNOS expression. Moreover, melatonin was found to be efficiently oxidized to AFMK by macrophages [91]. AMK was reported to downregulate COX-2—but not COX-1—expression in

Melatonin plays important roles in neurogenesis, neuroprotection, maintenance of oxidant/ antioxidant balance, and modulation of cardiovascular and/or immune system. It also exerts a direct antioxidant effect on tissues/organs and antiapoptotic effects on cells [9]. Melatonin has been investigated in a wide range of diseases, such as neurodegenerative, cardiovascular,

Melatonin is a ubiquitously acting direct free radical scavenger and also an indirect antioxidant. Melatonin and its metabolites are efficient in scavenging ROS and RNS. It plays an effective role


[97]. Melatonin decreases the Cu(II)/H<sup>2</sup>

72 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

nervous system [42].

Excessive and/or sustained increase in ROS generation plays a pivotal role in the initiation, progression, and clinical consequences of cardiovascular diseases (CVDs) [64, 124]. Clinically, melatonin is being increasingly recognized in the pathophysiology of CVD. Low levels of serum melatonin as well as its urinary metabolite, 6-sulphatoxymelatonin, have been reported in various CVDs including coronary heart disease, angina, congestive heart failure, and myocardial infarcts [125]. Melatonin plays an important role in the regulation of several parameters of the cardiovascular system, including blood pressure, and is considered to be a putative antihypertensive agent [126]. It may have cardio-protective properties via its direct free radical scavenger activity and its indirect antioxidant activity together with its significant anti-inflammatory properties [127, 128]. Mitochondrial respiration, mainly at the level of complex I and complex III, is an important source of ROS generation and hence a potential contributor of cardiac reperfusion injury [129]. Most of the beneficial actions of melatonin at the heart level may depend on its effect on mitochondrial bioenergetics mediated through various mechanisms including general antioxidant actions at the level of ETC dysfunction, electron leakage, and mitochondrial oxidative damage and also through a direct action of melatonin on mitochondrial permeability transition pore opening [127]. It was reported that melatonin protects against mitochondrial dysfunction associated with cardiac ischemia reperfusion, by preventing alterations to several parameters involved in mitochondrial bioenergetics [17].

Melatonin may also exhibit anticancer and protective oncostatic activity through several mechanisms, including inhibition of cancer cell proliferation, decrease in oxidative stress, and increase in immune system activity [130, 131]. Oxidative stress has complex and different effects on each type of cancer development [132]. Oxidation of cellular lipids and proteins can adversely affect several steps of the carcinogenic process through changes in a variety of cell regulatory functions, including signal transduction and gene expression. ROS are postulated to be involved in carcinogenesis process, especially in the stages of initiation and promotion [133]. It appears that the DNA damage is predominantly linked with the initiation process [132]. Free radicals and ROS generated by environmental carcinogens, or by metabolic alterations, cause DNA damage and genetic instability [134]. Furthermore, DNA damage, apoptosis resistance, enhanced proliferation, mutation, COX-2 upregulation, oxidative stress, tumor vascularity, and metastatic potential may be caused by nitric oxide synthase overexpression and increased nitric oxide and other RNS productions [132]. A growing body of evidence implicates melatonin's antioxidant/free radical scavenging actions in the inhibition of cancer development and growth [75]. Melatonin is a powerful scavenger of ROS, such as hydroxyl radical, peroxyl radical, singlet oxygen, and nitric oxide, as well as a stimulator of the antioxidant enzymes, SOD, GPx, and CAT, all leading to a decrease in DNA damage [135]. Additionally, this indole stimulates antioxidant enzymes that remove ROS before they can inflict damage and aids in the repair of damaged DNA [136]. Melatonin could be an excellent candidate for the prevention and treatment of several cancers, such as breast cancer, prostate cancer, gastric cancer, and colorectal cancer [137].

enzymes; improves mitochondrial function, hence reducing radical formation; and reduces metal-induced toxicity. Results from previous studies support these effects on several diseases including cancer, diabetes, neurodegenerative, cardiovascular, liver; and kidney diseases.

An Overview of Melatonin as an Antioxidant Molecule: A Biochemical Approach

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75

Department of Biochemistry, Faculty of Pharmacy, Gazi University, Etiler, Ankara, Turkey

[1] Lerner AB, Case JD, Takahashi Y. Isolation of melatonin, a pineal factor that lightens

[2] Eghbal MA, Eftekhari A, Ahmadian E, Yadollah Azarmi Y, Parvizpur A. A review of biological and pharmacological actions of melatonin: Oxidant and prooxidant proper-

[3] Comai S, Gobbi G. Unveiling the role of melatonin MT2 receptors in sleep, anxiety and other neuropsychiatric diseases: A novel target in psychopharmacology. Journal of

[4] Yin J, Jin X, Shan Z, Li S, Huang H, Li P, Peng X, Peng Z, Yu K, Bao W, Yang W, Chen X, Liu L. Relationship of sleep duration with all-cause mortality and cardiovascular events: A systematic review and dose-response meta-analysis of prospective cohort studies.

[5] Altman NG, Izci-Balserak B, Schopfer E, Jackson N, Rattanaumpawan P, Gehrman PR, Patel NP, Grandner MA. Sleep duration versus sleep insufficiency as predictors of car-

[6] Cappuccio FP, Cooper D, D'Elia L, Strazzullo P, Miller MA. Sleep duration predicts cardiovascular outcomes: A systematic review and meta-analysis of prospective studies.

[7] Pandi-Perumal SR, Trakht I, Srinivasan V, Spence DW, Maestroni GJ, Zisapel N, Cardinali DP. Physiological effects of melatonin: Role of melatonin receptors and signal

melanocytes. Journal of the American Chemical Society. 1958;**80**:2587

ties. Pharmaceutical Bioprocessing. 2016;**4**(4):069-081

Journal of the American Heart Association. 2017;**6**(9):1-15

diometabolic health outcomes. Sleep Medicine. 2012;**13**(10):1261-1270

transduction pathways. Progress in Neurobiology. 2008;**85**(3):335-353

Psychiatry & Neuroscience. 2014;**39**(1):6-21

European Heart Journal. 2011;**32**(12):1484-1492

**Conflict of interest**

**Author details**

**References**

Aysun Hacışevki\* and Burcu Baba

The authors do not have any conflict of interest to declare.

\*Address all correspondence to: aysunsevki@gmail.com

A variety of antioxidants protect the liver from free radical-mediated damage, one of the best of which is melatonin. Clinical studies have confirmed that melatonin protects the liver from nonalcoholic liver disease and also during the surgical procedure of partial liver resection [138]. Melatonin is a well-known natural antioxidant and has many bioactivities. Melatonin exerts antioxidant effects in hepatocytes and epithelium of the liver by reducing lipid peroxidation and increasing the level of reduced liver glutathione. Melatonin is a highly valuable OH and H<sup>2</sup> O2 scavenger, during its metabolism to AFMK. It also induces several antioxidative enzymes such as glutathione peroxidase, glutathione reductase, and SOD and increases the synthesis of GSH [2, 88]. Melatonin exhibits potent anti-inflammatory, antioxidant, and fibrosuppressive activities against thioacetamide-induced hepatic fibrogenesis via the suppression of oxidative stress, DNA damage, pro-inflammatory cytokines, and fibrogenic gene transcripts [139]. Melatonin protects against lipid-induced mitochondrial dysfunction in hepatocytes and inhibits stellate cell activation during hepatic fibrosis in mice [140].

Inflammation and increased oxidative stress are also common features in chronic kidney disease patients [130, 141]. Oxidative stress and inflammation promote renal injury via damage to molecular components of the kidney by different mechanisms of action. ROS lead to the loss of significant functional properties, lipid peroxidation of cell membrane, decrease membrane viability, and cleavage, and cross-linking of renal DNA occurs leading to harmful mutations by oxidizing amino acids in the nephron. Furthermore, other ROS interactions in the nephron increase secondary radical production [130, 142]. Diabetes-associated hyperglycemia leads to mitochondrial ETC dysfunction culminating in a rise in ROS production [143]. Experimental evidence suggests that the indoleamine hormone melatonin is capable of influencing in development of diabetic complications by neutralizing the unnecessary ROS generation and protection of beta cells, as they possess low antioxidant potential and normalize redox state in the cell [144]. Melatonin acts as a cell survival agent by modulating autophagy in various cell types and under different conditions through amelioration of oxidative stress, ER stress, and inflammation [143].

### **7. Conclusion**

Melatonin is a circulating neurohormone secreted predominantly at night, thereby called as hormone of darkness. It can cross all physiological barriers to exert widespread regulatory effects on body tissues. Melatonin is a universal antioxidant with multifunctional activities such as anti-inflammatory, antiapoptotic, and antioxidant effects in addition to its function as a synchronizer of the biological clock and seasonal reproduction. Melatonin and its derivatives have been shown to be powerful direct free radical scavengers. Besides direct scavenging of ROS/RNS, melatonin also stimulates antioxidant enzymes; suppresses prooxidant enzymes; improves mitochondrial function, hence reducing radical formation; and reduces metal-induced toxicity. Results from previous studies support these effects on several diseases including cancer, diabetes, neurodegenerative, cardiovascular, liver; and kidney diseases.

### **Conflict of interest**

hydroxyl radical, peroxyl radical, singlet oxygen, and nitric oxide, as well as a stimulator of the antioxidant enzymes, SOD, GPx, and CAT, all leading to a decrease in DNA damage [135]. Additionally, this indole stimulates antioxidant enzymes that remove ROS before they can inflict damage and aids in the repair of damaged DNA [136]. Melatonin could be an excellent candidate for the prevention and treatment of several cancers, such as breast cancer, prostate

A variety of antioxidants protect the liver from free radical-mediated damage, one of the best of which is melatonin. Clinical studies have confirmed that melatonin protects the liver from nonalcoholic liver disease and also during the surgical procedure of partial liver resection [138]. Melatonin is a well-known natural antioxidant and has many bioactivities. Melatonin exerts antioxidant effects in hepatocytes and epithelium of the liver by reducing lipid peroxidation and increasing the level of reduced liver glutathione. Melatonin is a highly valuable

tive enzymes such as glutathione peroxidase, glutathione reductase, and SOD and increases the synthesis of GSH [2, 88]. Melatonin exhibits potent anti-inflammatory, antioxidant, and fibrosuppressive activities against thioacetamide-induced hepatic fibrogenesis via the suppression of oxidative stress, DNA damage, pro-inflammatory cytokines, and fibrogenic gene transcripts [139]. Melatonin protects against lipid-induced mitochondrial dysfunction in

Inflammation and increased oxidative stress are also common features in chronic kidney disease patients [130, 141]. Oxidative stress and inflammation promote renal injury via damage to molecular components of the kidney by different mechanisms of action. ROS lead to the loss of significant functional properties, lipid peroxidation of cell membrane, decrease membrane viability, and cleavage, and cross-linking of renal DNA occurs leading to harmful mutations by oxidizing amino acids in the nephron. Furthermore, other ROS interactions in the nephron increase secondary radical production [130, 142]. Diabetes-associated hyperglycemia leads to mitochondrial ETC dysfunction culminating in a rise in ROS production [143]. Experimental evidence suggests that the indoleamine hormone melatonin is capable of influencing in development of diabetic complications by neutralizing the unnecessary ROS generation and protection of beta cells, as they possess low antioxidant potential and normalize redox state in the cell [144]. Melatonin acts as a cell survival agent by modulating autophagy in various cell types and under different conditions through amelioration of oxidative stress, ER stress,

Melatonin is a circulating neurohormone secreted predominantly at night, thereby called as hormone of darkness. It can cross all physiological barriers to exert widespread regulatory effects on body tissues. Melatonin is a universal antioxidant with multifunctional activities such as anti-inflammatory, antiapoptotic, and antioxidant effects in addition to its function as a synchronizer of the biological clock and seasonal reproduction. Melatonin and its derivatives have been shown to be powerful direct free radical scavengers. Besides direct scavenging of ROS/RNS, melatonin also stimulates antioxidant enzymes; suppresses prooxidant

hepatocytes and inhibits stellate cell activation during hepatic fibrosis in mice [140].

scavenger, during its metabolism to AFMK. It also induces several antioxida-

cancer, gastric cancer, and colorectal cancer [137].

74 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

OH and H<sup>2</sup>

O2

and inflammation [143].

**7. Conclusion**

The authors do not have any conflict of interest to declare.

### **Author details**

Aysun Hacışevki\* and Burcu Baba

\*Address all correspondence to: aysunsevki@gmail.com

Department of Biochemistry, Faculty of Pharmacy, Gazi University, Etiler, Ankara, Turkey

### **References**


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[22] Bedrosian TA, Herring KL, Walton JC, Fonken LK, Weil ZM, Nelson RJ. Evidence for feedback control of pineal melatonin secretion. Neuroscience Letters. 2013;**542**:123-125

An Overview of Melatonin as an Antioxidant Molecule: A Biochemical Approach

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[23] Maitra S, Baidya DK, Khanna P. Melatonin in perioperative medicine: Current perspec-

[24] De Almeida EA, Di Mascio P, Harumi T, Spence DW, Moscovitch A, Hardeland R, Cardinali DP, Brown GM, Pandi-Perumal SR. Measurement of melatonin in body fluids: Standards, protocols and procedures. Child's Nervous System. 2011;**27**(6):879-891

[25] Ren W, Liu G, Chen S, Yin J, Wang J, Tan B, Wu G, Bazer FW, Peng Y, Li T, Reiter RJ, Yin Y. Melatonin signaling in T cells: Functions and applications. Journal of Pineal Research.

[26] Zawilska JB, Skene DJ, Arendt J. Physiology and pharmacology of melatonin in relation

[27] Slominski RM, Reiter RJ, Schlabritz-Loutsevitch N, Ostrom RS, Slominski AT. Melatonin membrane receptors in peripheral tissues: Distribution and functions. Molecular and

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[120] Adefegha SA, Oboh G. Water extractable phytochemicals from some Nigerian spices inhibit Fe2+-induced lipid peroxidation in Rat's brain–in vitro. Journal of Food

[121] Cardinali DP, Vigo DE, Olivar N, Vidal MF, Brusco LI. Melatonin therapy in patients

[122] Hardeland R. Melatonin, hormone of darkness and more: Occurrence, control mechanisms, actions and bioactive metabolites. Cellular and Molecular Life Sciences.

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**Chapter 4**

**Provisional chapter**

**Melatonin and Its Indisputable Effects on the Health**

**Melatonin and Its Indisputable Effects on the Health** 

Melatonin is a hormone synthesized from the amino acid tryptophan produced especially at night in the pineal gland and helps induce sleep. It is reported to play a role in preventing the production of free radicals and is thus a potent antioxidant. It can also enhance the function of the immune system and appears to have an antitumor effect. Melatonin secretion, mediated by photoperiod, directly influences reproductive function and dopamine which moves into frontal lobe regulating flow of information coming in from other areas of the brain. Additional side effects may be produced from treatment with melatonin and include stomach cramps, dizziness, headache, irritability, breast enlargement in men (called gynecomastia), and decreased sperm count. For clinical trials, the direct effect of exogenous melatonin administration on patients manifested with cancer should be studied to find its oncostatic effects on some cancers and provide information on its dosage and long-term safety. Moreover, mechanisms of action should

**Keywords:** melatonin, anti-aging, anticancer antiproliferative effect, geroprotector

Melatonin is a hormone (N-acetyl-5 methoxytryptamine) produced especially at night in the pineal gland which helps in the maintenance of the body's hormone balance and regulation, in immune system integrity, and in circadian rhythm (daily metabolic balance). This gland functions as a biological clock and time keeper of the brain by secreting melatonin and many other neuropeptides at night, helps to govern the sleep-wake cycle and, in animals, seasonal rhythms

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.79817

**State**

**State**

Hanan Farouk Aly and Maha Zaki Rizk

Hanan Farouk Aly and Maha Zaki Rizk

http://dx.doi.org/10.5772/intechopen.79817

**Abstract**

be further investigated.

**1. Hormone description**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **Melatonin and Its Indisputable Effects on the Health State Melatonin and Its Indisputable Effects on the Health State**

DOI: 10.5772/intechopen.79817

Hanan Farouk Aly and Maha Zaki Rizk Hanan Farouk Aly and Maha Zaki Rizk

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79817

#### **Abstract**

Melatonin is a hormone synthesized from the amino acid tryptophan produced especially at night in the pineal gland and helps induce sleep. It is reported to play a role in preventing the production of free radicals and is thus a potent antioxidant. It can also enhance the function of the immune system and appears to have an antitumor effect. Melatonin secretion, mediated by photoperiod, directly influences reproductive function and dopamine which moves into frontal lobe regulating flow of information coming in from other areas of the brain. Additional side effects may be produced from treatment with melatonin and include stomach cramps, dizziness, headache, irritability, breast enlargement in men (called gynecomastia), and decreased sperm count. For clinical trials, the direct effect of exogenous melatonin administration on patients manifested with cancer should be studied to find its oncostatic effects on some cancers and provide information on its dosage and long-term safety. Moreover, mechanisms of action should be further investigated.

