Indian Journal of Experimental Biology Vol. 40, June 2002, pp. 668-679

Melatonin oxidative stress and neurodegenerative diseases*

Venkatramanujan Srinivasant

Department of Physiology, PSG Institute"of Medical Sciences and Research, Coimbatore 641 004, India E-mail:saisrinivasaya@yahoo.com

Oxidative Stress is implicated as one of the primary factors that contribute to the development of neurodegenerative diseases like Alzheimer's Disease, Parkinsoni sm and neurological conditions like epi leptic seizures, stroke. brain damage, neurotrauma etc. The increased formation and release of oxygen free radicals coupled with the rather low antioxidative po­tential of the central nervous sys tem are the major reasons that account for the enhanced oxidative stress seen in neuronal cells. In addition to this, brain is also enriched with polyunsaturated fatty ac ids that render neuronal cells eas il y vulnerable to ox idati ve attack. The fac t that there is increased incidence of neurodegenerative disorders in aged individuals. has prompted many investigators to search for a common factor whose progressive decline with increase in age could account for in­creased oxidative stress resulting in senescence and age associated degenerative diseases. Since melatonin. the hormone se­creted from the pineal gland has a remarkable anti-oxidant property and whose rate of production declines wi th increase in age, has prompted many to suggest that this hormone plays a crucial role in the genesis of neurodegenerati ve diseases. Mela-

tonin cannot only scavenges oxygen free radicals like super ox ide rad ical (0;), hydroxyl radical (.OH), peroxyl radical

(LOO,) and peroxynitrite anion (ONOO'), but can also enhance the antioxidative potential of the cell by stimulating the syn­thesis of antioxidative enzymes like super oxide dismutase (SOD), glu tathione peroxidase (GPX), and also the enzymes that are involved in the synthesis of glutathione. In many instances, melatonin increases the expression of m RNA's of the an ti­oxidative enzy mes. Melatonin administration has been shown to be effective in counteracting the neurodegcnerative condi­tions both in experimental models of neurodegenerative diseases and in patients suffering from such diseases. A disturbance of melatonin rhythm and secretion also has been noted in patients suffering from certain neurodegenerati ve diseases. From all these, it is evident that melatonin has a neuroprotective role.

Morphological, biochemical and molecular studies undertaken in recent years both in experimental ani­mals and in man have shown that oxidative stress plays a primary role in the development of degenera­tive changes in cells and tissues of our body. The highest degree of oxidative damage usually occurs in organs like brain, heart, and skeleton muscle since these organs are composed primarily of post-mitotic cells. The progressive and irreversible accrual of mo­lecular oxidative damage is the primary cause for age­ing and age associated degenerative disorders I . The central nervous system shows increased susceptibility to oxidative stress because of its high oxygen con­sumption rate (20% of the total oxygen inhaled by the body) that accounts for the increased generation of oxygen free radicals and reactive oxygen substrates like super oxide radical (0;), singlet oxygen (i02)

hydrogen peroxide (H20 2), and hydroxyl radical (.OH).

*This paper is dedicated to Sri Sathya Sai,the Universal Teacher for mankind.

tpresent address: Department of Physiology, School of Medical Sciences-PPSP, University Sains Malaysia, 16 150, Kubang Kerian, Kota Bharu, Kelantan, Malaysia

Since all the cells and tissues of our body are also equipped with anti oxidative enzymes like super oxide dismutase (SOD), glutothione peroxidase (GPX), glu­tothione reductase (GRd) and substances like reduced glutathione (GSH), they dispose the free radicals as and when they are generated thereby protecting the cells and tissues from the oxidative attack. Normally a balance is maintained between the oxidative attack of the free radicals and the anti oxidative defense system prevailing in the cells and tissues of our body. But the balance is tilted more towards the generation of free radicals, then degenerative changes causing many degenerative diseases. Brain has a low level of anti­oxidative defense system. The concentration of vari­ous antioxidative enzymes like SOD, GPX, GRd, catalase2 is low in brain. The glutathione (GSH), con­centration is also very much reduced in the brain when compared to other organs in the body". In addi­tion to these factors, brain has high iron and ascorbate content in certain regions, which provide favourable environment for generation of oxygen free radicals. Brain is also enriched with polyunsaturated fatty acids (PUFA) that render them susceptible to oxidati ve

SRINIVASAN: MELATONIN OXIDATIVE STRESS & NEURODEGENERATIVE DISEASES 669

attack. The interplay of all these factors that contrib­ute to enhance the oxidative stress are outlined in Fig. 1. The increased level of oxidative stress seen in the brain is thus the major contributory factor for the de­velopment of neurodegenerative diseases like Alz­heimer's disease and Parkinsonism in aged individu­als4

