REVIEW OF LITERATURE

CHAPTER III

MELATONIN AND ANTIOXIDANT DEFENSE

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1. Introduction

Oxidative stress has been implicated in the pathogenesis of many diseases hence much research has recently been focused on use of antioxidants as therapeutics. In addition to the primary defense against ROS by antioxidant enzymes (Le. superoxide dismutase, catalase, hemeperoxidase and glutathione peroxidase), secondary defense against ROS is also offered by small molecules which react with radicals to produce another radical compound, the 'scavengers'. When these scavengers produce a lesser harmful radical species, they are called 'antioxidants'. For example, a-tocopherol, ascorbate and reduced glutathione (GSH) may act in combination to act as cellular antioxidants. a-Tocopherol, present in the cell membrane and plasma lipoproteins, functions as a chain-breaking antioxidant. Once the tocopherol radical is formed, it can migrate to the membrane surface and is reconverted to a-tocopherol by reaction with ascorbate or GSH. The resulting ascorbate radical can regenerate ascorbate by reduction with GSH, which can also directly scavenge ROS, and the resulting GSSG can regenerate GSH through NADPH-glutathione reductase system (Fig. I). However, ascorbate, in addition to its antioxidant capacity, at low concentrations and in the presence of catalytic metal ions(copper and iron) may generate ·OH by virtue of its metal reducing capacity similar to O2-- (I) together with generation of H20 2 (2).

RedU<:ed Glutlthlone (2 GSH)

NADP'~I' \ Glutathione reducl8se

~ Glutathione peroltid85e

NAOPH+W / "

(GSSG) OJIldized Glutathione

7~@ Figure 1. Glutathione Oxidation Reduction (Redox) Cycle. One molecule of hydrogen peroxide is reduced to 2 molecules of water while 2 molecules of glutathione (GSH) are oxidized in a reaction catalyzed by the selenoenzyme, glutathione peroxidase. Oxidized glutathione (GSSG) may be reduced by the flavin adenine dinucleotide (F AD)-dependent enzyme, glutathione reductase.

Melatonin, 5-methoxy-N-acetyltryptamine, is a hormone found in all living creatures from algae (3) to humans, at levels that vary in a diurnal cycle. In higher animals melatonin is produced by pinealocytes in the pineal gland (located in the brain) and also by the retina and gastro-intestinal tract. Melatonin is considered to be a powerful antioxidant and extensive efforts are given to make this molecule as a therapeutically viable molecule to treat different human diseases originated from oxidative stress

65

Melatonin plays important role in regulating sleep-wake cycles circadian rhythms. At night melatonin is produced to help our bodies regulate our sleep-wake cycles. The amount of melatonin produced by our body seems to lessen, as we get older. Melatonin has been called "the hormone of darkness.

2. Synthesis and metabolism of melatonin

Originally, melatonin was believed to be synthesized exclusively in pineal gland of vertebrates. Studies have shown that many extrapineal tissues and organs have the capacity to synthesize melatonin. These include retina, ciliary body, lens, harderian gland, brain, thymus, airway epithelium, bone marrow, gut, ovary, testicle, placenta, lymphocytes and skin (4). Melatonin is synthesized from tryptophan in pineal parenchymal cells. Tryptophan is the least abundant of essential amino acids in normal diets. It is converted to another amino acid, 5-hydroxytryptophan, through the action of the enzyme tryptophan hydroxylase and then to 5-hydroxytryptamine (serotonin) by the enzyme aromatic amino acid decarboxylase. Serotonin concentrations are higher in the pineal than in any other organ or in any brain region. They exhibit a striking diurnal rhythm remaining at a maximum level during the daylight hours and falling by more than 80% soon after the onset of darkness as the serotonin is converted to melatonin, 5-hydroxytryptophol and other methoxyindoles. Serotonin's conversion to melatonin involves two enzymes that are characteristic of the pineal: SNAT (serotonin-N-acetyltransferase) which converts the serotonin to N-acetylserotonin, and HIOMT (hydroxyindole-O-methyltrasferase) which trasfers a methyl group from S-adenosylmethionine to the 5-hydroxyl of the N-acetylserotonin (Fig. 2). The activities of both enzymes rise soon after the onset of darkness (5).