**Keywords:** melatonin, anti-aging, anticancer antiproliferative effect, geroprotector

### **1. Hormone description**

Melatonin is a hormone (N-acetyl-5 methoxytryptamine) produced especially at night in the pineal gland which helps in the maintenance of the body's hormone balance and regulation, in immune system integrity, and in circadian rhythm (daily metabolic balance). This gland functions as a biological clock and time keeper of the brain by secreting melatonin and many other neuropeptides at night, helps to govern the sleep-wake cycle and, in animals, seasonal rhythms

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of migration, mating, and hibernation. Secretion of melatonin is stimulated by the dark and inhibited by light. Melatonin levels start to be released at sunsets, where neural signals are triggered which stimulate the pineal gland to begin releasing the hormone.

**2. Biological functions**

and the frequency and duration of menstrual cycles [1].

be particularly important in protecting brain cells [1].

radicals such as H2

(ONOO<sup>−</sup>

O2

**3. Melatonin as an activity enhancer of antioxidative enzymes**

, •OH, singlet oxygen (1

Two decades ago, melatonin was found to be a free radical scavenger [2]. However, abundant data ascertaining its ability to reduce oxidative stress have rapidly accumulated [3, 4]. The efficacy of melatonin in functioning in this subject is related to its direct free radical scavenging actions. Owing to its chemical formula, melatonin can interact with various forms of free

O2

degradation are potent antioxidants such as *N*1-acetyl-5-methoxykynuramine (AMK) or *N*1 acetyl-*N*2-formyl-5-methoxykynuramine (AFMK) [4]. Moreover, investigations on free radicals produced as a result of UV exposure, showed that by using cell-free melatonin-containing systems exposed to UV radiation (UVB: 60%, UVA: 30%) four metabolites were identified by HPLC and LC–MS: 2-OH-melatonin, 4-OH-melatonin, 6-OH-melatonin and AFMK [5]. Since these metabolites are potent antioxidants, this may suggest that, unlike other classic antioxidants, they do not induce prooxidant reactions. In addition, melatonin acts as a potent antioxidant through enhancing activity of antioxidant enzymes [6]. It should be noted that not only enzyme activity, but also gene transcription of antioxidant enzymes such as manganese

) and peroxyl radical (LOO•) [4]. The main photoproduct metabolites of melatonin

), superoxide anion (•O2

−

Melatonin and Its Indisputable Effects on the Health State

http://dx.doi.org/10.5772/intechopen.79817

89

), peroxynitrite anion

Melatonin, stimulated by darkness and inhibited by light, is involved in synchronizing the body's hormone secretions and in regulating their levels, setting the brain's internal biological clock and hence controlling circadian rhythms (daily biorhythms) or sleep-wake. Melatonin regulates many neuroendocrine functions and can inhibit secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary gland. When the timing or intensity of melatonin peak is disrupted (as in aging, stress, jet-lag, or artificial jet-lag syndromes), the biological clock is upset and many physiological and mental functions are adversely affected including the ability to think, remember, and make sound decisions can be profoundly hampered. Melatonin is also controls the timing and release of female reproductive hormones and hence helps determine when menstruation begins and ends (menopause),

In addition to its hormone actions, melatonin also has strong antioxidant properties and may scavenge and eliminate cell-damaging free radicals. The latter are chemical constituents that have an unpaired electron and cause lipid peroxidation, DNA damage and protein oxidation. Besides, it inhibits nitric oxide synthetase enzyme leading to reduction in the formation of peroxynitrite in tissues of brain. Melatonin stimulates the activities of antioxidant enzymes; glutathione peroxidase, superoxide dismutase and catalase. Melatonin is twice as effective at protecting cell membranes from lipid peroxidation as vitamin E and is five times more effective than glutathione for neutralizing hydroxyl radicals. As an antioxidant, it possibly helps to control or delay the development of heart disease, cancer and other conditions and may be effective in destroying malignant cells when combined with certain anticancer drugs. Since glutathione concentrations are not very high in the brain, both melatonin and adenosine may

Melatonin is synthesized from the amino acid tryptophan. Tryptophan (l-tryptophan) is an essential amino acid formed from proteins during digestion by the action of proteolytic enzymes. Tryptophan is converted to serotonin, a brain chemical involved with mood during the day and the latter finally converted to the indole melatonin (**Figures 1** and **2**). Melatonin occurs naturally in some foods but in fairly small amounts. Of all the plant-based foods, oats, sweet corn and rice are the richest source in melatonin, containing between 1000 and 1800 picograms while ginger, tomatoes, bananas and barley levels amount to 500 picograms per gram. In the human population, melatonin levels are highest in children and middle-aged adults and usually about 5–25 micrograms of melatonin are secreted each night. This amount tends to decline with age, a possible link with an age-related rise in difficulty sleeping and in the production of free radicals [1]. Synthetic melatonin and melatonin derived from bovine pineal glands are available as dietary supplements over-the-counter.

**Figure 1.** Chemical structure of Tryptophan (A) and Melatonin (B).

**Figure 2.** The biosynthesis and metabolism process of melatonin. www.impactjournals.com/oncotarget.

### **2. Biological functions**

of migration, mating, and hibernation. Secretion of melatonin is stimulated by the dark and inhibited by light. Melatonin levels start to be released at sunsets, where neural signals are

Melatonin is synthesized from the amino acid tryptophan. Tryptophan (l-tryptophan) is an essential amino acid formed from proteins during digestion by the action of proteolytic enzymes. Tryptophan is converted to serotonin, a brain chemical involved with mood during the day and the latter finally converted to the indole melatonin (**Figures 1** and **2**). Melatonin occurs naturally in some foods but in fairly small amounts. Of all the plant-based foods, oats, sweet corn and rice are the richest source in melatonin, containing between 1000 and 1800 picograms while ginger, tomatoes, bananas and barley levels amount to 500 picograms per gram. In the human population, melatonin levels are highest in children and middle-aged adults and usually about 5–25 micrograms of melatonin are secreted each night. This amount tends to decline with age, a possible link with an age-related rise in difficulty sleeping and in the production of free radicals [1]. Synthetic melatonin and melatonin derived from bovine

triggered which stimulate the pineal gland to begin releasing the hormone.

88 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

pineal glands are available as dietary supplements over-the-counter.

**Figure 1.** Chemical structure of Tryptophan (A) and Melatonin (B).

**Figure 2.** The biosynthesis and metabolism process of melatonin. www.impactjournals.com/oncotarget.

Melatonin, stimulated by darkness and inhibited by light, is involved in synchronizing the body's hormone secretions and in regulating their levels, setting the brain's internal biological clock and hence controlling circadian rhythms (daily biorhythms) or sleep-wake. Melatonin regulates many neuroendocrine functions and can inhibit secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary gland. When the timing or intensity of melatonin peak is disrupted (as in aging, stress, jet-lag, or artificial jet-lag syndromes), the biological clock is upset and many physiological and mental functions are adversely affected including the ability to think, remember, and make sound decisions can be profoundly hampered. Melatonin is also controls the timing and release of female reproductive hormones and hence helps determine when menstruation begins and ends (menopause), and the frequency and duration of menstrual cycles [1].

In addition to its hormone actions, melatonin also has strong antioxidant properties and may scavenge and eliminate cell-damaging free radicals. The latter are chemical constituents that have an unpaired electron and cause lipid peroxidation, DNA damage and protein oxidation. Besides, it inhibits nitric oxide synthetase enzyme leading to reduction in the formation of peroxynitrite in tissues of brain. Melatonin stimulates the activities of antioxidant enzymes; glutathione peroxidase, superoxide dismutase and catalase. Melatonin is twice as effective at protecting cell membranes from lipid peroxidation as vitamin E and is five times more effective than glutathione for neutralizing hydroxyl radicals. As an antioxidant, it possibly helps to control or delay the development of heart disease, cancer and other conditions and may be effective in destroying malignant cells when combined with certain anticancer drugs. Since glutathione concentrations are not very high in the brain, both melatonin and adenosine may be particularly important in protecting brain cells [1].

### **3. Melatonin as an activity enhancer of antioxidative enzymes**

Two decades ago, melatonin was found to be a free radical scavenger [2]. However, abundant data ascertaining its ability to reduce oxidative stress have rapidly accumulated [3, 4]. The efficacy of melatonin in functioning in this subject is related to its direct free radical scavenging actions. Owing to its chemical formula, melatonin can interact with various forms of free radicals such as H2 O2 , •OH, singlet oxygen (1 O2 ), superoxide anion (•O2 − ), peroxynitrite anion (ONOO<sup>−</sup> ) and peroxyl radical (LOO•) [4]. The main photoproduct metabolites of melatonin degradation are potent antioxidants such as *N*1-acetyl-5-methoxykynuramine (AMK) or *N*1 acetyl-*N*2-formyl-5-methoxykynuramine (AFMK) [4]. Moreover, investigations on free radicals produced as a result of UV exposure, showed that by using cell-free melatonin-containing systems exposed to UV radiation (UVB: 60%, UVA: 30%) four metabolites were identified by HPLC and LC–MS: 2-OH-melatonin, 4-OH-melatonin, 6-OH-melatonin and AFMK [5]. Since these metabolites are potent antioxidants, this may suggest that, unlike other classic antioxidants, they do not induce prooxidant reactions. In addition, melatonin acts as a potent antioxidant through enhancing activity of antioxidant enzymes [6]. It should be noted that not only enzyme activity, but also gene transcription of antioxidant enzymes such as manganese superoxide dismutase (Mn-SOD), copper-zinc superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPx) and gamma-glutamyl cysteine synthetase (γ-GCS) were maintained by melatonin n brain of rat [4]. This continuing enhancement proposed a possible role of activation of melatonin receptors to modulate antioxidant enzymes regulation following stress signals [7]. Actually, there are some suggestions that melatonin-modified antioxidant enzymes expression following signal pathways of membrane, cytosolic and nuclear receptors [8].

data on the effect of melatonin on longevity supports its geroprotective effect. We believe that melatonin biosafety is important for the study of it's the long-term effects at different doses and in different strains and species (e.g., in rats). In adequately designed work (50 rats in each group), small doses of melatonin were supplemented at the night (2.5–3 mg/kg), slow the starting of age-associated disorders in function of estrous and elevated the animals survival. There are multiple evident on the melatonin suppressive effect on the growth of impulsive mammary carcinogenesis and that initiated in mice and rats as a results of chemicals and radiation [13–15], carcinogenesis induced in colon of rats by 1,2-dimethyl hydrazine [16], while, carcinogenesis induced in uterine cervix and vagina of mice by DMBA [16] and liver cancer induced in rats by N-nitrosodiethylamine [17]. In these cases, it was observed that melatonin exerted a positive effect in the treatment of advanced cancer patients [18]. Melatonin has a dual effect: it is potent geroprotector, suppressor of tumor growth in vivo and in vitro. There are no discrepancies between results of the carcinogenic and anticarcinogenic potential of melatonin since previous reports showed that other antioxidants, such as α-tocopherol has geroprotector and tumorigenic effects and could be powerful anticarcinogens as well. The data of melatonin supplementation to perimenopausal women are hopeful [19]. Simultaneously, there are actual results on the unfavorable impacts of melatonin [12] such as melatonin may produce infertility, damage of retina and hypothermia, it stimulates high blood pressure, diabetes, and cancer by suppressing sex drive in males. It was remark that melatonin may be harmful for people with cardiovascular risk factors and it should not be obtain by individuals who have

Melatonin and Its Indisputable Effects on the Health State

http://dx.doi.org/10.5772/intechopen.79817

91

immune-system or mental disorders, or by people administered steroids.

**6. Melatonin as a protector against UV-induced skin aging**

apoptosis cells [21].

vived (140 mJ/cm2

Because of its properties as wide antioxidant and scavenger of free radical, melatonin may act as a preventative factor against damage induced by UV in the skin [5]. Clinically melatonin is worthy to protect damage of sun when it is taken before irradiation of UV [20]. These effects of melatonin as a protective agent versus damage induced by UV have in vitro studies powerful support [20, 21]. Melatonin counteracting the formation of polyamine levels so it enhances cell viability in UV-irradiated fibroblasts, and malondialdehyde accumulation while inhibited

Regarding to Ryoo et al. [22] study in fibroblasts exposed to UV, only 56% of the cells sur-

1 nM melatonin which was paralleled with marked decrease in malondialdehyde and death of cells. Other experimental comparative study using fibroblasts treated by UV declared identical correlation in viability of cells using 100 nM melatonin [21]. Additionally, melatonin is considered as a powerful anti-apoptotic compound that inhibited caspase 9 and caspase 3 by suppresses mitochondria-dependent (intrinsic) apoptosis however, it does not inhibit receptor-dependent (extrinsic) pathway of apoptosis mediated by caspase 8 "[23]. UV irradiation is considered an immediate agent acted directly on skin, the oxidative stress resulting in all known successive, destruction events in the skin can distinctly, only be antagonized by antioxidants, melatonin which is already found at the target sites and at the same time of

), while the survival rate of cells reached to 92.50% when preincubated with

### **4. The melatoninergic antioxidative system (MAS) of the skin**

It should be mentioned that synthesis of melatonin is not only confined to the pineal gland, but also extends to various other organs including the skin [4]. After exposure to UV, melatonin is metabolized in the skin and in turn causes production of antioxidant melatonin metabolites in human keratinocytes. This antioxidant cascade can be suggested for the skin, the same as in several described melatonin-related antioxidant cascades in chemical or other tissue homogenate systems [9]. This cascade has been defined as the melatoninergic antioxidative system (MAS) of the skin (**Figure 2**) forming an important barrier organ and protecting it against UV-induced oxidative stress-mediated damaging events on the nuclear, subcellular, protein and cell morphology level [5]. Melatonin forms a defense mechanism against the multifaceted threats of environmental stress, especially UV, to which the skin is life-long exposed. Owing to its chemical structure, melatonin as well as its metabolites are strongly lipophilic, which renders them easily diffusible in every skin and cell compartment, therefore penetrating beyond the epidermis, namely to the dermis and the hair follicle [10]. When the skin is exposed to UV irradiation, the reactive hydroxyl radical is generated in the skin and reacts directly with melatonin [11]. The scavenging effect of melatonin to hydroxyl radicals causes decrease in the lipid peroxides, oxidation of protein and damage of mitochondrial DNA. Thus, the cascade of melatoninergic antioxidative is significant in decreasing the free radicals emerging from radiation of UV and subsequently performs, a very hopeful strategy to keep the skin versus stressor factor of environmental condition as well as causative agent for aging of skin and promotion of tumor.

### **5. Anti-aging**

Another functional importance of melatonin is its potency to enhance, augment or neutralize the negative effects that stress, drugs and infections have on the body's immune system. The decrease in melatonin secretion by age is so reliable that blood melatonin levels have been proposed as a measurement of biological age.

Melatonin is critical for the regulation of circadian and seasonal changes in various aspects of physiology and neuroendocrine functions [12]. The reduction in life span was detected in rats as a result of a pinealectomy [12], whereas prolongation in the life span occurs upon transfer of pineal gland grafting from young mice into thymus of old mice or into pinealectomized old mice which reveals the ability of melatonin to extend life span [12]. Analysis of available data on the effect of melatonin on longevity supports its geroprotective effect. We believe that melatonin biosafety is important for the study of it's the long-term effects at different doses and in different strains and species (e.g., in rats). In adequately designed work (50 rats in each group), small doses of melatonin were supplemented at the night (2.5–3 mg/kg), slow the starting of age-associated disorders in function of estrous and elevated the animals survival. There are multiple evident on the melatonin suppressive effect on the growth of impulsive mammary carcinogenesis and that initiated in mice and rats as a results of chemicals and radiation [13–15], carcinogenesis induced in colon of rats by 1,2-dimethyl hydrazine [16], while, carcinogenesis induced in uterine cervix and vagina of mice by DMBA [16] and liver cancer induced in rats by N-nitrosodiethylamine [17]. In these cases, it was observed that melatonin exerted a positive effect in the treatment of advanced cancer patients [18]. Melatonin has a dual effect: it is potent geroprotector, suppressor of tumor growth in vivo and in vitro. There are no discrepancies between results of the carcinogenic and anticarcinogenic potential of melatonin since previous reports showed that other antioxidants, such as α-tocopherol has geroprotector and tumorigenic effects and could be powerful anticarcinogens as well. The data of melatonin supplementation to perimenopausal women are hopeful [19]. Simultaneously, there are actual results on the unfavorable impacts of melatonin [12] such as melatonin may produce infertility, damage of retina and hypothermia, it stimulates high blood pressure, diabetes, and cancer by suppressing sex drive in males. It was remark that melatonin may be harmful for people with cardiovascular risk factors and it should not be obtain by individuals who have immune-system or mental disorders, or by people administered steroids.

