Melatonin is a ubiquitous molecule, synthesised primarily in the pineal gland, secondary sources are in the retina, the gastrointestinal tract, skin, bone marrow and lymphocytes.1
It was first characterised after its isolation from bovine pineal glands and structurally identified in 1958 by Lerner et al.2
The structure of melatonin (Fig. 1) explains its diversity with regards to its functions. The two functional groups define the molecule's amphiphilitic nature and specificity of receptor binding.3
(Hardeland R. et al, 2005)3
Due to melatonin being both lipid and water soluble, it is not confined to one cellular compartment. Exogenously added melatonin can readily pass through the blood-brain barrier and be easily distributed to all subcellular compartments, which makes this molecule very versatile.4
Melatonin has various functions; it is important as an indicator of time and date and is considered to be the body's chronological pacemaker.1 It is also known to be a very effective antioxidant, has immune-enhancing properties, is cytoprotective, has anti-apoptotic signalling function as well as oncostatic properties.1
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The involvement of melatonin in such a broad range of vital functions in the body makes it pharmacologically a very interesting compound, but melatonin is sold as a food supplement and is non-patentable according to the US FDA as a result. Therefore melatonergic agonists or melatonin analogs that are patentable are of greater interest to the pharmacological industry.5
Pharmacological and/or biological activity
A simple overview of melatonin metabolism is given in figure 2 below.
Fig. 2 Melatonin biosynthesis and metabolism.
(Pandi-Perumal et al., 2006)1
In pinealocytes (cells of the pineal gland) tryptophan is converted to serotonin via 5-hydroxytryptophan after which it is acetylated to form N-acetylserotonin by arylakylamine N-acetyltransferase (AA-NAT). By the action of Hydroxyindole-O-methyltransferase (HIOMT) N-Acetylserotonin can be converted to melatonin.1 Alternatively, melatonin can be formed by N-acetylation of 5-methoxytryptamine. In most articles AA-NAT is said to be the rate-limiting enzyme3,7,8, but it is suggested that HIOMT might be a rate limiting enzyme in some cases6.
Melatonin biosynthesis is largely regulated by the light/dark cycle via the suprachiasmatic nuclei (SCN) in the hypothalamus. Specialised neurons in the eyes respond to light and transfer the message to the SCN. The message is transduced in a circuitous pathway to the pineal gland. Melatonin synthesis is triggered by darkness. Norepinephrine is secreted during nighttime and couples to beta-adrenergic receptors. This results in cAMP formation and eventually stimulation of arylalkylamine-N-acetyltransferase (AA-NAT).7
Extrapineal melatonin is not regulated by circadian rhythm, however and it is hypothesised that it is produced as a means of protection in response to certain stressors, eg. Ultraviolet radiation, pollutants, infections etc. that may result in oxidative stress or inflammation.6
Circulating melatonin is mainly metabolised by cytochrome P450 enzymes, CYP1A2, CYP1A1 and CYP2C19 in the liver or CYP1B1 at extrahepatic sites.6 Resulting 6-hydroxymelatonin by CYP1A, CYP1A2 or CYP1B1 can be conjugated with sulfate (and glucoronide to a lesser extent) to form a more hydrophilic compound, 6-sulfatoxymelatonin (aMT6S), which can be excreted in urine by the kidney.1 CYP2C19 or CYP1A2 are cytochromes involved in the demethylation of melatonin to N-acetylserotonin.
6-Hydroxymelatonin is not only formed through enzymatic means as stated above, but also through the interaction of melatonin with reactive oxygen species (ROS) and reactive nitrogen species (RNS).6
Melatonin has the ability to neutralise free radicals, ROS and RNS as well as stimulate antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd).4,9
Other metabolites of melatonin include 2-hydroxymelatonin, which is postulated to be a product of melatonin's reaction with ROS/RNS,6 as well as cyclic 3-hydroxymelatonin (C3-OHM), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N-acetyl-5-methoxykynuramine (AMK). The parent hormone, melatonin, and its metabolites are important in protecting cells from damage by free radicals and reactive oxygen and reactive nitrogen species.