In the brain, a substantial fraction of melatonin is metabolized to kynuramine derivatives. This is of interest as the antioxidant and anti-inflammatory properties of melatonin are shared by these metabolites, NI-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and, with considerably higher efficacy, N I-acetyl-5-methoxykynuramine (AMK). AFMK is produced by numerous nonenzymatic and enzymatic mechanisms. Its formation by myeloperoxidase appears to be important in quantitative terms (Fig. 3).

In as much as melatonin diffuses through biological membrane with ease, it can exert actions in almost every cell in the body. Some of its effects are receptor mediated while others are receptor independent (Fig. 4). Melatonin is involved in various physiological functions and major actions are mediated by the membrane receptor MTI and MT2 (Fig. 4). They belong to the superfamily of G-protein coupled receptors containing the typical seven trans-membrane domains. These receptors are responsible for chronobiological effects at the SCN, the circadian pacemaker.

3. Role of melatonin in different diseases

Melatonin is effective against wide variety of diseases. Neurodegenerative diseases are a group of chronic and progressive diseases that are characterized by selective and often symmetric loss of neurons in motor, sensory and cognitive systems. Clinically relevant examples of these disorders are Alzheimer's disease (AD), Parkinson's disease, Huntington's chorea and amyotrophic lateral sclerosis (6). Although the origin of neurodegenerative diseases mostly remains undefined, three major and frequently inter-related processes (glutamate excitotoxicity, free radical-mediated nerve injury and mitochondrial dysfunction) have been identified as common pathophysiological

HO~NH2

~N)J H

Serotonin

! Arylalkylamine

Ho~y:n ~~I)J 0

N H

N-Acetylserotonin

HYdrOXYindOle.o-nlethYI_! i transferase (HIOMT) CYP2C19 (or C;P1A2)

H,co~yCH'

CYP1A2, CYP1A1 or CYP181

N H

Melatonin

4 1atonin deacetylase or

M.NAT Aryl acylamidases

H,co'O=:)NH, H

5-Methoxytryptami ne

66

Figure. 2. Fonnation of melatonin, its major pathways ofindolic catabolism, and interconversions between bioactive indoleamines. CYP, cytochrome P450 isofonns (hydroxy lases and demethylases) (7).

mechanisms leading to neuronal death. In the context of oxidative stress, the brain is particularly vulnerable to injury because it is enriched with phospholipids and proteins that are sensitive to oxidative damage and has a rather weak antioxidative defense system. In the case of AD, the increase in p-amyloid protein- or peptide-induced oxidative stress (8), in conjunction with decreased neurotrophic support, contributes significantly to the pathophysiology of the disease.

H20 + H"

H H'CO~NyCH'

H ~ 2.0H MelatonIn ~

OH

numerous reactions'

H'CO~ ~I ::::....

/ ~ oJCH ,

o 0 Cyclic 3.hydrOl'y,

~• II ) melatonIn (c30HM)

H,CO ~ I '<::: N CH) .0 H

NH-CHO N'.Acelyl·N'·formyl.5.methoxykynuramine (AFMK)

H20 H,O, Hemoperoxidase

2 H,O

HzO

CO,

H'CO±~~CH' .0 NH N'.Acetyl·5.methoxy.

J kynuramlOe (AMK) R·

/" CO,'· + ·NO: _ ONOOCO.,'

·NO

H20 RH

(

Heo,'

H'C~~~CH' ~N'~

HlCO~O ~ '<::: N~ I! H CH, .;::::'

NH,

NO, N'-Acetyl-5-melhoxy-3·nltrokynuramine (AMNK) • 3.nitro.AMK

H 3·Acetamldomethyl·6-methoxy· cinnolinone (AMMe)

67

Figure 3. The kynuric pathway of melatonin metabolism, including recently discovered metabolites formed by interaction ofN I-acetyl-5-methoxykynuramine (AMK) with reactive nitrogen species_ *Mechanisms of N I -acetyl-N2-formyl-5-methoxykynuramine (AFMK) formation (I) enzymatic: indoleamine 2,3 dioxygenase, myeloperoxidase; (2) pseudoenzymatic: oxoferrylhemoglobin, hemin; (3) photocatalytic: protoporphyrinyl cation radicals + 0 3 0-, 02(lDg), 02 + UV; (4) reactions with oxygen radicals: oOH + O2 0_, C03o- + 0 2 0 -; and (5) ozonolysis (7).