### **6. Melatonin as a protector against UV-induced skin aging**

superoxide dismutase (Mn-SOD), copper-zinc superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPx) and gamma-glutamyl cysteine synthetase (γ-GCS) were maintained by melatonin n brain of rat [4]. This continuing enhancement proposed a possible role of activation of melatonin receptors to modulate antioxidant enzymes regulation following stress signals [7]. Actually, there are some suggestions that melatonin-modified antioxidant enzymes expression following signal pathways of membrane, cytosolic and nuclear receptors [8].

It should be mentioned that synthesis of melatonin is not only confined to the pineal gland, but also extends to various other organs including the skin [4]. After exposure to UV, melatonin is metabolized in the skin and in turn causes production of antioxidant melatonin metabolites in human keratinocytes. This antioxidant cascade can be suggested for the skin, the same as in several described melatonin-related antioxidant cascades in chemical or other tissue homogenate systems [9]. This cascade has been defined as the melatoninergic antioxidative system (MAS) of the skin (**Figure 2**) forming an important barrier organ and protecting it against UV-induced oxidative stress-mediated damaging events on the nuclear, subcellular, protein and cell morphology level [5]. Melatonin forms a defense mechanism against the multifaceted threats of environmental stress, especially UV, to which the skin is life-long exposed. Owing to its chemical structure, melatonin as well as its metabolites are strongly lipophilic, which renders them easily diffusible in every skin and cell compartment, therefore penetrating beyond the epidermis, namely to the dermis and the hair follicle [10]. When the skin is exposed to UV irradiation, the reactive hydroxyl radical is generated in the skin and reacts directly with melatonin [11]. The scavenging effect of melatonin to hydroxyl radicals causes decrease in the lipid peroxides, oxidation of protein and damage of mitochondrial DNA. Thus, the cascade of melatoninergic antioxidative is significant in decreasing the free radicals emerging from radiation of UV and subsequently performs, a very hopeful strategy to keep the skin versus stressor factor of environmental condition as well as causative agent

Another functional importance of melatonin is its potency to enhance, augment or neutralize the negative effects that stress, drugs and infections have on the body's immune system. The decrease in melatonin secretion by age is so reliable that blood melatonin levels have been

Melatonin is critical for the regulation of circadian and seasonal changes in various aspects of physiology and neuroendocrine functions [12]. The reduction in life span was detected in rats as a result of a pinealectomy [12], whereas prolongation in the life span occurs upon transfer of pineal gland grafting from young mice into thymus of old mice or into pinealectomized old mice which reveals the ability of melatonin to extend life span [12]. Analysis of available

**4. The melatoninergic antioxidative system (MAS) of the skin**

90 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

for aging of skin and promotion of tumor.

proposed as a measurement of biological age.

**5. Anti-aging**

Because of its properties as wide antioxidant and scavenger of free radical, melatonin may act as a preventative factor against damage induced by UV in the skin [5]. Clinically melatonin is worthy to protect damage of sun when it is taken before irradiation of UV [20]. These effects of melatonin as a protective agent versus damage induced by UV have in vitro studies powerful support [20, 21]. Melatonin counteracting the formation of polyamine levels so it enhances cell viability in UV-irradiated fibroblasts, and malondialdehyde accumulation while inhibited apoptosis cells [21].

Regarding to Ryoo et al. [22] study in fibroblasts exposed to UV, only 56% of the cells survived (140 mJ/cm2 ), while the survival rate of cells reached to 92.50% when preincubated with 1 nM melatonin which was paralleled with marked decrease in malondialdehyde and death of cells. Other experimental comparative study using fibroblasts treated by UV declared identical correlation in viability of cells using 100 nM melatonin [21]. Additionally, melatonin is considered as a powerful anti-apoptotic compound that inhibited caspase 9 and caspase 3 by suppresses mitochondria-dependent (intrinsic) apoptosis however, it does not inhibit receptor-dependent (extrinsic) pathway of apoptosis mediated by caspase 8 "[23]. UV irradiation is considered an immediate agent acted directly on skin, the oxidative stress resulting in all known successive, destruction events in the skin can distinctly, only be antagonized by antioxidants, melatonin which is already found at the target sites and at the same time of exposure to UV ([23]. Besides, there is clear evidence that the preventive actions of melatonin against photobiological distraction are ameliorated by the powerful antioxidative characteristic of this compound. Photodamage, is tightly connected with UV-induced generation of ROS and it was shown that melatonin is a markedly powerful free radicals scavenger compared with vitamin C or Trolox, a vitamin E analog [21].

an inverse relationship was demonstrated between risk of breast cancer and the highest levels of aMT6s in urine [30, 31]. In controversy, in case–control study declared that high aMT6s level in urine level was markedly linked with a low breast cancer risk [26]. However, it was demonstrated that, no confirmation was detected between level of melatonin and its association with risk of breast cancer in four case–control studies. Regardless of menopausal status, there is no statistically significant differences was detected in urinary aMT6s level between British women with breast cancer and healthy one in a prospective nested case–control [26]. In postmenopausal women, there was no suggestion that high melatonin levels in urine were inversely associated with risk of breast cancer [32]. In the study of Brown et al. [33], did not document an overall correlation between melatonin levels in urine and the onset risk of breast cancer [33]. Simultaneously, no markedly relation was detected between level of aMT6s and

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In other types of cancer, it was found that, the men with low level of aMT6s in the urine below the median first morning connected with a four time increase in risk of in comparison with those with levels above the median. In addition, a case–control study showed that patients with high melatonin-sulfate levels or a high melatonin-sulfate/cortisol ratio were less likely to have prostate cancer or advanced stage prostate. It was found that the serum melatonin levels in women with ovarian cancer were significantly lower compared with control subjects (*p* < 0.05), demonstrating that decline in circulating melatonin level might contribute to the

It is worth mentioning that, the assessment melatonin levels are not equals, since concentrations of melatonin were determined in various sample as urine, plasma or serum. Also, the concentration of melatonin in human modifies with circadian rhythm; however, it has not been demonstrated which the best time for the sample collection could demonstrate the effects of melatonin. These variations might incorporate in the discrepancy of researches.

Research relating melatonin's effects on breast cancer is the serious, may be due to that melatonin has reported to attenuate various endocrine physiological biomarkers. Novel works showed that melatonin exhibited antiproliferative action against *in vitro* cell line of breast cancer [26], and suppressed mammary tumors development in rats [26]. Several melatonin mechanisms as anticancer were identified as it is apoptosis inducer [36], antiestrogenic effect through signaling pathway of ERα and decreased activity of aromatase enzyme [36], attenuation of receptors of melatonin [26], suppression on invasion [37] and angiogenesis [38].

Prostate cancer is the second most cancer type recorded and the fifth leading cause of cancer mortality in men [26]. It was found that melatonin at pharmacological concentrations could inhibit cell growth of both androgen-dependent and androgen-independent prostate cancer

One of the leading causes of death among women with genital tract disorders is ovarian cancer [39]. Even though various surgical techniques and chemotherapies have been useful for treatment of ovarian carcinoma, the prognosis remains lacking [40]. In recent years, a few

Cervical cancer is considered the principle leading reason of female tumor worldwide, [41].

studies have reported the anticancer effect of melatonin on cancer of ovary.

The melatonin effect on cervical cancer has been detected in insufficient works.

risk of breast cancer (either totally or by status of menopausal) [34].

pathogenesis of ovarian cancer in a retrospective study [35].

[26], through a range of mechanisms.

#### **7. Anti-cancer, immunity and reproduction**

Tumor growth is showed to be inhibited by melatonin. Melatonin may be of a great value in patients with untreatable metastatic cancer, especially in ameliorating their life quality. Several experiments showed that the levels of melatonin may be connected with risk of breast cancer. Some chemotherapy drugs used to treat breast cancer may be enhanced also by melatonin. Supplementation of melatonin reduces luteinizing hormone concentrations leading to inhibition of ovulation in humans. Further, melatonin administration may aid menopausal women by eliciting and sustaining sleep. Levels of melatonin may have a role in the anorexia symptoms. It was found that melatonin was used in seasonal affective disorder (SAD), due to the disorder is considered to be produced by melatonin release at an inadequate time [1].

Melatonin immunopharmacological activity has been indicated in different models. Melatonin treatment elevates antibodies production of sheep erythrocytes and immune response to primary immunization with T-dependent antigens [24]. Melatonin is involved in complicated relationships between the endocrine and nervous systems [25]. There are membrane receptors of melatonin on helper (Th). Melatonin receptors activation results to an elevate the production of Th1 cytokines, such as *γ*-interferon, interleukin-1, and opioid cytokines (interleukin-4 and dinorphine) [25]. At physiological concentrations of melatonin, it induces release of interleukins-1, -6 and -12 in monocytes of human. These mediators can protect stress-stimulated immunodepression maintaining mice from virus- and bacteria caused lethal diseases [25]. It is useful noticing that *γ*-interferon and colony-inducing factors (CSFs) can stimulate melatonin release in the pineal gland [25].

It is well accepted that melatonin is considered not only a hormone, but also protector for cells [26], implicated in modulation of immune system, processes of antioxidant and hematopoiesis [27]. Moreover, melatonin has a powerful oncostatic characters, through receptor-dependent and -independent mechanisms [28]. The melatonin receptors MT1 (encoded by *MTNR1A*) and MT2 (encoded by *MTNR1B*) are associated with the G-protein-coupled receptor (GPCR) group [26], and are mainly responsible for mediating melatonin downstream effects [29]. The antiproliferative effects melatonin may be due to melatonin–stimulated suppression in the uptake of linoleic acid [26]. Melatonin also demonstrated the probability to be used as adjuvant in therapies of cancer, through augmentation the effects of therapeutic drugs and decreased chemotherapies or radiation side effects [26].

Some studies recommended an inverse association between circadian melatonin level and breast cancer incidence. In addition, it was demonstrated high melatonin level up to ≤39.5 pg/mL in female showed high risk for breast cancer than females had high melatonin level > 39.5 pg/mL in a case–control study. Besides, in another five prospective case control, an inverse relationship was demonstrated between risk of breast cancer and the highest levels of aMT6s in urine [30, 31]. In controversy, in case–control study declared that high aMT6s level in urine level was markedly linked with a low breast cancer risk [26]. However, it was demonstrated that, no confirmation was detected between level of melatonin and its association with risk of breast cancer in four case–control studies. Regardless of menopausal status, there is no statistically significant differences was detected in urinary aMT6s level between British women with breast cancer and healthy one in a prospective nested case–control [26]. In postmenopausal women, there was no suggestion that high melatonin levels in urine were inversely associated with risk of breast cancer [32]. In the study of Brown et al. [33], did not document an overall correlation between melatonin levels in urine and the onset risk of breast cancer [33]. Simultaneously, no markedly relation was detected between level of aMT6s and risk of breast cancer (either totally or by status of menopausal) [34].

exposure to UV ([23]. Besides, there is clear evidence that the preventive actions of melatonin against photobiological distraction are ameliorated by the powerful antioxidative characteristic of this compound. Photodamage, is tightly connected with UV-induced generation of ROS and it was shown that melatonin is a markedly powerful free radicals scavenger compared

Tumor growth is showed to be inhibited by melatonin. Melatonin may be of a great value in patients with untreatable metastatic cancer, especially in ameliorating their life quality. Several experiments showed that the levels of melatonin may be connected with risk of breast cancer. Some chemotherapy drugs used to treat breast cancer may be enhanced also by melatonin. Supplementation of melatonin reduces luteinizing hormone concentrations leading to inhibition of ovulation in humans. Further, melatonin administration may aid menopausal women by eliciting and sustaining sleep. Levels of melatonin may have a role in the anorexia symptoms. It was found that melatonin was used in seasonal affective disorder (SAD), due to the disorder is considered to be produced by melatonin release at an inadequate time [1].

Melatonin immunopharmacological activity has been indicated in different models. Melatonin treatment elevates antibodies production of sheep erythrocytes and immune response to primary immunization with T-dependent antigens [24]. Melatonin is involved in complicated relationships between the endocrine and nervous systems [25]. There are membrane receptors of melatonin on helper (Th). Melatonin receptors activation results to an elevate the production of Th1 cytokines, such as *γ*-interferon, interleukin-1, and opioid cytokines (interleukin-4 and dinorphine) [25]. At physiological concentrations of melatonin, it induces release of interleukins-1, -6 and -12 in monocytes of human. These mediators can protect stress-stimulated immunodepression maintaining mice from virus- and bacteria caused lethal diseases [25]. It is useful noticing that *γ*-interferon and colony-inducing factors (CSFs) can stimulate melatonin

It is well accepted that melatonin is considered not only a hormone, but also protector for cells [26], implicated in modulation of immune system, processes of antioxidant and hematopoiesis [27]. Moreover, melatonin has a powerful oncostatic characters, through receptor-dependent and -independent mechanisms [28]. The melatonin receptors MT1 (encoded by *MTNR1A*) and MT2 (encoded by *MTNR1B*) are associated with the G-protein-coupled receptor (GPCR) group [26], and are mainly responsible for mediating melatonin downstream effects [29]. The antiproliferative effects melatonin may be due to melatonin–stimulated suppression in the uptake of linoleic acid [26]. Melatonin also demonstrated the probability to be used as adjuvant in therapies of cancer, through augmentation the effects of therapeutic drugs and

Some studies recommended an inverse association between circadian melatonin level and breast cancer incidence. In addition, it was demonstrated high melatonin level up to ≤39.5 pg/mL in female showed high risk for breast cancer than females had high melatonin level > 39.5 pg/mL in a case–control study. Besides, in another five prospective case control,

with vitamin C or Trolox, a vitamin E analog [21].

92 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

release in the pineal gland [25].

decreased chemotherapies or radiation side effects [26].

**7. Anti-cancer, immunity and reproduction**

In other types of cancer, it was found that, the men with low level of aMT6s in the urine below the median first morning connected with a four time increase in risk of in comparison with those with levels above the median. In addition, a case–control study showed that patients with high melatonin-sulfate levels or a high melatonin-sulfate/cortisol ratio were less likely to have prostate cancer or advanced stage prostate. It was found that the serum melatonin levels in women with ovarian cancer were significantly lower compared with control subjects (*p* < 0.05), demonstrating that decline in circulating melatonin level might contribute to the pathogenesis of ovarian cancer in a retrospective study [35].

It is worth mentioning that, the assessment melatonin levels are not equals, since concentrations of melatonin were determined in various sample as urine, plasma or serum. Also, the concentration of melatonin in human modifies with circadian rhythm; however, it has not been demonstrated which the best time for the sample collection could demonstrate the effects of melatonin. These variations might incorporate in the discrepancy of researches.

Research relating melatonin's effects on breast cancer is the serious, may be due to that melatonin has reported to attenuate various endocrine physiological biomarkers. Novel works showed that melatonin exhibited antiproliferative action against *in vitro* cell line of breast cancer [26], and suppressed mammary tumors development in rats [26]. Several melatonin mechanisms as anticancer were identified as it is apoptosis inducer [36], antiestrogenic effect through signaling pathway of ERα and decreased activity of aromatase enzyme [36], attenuation of receptors of melatonin [26], suppression on invasion [37] and angiogenesis [38].

Prostate cancer is the second most cancer type recorded and the fifth leading cause of cancer mortality in men [26]. It was found that melatonin at pharmacological concentrations could inhibit cell growth of both androgen-dependent and androgen-independent prostate cancer [26], through a range of mechanisms.

One of the leading causes of death among women with genital tract disorders is ovarian cancer [39]. Even though various surgical techniques and chemotherapies have been useful for treatment of ovarian carcinoma, the prognosis remains lacking [40]. In recent years, a few studies have reported the anticancer effect of melatonin on cancer of ovary.

Cervical cancer is considered the principle leading reason of female tumor worldwide, [41]. The melatonin effect on cervical cancer has been detected in insufficient works.

Visceral obesity is a risk factor of endometrial cancer, as it is associated with chronic inflammatory process [42]. Ciortea *et al*. [42] reported that the combinational treatment of melatonin and estrogen in ovariectomized rats was linked with lower body weight, less intra-retroperitoneal fat, reduction in endometrial proliferation, and less appearance of cellular atypia compared with estrogen replacement treatment. These results show that melatonin supplementation could be used in the prophylaxis of endometrial cancer in menopause women [42].