.s. With the arrival of 21st century, there will be

more number of people over 60 years and above due to the enhanced life span of human beings caused by the effective implementation of various health care programmes. But the prevention of neurodegenerative diseases has become a major problem that has to be tackled effectively both by governmental and non­governmental organizations that are involved in main­taining the health care of the society. Intake of. diet rich in vegetables and fruits has been recommended as a precautionary measure for preventing the possible occurrence of the various neurodegenerative diseases in the elderl/ . But the availability of sufficient quan­tity of fruits and vegetables for consumption by all members of the society is a major question. Supply of natural antioxidants like vitamin E, and vitamin C is also effective for preventing the neurodegenerative diseases. Recently the pineal hormone melatonin has assumed major significance as it is endogenously syn­thesized in the body and its decline in production with increase in age has been suggested as one of the major reason for the occurrence of neurodegenerative dis­eases, since melatonin has been demonstrated to have a great antioxidative potential by acting both as a free radical scavenger and preventive antioxidant4•

6-12

•

Hence, the age-associated occurrence of neurodegen­erative diseases has been attributed to the decline pro­duction of melatonin I3

.14

. If the melatonin deficiency is the primary cause for the development of neurode­generation, the intake of melatonin in large doses

Pro oxidant factors in brain Antiodative Potential of brain

1. High Oxygen utilization. 1. Low Glutathione Peroxidase

2. Enrichment with Polyunsaturated 2. Low Catalase. 3. Low Superoxide dismutase

fatty acids. 4. Glutathione.

3. High Iron and ascorbate content 5. Vitamin E. 6. Melatonin (Oedines with age) y 'o,,~ •• ,,_ rn';~' 'OOO""'!"', 01-1,-. H,O,. r--J

OH. LOO • ONOO )

t I Increased oxidative stress I

... Neuronal degeneration

Fig. 1-Brain and oxidative damage

should be really beneficial not only for arresting the progression of neurodegenerative changes, but also for preventing the occurrence of neurodegeneration in the elderly individuals.

This has been demonstrated in several experimental models of neurodegenerative diseases IS

.16

• This review summarizes various evidences obtained from large number of studies that indicate the role of oxidative stress in the genesis of the neurodegenerative diseases and how melatonin as an effective antioxidant is able to counteract the neurodegenerative changes induced by oxidative stress. The usefulness of this substance as a possible therapeutic agent for treating neurode­generative diseases like Alzheimer's disease and Parkinsonism and other neurological conditions are discussed.

Free radicals and oxidative stress Most of the oxygen taken up by the cells of our

body is converted into H20 during mitochondrial res­piration. However, a small percentage of oxygen (less than 5%) is converted into reactive oxygen substrates (ROS) like O2-, H20 2, 102, and ·OH radicals. These substances are highly toxic in nature and if allowed to accumulate, they can destroy all the macromolecules of the cells like lipids, proteins, mitochondrial and nuclear DNA molecules causing severe 'oxidative stress 17. When oxygen accepts a single electron, it forms a superoxide radical (0 ~). Once superoxide

radical is formed, it quickly undergoes dismutation to form H20 2 by a family of enzymes known as superox­ide dismutases (SOD). H20 2 can also be formed by other enzymes. In dopaminergic neurons, oxidation of dopamine by the enzyme mono amine oxidase (MAO) generates hydrogen peroxide. The increased destruc­tion of dopamine by MAO is the major reason for the degeneration of dopaminergic neurons resulting in neuropathological and biochemical changes that are seen in Parkinsonism l8

. Hydrogen peroxide is con­verted into a highly reactive radical (.OH) in the pres­ence of transition metal ions like copper (Cu2+), iron (Fe2+) through Fenton reaction or Haber-weiss reac­tion 17 (Fig. 2). H20 2 is normally disposed as H20 and O2 by two important antioxidative enzymes like glu­tathione peroxidase (GPX) and catalase. In the brain, GPX is the major enzyme that plays a dominant role in disposing H20 2; catalase is found in low concentra­tions l9

. Glutathione peroxidase utilizes H20 2 and other hydro peroxides as substrates in the process of conversion of reduced glutathione (GSH) into its

670 INDIAN J EXP BIOL, JUNE 2002

disulphide (GSSG) and thereby most of the H20 2 is disposed off. The hydroxyl radicals (.OH) that are formed through fenton reaction are highly destructive in nature and can destroy lipids, proteins and DNA molecules of the cells. It also removes H+ ions from the polyunsaturated fatty acid molecules (PUFA) of the cell membrance and generates peroxyl (LOO-) and peroxynitrite anions, which can further substract H+ ions causi ng extensive damage of the lipid molecules of the cell membrane. Thus, lipid peroxidation in­duced by .OH radicals will become self-propagating in nature that can eventually lead to destruction of all lipid in the cell membrane. Vitamin E acts both as a peroxyl radical scavenger and as a chain breaking

1 MOLECULAR OXYGEN (0,) 1 _ I ('0.) SINGLET OXYGEN

(Mitochondrial Electron 1 Leak and Dopamine --.

Oxidation)

Schematic Daigram illustrating the formation of Oxygen free rad icals.

Fig. 2-Free radical generation

o II C

antioxidant and has been shown to e beneficial in arresting neurodegenerative changes lo

. A typical cell in the rat undergoes nearly 100,000 ROM attack on DNA per day', resulting in both mutation of DNA molecules and DNA damage. In recent years, nitric oxide (NO) has also been shown to act as a free radi­cal. Under normal physiological conditions, nitric ox­ide acts as a messenger molecule. But when its con­centration increases intracellularly, it exerts toxic ef­fects causing neuronal degeneration and neuronal death20

.