Circadian pacemaker: Suprachias-

matic nucleus

1 Overt rhythms:

Seasonal breeding

(hypothalamus and other

organs relevant to reproduction)

Vasomotor control:

Constriction via MT1

Dilation via MT2

Immune system (B cells, T cells,

NK cells, thymocytes.

bone marrow)

Resetting via MT2 • Inhibition via MT1

Direct transmission of signal darkness (via MT1. MT2. RORa.RZRp. other rocop- Scavenging of tors?) reactive oxygen

species (ROS). reactive nitrogen

species (RNS) and organic

radicals'

l t

Inhibition and downregulatlon of

CNS: Antiexcitatory effects, avoidance of

Ca2' overload

Elimination of toxic quinones:

Binding to quinone reductase 2

Cytoskeletal effects: Binding to cal-

modulin, activation of protein kinase C

Upregulatlon of anti-oxidant and down· regulation of proox·

idant enzymes-

Attenuation of mitochondrial

electron leakage

! Decrease of free

radicals and other

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cyclooxygenase 2 ..!=======-____ -+ oxidants '------'-'---' ~ '------_ .... Figure. 4. The pleiotropy of melatonin: an overview of several major actions. AFMK, NI-acetyl-N2-formyl-5-methoxykynuramine; AMK, NI-acetyl-5-methoxykynuramine; c30HM, cyclic 3-hydroxymelatonin; MTI, MT2, melatonin membrane receptors I and 2; mtPTP, mitochondrial permeability transition pore; RORa, RZRI3, nuclear receptors of retinoic acid receptor superfamily. ·Several reactive oxygen species (ROS) scavenged by melatonin: -OH, C03~, 0 3, in catalyzed systems also 02~ species reactive nitrogen species (RNS) scavenged by melatonin: -NO, -N02 (in conjunction with -OH or C03~), perhaps peroxynitrite (ONOO-) ;organic radicals scavenged by melatonin: protoporphyrinyl cation radicals, 2,2¢-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) cation radicals, substituted anthranylyl radicals, some peroxyl radicals; radical scavenging by c30HM, AFMK and AMK. ··Antioxidant enzymes up-regulated by melatonin: glutathione peroxidase (GPx) (consistently in different tissues), glutathione reductase (GRoad), c-glutamylcysteine synthase, glucose 6-phosphate dehydrogenase; hemoperoxidase / catalase, Cu-, Zn- and Mn-superoxide dismutases (SODs) (extent of stimulation cell type-specific, sometimes small) ; pro-oxidant enzymes down-regulated by melatonin: neuronal and inducible nitric oxide synthases, 5- and 12-lipoxygenases (7).

AD has been also related to mitochondrial dysfunction (9). Collectively, most evidence convincingly supports the notion that the neural tissue of AD patients is subjected to an increased oxidative stress (10). Therefore, attenuation or prevention of oxidative stress by administration of suitable antioxidants should be a possible basis for the strategic treatment of AD (7). Melatonin appears to have some use against insomnia. It has been studied for the treatment of cancer (11) immune disorders (12), depression( 13), and sexual dysfunction (14). Melatonin receptors appear to be important in mechanisms of learning and memory (15). Melatonin has been shown to prevent the hyperphosphorylation of the tau protein. Hyperphosphorylation of tau protein can result in the formation of neurofibrillary tangles, a pathological feature seen in Alzheimer's disease. Thus, melatonin may be effective for treating Alzheimer's Disease (16). Several clinical studies indicate that supplementation with melatonin is an effective preventative treatment for migraine sufferers (17). There may be other, far-reaching therapeutic uses for melatonin, such as

69

in the treatment HIV (18), and other viral diseases (19). Melatonin as a supplement is as a natural aid to better sleep. Dosages are designed to raise melatonin levels for several hours to enhance quality of sleep, but some studies suggest that smaller doses are just as effective at improving sleep quality (20). A number of studies indicate melatonin supplementation helps to reduce the age-related decline in hormone production from the thyroid and pituitary glands. Melatonin is involved in the regulation of body weight, and may be helpful in treating obesity (especially when combined with calcium) (21). Several controlled studies in patients with high blood pressure report small reductions in diastolic and systolic blood pressure when taking melatonin by mouth (orally) or inhaled through the nose (intranasally). A summary of role of melatonin has been depicted in Fig 4.