951,600 new cases and 723,100 deaths from gastric cancer in 2012 worldwide [26]. Melatonin has been reported to inhibit gastric cancer through various mechanisms in numerous studies. Pancreatic cancer is a highly fatal disease with a relatively low 5-year survival rate [48]. It responds poorly to radiotherapy and chemotherapy because the tumor cells are challenging

Melatonin and Its Indisputable Effects on the Health State

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95

Colorectal cancer is one of the major causes responsible for cancer death worldwide [26], and in several studies, melatonin recorded anticancer potency for various colorectal cancers. Overall, melatonin could be a new tempting therapeutic strategy for colorectal cancer, since it could regulate carcinogenesis, development, and progression of colorectal cancer. The underlying mechanisms involve multiple signaling pathways, including regulation of Ca MKII,

The presenting results on the melatonin genomic effect is rather few. In a study of cytogenecity, it was found that a decrease in the gene activity of ribosomes as a result of a pinealectomy in rats [12]. Menendez-Pelaez et al. [50] declared that melatonin treatment reduces mRNA level in the synthesis porphyrin, and 5- aminolevulinate synthase, in the Syrian hamsters Harderian glands. Melatonin declines mRNA levels of histone H4 and stopped age-attributed mRNA Bcl-2 reduction, in mice thymocytes [12]. Also, melatonin elevated some antioxidant enzymes mRNA (Mn-SOD, Cu,Zn-SOD) in Syrian hamsters Harderian gland [12]. Supplementation of melatonin produced significant enhancement in relative levels of mRNA for Mn-SOD, Cu,Zn-SOD and glutathione peroxidase in cerebral cortexes of rat [12]. Melatonin (1 nM) markedly modulates the mRNA of gonadotropin-releasing hormone. It was observed that melatonin regulate transforming growth factor-*α* gene expression level, macrophage-colony stimulating factor (M-CSF), tumor necrosis factor-*α* (TNF*α*) the stem cell factor in PEC, and interleukin-1*β*, M-CSF, TNF*α*, interferon-*γ*, and the stem cell factor in splenocytes [12]. These results are appropriate with results that the SCN is the main site for the exogenous melatonin effect on the amplitude rhythm of the endogenous melatonin [51]. Medication with melatonin suppressed the development of mammary tumor and regulated *HER-2/neu* onco gene in trans-

Marked melatonin effect was noticed on some oncogenesis- associated genes expression [12]. Further, myeloblastosis oncogene-like 1 (Mybl1) expression was adjusted by melatonin. On the other hand, melatonin showed a great effect on a large number of genes attributed to exchange of calcium, as cullins, Kcnn4 and Dcamkl1, calmodulin, calbindin, Kcnn2 and Kcnn4. Meanwhile, cullin-1 expression in the heart of mouse is down regulated, that of cullin-5 is significantly upregulated, and cullins-2 and -3 expression are significantly not deformed. Six members of cullin family are included, and are implicated in ubiquinone-mediated protein destruction necessary for cell-cycle through the G1 and S phases. Nevertheless, cullin-1, but not other members of the cullin family, is responsible for cell proliferation and differentiation [53]. It is believe that melatonin may effect on expansion of tumor by intermediating with binding of calcium and preventing the MAPs/calmodulin and tubulin/calmodulin complexes formation to stop degradation of cytoskeletal [54]. Peutz-Jeghers syndrome, which is

ET-1, Nrf2 signaling pathways, and induction of aberrant crypt foci (ACF).

**9. Effect of melatonin on gene expression**

to apoptosis [49].

genic *HER-2/neu* mice [52].

### **8. Oral cancer**

Oral cancer is a common type of human head and neck cancers, and the majority of the cases involve oral squamous cell carcinoma [26]. In several *in vitro* studies, melatonin has shown remarkable effect on oral cancer.

It was reported that melatonin presented effect on oral cancer cell lines as an anti-metastatic action (HSC-3 and OECM-1), through modulation of expression and activity of MMP-9, which was occurred by decreasing acetylation of histone [26]. Also, melatonin could minimize SCC9 and SCC25 cell lines viabilities (both tongue carcinoma), and exhibit suppressive effect on the pro-metastatic *ROCK- 1*gene expression and *HIF-1α* pro-angiogenic genes as well as *VEGF* in SCC9 cell line [43]. Overall, inhibitory effect of melatonin was demonstrated on some oral cancer cells, and its mechanisms of action mainly involved inhibitory effect on metastasis and its related angiogenesis.

Cancer of liver is considered the common reason of death globally, and hepatocellular carcinoma (HCC) is contributed to the most common type of cancer (70–80%), occurrence in developing countries [44]. Treatment with surgery still remains the most pronounced way for HCC patients, however it is only occurs in a few cases, thus it is necessary to find efficient chemotherapeutic drug [45]. Hence, several studies pointed out to the effects of melatonin on hepatocellular carcinoma. Melatonin modulates the changes produced by N-nitrosodiethylamine-initiated cancer of liver and ameliorates biomarkers of liver enzymes (ALT, AST), levels of antioxidant, as well as the disturbance in circadian clock in mice [46, 47].

SO, melatonin exerts its anti-liver cancer effects mainly due to its anti- pro-apoptotic activity (via COX-2/PI3K/AKT pathway attenuation, modulates the ratio of Bcl-2/Bax, as well as it activates ER stress), anti-angiogenesis and anti-invasive effects.

Renal cancer is considered the third high cancer accounts for 3% with predominance of a male (3 male/1 female) [26].

Lung cancer is a principal cause of cancer-related death. For instance, lung cancer is the second most frequent type of cancer in males with approximately 17,330 new cases identified in 2016 in Brazil [26]. Non-small-cell lung cancer (NSCLC) is a main form of cancer of lung [26], and the literatures have suggested that the disturbance of rhythm of melatonin could elevate the incidence of NSCLC [26]. In different researches, melatonin due to mainly because melatonin showed to enhance the effects of it enhances radiotherapy and chemotherapeutic drugs.

Gastric cancer causes a mortality rate ranking second among malignant tumors worldwide one of the most frequent forms of cancer worldwide [26]. It was recorded that there were 951,600 new cases and 723,100 deaths from gastric cancer in 2012 worldwide [26]. Melatonin has been reported to inhibit gastric cancer through various mechanisms in numerous studies.

Pancreatic cancer is a highly fatal disease with a relatively low 5-year survival rate [48]. It responds poorly to radiotherapy and chemotherapy because the tumor cells are challenging to apoptosis [49].

Colorectal cancer is one of the major causes responsible for cancer death worldwide [26], and in several studies, melatonin recorded anticancer potency for various colorectal cancers. Overall, melatonin could be a new tempting therapeutic strategy for colorectal cancer, since it could regulate carcinogenesis, development, and progression of colorectal cancer. The underlying mechanisms involve multiple signaling pathways, including regulation of Ca MKII, ET-1, Nrf2 signaling pathways, and induction of aberrant crypt foci (ACF).

### **9. Effect of melatonin on gene expression**

Visceral obesity is a risk factor of endometrial cancer, as it is associated with chronic inflammatory process [42]. Ciortea *et al*. [42] reported that the combinational treatment of melatonin and estrogen in ovariectomized rats was linked with lower body weight, less intra-retroperitoneal fat, reduction in endometrial proliferation, and less appearance of cellular atypia compared with estrogen replacement treatment. These results show that melatonin supplementation

Oral cancer is a common type of human head and neck cancers, and the majority of the cases involve oral squamous cell carcinoma [26]. In several *in vitro* studies, melatonin has shown

It was reported that melatonin presented effect on oral cancer cell lines as an anti-metastatic action (HSC-3 and OECM-1), through modulation of expression and activity of MMP-9, which was occurred by decreasing acetylation of histone [26]. Also, melatonin could minimize SCC9 and SCC25 cell lines viabilities (both tongue carcinoma), and exhibit suppressive effect on the pro-metastatic *ROCK- 1*gene expression and *HIF-1α* pro-angiogenic genes as well as *VEGF* in SCC9 cell line [43]. Overall, inhibitory effect of melatonin was demonstrated on some oral cancer cells, and its mechanisms of action mainly involved inhibitory effect on metastasis and

Cancer of liver is considered the common reason of death globally, and hepatocellular carcinoma (HCC) is contributed to the most common type of cancer (70–80%), occurrence in developing countries [44]. Treatment with surgery still remains the most pronounced way for HCC patients, however it is only occurs in a few cases, thus it is necessary to find efficient chemotherapeutic drug [45]. Hence, several studies pointed out to the effects of melatonin on hepatocellular carcinoma. Melatonin modulates the changes produced by N-nitrosodiethylamine-initiated cancer of liver and ameliorates biomarkers of liver enzymes (ALT, AST), levels of antioxidant, as well as the disturbance in circadian clock in mice [46, 47].

SO, melatonin exerts its anti-liver cancer effects mainly due to its anti- pro-apoptotic activity (via COX-2/PI3K/AKT pathway attenuation, modulates the ratio of Bcl-2/Bax, as well as it

Renal cancer is considered the third high cancer accounts for 3% with predominance of a male

Lung cancer is a principal cause of cancer-related death. For instance, lung cancer is the second most frequent type of cancer in males with approximately 17,330 new cases identified in 2016 in Brazil [26]. Non-small-cell lung cancer (NSCLC) is a main form of cancer of lung [26], and the literatures have suggested that the disturbance of rhythm of melatonin could elevate the incidence of NSCLC [26]. In different researches, melatonin due to mainly because melatonin showed to enhance the effects of it enhances radiotherapy and chemotherapeutic drugs.

Gastric cancer causes a mortality rate ranking second among malignant tumors worldwide one of the most frequent forms of cancer worldwide [26]. It was recorded that there were

activates ER stress), anti-angiogenesis and anti-invasive effects.

could be used in the prophylaxis of endometrial cancer in menopause women [42].

**8. Oral cancer**

remarkable effect on oral cancer.

94 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

its related angiogenesis.

(3 male/1 female) [26].

The presenting results on the melatonin genomic effect is rather few. In a study of cytogenecity, it was found that a decrease in the gene activity of ribosomes as a result of a pinealectomy in rats [12]. Menendez-Pelaez et al. [50] declared that melatonin treatment reduces mRNA level in the synthesis porphyrin, and 5- aminolevulinate synthase, in the Syrian hamsters Harderian glands. Melatonin declines mRNA levels of histone H4 and stopped age-attributed mRNA Bcl-2 reduction, in mice thymocytes [12]. Also, melatonin elevated some antioxidant enzymes mRNA (Mn-SOD, Cu,Zn-SOD) in Syrian hamsters Harderian gland [12]. Supplementation of melatonin produced significant enhancement in relative levels of mRNA for Mn-SOD, Cu,Zn-SOD and glutathione peroxidase in cerebral cortexes of rat [12]. Melatonin (1 nM) markedly modulates the mRNA of gonadotropin-releasing hormone. It was observed that melatonin regulate transforming growth factor-*α* gene expression level, macrophage-colony stimulating factor (M-CSF), tumor necrosis factor-*α* (TNF*α*) the stem cell factor in PEC, and interleukin-1*β*, M-CSF, TNF*α*, interferon-*γ*, and the stem cell factor in splenocytes [12]. These results are appropriate with results that the SCN is the main site for the exogenous melatonin effect on the amplitude rhythm of the endogenous melatonin [51]. Medication with melatonin suppressed the development of mammary tumor and regulated *HER-2/neu* onco gene in transgenic *HER-2/neu* mice [52].

Marked melatonin effect was noticed on some oncogenesis- associated genes expression [12]. Further, myeloblastosis oncogene-like 1 (Mybl1) expression was adjusted by melatonin. On the other hand, melatonin showed a great effect on a large number of genes attributed to exchange of calcium, as cullins, Kcnn4 and Dcamkl1, calmodulin, calbindin, Kcnn2 and Kcnn4. Meanwhile, cullin-1 expression in the heart of mouse is down regulated, that of cullin-5 is significantly upregulated, and cullins-2 and -3 expression are significantly not deformed. Six members of cullin family are included, and are implicated in ubiquinone-mediated protein destruction necessary for cell-cycle through the G1 and S phases. Nevertheless, cullin-1, but not other members of the cullin family, is responsible for cell proliferation and differentiation [53]. It is believe that melatonin may effect on expansion of tumor by intermediating with binding of calcium and preventing the MAPs/calmodulin and tubulin/calmodulin complexes formation to stop degradation of cytoskeletal [54]. Peutz-Jeghers syndrome, which is associated with high risk of tumor development in multiple localizations is associated with at least one of these, Stk11 kinase with an unclear function, has anticarcinogenic effects and mutations [55]. Eventually, these data present undeviating evidence for the different effect of melatonin on the expression of different genes *in vivo*. Specific gene expression profiles are connected with the aging process in animals and humans [12]. Lund et al. [56] have detected a reduction in gene expression of heat shock protein while an elevation in the insulin-like genes expression, resulting in a decline in gene expression of insulin signaling during aging. Pletscher et al. [57], showed that down regulation of a large number of genes implicated in cell growth and maintenance following caloric restriction. Weindruch et al. [58], declared that in mice, the process of aging is describe by the high level of reactive oxygen species in both the skeletal muscle and brain, inhibition in the genes expression of biosynthetic enzyme and genes implicated in turnover of protein. Hence, caloric restriction stimulated genes are implicated in the metabolism of fatty acid, glycolysis, and gluconeogenesis. Presented results on the melatonin effects on gene expression, mainly genes of mitochondria, suggest that some of them may be accountable for the hormone capacity to block disorders resulting from aging. Further studies are need in this direction.

actually decreases circulating levels of melatonin and reconstruct the suprachiasmatic nuclei circadian pacemaker, leading to the elevation in risk of breast cancer, which may be due to down-regulating gonadal synthesis of steroids, by acting on receptor sites within the neuroendocrine reproductive axis or altered estrogen receptor function [59]. Consequently, in the

Melatonin and Its Indisputable Effects on the Health State

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97

Disorder in the system of dopamine has also been noticeably associated with psychosis and schizophrenia. Dopamine proceeds in the frontal lobe and regulating the information coming in from other parts of the brain. Normalization in the dopamine flow may produce interrupted or discontinuous cogitation as in schizophrenia. Schizophrenia is described by both 'positive '(additional experience and behavior) and 'negative symptoms' (lack in experience or behavior). Symptoms of positive response are classifying under the psychosis term and identically involve disorders of illusions, deliriums, and intellect. Symptoms of negative response may involve unsuitable emotional manifestation, lack of speech and stimulus. Some drugs, as cocaine, prevent dopamine return into the brain, coherently, dopamine buildup in

right circumstances, melatonin may be quite beneficial for reproductive health.

the synapse, producing drug-initiated psychosis or schizophrenia [1].

breast in the men and reduction in the count of sperm.

melatonin with these drugs to prevent melatonin overdose.

dination, elevated blood pressure, diarrhea, and convulsions [1].

**13. Discussion and conclusion**

**12. Contraindications, interactions, precautions and side effects**

Melatonin can produce sleepiness if given during the day. Additional, side effects that have been documented upon melatonin supplementation including cramps of stomach, vertigo, a continuous pain in the head, touchiness, moodiness, reduced sexual desire, enlargement of

So, melatonin should not take during operating machine or drive. Further, melatonin could interrupt with human fertility and also melatonin should be not used for pregnant or nursing women. Utilization of melatonin by person who already have an augmentation level of melatonin as children, teenagers, pregnant and lactating women can result in melatonin overdose. MAOI drugs inhibit melatonin breakdown from the body, so people should not take

Melatonin causes drug–drug interaction with antidepressants, such as Prozac (inhibitor of serotonin) or Nardil (inhibitor of monoamine oxidase). Melatonin Interaction with these kinds of drugs can produce heart attack, confusion, sweating, shaking, and fever, lack of coor-

Melatonin is considered as a potent geroprotector, anticarcinogen, and inhibitor of tumor growth *in vivo* and *in vitro*, and in some models it may induce tumors and promote tumor growth. An important mechanism of melatonin is its impact on hemopoiesis involves the

**11. Dopamine and psychosis**

### **10. Melatonin and reproduction**

Pattern of melatonin secretion, mediated by photoperiod, directly affect reproductive function which was recorded in several evidence-based researches. The daily light/dark (LD) cycle is considered the main physiological melatonin role, so, the variation in the duration of signal of melatonin occurs in attribution to the night length. The variation in melatonin signal duration is used to synchronize neuroendocrine rhythms with the annual variation in day-length in seasonal mammals. In addition, fetal and newborn animals use the maternal signal of melatonin to entrain endogenous circadian rhythms before direct photic information is presented. It was found that, very marked effect for exogenous melatonin was detected in modulating reproductive function in different organisms, depending on the animal age, melatonin supplementation time [59].