The excitatory neuro transmitter glutamate inter­acts with N-methyl-D-aspartate (NMDA) receptor present on the postsynaptic cell. But under certain ischemic conditions, the enhanced release of the glu­tamate causes overactivation of NMDA receptors re­sulting in increased calcium (Ca2+) influx causing overstimulation of nitric oxide synthetase (NOS) ac­tivity. This leads to the generation of more number of nitric oxide molecules. Nitric oxide interacts with su­per oxide radicals causing formation of peroxynitrite anions (ONOO-) that cause heavy damage to lipid molecules of the cell membranes. Multiple studies have shown increased oxidation of proteins, lipids and DNA molecules in the brain of patients suffering from Alzheimer's disease, Parkinsonism, Amyotrophic lat­eral sclerosis, etc8. I 8.22.25 .

~"OH VI NIH 2

TRYPTOPHAN HYDROXYLASE ~I'" ~.) NH2

N

TRYPTOPHAN 5-HYDROXY TRYPTOPHAN

HOM HO // I N-ACETYL .., I

~ I· TRANSFERASE" N NH2 7" \~"

ACETYL COA

SEROTONIN N-ACE TYL SEROTONIN MELATONIN

Fig. 3-Biosynthetic pathway of melatonin

SRINIVASAN: MELATONIN OXIDATIVE STRESS & NEURODEGENERATIVE DISEASES 671

Melatonin as a free radical scavenger and an anti· oxidant

Melatonin (N-acetyl 5-methory tryptamine) is a hormone formed mainly in the pineal gland of all ver­tebrates including man. It is both lipid and water­soluble and has a molecular weight of 232. The bio­synthetic scheme of melatonin is shown in Fig. 3. The rate of production and blood levels of melatonin are very high, nearly 5 to 15 times during night. Physio­logical and pharmacological actions of melatonin in regulating sleep, sleep-wake fullness rhythm, biologi­cal rhythm disorders like jet lag, shift-work disorder, seasonal affective disorder (SAD) have been re­ported26.28. The preservation of normal melatonin rhythm and its rate of production are essential for ar­resting the age-associated degenerative changes oc­curring in cells and tissues of the body29. The age­associated decline in melatonin production and the attenuation of melatonin rhythm are the major con­tributory factors for the increased level of oxidative stress and the associated degenerative changes seen during old age l4

• Individuals of the same chronologi­cal age may exhibit variations in the degree of senes­cence associated functional impairment ' and since there are variations of melatonin levels in the same age group, it is likely that the physiological age is determined by one's own melatonin production and variations in the occurrence of degenerative changes seen in cells and tissues of the body can be attributed to the variations in the level of melatonin synthesis and release. Dietary restriction delays the age­associated drop in melatonin synthesis. The key en­zyme that is involved in melatonin production namely N-acetyl transferase (NAT) is increased by dietary restriction30. Needless to point out here that dietary restriction also increases the life span of individuals. Melatonin enters easily through the cell membranes of all cells and distributes itself freely in the cytosol and nuclear components of the cells. This subcellular dis­tribution of melatonin has been studied in tissues like NB 41 A3 neuroblastoma cells of the mouse31 . This study is very important, since it reveals the ubiquitous

IMETHOXY GROUP)

H

H

l ' 2 ' 3' 4' 5' 3 CH, CH,N·C·CH3

i'l'-- ----,'f' I I (ACETYL GROUP(

1 N I H

HO (SIDE CHAIN)

Fig. 4-Structure of melatonin

distribution of melatonin within the cells. The hy­droxyl radicals which are very destructive in nature can travel only for a few molecular distance before it inflicts damage on nucleus or on cell membranes. Me­latonin by being present in the cell membrances, cyto­sol, and nucleus is able to neutralize the hydroxyl radicals as and when they are generated in any com­partments of the cells. Highest concentration of mela­tonin has been shown to be present within the nuclei of all the cells showing thereby that melatonin is very effective in protecting the nucleus from oxidative at­tack32

. Melatonin is a efficient scavenger of all free radicals like O2', H20 2, 102, 'OH and LOO. This has been well demonstrated in several experimental model systems. Tan et at. 33 provided first evidence to show that melatonin can scavenge .OH radicals . In their in vitro study they found that melatonin has been shown to be 5 times superior to glutathione (GSH) in scavenging .OH radicals. Both methoxy group at posi­tion 5 of the indole nucleus and the acetyl group on the side chain (Fig. 4) are both essential for .OH scav­enging activity. Melatonin reduces chromium VI re­lated .OH generation and has been shown to protect against the DNA strand breaks, lipid peroxidation and cytotoxicity caused by chromium34. The hydroxyl radical scavenging activity of melatonin has since been confirmed in other in vitro model systems wherein terephthalic acid (THA) was used, as THA form adduct with .OH radical. The THA-.OH forma­tion was reduced when melatonin was added in a dose-dependent manner35. Melatonin donates an elec­tron to scavenge .OH radical and becomes indolyl cation radical that in turn neutralizes superoxide radical (02')33.