4. Antioxidant roles of melatonin

Melatonin is a powerful antioxidant that can easily cross cell membranes and the blood-brain barrier (22). Melatonin, once oxidized, cannot be reduced to its former state because it forms several stable end-products upon reacting with free radicals. Therefore, it has been referred to as a terminal (or suicidal) antioxidant (23). In animal models, melatonin has been demonstrated to prevent the damage to DNA by some carcinogens, stopping the mechanism by which they cause cancer (24). It has been proposed that antioxidant properties of melatonin may be protective (2S).

In the pathogenesis of acute liver injury, oxidative stress and its consequent lipid peroxidation plays important role. Melatonin at dose 100 mglkg body weight effectively decreases hepatic injury in male Wistar rats (26). The protective actions of melatonin may be due to the molecule itself and to its metabolites. The efficacy of melatonin in reducing oxidative stress is increased by the metabolites that produce while scavenging free radical (26). When the antioxidative effects of melatonin was compare with taurine and S-hydroxytryptophan against hyperglycemia-induced kidney-cotex tubules injury, it was found that Melatonin is much more effective than later one (27). When the effect of melatonin was compared with Vitamin E on the cholestasis syndrome and their protective effective on liver injury, it was found that malatonin (SOO~glkg daily) offered significantly better protection against cholestasis and a superior protective effect on the hepatic injury than did Vitamin E (IS mglkg daily)(28). Melatonin regulates glutathione redox status in brain and liver mitochondria, correcting it when it is disrupted by oxidative stress. Also melatonin has a physiological role on mitochondrial homeostasis, a role not sustained by the other endogenous antioxidant such as Vitamin C and Vitamin E (29). Although ascorbate and Vitamin E have important antioxidant properties, but result shows Melatonin as a better endogeneous antioxidant than Vitamin C and Vitamin E in t-butyl hydroperoxide induced mitochondrial oxidative stress (29).

New antioxidants or free radical scavengers should be of high potency, low toxicity, and easy permeability to cellular and subcellular compartments. Melatonin appears to fulfill most of these criteria. This compound has been shown to directly scavenge free radicals such as peroxynitrites, hydroxyl and peroxyl radicals, while its lipophilic nature allows its rapid access to cellular and subcellular membranes compartments. Melatonin has been found to be effective in protecting against pathological states characterized by an increase in basal rate of ROS production. Melatonin has been shown to effectively protect against ischemic-reperfusion myocardial damage (30). The importance of melatonin as antioxidant depends on several characteristics: its lipophilic and hydrophilic nature, its ability to pass all bio-barriers with ease, and its availability to all tissues and cells. Melatonin distributes in all cell compartments, being especially high in the nucleus and mitochondria. Melatonin maintains membrane function and permeability by

70

preventing lipid peroxidation and increasing its fluidity. It maintains mitochondrial function by reducing hydroperoxide levels and maintaining GSH homeostasis in both normal conditions and under oxidative stress.