The data presented above exhibited that the antigonadal effects of melatonin in humans are apparently much less significant than in some seasonally breeding mammalian species. This is due to humans are not 'seasonally breeding'. Recently, accumulated evidences declared the efficacy of melatonin in attenuation the reproductive function in human. The suppressive effect of melatonin at the level of CNS have decreased daring growth. During development of human, such suppressive action of melatonin on GnRH function gradually reduced due to a down regulation in the functional of melatonin receptors expression. In other adult rodents, melatonin does not have noticeable action on the functioning of pituitary, whereas the association between the release of melatonin release and the functions of hypothalamic, involving the release of GnRH, are right. These actions are markedly significant in coinciding the external photoperiods and functions of reproduction through well not characterized mechanisms. The circadian rhythm regulated genes are considered seriously players in regulation of gene throughout different organism, especially for regulatory genes of cell-cycle and apoptotic genes. Melatonin may have also ameliorating effectiveness against human disorders attributed to reproductive function. Such as illumination intensity during the night actually decreases circulating levels of melatonin and reconstruct the suprachiasmatic nuclei circadian pacemaker, leading to the elevation in risk of breast cancer, which may be due to down-regulating gonadal synthesis of steroids, by acting on receptor sites within the neuroendocrine reproductive axis or altered estrogen receptor function [59]. Consequently, in the right circumstances, melatonin may be quite beneficial for reproductive health.

### **11. Dopamine and psychosis**

associated with high risk of tumor development in multiple localizations is associated with at least one of these, Stk11 kinase with an unclear function, has anticarcinogenic effects and mutations [55]. Eventually, these data present undeviating evidence for the different effect of melatonin on the expression of different genes *in vivo*. Specific gene expression profiles are connected with the aging process in animals and humans [12]. Lund et al. [56] have detected a reduction in gene expression of heat shock protein while an elevation in the insulin-like genes expression, resulting in a decline in gene expression of insulin signaling during aging. Pletscher et al. [57], showed that down regulation of a large number of genes implicated in cell growth and maintenance following caloric restriction. Weindruch et al. [58], declared that in mice, the process of aging is describe by the high level of reactive oxygen species in both the skeletal muscle and brain, inhibition in the genes expression of biosynthetic enzyme and genes implicated in turnover of protein. Hence, caloric restriction stimulated genes are implicated in the metabolism of fatty acid, glycolysis, and gluconeogenesis. Presented results on the melatonin effects on gene expression, mainly genes of mitochondria, suggest that some of them may be accountable for the hormone capacity to block disorders resulting from aging.

Pattern of melatonin secretion, mediated by photoperiod, directly affect reproductive function which was recorded in several evidence-based researches. The daily light/dark (LD) cycle is considered the main physiological melatonin role, so, the variation in the duration of signal of melatonin occurs in attribution to the night length. The variation in melatonin signal duration is used to synchronize neuroendocrine rhythms with the annual variation in day-length in seasonal mammals. In addition, fetal and newborn animals use the maternal signal of melatonin to entrain endogenous circadian rhythms before direct photic information is presented. It was found that, very marked effect for exogenous melatonin was detected in modulating reproductive function in different organisms, depending on the animal age,

The data presented above exhibited that the antigonadal effects of melatonin in humans are apparently much less significant than in some seasonally breeding mammalian species. This is due to humans are not 'seasonally breeding'. Recently, accumulated evidences declared the efficacy of melatonin in attenuation the reproductive function in human. The suppressive effect of melatonin at the level of CNS have decreased daring growth. During development of human, such suppressive action of melatonin on GnRH function gradually reduced due to a down regulation in the functional of melatonin receptors expression. In other adult rodents, melatonin does not have noticeable action on the functioning of pituitary, whereas the association between the release of melatonin release and the functions of hypothalamic, involving the release of GnRH, are right. These actions are markedly significant in coinciding the external photoperiods and functions of reproduction through well not characterized mechanisms. The circadian rhythm regulated genes are considered seriously players in regulation of gene throughout different organism, especially for regulatory genes of cell-cycle and apoptotic genes. Melatonin may have also ameliorating effectiveness against human disorders attributed to reproductive function. Such as illumination intensity during the night

Further studies are need in this direction.

96 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

**10. Melatonin and reproduction**

melatonin supplementation time [59].

Disorder in the system of dopamine has also been noticeably associated with psychosis and schizophrenia. Dopamine proceeds in the frontal lobe and regulating the information coming in from other parts of the brain. Normalization in the dopamine flow may produce interrupted or discontinuous cogitation as in schizophrenia. Schizophrenia is described by both 'positive '(additional experience and behavior) and 'negative symptoms' (lack in experience or behavior). Symptoms of positive response are classifying under the psychosis term and identically involve disorders of illusions, deliriums, and intellect. Symptoms of negative response may involve unsuitable emotional manifestation, lack of speech and stimulus. Some drugs, as cocaine, prevent dopamine return into the brain, coherently, dopamine buildup in the synapse, producing drug-initiated psychosis or schizophrenia [1].

### **12. Contraindications, interactions, precautions and side effects**

Melatonin can produce sleepiness if given during the day. Additional, side effects that have been documented upon melatonin supplementation including cramps of stomach, vertigo, a continuous pain in the head, touchiness, moodiness, reduced sexual desire, enlargement of breast in the men and reduction in the count of sperm.

So, melatonin should not take during operating machine or drive. Further, melatonin could interrupt with human fertility and also melatonin should be not used for pregnant or nursing women. Utilization of melatonin by person who already have an augmentation level of melatonin as children, teenagers, pregnant and lactating women can result in melatonin overdose. MAOI drugs inhibit melatonin breakdown from the body, so people should not take melatonin with these drugs to prevent melatonin overdose.

Melatonin causes drug–drug interaction with antidepressants, such as Prozac (inhibitor of serotonin) or Nardil (inhibitor of monoamine oxidase). Melatonin Interaction with these kinds of drugs can produce heart attack, confusion, sweating, shaking, and fever, lack of coordination, elevated blood pressure, diarrhea, and convulsions [1].

### **13. Discussion and conclusion**

Melatonin is considered as a potent geroprotector, anticarcinogen, and inhibitor of tumor growth *in vivo* and *in vitro*, and in some models it may induce tumors and promote tumor growth. An important mechanism of melatonin is its impact on hemopoiesis involves the stimulation of melatonin on opioid receptors of bone marrow. Hence, we confirm further experimental studies and clinical trials which are necessary to estimate both the effectiveness and the safety for humans. Some antioxidants, including natural ones (e.g., *α*-tocopherol), have both geroprotector and tumorigenic potential and could be potent anticarcinogens as well. The results of administration of melatonin to perimenopausal women are promising. There are no contradictions between data on the carcinogenic and anticarcinogenic potential of melatonin but there are real data on the adverse effects of melatonin. Melatonin might own some ameliorating actions on human disorders that are contributed to the reproductive function. Such as lighting intensity during the night decreased the levels of circulating melatonin resulting in high risk of breast cancer [60]. Therefore, in the optimum condition, melatonin may have significant beneficial reproductive effects.

**References**

[1] Available from: http://www.Vitamins and Health

sensitivity assay. Life Sciences. 2000;**67**:2953-2960

European Journal of Cancer. 1999;**35**:1688-1692

[2] Bartsch H, Buchberger A, Franz H, Bartsch C, Maidonis I, Mecke D, Bayer E. Effect of melatonin and pineal extracts on human ovarian and mammary tumor cells in a chemo-

Melatonin and Its Indisputable Effects on the Health State

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99

[3] Lissoni P, Barni S, Mandala M, Ardizzoia A, Paolorossi F, Vaghi M, Longarini R, Malugani F, Tancini G. Decreased toxicity and increased efficacy of cancer chemotherapy using the pineal hormone melatonin in metastatic solid tumor patients with poor clinical status.

[4] Di Bella G, Mascia F, Gualano L, Di Bella L. Melatonin anticancer effects: Review. International Journal of Molecular Sciences. 2013;**14**:2410-2430. DOI: 10.3390/ijms14022410

[5] Lissoni P, Bolis S, Brivio F, Fumagalli L. A phase II study of neuroimmunotherapy with subcutaneous low-dose IL-2 plus the pineal hormone melatonin in untreatable advanced

[6] Eck-Enriquez K, Kiefer TL, Spriggs LL, Hill SM. Pathways through which a regimen of melatonin and retinoic acid induces apoptosis in MCF-7 human breast cancer cells.

[7] Hill SM, Blask DE, Xiang S, Yuan L, Mao L, Dauchy RT, Dauchy EM, Frasch T, Duplesis T. Melatonin and associated signaling pathways that control normal breast epithelium and breast cancer. Journal of Mammary Gland Biology and Neoplasia. 2011;**16**:235-245

[8] SoybIr G, Topuzlu C, Odaba SO, Dolay K, BIlIr A, Koksoy F. The effects of melatonin on

[9] García-Pergañeda A, Pozo D, Guerrero JM, Calvo JR. Signal transduction for melatonin in human lymphocytes: Involvement of a pertussis toxin-sensitive G protein. Journal of

[10] Dong C, Yuan L, Dai J, Lai L, Mao L, Xiang S, Rowan B, Hill SM. Melatonin inhibits mitogenic cross-talk between retinoic acid-related orphan receptor alpha (RORalpha)

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Epidemiological studies concerning the association between body circadian melatonin levels and cancer incidence led to controversial results, which were either significant association or no association at all. The effects of melatonin on cancers have been investigated, with a focus on hormone-dependent cancers. Different experimental works have suggested the ameliorative effect of melatonin in numerous types of metastatic tumors, including breast, ovarian, prostate, oral, gastric, and colorectal cancers. The mechanisms contributed with this improvement role of melatonin include various pathways of molecular origin, which are implicated with the activity of antioxidant enzymes, attenuation of MT1 and MT2 melatonin receptors, apoptosis regulation, metabolism of tumor, angiogenesis inhibition, invasion and metastasis, and initiation of epigenetic alteration. In different clinical trials, melatonin exhibited the capability to augment the treatment effect of chemotherapeutic drugs, and might help in enhancing the cancer patient's life quality. Collectively, melatonin is considered a promising hormone for cancers prevention and treatment. So, it could be concluded that extensive future work may be occur which involves the effect of melatonin on autophagy and mitophagy, other mechanisms of molecular origin implicated in its anticancer effect. Melatonin improves also chemotherapeutic drugs, which should be further determined on a large scale of drugs. The oncostatic effects of melatonin on some type of cancers, dosage and safety of long-term supplementation of melatonin must be also further elucidated.

### **Conflict of interest**

The authors declared no conflict of interest.

### **Author details**

Hanan Farouk Aly\* and Maha Zaki Rizk

\*Address all correspondence to: hanan\_abdallah@yahoo.com

Therapeutic Chemistry Department, National Research Centre, Giza-Dokki, Egypt

### **References**

stimulation of melatonin on opioid receptors of bone marrow. Hence, we confirm further experimental studies and clinical trials which are necessary to estimate both the effectiveness and the safety for humans. Some antioxidants, including natural ones (e.g., *α*-tocopherol), have both geroprotector and tumorigenic potential and could be potent anticarcinogens as well. The results of administration of melatonin to perimenopausal women are promising. There are no contradictions between data on the carcinogenic and anticarcinogenic potential of melatonin but there are real data on the adverse effects of melatonin. Melatonin might own some ameliorating actions on human disorders that are contributed to the reproductive function. Such as lighting intensity during the night decreased the levels of circulating melatonin resulting in high risk of breast cancer [60]. Therefore, in the optimum condition, melatonin

Epidemiological studies concerning the association between body circadian melatonin levels and cancer incidence led to controversial results, which were either significant association or no association at all. The effects of melatonin on cancers have been investigated, with a focus on hormone-dependent cancers. Different experimental works have suggested the ameliorative effect of melatonin in numerous types of metastatic tumors, including breast, ovarian, prostate, oral, gastric, and colorectal cancers. The mechanisms contributed with this improvement role of melatonin include various pathways of molecular origin, which are implicated with the activity of antioxidant enzymes, attenuation of MT1 and MT2 melatonin receptors, apoptosis regulation, metabolism of tumor, angiogenesis inhibition, invasion and metastasis, and initiation of epigenetic alteration. In different clinical trials, melatonin exhibited the capability to augment the treatment effect of chemotherapeutic drugs, and might help in enhancing the cancer patient's life quality. Collectively, melatonin is considered a promising hormone for cancers prevention and treatment. So, it could be concluded that extensive future work may be occur which involves the effect of melatonin on autophagy and mitophagy, other mechanisms of molecular origin implicated in its anticancer effect. Melatonin improves also chemotherapeutic drugs, which should be further determined on a large scale of drugs. The oncostatic effects of melatonin on some type of cancers, dosage and safety of long-term

may have significant beneficial reproductive effects.

98 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

supplementation of melatonin must be also further elucidated.

\*Address all correspondence to: hanan\_abdallah@yahoo.com

Therapeutic Chemistry Department, National Research Centre, Giza-Dokki, Egypt

**Conflict of interest**

**Author details**

The authors declared no conflict of interest.

Hanan Farouk Aly\* and Maha Zaki Rizk


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**Chapter 5**

**Provisional chapter**

**Melatonin and Exercise: Their Effects on**

**Melatonin and Exercise: Their Effects on** 

DOI: 10.5772/intechopen.79561

Melatonin as an omnipresent molecule is secreted by the pineal gland. It is a strong free radical scavenger, which reduces nitric oxide (NO) generation within mitochondria. Exercise has great impacts on many body's homeostatic systems. Most human's organisms display rhythms and have 24 hours environmental cycles, which are called circadian rhythm. Melatonin is one of the circadian rhythm generator in various physiological variables. Exercises could regulate plasma melatonin levels. Melatonin scavenges reactive oxygen spices (ROS) and reactive nitrogen spices (RNS) and acts as the antioxidant cascade. It not only decreases the exercise induced-oxidative stress in the muscles but also enhances muscle antioxidant enzymes, such as superoxide dismutase. Body lipids and unsaturated fatty acids are prone to oxidation, while the free radicals penetrate into bilayer membrane structure lipid peroxidation is going to happen. Malondialdehyde (MDA) is created by free radicals, and it is one of the most frequent marker of lipid peroxidation. Exercise, its duration, and time of the day have immediate and or delayed effects on melatonin secretion. The combination of aerobic exercise and melatonin reduces the exercise induced-free radicals agents. Melatonin supplementation, especially while it combined with aerobic training, could decrease the lipid peroxidation and malondialdehyde. Melatonin could impede exercise-induced ROS, increase body health, and exercise-related adaptation.

**Keywords:** exercise, health, lipid peroxidation, malondialdehyde, melatonin,

Melatonin is an omnipresent molecule that has various functional activities in plants and animals [1]. It is possibly associated with longevity in which their dysfunction is what

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Malondialdehyde and Lipid Peroxidation**

**Malondialdehyde and Lipid Peroxidation**

Mahsa Rastegar Moghaddam Mansouri, Sadegh Abbasian and Mohammad Khazaie

Mahsa Rastegar Moghaddam Mansouri, Sadegh Abbasian and Mohammad Khazaie

http://dx.doi.org/10.5772/intechopen.79561

**Abstract**

reactive oxygen spices

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **Melatonin and Exercise: Their Effects on Malondialdehyde and Lipid Peroxidation Melatonin and Exercise: Their Effects on Malondialdehyde and Lipid Peroxidation**

DOI: 10.5772/intechopen.79561

Mahsa Rastegar Moghaddam Mansouri, Sadegh Abbasian and Mohammad Khazaie Mahsa Rastegar Moghaddam Mansouri, Sadegh Abbasian and Mohammad Khazaie

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79561

#### **Abstract**

Melatonin as an omnipresent molecule is secreted by the pineal gland. It is a strong free radical scavenger, which reduces nitric oxide (NO) generation within mitochondria. Exercise has great impacts on many body's homeostatic systems. Most human's organisms display rhythms and have 24 hours environmental cycles, which are called circadian rhythm. Melatonin is one of the circadian rhythm generator in various physiological variables. Exercises could regulate plasma melatonin levels. Melatonin scavenges reactive oxygen spices (ROS) and reactive nitrogen spices (RNS) and acts as the antioxidant cascade. It not only decreases the exercise induced-oxidative stress in the muscles but also enhances muscle antioxidant enzymes, such as superoxide dismutase. Body lipids and unsaturated fatty acids are prone to oxidation, while the free radicals penetrate into bilayer membrane structure lipid peroxidation is going to happen. Malondialdehyde (MDA) is created by free radicals, and it is one of the most frequent marker of lipid peroxidation. Exercise, its duration, and time of the day have immediate and or delayed effects on melatonin secretion. The combination of aerobic exercise and melatonin reduces the exercise induced-free radicals agents. Melatonin supplementation, especially while it combined with aerobic training, could decrease the lipid peroxidation and malondialdehyde. Melatonin could impede exercise-induced ROS, increase body health, and exercise-related adaptation.