In addition to this, direct free radical scavenging activity; melatonin also enhances the synthesis of an­tioxidative enzymes like SOD) I. The quick disposal of H20 2 is also carried out by increased synthesis of other antioxidative enzymes like glutathione peroxi­dase (GPX) and catalase36. The stimulatory effect of melatonin on glutathione peroxidase seems to be the physiological role of melatonin in all cells of our body. This has been proved in birds where the noctur­nal increase of glutathione peroxidase is prevented by illumination37

, a procedure that normally blocks mela­tonin synthesis.

Pinealeactomy also exaggerates the oxidative dam­age induced by free radical generating agents38, thereby substantiating the fact that melatonin is a physiological free radical scavenger and antioxidant.

672 INDIAN J EX? BIOl, JUNE 2002

The total antioxidant status of the blood correlates well with endogenous melatonin level in the blood39

.

From these studies, it is clear that melatonin can dis­pose all the free radicals like hydroxyl (.OH), super­oxide(02), peroxyl(LOO-) and peroxy nitrate anion (ONOO-). In scavenging peroxyl radicals also, 5-methoxy group and the side chain of the melatonin molecules seems to be essential. Pieri et al.4 1 tested the ability of the melatonin to scavenge peroxyl radi­cals. In their study they used peroxyl radical initiator 2,2'-azo-bis (2-amidino propane) dihydrochloride (AAPH) to induce lysis of human erythrocytes and then compared the efficiency of melatonin, vitamin E (Trolox), Vitamin C, GSH, and mannitol. Their re­sults revealed that melatonin is superior to all other free radical scavengers in neutralizing peroxyl radicals.

As pointed out earlier, nitric oxide (NO-), the physiological messenger molecule forms peroxynitrite anion (ONOO-) when it interacts with superoxide radical (02-) (Fig. 2) . The formation of nitric oxide depends upon the activity of the enzyme nitric oxide synthetase (NOS), which is classified as a pro oxidant enzyme. Nitric oxide itself is not highly toxic but per­oxynitri te anion can ruthlessly destroy all the macro­molecules of the cell causing cell death42. Bettahi et al.43 have reported that melatonin at physiological concentrations greatly reduces the NOS activity in rat cerebellum and hypothalamus. The potency of mela­tonin in curtailing the formation of peroxynitrite radi­cal is as effective as other peroxynitrite scavengers like GSH and cysteine9

_ Oxidation of membrane lipid and protein molecules affects the functional dynamics of plasma membrane causing increased rigidity. Mela­tonin prevents this reduction of membrane fluidity caused by lipid peroxidation. The stabilizing action of melatonin in keeping the fluid nature of membrane has been attributed to its ability to scavenge free radi­cals 10. Melatonin prevented the neuronal death in­duced by the cytotoxic effects of singlet oxygen. Singlet oxygen is known to playa role in neurodegen­erarative diseases like Parkinsonism. It is generated when hydrogen peroxide reacts with peroxynitrite or when superoxide reacts with nitric oxide. Hence, the findings that melatonin is able to quench singlet oxy­gen and prevents the neuronal death as evidenced by the marked reduction of DNA fragmentation44, is a good indication for employing melatonin as a thera­peutic tool for treating neurodegenerative diseases. We have already seen that melatonin enhances the synthesis of antioxidative enzymes like SOD, GPX,

glutathione reductase and substances like reduced glu­tathione. Recently by using ECV 304 human endothe­lial cells, Urata et al.45 demonstrated that melatonin induces the expression of y glutamyl cysteine syn­thetase (y -GCS), the rate limiting enzyme in glu­tathione (GSH) synthesis. One micromolar .melatonin has been shown to increase the expression of y GCS m RNA followed by increase in GSH concentration within 24 hr. As RZRlROR a nucle~ r orphan recep­tors appears to be the natural ligand for melatonin46, melatonin induced the formation of this enzyme by acting on these receptors. It induced the expression of y GCS by stimulating the DNA binding activator pro­tein 1 (AP-l) and retinoid z receptor/retinoid related orphan receptor a activity in ECV 304 cells45

_ Thus melatonin increases the antioxidative activity by its transcriptional action. From all these discussions it is clear that melatonin because of its free radical scav­enging and antioxidant role has a definite neuro pro­tective role.

Neurodegenerative diseases The occurrence of age-associated neurodegenera­

tive diseases like Alzheimer's disease (AD) and Park­insonism (PD) and other neurological conditions like amyotrophic lateral sclerosis, traumatic brain injury, stroke, epilepsy, brain damage associated with meta­bolic disorders, hyperbaric hyperexia, Huntington's chorea are all due to effects of increased oxidative stress on protein, lipid and DNA molecules of neu­ronal cells. As melatonin has been demonstrated as an effective antioxidant and a free radical scavenger, the role of this hormone not only in arresting the oxida­tive stress induced neuronal damage, but also in pre­venting the possible occurrence of neurodegenerative diseases is discussed below.