5. Anti apoptotic role of melatonin

Most of the beneficial consequences resulting from melatonin administration may depend on its effects on mitochondrial physiology. The physiological effects of melatonin on normal mitochondria, its role to prevent mitochondrial impairment, energy failure, and apoptosis in oxidatively-damaged mitochondria, and the beneficial effects of the administration of melatonin in experimental and clinical diseases involving mitochondrial dysfunction and cell death, are revised (31). Effect of daily melatonin supplementation on liver apoptosis induced by aging in rats was studied and found that Melatonin reduces the apoptotic liver changes induced by aging via inhibition of the intrinsic pathway ofapoptosis. (32). Also melatonin inhibits neural apoptosis induced by homocysteine in hippocampus of rats via inhibition of cytochrome c translocation and caspase-3 activation and by regulating pro- and anti-apoptotic protein levels (33). Melatonin prevents mitochondrial as well as ER pathway of apoptosis (34). Among the non-neurological functions of melatonin, much attention is being directed to the ability of melatonin to modulate the immune system, whose cells possess melatonin-specific receptors and biosynthetic enzymes (35). Melatonin protected against LPS-induced liver damage in d-galactosamine-sensitized mice through its strong ROS-scavenging, anti inflammatory and antiapoptotic effects (36). Melatonin is more effective than taurine and 5-hydroxytryptophan against hyperglycemia-induced kidney-cortex tubules injury (27). Melatonin diminished the intestinal oxidative stress and apoptotic damage induced by endotoxemia in infant rats. (37). Melatonin treatment protects cardiolipin from ROS and this suggests a possible link with the reduction of the apoptotic phenomenon (38). Melatonin administration exerts beneficial effects in inflammatory bowel disease by modulating signal transduction pathways (39). Recently, mitochondria, which are implicated in the intrinsic pathway of apoptosis, have been identified as a target of Melatonin action.!t is known that melatonin scavenges oxygen and nitrogen-based reactants generated in mitochondria. This limits the loss of the intra mitochondrial glutathione and lowers mitochondrial protein damage, improving electron transport chain (ETC) activity and reducing mt-DNA damage. Melatonin also increases the activity of the complex I and complex IV of the ETC, thereby improving mitochondrial respiration and increasing A TP synthesis under normal and stressful conditions. These effects reflect the ability of melatonin to reduce the harmful reduction in the mitochondrial membrane potential that may trigger mitochondrial transition pore (MTP) opening and the apoptotic cascade (Fig 5) (40). In addition, a reported direct action of melatonin in the control of currents through the MTP opens a new perspective in the understanding of the regulation of apoptotic cell death by the indoleamine (40). Electron microscopic studies demonstrated that treatment with Melatonin restored to near normal the ischemialreperfusion-induced disorganization of mitochondrial structure (41). Melatonin is also effective in aging as it reduced the deteriorative oxidative changes in mitochondria during aging. During aging melatonin actually reduces oxidant damage and promotes mitochondrial respiration by stimulating electron transport and ATP production in the inner-mitochondrial membrane (42).

Cardiolipin, a phospholipid localized almost exclusively within the mitochondrial inner membrane, is particularly rich in unsaturated fatty acids. Thus, mitochondrial cardiolipin molecules are a possible early target of ROS attack, either because of their high content of unsaturated fatty acids or because of their location in the inner mitochondrial membrane near to the site of ROS production, mainly at the level of complex I and III of the respiratory chain.

............. / '-----~

J "

~. I

~ o o _._.- ......

/O"·'~ I \ I j \ 0 '0 ...... .' ., / ..... .-

- - ~ 0 o

' .. ,.. , ".~ ~

.. ~. ," ' . . :~~. \'

Melatonin

/ HP2/

1 ~=~ [mROS] 1

l exposure of PS

l mito Can overload

mito 6, depolarization openi ng of the MPT pore

! cytochrome c release

J activation cI caspase 3

l positive YOPRO-1 staini ng of

earlyapoptotic nucleus. , condensation and karyorrhexis

of the nucleus , apoptotic fragmentation

of nuclear DNA.

71

Figure. 5 Schematic diagram of the mechanisms of action of melatonin at the mitochondrial level in limiting oxidative stress-induced apoptosis. The indole significantly reduces mitochondrial ROS fonnation induced by H20 2• Via its potent inhibitory effect on mitochondrial ROS generation, melatonin prevents the externalization of PS, mitochondrial calcium overload, mitochondrial membrane potential depolarization, and the opening of the MPT. Subsequently, melatonin blocks MPT-dependent cytochrome c release, downstream activation of caspase 3, and the condensation and karyorrhexis of the nuclei and apoptotic fragmentation of nuclear DNA.

This phospholipid plays a pivotal role in mitochondrial bioenergetics, optimizing the activity of the respiratory chain complexes as well as of anion carrier proteins. More recently an involvement of cardiolipin in the execution phase of the apoptotic process has been suggested. The effect of melatonin on mitochondrial cardiolipin content was tested in ischemic-reperfused heart. The content of cardiolipin was dramatically reduced in mitochondria from ischemic-reperfused rat heart compared with the control heart. Melatonin treatment significantly prevented this cardiolipin loss (30).

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