**Keywords:** exercise, health, lipid peroxidation, malondialdehyde, melatonin, reactive oxygen spices

#### **1. Introduction**

Melatonin is an omnipresent molecule that has various functional activities in plants and animals [1]. It is possibly associated with longevity in which their dysfunction is what

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

initiates the aging process [2, 3]. Melatonin was secreted by pinecone-like gland that is deeply placed in the brain which is called the "pineal gland." This gland has sympathetic innervation as its main sources due to its special location in the brain [2]. The pineal gland is a tranquilizing organ leading to a high melatonin production in response to darkness. Serotonin (made from tryptophan) through the cascade of enzymatic reactions produces melatonin with chemical name N-acetyl-5-methoxy tryptamine. This gland is located outside the blood–brain barrier (BBB) and has large uptake of tryptophan which produces high melatonin levels [2]. This hormone is mostly secreted at night and results in sleep regulation, signaling the time of the day that acts as chronological pacemaker, and participates in various physiological functions.

condition with three dimensions as physical, social, and psychological. To clarify the benefit of exercise and physical activity, we have to define the physical inactivity which refers to those physical activities less than what is required for optimal health and prevention of pre-

Melatonin and Exercise: Their Effects on Malondialdehyde and Lipid Peroxidation

http://dx.doi.org/10.5772/intechopen.79561

107

There are huge differences between physical activity and physical fitness which should not be interchangeably used. According to CDC physical fitness is the ability to carry out daily tasks without fatigue and of course with plenty of energy to enjoy leisure time and respond to emergencies. Physical fitness has a number of components consisting of cardiorespiratory endurance, skeletal muscle endurance, skeletal muscle strength, skeletal muscle power, flexibility, balance, speed of movement, reaction time, and body composition. Physical activity according to above definition refers to any bodily movement produced by skeletal muscle

Muscular strength is an essential factor for health and functional ability and consequently increases life quality. The importance of progressive resistance exercise had been clarified seen in World War II which was recommended for veteran rehabilitation. The important point of all resistance training is that the training must be "progressive" and progression is the act of advancing toward a specific goal which leads to continued improvement in muscle ability. Muscle development is under the effects of program variables such as exercise selection and

Physical activity and exercises are so important strategies to face chronic conditions, lower cancer risk, and synchronize the circadian system. Increasing physical activity and taking part in aerobic endurance activities, resistance training, and flexibility exercises have been shown to decrease the risk of several chronic diseases such as coronary heart disease, obesity, diabetes, low back pain, osteoporosis, and sarcopenia [12, 14–16]. Physical activities and exercises are nonphotic signals that entrain human circadian clock [16]. Physical activity could increase

Cardiorespiratory fitness also is the capacity of the cardiovascular (both heart and vessels) and respiratory (lungs) systems to supply oxygen to the blood and consequently to the working skeletal muscles and the capacity of the muscles to use oxygen to produce energy for movement. The best test to determine the cardiorespiratory fitness is the maximum aerobic fitness; moreover in the human studies, length of time running or cycling in standardized test

Strength fitness is also another health component that is defined as the capacity of the skeletal muscle to move an external load. Balance is defined as the ability to control the body during the body movement, and flexibility is also defined as range of joint motion or range of motion

To continue the importance of the combination of the exercise and melatonin on the body system, we have to deliberate some fundamental concepts of training. Overload as the first concept is the "gradual increase of stress placed upon the body during exercise training." Specificity of training is that training should be specific to the body needs or both movement patterns and force-velocity characteristics. Also, the good training program should have

the melatonin levels, decrease estrogen production, and improve fat metabolism [15].

could examine the physiological/biochemical/psychological exhaustion [12].

order, number of sets and repetitions, and rest period length [14].

mature death [12, 13].

contraction [12, 13].

(ROM) [12, 14].

### **2. The melatonin advantages for the body**

Melatonin is a strong free radical scavenger which reduces nitric oxide (NO) generation within mitochondria. NO strongly interferes with components of the respiratory chain in the mitochondria [4, 5]. Melatonin has two direct and indirect antioxidant capacities, which directly scavenges free radicals and indirectly regulates the activity of antioxidant enzymes. When melatonin interacts with the toxic reactants, several metabolites are generated which per se act as direct free radical scavengers such as N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) [6]. Melatonin impacts toxic radicals and breaks them down, eliminates reactive oxygen species-induced H2 O2 (one of the most important reactive oxygen species or ROS), acts on uncoupling proteins (UCPs), and decreases body heat production [7, 8]. Moreover, as melatonin decreases NO generation, melatonin leads to mitochondria function development, and due to increase in the mitochondria respiration, the ATP production and electron transportation increase as well [5, 9, 10].

To have a glance at the other importance of melatonin, we pointed to neurodegenerative diseases. Melatonin production in aged individuals declines and is known as the primary contributing factor for aged-related neurodegenerative diseases. Also, hypoxia, hypoglycemia, viruses, drug neurotoxicity, radiation, or noxious substances all could produce neural damage. Through antioxidant effects of melatonin, it has been proposed as a neuroprotective agent. Over the melatonin therapeutic value, it is used as treatment of Alzheimer disease (AD), Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), stroke, and brain trauma. Neurodegenerative disorders mostly happen due to free radical-mediated damage and mitochondria dysfunction as common pathophysiological mechanism [11].

#### **3. What is exercise, and how does it affect the melatonin?**

According to the Center for Disease Control and Prevention (CDC), exercise is a subcategory of physical activity that is planned, structured, repetitive, and purposive that improves or maintains one or more components of physical fitness. Physical activity also refers to any bodily movement which is produced by the skeletal muscle contraction that increases energy expenditure more than basal level and enhances health. Furthermore, health is a human condition with three dimensions as physical, social, and psychological. To clarify the benefit of exercise and physical activity, we have to define the physical inactivity which refers to those physical activities less than what is required for optimal health and prevention of premature death [12, 13].

initiates the aging process [2, 3]. Melatonin was secreted by pinecone-like gland that is deeply placed in the brain which is called the "pineal gland." This gland has sympathetic innervation as its main sources due to its special location in the brain [2]. The pineal gland is a tranquilizing organ leading to a high melatonin production in response to darkness. Serotonin (made from tryptophan) through the cascade of enzymatic reactions produces melatonin with chemical name N-acetyl-5-methoxy tryptamine. This gland is located outside the blood–brain barrier (BBB) and has large uptake of tryptophan which produces high melatonin levels [2]. This hormone is mostly secreted at night and results in sleep regulation, signaling the time of the day that acts as chronological pacemaker, and participates in vari-

Melatonin is a strong free radical scavenger which reduces nitric oxide (NO) generation within mitochondria. NO strongly interferes with components of the respiratory chain in the mitochondria [4, 5]. Melatonin has two direct and indirect antioxidant capacities, which directly scavenges free radicals and indirectly regulates the activity of antioxidant enzymes. When melatonin interacts with the toxic reactants, several metabolites are generated which per se act as direct free radical scavengers such as N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) [6]. Melatonin impacts toxic radicals and breaks them down, eliminates reactive oxygen species-induced H2

(one of the most important reactive oxygen species or ROS), acts on uncoupling proteins (UCPs), and decreases body heat production [7, 8]. Moreover, as melatonin decreases NO generation, melatonin leads to mitochondria function development, and due to increase in the mitochondria

To have a glance at the other importance of melatonin, we pointed to neurodegenerative diseases. Melatonin production in aged individuals declines and is known as the primary contributing factor for aged-related neurodegenerative diseases. Also, hypoxia, hypoglycemia, viruses, drug neurotoxicity, radiation, or noxious substances all could produce neural damage. Through antioxidant effects of melatonin, it has been proposed as a neuroprotective agent. Over the melatonin therapeutic value, it is used as treatment of Alzheimer disease (AD), Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), stroke, and brain trauma. Neurodegenerative disorders mostly happen due to free radical-mediated damage and mito-

According to the Center for Disease Control and Prevention (CDC), exercise is a subcategory of physical activity that is planned, structured, repetitive, and purposive that improves or maintains one or more components of physical fitness. Physical activity also refers to any bodily movement which is produced by the skeletal muscle contraction that increases energy expenditure more than basal level and enhances health. Furthermore, health is a human

respiration, the ATP production and electron transportation increase as well [5, 9, 10].

chondria dysfunction as common pathophysiological mechanism [11].

**3. What is exercise, and how does it affect the melatonin?**

O2

ous physiological functions.

**2. The melatonin advantages for the body**

106 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

There are huge differences between physical activity and physical fitness which should not be interchangeably used. According to CDC physical fitness is the ability to carry out daily tasks without fatigue and of course with plenty of energy to enjoy leisure time and respond to emergencies. Physical fitness has a number of components consisting of cardiorespiratory endurance, skeletal muscle endurance, skeletal muscle strength, skeletal muscle power, flexibility, balance, speed of movement, reaction time, and body composition. Physical activity according to above definition refers to any bodily movement produced by skeletal muscle contraction [12, 13].

Muscular strength is an essential factor for health and functional ability and consequently increases life quality. The importance of progressive resistance exercise had been clarified seen in World War II which was recommended for veteran rehabilitation. The important point of all resistance training is that the training must be "progressive" and progression is the act of advancing toward a specific goal which leads to continued improvement in muscle ability. Muscle development is under the effects of program variables such as exercise selection and order, number of sets and repetitions, and rest period length [14].

Physical activity and exercises are so important strategies to face chronic conditions, lower cancer risk, and synchronize the circadian system. Increasing physical activity and taking part in aerobic endurance activities, resistance training, and flexibility exercises have been shown to decrease the risk of several chronic diseases such as coronary heart disease, obesity, diabetes, low back pain, osteoporosis, and sarcopenia [12, 14–16]. Physical activities and exercises are nonphotic signals that entrain human circadian clock [16]. Physical activity could increase the melatonin levels, decrease estrogen production, and improve fat metabolism [15].

Cardiorespiratory fitness also is the capacity of the cardiovascular (both heart and vessels) and respiratory (lungs) systems to supply oxygen to the blood and consequently to the working skeletal muscles and the capacity of the muscles to use oxygen to produce energy for movement. The best test to determine the cardiorespiratory fitness is the maximum aerobic fitness; moreover in the human studies, length of time running or cycling in standardized test could examine the physiological/biochemical/psychological exhaustion [12].

Strength fitness is also another health component that is defined as the capacity of the skeletal muscle to move an external load. Balance is defined as the ability to control the body during the body movement, and flexibility is also defined as range of joint motion or range of motion (ROM) [12, 14].

To continue the importance of the combination of the exercise and melatonin on the body system, we have to deliberate some fundamental concepts of training. Overload as the first concept is the "gradual increase of stress placed upon the body during exercise training." Specificity of training is that training should be specific to the body needs or both movement patterns and force-velocity characteristics. Also, the good training program should have variation to support the training needs to remain optimal. In this case training periodization is defined as utilizes variation in training program design [14]. Exercise and physical activity can also act as preventer of chronic disease and regulate body systems. Regular activity and structured exercises are related to vast health benefits and body hormone regulation [12, 17, 18].

responses, hormonal and biochemical disturbances, and metabolic and defensive changes [6]. Acute exercise increases oxygen consumption much more than rest time which makes muscles prone to oxidative stress. Oxygen molecule is a radical species per se and also results in generation of various free radicals, and those free radical species that are forming due to oxygen and nitrogen are the most important in the living organisms [23]. While subjects have routine life pattern, their energy demands and blood vessel contribution are balanced. During the acute or resistance exercise training, oxygen consumption of both striated and smooth muscles increases dramatically which leads to increase in reactive oxygen/nitrogen species that is called RONS. While exercise-derived RONS are generated, the body antioxidant defense system starts to work and protects cells and tissues against free radicals. The imbalance between body antioxidant defense system and RONS is called oxidative stress [24]. Low levels of ROS regulate muscle force through calcium release mechanism, and influencing myofilament structure creates adaptive response to training. However, high levels of ROS

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Melatonin due to its scavenging ability could directly interact with a variety of oxygen- and nitrogen-based radicals. It is an antioxidant that regulates the activities of other antioxidant enzymes [6, 25]. Strenuous sport activities lead to acute muscle injuries that are indicated by muscle soreness, prolong loss of muscle function, and leakage of muscle proteins. Acute exercise-induced muscle injuries consist of 30–67% of athletic injuries, while exercise-induced severe muscle injuries could impede the athletic progression. Muscle damages result in multidimensional changes in muscle tissue such as inflammation that per se encourage the free radical production and muscle atrophy [26, 27]. This condition could be reduced with optimal nutrition mostly by increasing dietary content of nutritional antioxidant. Melatonin as one of the body-secreted natural antioxidants could cross all barriers and reduce the oxidative damage in almost every environment in the body. Intensive exercises cause abundant changes in immunity and also change the carbohydrate and lipid metabolism that make athletes vulnerable to infection. In this case melatonin protects heart muscle cells and other body parts from exercise-induced inflammation [6, 27]. As previously mentioned, strenuous exercise muscle injuries lead to protein degradation that encourages muscle injury. Whenever exerciseinduced protein degradation and consequent muscle atrophy are limited, the extent of muscle injuries could be blunted. Melatonin is one of the most effective factors that could limit muscle injuries. It was shown that melatonin inhibited the nuclear factor kappa-B (NFKB) activation that prevents the cytokine-induced atrophy and thus muscle injury. Melatonin also decreases proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) expression within the muscle. Also, muscle atrophy F-box (MAFBX) and muscle RING finger 1 (MURF-1) are inhibited by melatonin. MAFBX is a muscle-specific ubiquitin ligase that mediates the degradation of muscle-specific transcription factor MyoD. MURF-1 also is a member of ubiquitin ligase family which interacts with the giant protein titin in the muscle and is called titin-associated protein that expert antihypertrophic activity [28, 29]. In this case melatonin through the elevation of the expression of the muscle Akt could reduce the ratio of the MAFBX/MURF-1 and inhibits the breakdown of the structural muscle protein such as myosin heavy chain (**Figure 1**). The functional role of the melatonin and strenuous muscle injury prevention is not fully understood, but the melatonin effects on the muscle cytokines, NFKB activation, muscle Akt elevation, and consequent decline in the MAFBX/MURF-1 could open

reduce force production and result in muscular fatigue [25].

new points of view to the melatonin protection mechanism [26].

Exercise has great impacts on many body's homeostatic systems. Most human organisms display rhythms in their physiology and have 24 hour environmental cycle which is called circadian rhythm. Melatonin is one of the circadian rhythm generators in various physiological variables. Also, there are some voluntary rhythm modifiers such as activity or physical exercise; meal time can also act as circadian rhythm modifier signal [16]. Physical activity and exercise are nonphotic signals that regulate the human circadian clock and synchronize circadian system. Melatonin is one of the main signals of body clock that is commonly measured to report the effects of exercise on the circadian clock. Noteworthy almost different kinds of exercises and physical activities both acute and chronic could modify plasma melatonin levels. Meanwhile, endogenous profile of melatonin has different responses such as increase, decrease [19], or even unaffected to exercises that time of the day, and lighting condition is the most effective item on the melatonin secretion cycle even in response to exercises. As an overall consensus, those exercises which have been done at night or in the dark whether of moderate or high intensity result in delay in melatonin secretion. Indeed, the age does not influence the exercise-induced circadian rhythm of melatonin as nocturnal hormone. Besides the effects of exercises on the different phases of melatonin secretion, the exercises also transiently affect the melatonin levels. The mechanism of this transient changes in melatonin levels could be due to the circadian phase at that time exercise was undertaken. A bout of exercise increases the plasma melatonin levels, while regular training and exercises attenuate the melatonin [16]. There are several potent physiological factors that describe the melatonin changes. Melatonin acts as an antioxidant, and exercise especially strenuous exercise increases oxidative stress, hence melatonin which is secreted by the human body or even ingested capable of protecting against potential molecular damage [6]. It was reported that proximately after exercise melatonin levels increase and following 1 hour after physical activity, it returns to pre-exercise level [20]. Melatonin levels of trained individuals is higher in the morning compared to the evening, but in the following 3 weeks of hard training, the evening melatonin levels were higher than morning levels. Interestingly both morning and evening levels of melatonin decreased compared to pre-3 weeks of hard training. Well-trained individuals show an adaptive response to each training that they take part as their oxidative stress regulates and diurnal melatonin levels temporarily increase [16, 21]. Total mechanisms of the melatonin alteration following exercises remained unknown, but exercise-induced absolute rise in melatonin levels is more pronounced in the morning compared to the evening [22].