Alzheimer's disease Alzheimer's disease is a age-associated neurodege­

nerative disease that is characterized by the progres­sive loss of cognitive function, loss of memory and other neurobehavioural manifestations that are the result of extensive neuronal damage seen in this dis­order. In spite of number of studies undertaken in Alzheimer's disease (AD), there is no uniformity of opinion with regard to its etiology and pathogenesis_ Numerous mechanisms like genetic factors, calcium dysregulation, chronic inflammation that is associated with cytokine release and trace element neurotoxicity have all been suggested as triggering factors for

SRINIV ASAN: MELATONIN OXIDATIVE STRESS & NEURODEGENERATIVE DISEASES 673

induction of neuronal damage. But the most accepted, reasonable and well documented explanation is the excessive generation of oxygen free radicals seen in this disease. The brains of Alzhemier's patients' show

the deposition of amyloid ~ protein (senile plaques) formation of neuro fibrillary tangles and extensive loss of neurons in hippocampal and cortical areas. The deposition of amyloid ~ protein generates release of oxygen free radicals that causes extensive damage to the neuronal tissues47

. Studies undertaken in the brains of patients suffering from Alzheimer's disease have revealed extensive lipid, protein and DNA oxi­dation in the neuronal cells48

. Increased protein oxida­tion eventually leads to loss of enzyme function . Loss of glutamine synthetase and creatine kinase activity has been demonstrated in neocortical and hippocam­pal regions of AD patients49

.

Glycation of protein results in the formation of ad­vance glycation end-products (AGE) which by modi­

fying A ~ protein induces further release of oxygen free radicals that leads to further neuronal degenera­tion48

• Increased lipid peroxidation as evidenced by the formation of thiobarbituric acid reactive sub­stances (TBARS) has been found in the frontal lobes of AD patients50

. Glutathione enzymes that are in­volved in the inactivation of toxic products of oxygen metabolism such as 4-hydroxynonenal (HNE) are also decreased in various regions of the brain51

• HNE is a highly reactive a~ aldehyde that can inhibit glycoly­sis, DNA, RNA and protein synthesis. Increased ac­cumulation of HNE accounts for most of the neuronal loss seen in AD patients. By inhibiting Na+/K+ ATPase pump, HNE causes increased calcium influx into neu­ronal cells resulting in increased generation of oxygen free radicals51

• Neuroinflammation induced by proin­f1ammatory cytokines also enhances oxygen free radi­cal formation and reactive nitric oxide species (RNS). One such product 3-nitro-tyrosine has been demon­strated in affected regions of the Alzheimer's brain. Administration of a-phenyl-tert-butyl nitrone (PBN) in the Mongolian gerbils and rats revealed that PBN can decrease the level of oxidized protein . PBN also reduces the nitric oxide (NO) formation by inhibiting the enzyme, inducible nitric oxide synthetase (fNOS) and thereby reduces the formation of neuroinflamma­tory products. PBN inhibits H20 2 and IL-l ~ mediated p38 activation. The enhancement of p38 activation and its inhibition by PBN supports the concept that oxidative stress is involved in the pathogenesis of Alzheimer's disease52

.

Having convinced about the possible role of oxida­tive stress as the main triggering factor in Alzheimer's disease, Pappolla et al. 53 studied the efficacy of mel a­tonin in arresting the neuropathological changes seen in experimental models of neurodegenerative dis­eases. Using in murine neuroblastoma cells (N2 a) they demonstrated that incubation of these cells with both amyloid ~ protein and melatonin caused signifi­cant reduction in the degree of lipid peroxidation . In addition to this, the features of apoptosis like cellul ar shrinkage, formation of membrane blebs were also effectively prevented by melatonin. Melatonin acted by scavenging oxygen free radicals released by the deposition of amyloid ~ protein and those caused by the increased calcium influx into the neuronal cells. The survival rate of the neuronal cells was also in­creased significantly by the presence of melatonin in the incubation medium. These studies show that mela­tonin has a definite neuroprotective role even in the presence of amyloid ~ protein53

. Melatonin not only prevents the release of oxygen free radicals induced by amyloid ~ protein but also inhibits the formation of amyloid ~ protein itself from its precursor amyloid ~ precursor protein (A~ pp)54. In addition to amyloid ~ protein, certain trace metals are also involved in Alz­heimer's disease. Melatonin administration has been shown to reduce aluminum ion induced lipid peroxi­dation55

. Melatonin binds with aluminum ion and thereby prevents it from inducing neurotoxicity. All these studies show that melatonin is essential for ar­resting neurodegenerative changes. Decreased secre­tion of melatonin as it occurs in some individuals may predispose them to increased oxidative attack causing neuronal degeneration . Indeed, studies of melatonin rhythm and secretion in AD patients have shown that the rhythm is disturbed in AD patients. Phase advance of peak nocturnal melatonin rise by 3 hr has been documented in AD patients showing thereby the pres­ence of timing abnormality of melatonin secretion in AD patients57

. High prevalence of lack of melatonin rhythm (nocturnal rise) has been reported in hospital­ized AD patients56

. These are some of the evidences to show that dysregulation of melatonin secretion plays a definite role in the pathogenesis of Alz­heimer's disease.