### **4. Exercises on the melatonin**

#### **4.1. Acute and strenuous exercises**

Acute sport trainings and resistance exercises lead to change in the energy demands and are strong stimulation of muscle tissue. Intense exercises produce free radicals, inflammatory responses, hormonal and biochemical disturbances, and metabolic and defensive changes [6]. Acute exercise increases oxygen consumption much more than rest time which makes muscles prone to oxidative stress. Oxygen molecule is a radical species per se and also results in generation of various free radicals, and those free radical species that are forming due to oxygen and nitrogen are the most important in the living organisms [23]. While subjects have routine life pattern, their energy demands and blood vessel contribution are balanced. During the acute or resistance exercise training, oxygen consumption of both striated and smooth muscles increases dramatically which leads to increase in reactive oxygen/nitrogen species that is called RONS. While exercise-derived RONS are generated, the body antioxidant defense system starts to work and protects cells and tissues against free radicals. The imbalance between body antioxidant defense system and RONS is called oxidative stress [24]. Low levels of ROS regulate muscle force through calcium release mechanism, and influencing myofilament structure creates adaptive response to training. However, high levels of ROS reduce force production and result in muscular fatigue [25].

variation to support the training needs to remain optimal. In this case training periodization is defined as utilizes variation in training program design [14]. Exercise and physical activity can also act as preventer of chronic disease and regulate body systems. Regular activity and structured exercises are related to vast health benefits and body hormone regulation [12, 17, 18].

108 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

Exercise has great impacts on many body's homeostatic systems. Most human organisms display rhythms in their physiology and have 24 hour environmental cycle which is called circadian rhythm. Melatonin is one of the circadian rhythm generators in various physiological variables. Also, there are some voluntary rhythm modifiers such as activity or physical exercise; meal time can also act as circadian rhythm modifier signal [16]. Physical activity and exercise are nonphotic signals that regulate the human circadian clock and synchronize circadian system. Melatonin is one of the main signals of body clock that is commonly measured to report the effects of exercise on the circadian clock. Noteworthy almost different kinds of exercises and physical activities both acute and chronic could modify plasma melatonin levels. Meanwhile, endogenous profile of melatonin has different responses such as increase, decrease [19], or even unaffected to exercises that time of the day, and lighting condition is the most effective item on the melatonin secretion cycle even in response to exercises. As an overall consensus, those exercises which have been done at night or in the dark whether of moderate or high intensity result in delay in melatonin secretion. Indeed, the age does not influence the exercise-induced circadian rhythm of melatonin as nocturnal hormone. Besides the effects of exercises on the different phases of melatonin secretion, the exercises also transiently affect the melatonin levels. The mechanism of this transient changes in melatonin levels could be due to the circadian phase at that time exercise was undertaken. A bout of exercise increases the plasma melatonin levels, while regular training and exercises attenuate the melatonin [16]. There are several potent physiological factors that describe the melatonin changes. Melatonin acts as an antioxidant, and exercise especially strenuous exercise increases oxidative stress, hence melatonin which is secreted by the human body or even ingested capable of protecting against potential molecular damage [6]. It was reported that proximately after exercise melatonin levels increase and following 1 hour after physical activity, it returns to pre-exercise level [20]. Melatonin levels of trained individuals is higher in the morning compared to the evening, but in the following 3 weeks of hard training, the evening melatonin levels were higher than morning levels. Interestingly both morning and evening levels of melatonin decreased compared to pre-3 weeks of hard training. Well-trained individuals show an adaptive response to each training that they take part as their oxidative stress regulates and diurnal melatonin levels temporarily increase [16, 21]. Total mechanisms of the melatonin alteration following exercises remained unknown, but exercise-induced absolute rise in melatonin levels is more pronounced in the morning com-

Acute sport trainings and resistance exercises lead to change in the energy demands and are strong stimulation of muscle tissue. Intense exercises produce free radicals, inflammatory

pared to the evening [22].

**4. Exercises on the melatonin**

**4.1. Acute and strenuous exercises**

Melatonin due to its scavenging ability could directly interact with a variety of oxygen- and nitrogen-based radicals. It is an antioxidant that regulates the activities of other antioxidant enzymes [6, 25]. Strenuous sport activities lead to acute muscle injuries that are indicated by muscle soreness, prolong loss of muscle function, and leakage of muscle proteins. Acute exercise-induced muscle injuries consist of 30–67% of athletic injuries, while exercise-induced severe muscle injuries could impede the athletic progression. Muscle damages result in multidimensional changes in muscle tissue such as inflammation that per se encourage the free radical production and muscle atrophy [26, 27]. This condition could be reduced with optimal nutrition mostly by increasing dietary content of nutritional antioxidant. Melatonin as one of the body-secreted natural antioxidants could cross all barriers and reduce the oxidative damage in almost every environment in the body. Intensive exercises cause abundant changes in immunity and also change the carbohydrate and lipid metabolism that make athletes vulnerable to infection. In this case melatonin protects heart muscle cells and other body parts from exercise-induced inflammation [6, 27]. As previously mentioned, strenuous exercise muscle injuries lead to protein degradation that encourages muscle injury. Whenever exerciseinduced protein degradation and consequent muscle atrophy are limited, the extent of muscle injuries could be blunted. Melatonin is one of the most effective factors that could limit muscle injuries. It was shown that melatonin inhibited the nuclear factor kappa-B (NFKB) activation that prevents the cytokine-induced atrophy and thus muscle injury. Melatonin also decreases proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) expression within the muscle. Also, muscle atrophy F-box (MAFBX) and muscle RING finger 1 (MURF-1) are inhibited by melatonin. MAFBX is a muscle-specific ubiquitin ligase that mediates the degradation of muscle-specific transcription factor MyoD. MURF-1 also is a member of ubiquitin ligase family which interacts with the giant protein titin in the muscle and is called titin-associated protein that expert antihypertrophic activity [28, 29]. In this case melatonin through the elevation of the expression of the muscle Akt could reduce the ratio of the MAFBX/MURF-1 and inhibits the breakdown of the structural muscle protein such as myosin heavy chain (**Figure 1**). The functional role of the melatonin and strenuous muscle injury prevention is not fully understood, but the melatonin effects on the muscle cytokines, NFKB activation, muscle Akt elevation, and consequent decline in the MAFBX/MURF-1 could open new points of view to the melatonin protection mechanism [26].

Melatonin and all its metabolites could scavenge ROS and reactive nitrogen species (RNS) and act as the antioxidant cascade. Melatonin not only decreases the exercise-induced oxidative stress in the muscles but also enhances muscle antioxidant enzymes such as superoxide dismutase. Moreover, melatonin reduces muscle inflammatory factors such as IL-6 and TNF-alpha [25–27]. Melatonin could successfully manage the strenuous exerciseinduced muscle damage through several ways. It increases the strength of injured muscle, reduces severity of the injury, increases number of satellite cells, inhibits NF-κB activation/ translocation, causes TNF-alpha and IL-6 to decline, and increases muscle Akt and thus decreases MAFBX/MuRF-1 ratio. Due to its broad-spectrum antioxidant, it could protect DNA, proteins, and biological membrane lipids from the effects of ROS and other oxidative

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Physical exercise is performing some activities to keep healthy weight, building and conserving healthy bone, muscle, and joints to develop physiological health. Exercise also promotes the immune system. Immune system responses to exercise are directly dependent on the intensity, duration, and body adaptive responses. It has been suggested that moderate exercise through endocrine hormone elevation could reverse immunosenescence. Moderate exercise training modulates exercise-induced ROS and DNA damage and regulates cytokine

Aerobic training and regular physical activity are highly associated with vast beneficial health issues including limiting cardiovascular disease (CVD), diabetes type 2, and age-related mortality. Aerobic training also induces vast acute and chronic adaptation in various physiological systems. Furthermore, physical inactivity is one of the four main causes of premature mortality. Although the aerobic physical activity increases in the current society, the level of the physical inactivity is still high [17, 31]. It was shown that physical activity could elevate melatonin levels, decrease estrogen production, improve fat metabolism, and reduce cancer risk as well. It has been reported that short- or even long-term physical activity has no substantial effects on the melatonin levels [15]. Moderate exercise, in another study, could modulate ROS, cytokines, and hormone levels that all affect apoptosis. Melatonin as one of this regulating hormones has diverse physiological aspects that it can counteract the immune depression following acute stress or aging and also upregulate TNF [30]. Exercise has immediate and/or delayed effects on melatonin secretion, in which duration, type of exercise, time of the day, fitness status, and age have also been identified as intervening factors in exercise-

Melatonin also plays an important role in the exercise-induced metabolic adaptation. Pinealectomized animals do not show adaptive metabolic changes due to aerobic training. Melatonin acts as a mediator between environment situation and physiological regulatory manner. Besides melatonin effects on the blood pressure and endocrine regulation, it also acts on the GLUT 4 (glucose transporter) gene expression (31). In this case aerobic training is coworker of melatonin in which both of them stimulate glucose uptake through

stress [6, 24, 26].

levels [30].

**4.2. Aerobic training**

induced changes in the melatonin levels (**Figure 2**).

**Figure 1.** Advantages of melatonin on the muscle injury that contribute to the strenuous exercise. Melatonin inhibits the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB) and translocates it that reduces the expression of tumor necrosis factor-alpha or TNF-alpha and interleukin-6 or IL-6 in exercise-induced injured muscle. Consequently melatonin could limit inflammation and increase the activation of the protein kinase B also known as Akt to control protein deprivation and downregulate the atrophy via NFKB/MAFBX/MURF-1/Akt pathway during injury. NF-κB, nuclear factor kappa-B; TNF-alpha, tumor necrosis factor-alpha; IL-6, interleukin-6; Akt, serine/threonine kinase; MAFBX, muscle atrophy F-box; and MuRF-1, muscle RING finger 1 [26].

Melatonin and all its metabolites could scavenge ROS and reactive nitrogen species (RNS) and act as the antioxidant cascade. Melatonin not only decreases the exercise-induced oxidative stress in the muscles but also enhances muscle antioxidant enzymes such as superoxide dismutase. Moreover, melatonin reduces muscle inflammatory factors such as IL-6 and TNF-alpha [25–27]. Melatonin could successfully manage the strenuous exerciseinduced muscle damage through several ways. It increases the strength of injured muscle, reduces severity of the injury, increases number of satellite cells, inhibits NF-κB activation/ translocation, causes TNF-alpha and IL-6 to decline, and increases muscle Akt and thus decreases MAFBX/MuRF-1 ratio. Due to its broad-spectrum antioxidant, it could protect DNA, proteins, and biological membrane lipids from the effects of ROS and other oxidative stress [6, 24, 26].

#### **4.2. Aerobic training**

**Figure 1.** Advantages of melatonin on the muscle injury that contribute to the strenuous exercise. Melatonin inhibits the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB) and translocates it that reduces the expression of tumor necrosis factor-alpha or TNF-alpha and interleukin-6 or IL-6 in exercise-induced injured muscle. Consequently melatonin could limit inflammation and increase the activation of the protein kinase B also known as Akt to control protein deprivation and downregulate the atrophy via NFKB/MAFBX/MURF-1/Akt pathway during injury. NF-κB, nuclear factor kappa-B; TNF-alpha, tumor necrosis factor-alpha; IL-6, interleukin-6; Akt, serine/threonine kinase;

MAFBX, muscle atrophy F-box; and MuRF-1, muscle RING finger 1 [26].

110 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

Physical exercise is performing some activities to keep healthy weight, building and conserving healthy bone, muscle, and joints to develop physiological health. Exercise also promotes the immune system. Immune system responses to exercise are directly dependent on the intensity, duration, and body adaptive responses. It has been suggested that moderate exercise through endocrine hormone elevation could reverse immunosenescence. Moderate exercise training modulates exercise-induced ROS and DNA damage and regulates cytokine levels [30].

Aerobic training and regular physical activity are highly associated with vast beneficial health issues including limiting cardiovascular disease (CVD), diabetes type 2, and age-related mortality. Aerobic training also induces vast acute and chronic adaptation in various physiological systems. Furthermore, physical inactivity is one of the four main causes of premature mortality. Although the aerobic physical activity increases in the current society, the level of the physical inactivity is still high [17, 31]. It was shown that physical activity could elevate melatonin levels, decrease estrogen production, improve fat metabolism, and reduce cancer risk as well. It has been reported that short- or even long-term physical activity has no substantial effects on the melatonin levels [15]. Moderate exercise, in another study, could modulate ROS, cytokines, and hormone levels that all affect apoptosis. Melatonin as one of this regulating hormones has diverse physiological aspects that it can counteract the immune depression following acute stress or aging and also upregulate TNF [30]. Exercise has immediate and/or delayed effects on melatonin secretion, in which duration, type of exercise, time of the day, fitness status, and age have also been identified as intervening factors in exerciseinduced changes in the melatonin levels (**Figure 2**).

Melatonin also plays an important role in the exercise-induced metabolic adaptation. Pinealectomized animals do not show adaptive metabolic changes due to aerobic training. Melatonin acts as a mediator between environment situation and physiological regulatory manner. Besides melatonin effects on the blood pressure and endocrine regulation, it also acts on the GLUT 4 (glucose transporter) gene expression (31). In this case aerobic training is coworker of melatonin in which both of them stimulate glucose uptake through

**Figure 2.** The effects of exercise time duration on the melatonin levels [30].

insulin-independent process and increase GLUT 4 protein expression. It was reported that those pinealectomized rats that undergo aerobic training did not show any metabolic development. So melatonin plays key role in metabolic adaptation in both adipose and muscle tissues. As it was mentioned, melatonin has circadian rhythm and regulates body clock; it also regulates energy metabolism circadian timing in which period of activity and adaptation to activity affect this timing [16, 31]. In one study the effects of melatonin supplementation on the aerobic exercise-induced adaptation were examined. For this purpose male wistar rats are divided into four groups: sedentary control, trained control, sedentary treated with melatonin, and trained treated with melatonin. Glucose tolerance, physical capacity, citrate synthesis, phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinas (MAPK), and GLUT4 were examined. Following the 8-week aerobic exercise training on treadmill, those trained animals that treated with melatonin showed better results in their measured parameters that are mentioned above. Briefly, melatonin supplementation plus aerobic training creates great metabolic adaptation and improves metabolism efficiency [31]. The combination of aerobic exercise and melatonin, also, reduces the exercise-induced free radical agents. Low to moderate levels of free radicals have regulatory roles, but their high levels create cellular damages and induce oxidative stress [24]. Melatonin elevation or melatonin supplementation especially while it is combined with aerobic training could decrease the lipid peroxidation and malondialdehyde—lipid peroxidation most frequent marker—in sedentary individuals [18]. Long-term aerobic training could manage the lipid profile of sedentary individuals. Meanwhile when combined with melatonin, the protective effects of aerobic training against free radicals advance, and the body antioxidant defense system improves [18].

Two central and direct ways guide the melatonin effects on the brown adipose tissue to increase exercise-energy expenditure. The nervous system is the central way that controls the melatonin through the sympathetic system. Sympathetic nerve-secreted norepinephrine controls the daily variation in melatonin synthesis [16]. Also, exercise leads to high increase in the activity of the sympathetic nervous system and catecholamine secretion which could modulate melatonin secretion [16]. Melatonin acts as antioxidant [6]. Its secretion is affected by daytime and especially the nervous system. Exercise training stimulates sympathetic nervous system and releases noradrenaline. Noradrenaline increases the tryptophan levels [30]. Tryptophan is uptaken by the pineal gland and decarboxylated to form serotonin (or 5-hydroxytryptamine). Serotonin during the daylight is stored in the pineal gland. Darkness causes noradrenaline to activate the enzymes (serotonin-N-acetyltransferase) and finally con-

**Figure 4.** Direct positive correlation between melatonin levels and exercise duration. As the training days progress, the

**Figure 3.** Tryptophan to melatonin cascade process. Tryptophan is the precursor of serotonin and melatonin [32].

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Also, in the direct way, protein kinase C (PKC) pathway leads to increase growth factor and mitochondria biogenesis [33]. Melatonin also could increase LDL receptor and inhibit choles-

Regarding long-term endurance training, the melatonin hormone reaches steady state. Furthermore, there is direct positive correlation between melatonin levels and exercise

vert serotonin into melatonin (**Figure 3**) [32].

melatonin levels increase [30].

terol synthesis which is even useful for control obesity [33].