Parkinsonism Parkinsonism (PO) is another major neuro degen­

erative disease caused by progressive degeneration of dopamine containing neurons in the substantia nigra. Though there are many theories to explain the patho-

674 INDIAN J EXP BIOL, JUNE 2002

physiology of PO, the free radical involvement has received special significance I8.58. Enhanced oxidative stress has been demonstrated in the brain of PO pa­tients59. In parkinsonism, hydrogen peroxide (H20 2) is generated both enzymatically due to increased activity of monoamine oxidase (MAO), as well as due to the autooxidation of dopamine itself causing selective destruction of dopaminergic neurons58. The initiating factor in Parkinsonism is only the increased release of oxygen free radical s6o. Mitochondrial oxidative phos­phorylation is inhibited lead ing to loss of energy pro­duction and consequent neuronal cell death . Miller et al.61 by employing oxygen radical absorbance ca­pacity (ORAC) assay system measured the oxidation of the fluorescent protein prophyridium cruentum ~ phycoerythrin (~-PE) in the presence of oxidizing agents like dopamine. Then they evaluated the effi­cacy of melatonin in thi s assay system and proved that melatonin significantly reduced the oxidation of ~-PE induced by the presence of dopamine. From their studies, they concluded that melatonin has a potential role in reducing dopaminergic cell destruction caused by the au toox idation of dopamine. Parkinsonism like animal models were also used for testing the potenti al ro le of melatonin in this disorder. The drug 1 methyl-4-pheny l 1,2,3,6 tetrahydropyridine (MPTP) when injected into animals causes increased lipid peroxida­tion in striatum, hippocampus and mid brain regions. The neuropathological sensory and motor distur­bances seen in these animals resembled wi th those seen in parkinsonism. MPTP is taken up by astrocytes and is metabolized into methyl-4-phenyl pyridinium ion (MPP+). This cation is taken up by dopamjnergic neurons where it generates oxygen free radicals caus­ing several toxic effects like ATP depletion, induction of apoptosis etc. Acuna-Castroviejo et at. 62 used this model of PO, to test the melatonin' s ability to reduce MPTP induced lipid peroxidation. They found that coadministration of melatonin along with MPTP sig­nificantly reduced the lipid peroxidation in striatum, hippocampal and mid brain regions. In another study, it was noted that melatonin when melatonin was added to serum-free culture medium, it sustained the life of dopaminergic neuronal cells for more than seven days. Since serum free media lacked growth factors, the neuronal cells degenerated within two to three days, but addition of melatonin compensated the lack of thi s Qrowth factors and increased the life of cultured dop; minergic neuronal cells63. The drug 6-hydroxy dopamine (6-0HD) selectively destroys

nigro-striatal system by generating oxygen free radi­cals in neuronal cells. It also reduces the concentra­tion of antioxidant enzymes like copper-zinc superox­ide dismutase (Cu-Zn SOD), manganese superoxide dismutase (Mn-SOD), and glutathione perox idase (GPX). But melatonin addition to the culture media containing 6 OHDA prevented the reduction in the concentration of these enzymes induced by 6 OHDA 64. Since 6 OHDA model is similar to the tough Parkinsonism, the study indicates the usefulness of melatonin in preventing the neuronal damage that can occur in Parkinsoni sm.

Brain Trauma, Stroke and Brain Damage The neuropathological changes observed during

brain ' injury, trauma, stroke, and epileptic associated brain damage have all been ascri bed to enhanced oxi­dative stress and related lipid protein and DNA mole­culesI5. 16.65. This has been well demonstrated in sev­eral experimental animal model systems. Kainic ac id (KA) is an agonist of NMDA receptor, and systemic administration of thi s drug resulted in brain injur/ 6

.

The excessive release of this exci tatory amino ac id neurotransmitter glutamate and oxidative stress are the two mechanisms through which kainate induces brai n injur/ 7-69. Kainic acid binds with and stimulates a sub type of ionotrophic receptor that results in trans membrane ionic imbalance causing increased calcium influx. This results in a cascade of events like activa­tion of protein kinases, phospholipases, proteases, nitric oxide synthetase, all leading into impairment of mitochondrial fuction l6 and release. of oxygen free radicals. These free radicals attack lipid protein and DNA molecules causing extensive lipid pe.roxidation, structural and functional changes of protein molecules causing loss of enzyme activity, DNA strand breaks, nuclear fragmentation and neuronal damage. Hence KA model of excitotoxicity is used as a model for studying oxidative stress induced neuronal degenera­tion. Potentiality of various antioxidants in arresting the neuropathological changes are also studied by us­ing this model only. As melatonin is a antioxidant, several investigators have analyzed the role of mela­tonin on morphological, biochemical and behavioural changes induced by kainic ac id administration.

In both in vitro and in invivo studies, melatonin has been shown to provide neuroprotection against KA induced excitotoxicity. By assessi ng the level of lipid peroxidation products, as endpoints, Melchiorri et at. 70.71 demonstrated that the presence of melatonin

SRINIVASAN: MELATONIN OXIDATIVE STRESS & NEURODEGENERATIVE DISEASES 675

in the incubation medium greatly reduced the kainate induced oxidative damage in the homogenates of cerebral cortex, cerebellum, hippocampus, hypo­thalamus and striatum. Giusti et al. 72 in their study on the neuroprotective role of melatonin in the kainite injected animals showed that brain damage in hippo­campal, amygdala and pyriform cortex were pre­vented by melatonin. They further showed that mela­tonin has antiapoptotic action and it is due to the anti­oxidative nature. In another study, it was demon­strated that melatonin reduced the hydrogen peroxide induced lipid peroxidation in different regions of the brain like frontal cortex, striatum, cerebellum, hippo­campus and hypothalamus as reflected by the reduced accumulation of lipid peroxidation products like MDA and 4-HAD. Oxidative apoptotic cell death in­duced directly by glutamate in cultured hippocampal cells (HT-22) and hippocampal brain slices were also reduced by melatonin administration73

.74

. The inten­sity of neuronal degeneration after administration of kainite was found to be significantly higher in pin­ealectomized than control ones showing thereby that endogenously synthesized melatonin can offer neuro­protective role38

. Harrell and Balagura75 while study­ing the influence of natural light and darkness on brain injury, showed that darkness occurring immedi­ately before injury or after injury had a potential bene­ficiary effect since it accelerated the speed of recov­ery from brain injury. The improvement was assessed by the enhancement of motor function. Manev and UZ76

, have postulated that progression of brain lesion and functional impairment will become worse if stroke occurs during daytime than during night hours. They reasoned that the increased production and se­cretion of melatonin at night hours will modulate the brain vulnerability to oxidative stress. The increased free radical scavenging activity and the increased syn­thesis of antioxidative enzymes induced by melatonin are supposed to be the major reasons for the increased neuroprotective ability of the brain during night hours77

•

Brain damage can also be induced experimentally by administration of 100% pure oxygen under great pressures, which releases large amount of oxygen free radicals. When rats were exposed to 100% pure oxy­gen at 4 atmospheric pressures, it caused significant increases of lipid peroxidation and neuronal damage in cerebral cortical, cerebellar, hippocampal, hypotha­lamic and striatal regions. The increase in the concen­tration of malondialdehyde and 4 hydroxyalkenals

were taken as evidences for enhanced lipid peroxida­tion in this study . When melatonin was administered prior to exposure of high oxygen pressure, it effec­tively prevented the hyperbaric hyuperiexia induced brain damage indicating thereby that melatonin be­cause of its free radical scavenging action counter­acted the oxidative damage induced by high oxygen pressure7

•

Metabolic disorders Acute intermittent porphyria is an inherited meta­

bolic disorder caused by increased formation and ex­cretion of 8 amino laevulinic acid (ALA). If ALA ac­cumulates in neural tissues, it undergoes enolization and subsequent iron catalysed oxidation generating oxygen free radicals. ALA induced lipid peroxidation in cerebral, cerebellar and straital brain homogenates and its subsequent prevention by the addition of mela­tonin was demonstrated in some studies7x 7

'l . In this context, it is interesting to note that in patients suffer­ing from this genetic disorder, acute intermittent por­phyria exhibit a low plasma melatonin levd".

Huntington chorea model Quinolinic acid (2,3-dicarboxylic ac id) a neuro ac­

tive metabolite of the tryptophan-kynurenine pathway is present in human brain and is implicated in the pathogenesis of a broad spectrum of neurodegenera­tive diseases. When injected into rats, it caused neu­rodegeneration similar to that caused by the injection of Kainic acid and Ibotenic acid. It caused extensive lipid peroxidation in the striatal, hippocampal, and globus pallidum regions. Increased levels of MDA+ and 4 HDA were demonstrated in these brain regions. But when melatonin was administered both before and after quinolinic acid administration, it decreased not only lipid peroxidation but also attenuated the neurobehavioural signs associated with quinolinic acid administration81

• Since quinoline is an endoge­nous ligand for NMDA receptor and is not metabo­lized ill the brain, the increased concentration of this substance in the synaptic cleft causes prolonged exci­tation of NMDA receptor resulting in increased cal­cium and associated free radical generation .- Mela­tonin by scavenging free radicals like hydroxyl (.OH), hydrogen peroxide (H20 2), peroxyl (LOO) and per­oxynitrite anions (.ONOO- ) and by stimulating the synthesis of anitoxidative enzymes, reduced the level of lipid peroxidation significantl/2. Since the neuronal damage induced by quinolinic acid is very similar to that observed in Huntington's chorea, this model is

676 INDIAN J EXP SIOL, JUNE 2002

Alzheimer's Disease Parkinsonism Neuronal Excitotoxicity Amyloid p peptide Deposition Dopamine Oxidation Glutamate-- NMDA Receptors

~ t ~

,I t Oxygen Free Radicals 1 It Oxygen Free Radicals j t Oxygen Free Radicals I I Increased Calcium

I I Increased Calcium I

I Influx Influxs I ~ ~

I H N E Formation ~ LIPID PEROXIDATION I w of .,j,

It c/2 J .1 t Oxygen Free Radicals I tca+

2 I 1 J

Increased Protein Oxidation Oxidative DNA Damage Organelle Destruction Loss of Enzyme Activies Apoptosis Energy Depletion