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**Figure 3.** Tryptophan to melatonin cascade process. Tryptophan is the precursor of serotonin and melatonin [32].

insulin-independent process and increase GLUT 4 protein expression. It was reported that those pinealectomized rats that undergo aerobic training did not show any metabolic development. So melatonin plays key role in metabolic adaptation in both adipose and muscle tissues. As it was mentioned, melatonin has circadian rhythm and regulates body clock; it also regulates energy metabolism circadian timing in which period of activity and adaptation to activity affect this timing [16, 31]. In one study the effects of melatonin supplementation on the aerobic exercise-induced adaptation were examined. For this purpose male wistar rats are divided into four groups: sedentary control, trained control, sedentary treated with melatonin, and trained treated with melatonin. Glucose tolerance, physical capacity, citrate synthesis, phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinas (MAPK), and GLUT4 were examined. Following the 8-week aerobic exercise training on treadmill, those trained animals that treated with melatonin showed better results in their measured parameters that are mentioned above. Briefly, melatonin supplementation plus aerobic training creates great metabolic adaptation and improves metabolism efficiency [31]. The combination of aerobic exercise and melatonin, also, reduces the exercise-induced free radical agents. Low to moderate levels of free radicals have regulatory roles, but their high levels create cellular damages and induce oxidative stress [24]. Melatonin elevation or melatonin supplementation especially while it is combined with aerobic training could decrease the lipid peroxidation and malondialdehyde—lipid peroxidation most frequent marker—in sedentary individuals [18]. Long-term aerobic training could manage the lipid profile of sedentary individuals. Meanwhile when combined with melatonin, the protective effects of aerobic training against free radicals advance, and the body antioxidant defense system

**Figure 2.** The effects of exercise time duration on the melatonin levels [30].

112 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

Two central and direct ways guide the melatonin effects on the brown adipose tissue to increase exercise-energy expenditure. The nervous system is the central way that controls the melatonin through the sympathetic system. Sympathetic nerve-secreted norepinephrine controls the daily variation in melatonin synthesis [16]. Also, exercise leads to high increase in the activity of the sympathetic nervous system and catecholamine secretion which could modulate melatonin secretion [16]. Melatonin acts as antioxidant [6]. Its secretion is affected by daytime and especially the nervous system. Exercise training stimulates sympathetic

improves [18].

**Figure 4.** Direct positive correlation between melatonin levels and exercise duration. As the training days progress, the melatonin levels increase [30].

nervous system and releases noradrenaline. Noradrenaline increases the tryptophan levels [30]. Tryptophan is uptaken by the pineal gland and decarboxylated to form serotonin (or 5-hydroxytryptamine). Serotonin during the daylight is stored in the pineal gland. Darkness causes noradrenaline to activate the enzymes (serotonin-N-acetyltransferase) and finally convert serotonin into melatonin (**Figure 3**) [32].

Also, in the direct way, protein kinase C (PKC) pathway leads to increase growth factor and mitochondria biogenesis [33]. Melatonin also could increase LDL receptor and inhibit cholesterol synthesis which is even useful for control obesity [33].

Regarding long-term endurance training, the melatonin hormone reaches steady state. Furthermore, there is direct positive correlation between melatonin levels and exercise duration (**Figure 4**) [30]. Following exercise melatonin gradually increases, and due to endurance training which should last for about 3 months, the melatonin reaches to steady state [30]. It was reported that the low-intensity aerobic training has better adaptation and lipid peroxidation prevention in sedentary individuals. Melatonin supplementation for about 2 months improves dyslipidemia, decreases LDL, and improves lipid metabolism [25].

radicals attack fatty acid molecule which detach hydrogen ion and create fatty acid radical. Due to reordering of double bond, two double bonds between carbon atoms contain and create conjugated diene. The diene structure reacts with oxygen molecule and creates lipoper-

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**Figure 7.** Malondialdehyde formation and metabolism process. Decomposition of arachidonic acid (AA) and

5,8,10-hepadecatrienoic acid (HHT) (blue pathway) or nonenzymatic process by lipid peroxidation-induced bicyclic endoperoxides (red pathway) generates malondialdehyde. Malondialdehyde could enzymatically be metabolized (green pathway); those key enzymes in both malondialdehyde formation and metabolism are cyclooxygenase [1], prostacyclin hydroperoxidase [2], thromboxane synthase [3], aldehyde dehydrogenase [4], decarboxylase [5], acetyl CoA synthase

(TXA2

) and 12-l-hydroxy-

PUFAs as side products of enzymatic process during the biosynthesis of thromboxane A2

[6], and tricarboxylic acid cycle [7] [38].

oxyl radical (**Figures 5** and **6**).

#### **5. What is lipid peroxidation, and what is malondialdehyde?**

Free radicals are those species that are created as a result of cellular oxygen consumption and are mediator of lipid peroxidation. Different elements and situations affect the lipid peroxidation such as heat, oxygen, and enzymes. Free radicals have one or even more than one free electron(s). All of these ingredients could damage the molecules' organisms and create oxidative stress. Body natural defense system impedes the oxidative stress; whenever an imbalance occurs between the free radical production and antioxidant defense system effectiveness, the oxidative stress happens [18, 34]. Lipids are one of the important agents either in food or body's biological system. Body lipids are prone to oxidation, and it would happen during several stages as food storage process or even in physiological/pathological conditions. Unsaturated fatty acids are prone to oxidation; while the free radicals penetrate into bilayer membrane structure, lipid peroxidation is going to happen [18, 35]. Oxygen and free radicals damage the unsaturated fatty acids under lipoperoxide formation. This compound is unstable and could break down into wide range of reactive species which bind to free amino groups and decrease the proteolytic degradation [34]. Free radical-induced lipid peroxidation happens in three stages: initiation, propagation, and termination [35]. In the first stage, free

**Figure 5.** The first stage (initiation stage) of lipid peroxidation process [36].

**Figure 6.** Structural formula of conjugated diene during lipid peroxidation [34].

radicals attack fatty acid molecule which detach hydrogen ion and create fatty acid radical. Due to reordering of double bond, two double bonds between carbon atoms contain and create conjugated diene. The diene structure reacts with oxygen molecule and creates lipoperoxyl radical (**Figures 5** and **6**).

duration (**Figure 4**) [30]. Following exercise melatonin gradually increases, and due to endurance training which should last for about 3 months, the melatonin reaches to steady state [30]. It was reported that the low-intensity aerobic training has better adaptation and lipid peroxidation prevention in sedentary individuals. Melatonin supplementation for about 2 months

Free radicals are those species that are created as a result of cellular oxygen consumption and are mediator of lipid peroxidation. Different elements and situations affect the lipid peroxidation such as heat, oxygen, and enzymes. Free radicals have one or even more than one free electron(s). All of these ingredients could damage the molecules' organisms and create oxidative stress. Body natural defense system impedes the oxidative stress; whenever an imbalance occurs between the free radical production and antioxidant defense system effectiveness, the oxidative stress happens [18, 34]. Lipids are one of the important agents either in food or body's biological system. Body lipids are prone to oxidation, and it would happen during several stages as food storage process or even in physiological/pathological conditions. Unsaturated fatty acids are prone to oxidation; while the free radicals penetrate into bilayer membrane structure, lipid peroxidation is going to happen [18, 35]. Oxygen and free radicals damage the unsaturated fatty acids under lipoperoxide formation. This compound is unstable and could break down into wide range of reactive species which bind to free amino groups and decrease the proteolytic degradation [34]. Free radical-induced lipid peroxidation happens in three stages: initiation, propagation, and termination [35]. In the first stage, free

improves dyslipidemia, decreases LDL, and improves lipid metabolism [25].

114 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

**5. What is lipid peroxidation, and what is malondialdehyde?**

**Figure 5.** The first stage (initiation stage) of lipid peroxidation process [36].

**Figure 6.** Structural formula of conjugated diene during lipid peroxidation [34].

**Figure 7.** Malondialdehyde formation and metabolism process. Decomposition of arachidonic acid (AA) and PUFAs as side products of enzymatic process during the biosynthesis of thromboxane A2 (TXA2 ) and 12-l-hydroxy-5,8,10-hepadecatrienoic acid (HHT) (blue pathway) or nonenzymatic process by lipid peroxidation-induced bicyclic endoperoxides (red pathway) generates malondialdehyde. Malondialdehyde could enzymatically be metabolized (green pathway); those key enzymes in both malondialdehyde formation and metabolism are cyclooxygenase [1], prostacyclin hydroperoxidase [2], thromboxane synthase [3], aldehyde dehydrogenase [4], decarboxylase [5], acetyl CoA synthase [6], and tricarboxylic acid cycle [7] [38].

In the second phase named propagation, the lipoperoxyl radical, the first phase product, reacts with other fatty acid molecules. Further this reaction a hydrogen atom detaches due to lipid hydroperoxide formation. Following propagation the last stage which is called termination occurs. In this phase enzymatic lipid peroxidation which catalyzes cyclooxygenase and lipoxygenase enzymes happens [34, 36].

decreases triglyceride and MDA [6]. Four-week melatonin supplementation followed by single exercise that lasts about 30 min decreases oxidative stress and MDA as well [42].

Melatonin and Exercise: Their Effects on Malondialdehyde and Lipid Peroxidation

http://dx.doi.org/10.5772/intechopen.79561

117

We would like to express our gratitude to many people who saw us through this book. The authors would like to acknowledge the help of all the people involved in this project. We would also like to show our gratitude to all those researchers and their pearls of wisdom and

Mahsa Rastegar Moghaddam Mansouri1,2\*, Sadegh Abbasian1,2 and Mohammad Khazaie2,3

2 Center of Sport Talent Identification and Development, Department of Education, Malard,

[1] Pandi-Perumal SR, Srinivasan V, Maestroni GJ, Cardinali DP, Poeggeler B, Hardeland R. Melatonin: Nature's most versatile biological signal? The FEBS Journal. 2006;**273**(13):

[2] Masters A, Pandi-Perumal SR, Seixas A, Girardin JL, McFarlane SI. Melatonin, the hormone of darkness: From sleep promotion to Ebola. Brain Disorders & Therapy. 2014;**4**(1):

[3] Oaknin-Bendahan S, Anis Y, Nir I, Zisapel N. Effects of long-term administration of melatonin and a putative antagonist on the ageing rat. Neuroreport. 1995;**6**:785-788 [4] Maldonado MD, Mora-Santos M, Naji L, Carrascosa-Salmoral MP, Naranjo MC, Calvo JR. Evidence of melatonin synthesis and release by mast cells. Possible modulatory role

on inflammation. Pharmacological Research. 2010;**62**(3):282-287

3 Faculty of Physical Education and Sport Sciences, University of Tehran, Tehran, Iran

\*Address all correspondence to: rastegar.moghadam.mansouri@gmail.com

**Acknowledgements**

**Conflict of interest**

**Author details**

Tehran, Iran

**References**

2813-2838

151-157

knowledge that help us in writing this chapter.

There is no conflict of interest for all authors.

1 2020 Olympic Sport School, Tehran, Iran

Lipid peroxidation leads to two results as structural damage into membrane and creates secondary products. Broken fatty acyl chains and lipid-lipid or even lipid-protein cross-links could damage membrane and affect biological systems and impair membrane function and enzymatic inactivation [36]. Malondialdehyde, which is known as MDA, is a three-carbon molecule that is created by free radicals, and it is not only a secondary product but also the most frequent marker of lipid peroxidation [18, 37]. MDA is generated by decomposition of arachidonic acid and polyunsaturated fatty acids (PUFAs) (**Figure 7**). MDA is stable and membrane permeable which may act as signaling messenger [38]. MDA is one the most popular oxidative stress markers, and due to its toxicity, it becomes very relevant to biomedical condition, and several technologies are used to determine MDA such as liquid chromatography-mass spectrometry (LC–MS) and several derivatization-based strategies [38].

There are multiple methods to prevent lipid peroxidation and MDA harmful effects that among them antioxidant usage is the most effective and suitable approach [39]. Antioxidants scavenge free radicals and inactivate peroxides and other ROS consequently could prevent or even delay oxidation process. The chemical structure, concentration, temperature, and type of oxidation substrate determine the efficiency of antioxidants. It means that to select the best antioxidant, we have to consider many points of view and take many factors into account [39]. Antioxidant defense system helps organism to battle with oxidative stress. This system has three lines of defense. In the first stage, ROS overproduction impedes. The second defense line is mainly created by enzymes, and in the third line of defense, molecules should scavenge ROS [24].

### **6. Conclusion**

Melatonin as pineal gland hormone is secreted according to circadian rhythm. Melatonin is also a strong antioxidant that could cross physiological barrier due to its amphiphilic feature, thereby decreasing oxidative damage. Melatonin has direct (direct scavenging of free radical and activate DNA reparation enzymes) and indirect (support superoxide dismutase or SOD) antioxidant capacities [24]. Amphiphilic feature of melatonin makes it strong scavenging factor that increases the efficiency of melatonin's radical scavenging which could pass between lipidic and aqueous phases. Melatonin also neutralizes singlet oxygen, peroxynitrite anion, and nitric oxide [24]. A wide range of biological systems such as linoleate model system or LDL contributes in melatonin antioxidant properties [39, 40]. Melatonin supplementation decreases the MDA and also lipid peroxidation [18]. In an experimental study, it was shown that melatonin supplementation during long-term aerobic exercise could diminish the exercise-induced lipid peroxidation and also malondialdehyde [18]. Exercise training generates almost twofold elevation in oxygen species, lipid peroxidation, and MDA levels [41]. Another report represented that melatonin administration 30 min before the exercise impressively decreases triglyceride and MDA [6]. Four-week melatonin supplementation followed by single exercise that lasts about 30 min decreases oxidative stress and MDA as well [42].

### **Acknowledgements**

In the second phase named propagation, the lipoperoxyl radical, the first phase product, reacts with other fatty acid molecules. Further this reaction a hydrogen atom detaches due to lipid hydroperoxide formation. Following propagation the last stage which is called termination occurs. In this phase enzymatic lipid peroxidation which catalyzes cyclooxygenase and

Lipid peroxidation leads to two results as structural damage into membrane and creates secondary products. Broken fatty acyl chains and lipid-lipid or even lipid-protein cross-links could damage membrane and affect biological systems and impair membrane function and enzymatic inactivation [36]. Malondialdehyde, which is known as MDA, is a three-carbon molecule that is created by free radicals, and it is not only a secondary product but also the most frequent marker of lipid peroxidation [18, 37]. MDA is generated by decomposition of arachidonic acid and polyunsaturated fatty acids (PUFAs) (**Figure 7**). MDA is stable and membrane permeable which may act as signaling messenger [38]. MDA is one the most popular oxidative stress markers, and due to its toxicity, it becomes very relevant to biomedical condition, and several technologies are used to determine MDA such as liquid chromatogra-

phy-mass spectrometry (LC–MS) and several derivatization-based strategies [38].

There are multiple methods to prevent lipid peroxidation and MDA harmful effects that among them antioxidant usage is the most effective and suitable approach [39]. Antioxidants scavenge free radicals and inactivate peroxides and other ROS consequently could prevent or even delay oxidation process. The chemical structure, concentration, temperature, and type of oxidation substrate determine the efficiency of antioxidants. It means that to select the best antioxidant, we have to consider many points of view and take many factors into account [39]. Antioxidant defense system helps organism to battle with oxidative stress. This system has three lines of defense. In the first stage, ROS overproduction impedes. The second defense line is mainly created by enzymes, and in the third line of defense, molecules should scavenge ROS [24].

Melatonin as pineal gland hormone is secreted according to circadian rhythm. Melatonin is also a strong antioxidant that could cross physiological barrier due to its amphiphilic feature, thereby decreasing oxidative damage. Melatonin has direct (direct scavenging of free radical and activate DNA reparation enzymes) and indirect (support superoxide dismutase or SOD) antioxidant capacities [24]. Amphiphilic feature of melatonin makes it strong scavenging factor that increases the efficiency of melatonin's radical scavenging which could pass between lipidic and aqueous phases. Melatonin also neutralizes singlet oxygen, peroxynitrite anion, and nitric oxide [24]. A wide range of biological systems such as linoleate model system or LDL contributes in melatonin antioxidant properties [39, 40]. Melatonin supplementation decreases the MDA and also lipid peroxidation [18]. In an experimental study, it was shown that melatonin supplementation during long-term aerobic exercise could diminish the exercise-induced lipid peroxidation and also malondialdehyde [18]. Exercise training generates almost twofold elevation in oxygen species, lipid peroxidation, and MDA levels [41]. Another report represented that melatonin administration 30 min before the exercise impressively

lipoxygenase enzymes happens [34, 36].

116 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

**6. Conclusion**

We would like to express our gratitude to many people who saw us through this book. The authors would like to acknowledge the help of all the people involved in this project. We would also like to show our gratitude to all those researchers and their pearls of wisdom and knowledge that help us in writing this chapter.

### **Conflict of interest**

There is no conflict of interest for all authors.

### **Author details**

Mahsa Rastegar Moghaddam Mansouri1,2\*, Sadegh Abbasian1,2 and Mohammad Khazaie2,3

\*Address all correspondence to: rastegar.moghadam.mansouri@gmail.com

1 2020 Olympic Sport School, Tehran, Iran

2 Center of Sport Talent Identification and Development, Department of Education, Malard, Tehran, Iran

3 Faculty of Physical Education and Sport Sciences, University of Tehran, Tehran, Iran

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**Section 2**

**Clinical Approaches and Health State**

**Modulation by Melatonin and Its Metabolites**