J, \ 1 J NEURONAL DEGENERATION

I I I

Fi g, 5-0xidative stress and neuronal degeneration

Table 1- Effects of melatonin on anti-ox idant and pro-oxidant enzymes

ACTION ON ENZYMES SUPEROXIDE DISMUTASE CATALASE GLUTATHIONE PEROXIDASE GLUTATHIONE REDUCTOS E y- GLUTAMYL CYSTEINE SYNTHETASE GLUCOSE-6-PHOSPHATE DEHYDROGENASE

NITRIC OXIDE SYNTHETASE

l' - INCREASE , -.It - DECREASE

useful for studying the neuropathology of Hunting­ton's chorea. The study points to the possibil ity that melatonin can be administered as a drug for treating free radical induced neurodegenerative conditions including Huntington 's Chorea83

. Recently melatonin has been detected in herbs, vegetables, and fruits 84

and it has been shown to act synergistically with Vi­tamin C, Vitamin E, and glutathione. Moreover, mela­tonin is a terminal antioxidant that does not undergo any redoxcycling and hence does not promote oxida­tion. It can trap free radicals or can trap or capture electrons to detoxify free radicals. The diverse mechanisms make melatonin as a unique broad­spectrum antioxidant that can be employed to treat

RESULTANT EFFECTS SUPEROXIDE RADICAL HYDROGEN PEROXIDE HYDROGEN PEROXIDE GLUTATHIONE

GLUTATHIONE

NADPH

NITRIC OXIDE PEROXY NITRITE

neurodegenerati ve diseases84.

Conclusion Although free radical hypothesis of ageing has

been proposed long back85, the role of oxidative stress

in the genesis of neurodegenerative diseases has been understood on ly recently. The high oxygen consump­tion rate coupled with low antioxidant potential of the brain are the main triggering factor for the enhanced release of oxygen free radicals in the brain. The pres­ence of increased iron concentration in certain regions of the brain favours the formation of iron ascorbate mixture that provides suitable medium for the genera­tion of free radicals .

Oxidative stress is implicated in the pathogenesis of the number of neurodegenerative diseases like Alz-

SRINIVASAN: MELATONIN OXIDATIVE STRESS & NEURODEGENERATIVE DISEASES 677

heimer's disease, Parkinsonism, Huntington 's Chorea, and neurological conditions like epileptic seizures, stroke, brain trauma, etc. An interaction of the various factors that are involved in the pathogenesis of these disorders is summarized in Fig. 5.

Although classical antioxidants like vitamin E, vitamin C can be used to treat the di sorders melatonin can be recommended because of its ubiquitous distri­bution in all compartments of the cells. This enables melatonin to prevent lipid oxidation or DNA damage very easily. Moreover, melatonin not only scavenges the free radicals like hyrdroxyl (.OH), peroxyl (LOO-), peroxynitrite anion (ONOO-), nitric oxide (NO.) but also can stimulate the synthesis of many antioxidative enzymes by its nuclear transcription action. Melatonin stimulates the synthesis of GSH-PX, GSH Rd, y-GCS , and catalases6. Table 1 summarizes the effect of mela­tonin on antioxidant and prooxidant enzymes. The indispensable nature of melatonin in providing anti­oxidative defense mechani sm has further been ex­plored in recent times and it was shown that pinealec­tomy in rats greatly accelerated the lipid peroxida­tion87 .

The finding that melaton in is also seen in high con­centrations in certain plants like peanut, soyabean, sunflower seed, almond and grapesS4 add further sup­port to the notion that melatonin is a natural antioxi­dant. This hormone is essential for maintaining the homeostasis of all cells and ti ssues in the bod/s.

Study of melatonin levels and rhythm in the body fluids of patients suffering from certain neuro degen­erative diseases reveal that there is a dysregulation of pineal gland function and melatonin secretion in these patientss9.9o.

Since the potential acute and chronic toxicity of melatonin appears to be low even in doses like I gmJday, the drug can be admini stered safely to pa­tients suffe ring from various neurodegenerative di s­eases. In the words of Prof. R. J. Reiter of the Univer­sity of Texas, Health Sciences Center, USA 'mela­tonin may find utility as a pharmacological agent in preventing and treating neurological disorders in which free radical formation is a pathogenic factor86, .

Acknowledgement Thanks are due to Prof. R. J. Reiter, (USA), Prof.

Paola S. Timiras (USA), Dr. H. Maniev, Dr. Bhatnagar, Dr. Shafii, and Dr. D. Kripke for reprints. The Managing trustee of PSGIMSR is also thanked. Thanks are due to Dr. Wan Mohd Suyuti Wan-Ismail,

Dean, PPSP, En Wan Suyuti W Ismail , Asst. Registrar, Mr. Ismail Ibrahim, Prof Harbinder Jeet Singh Dept. of Physiology, School of Medical Sciences, Uni versity Sains Malaysia for help and Dr. Urban J.A. D' Souza, Dept. of Physiology, School of Med ical Sciences, USM, Malaysia for software assistance.

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