Melatonin metabolism by cytochrome P450 enzymes
Pineal melatonin is metabolized to 6-hydroxymelatonin mainly and it was found that CYP1A1, CYP1A2, CYP1B1 and CYP2C19 are the enzymes responsible for the production of this metabolite.1,6,10
Melatonin can also be converted into N-acetylserotonin which is also a precursor for its synthesis (fig.1)1. Both 6-hydroxymelatonin and N-acetylserotonin can be excreted in the urine after conjugation with sulfate and/or glucoronide.
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A study conducted by Facciola et al. (2001)11 determined CYP1A2 to be the main cytochrome P450 enzyme responsible for 6-hydroxylation of melatonin.
In a study that was conducted by Ma et al. (2005)10, melatonin 6-hydroxylation and O-demethylation rates were measured to evaluate the potential role of 11 cDNA-expressed human cytochrome P450 enzymes in melatonin metabolism (figure 2)10. It was found that 6-hydroxylation was mainly carried out by CYP1A2, CYP1A1 and CYP1B1 and to a lesser extent CYP2C19 (fig.2 A) wherease O-demethylation occurred almost primarily due to the action of CYP2C19 and to a minimum degree by CYP1A2 (fig.2 B).
Figure 2 Relative rates of melatonin 6-hydroxylation (A) and O-demethylation (B)* by various cDNA-expressed CYPs
*a mistake was made on the Y-axis title of the graph obtained from the article and should be read MELATONIN O-DEMETHYLATION
Ma et. al (2005)10
Inhibitory action of melatonin on human cytochrome P450 enzymes CYP1A1, CYP1A2 and CYP1B1
In a recent study by Chang et al. (2010)12 the hypothesis that melatonin inhibits catalytic activity of CYP1A1, CYP1A2 and CYP1B1 along with CYP2A6 was tested, along with its effect on the alteration of human CYP1 gene expression and on the activity of the human aryl hydrocarbon receptor (AhR).
A significant result could prove useful during cancer therapy since these enzymes were found to be procarcinogen-bioactivating enzymes of benzo[a]pyrene or 7,12-dimethyl-benz[a]anthracene. Inhibition of expression or reduction in catalytic activity of these cytochrome P450 isozymes may reduce the production of carcinogenic metabolites from these substrates.
The results obtained (figure 3)12 indicate hat melatonin successfully reduces enzymatic activity of CYP1A1, CYP1A2 and CYP1B1, but not CYP2A6.
Figure 3 Effect of exogenously added melatonin on enzymatic activity of indicated CYPs
(Chang et al.)12
As described in the previous section, melatonin is metabolised by CYPs 1A1, 1A2 and 1B1. Therefore, its inhibitory effect may be due to it competing with the procarcinogenic substrate for the enzymes' active sites, although the exact mechanism of inhibition is unclear.
The study mentioned that endogenous melatonin does not exert inhibitory effects and resultingly a pharmacological potential in developing analogues of melatonin that could potentially inhibit these enzymes and prevent CYP1 mediated carcinogenesis is created.12
Activity of melatonin with AhR and melatonin's effect on CYP1 gene expression was found to be insignificant.
Fluvoxamine and melatonin
Fluvoxamine (FLU), an antidepressant, was found to increase serum melatonin levels and a study was conducted by von Bahr et al. (2000)13 to determine whether citalopram (CIT) also affects these levels.
The article concluded that CIT in fact does not have the same effect as FLU does. It did however establish a clear relationship between the levels of melatonin and concentrations of FLU in the plasma of the subjects.13
FLU is an inhibitor of CYP1A2 and CYP2C19 and thereby prevents these enzymes from metabolising melatonin into its metabolites; resultantly an increase in the levels of serum melatonin was witnessed.
Melatonin as a free radical scavenger and antioxidant
Melatonin and its metabolites have the potential to act as free radical scavengers and can neutralise reactive oxygen species (ROS) and reactive nitrogen species (RNS) as well as up-regulate antioxidative enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd).4
Byproducts of oxygen metabolism is summarised below (Figure 4)9
Figure 4 Byproducts of oxygen metabolism
Reiter et al. (2000)9
The free radical dioxide (O2-) can be generated due to cellular respiration, due to environmental factors or oxidative burst of macrophages. The toxicity of the O2- is considered to be low 9 but if it reacts with nitric oxide (NO•) it can produce ONOO- capable of doing molecular damage. SOD converts dioxide to hydrogen peroxide (H2O2) which is not truly a free radical, but if not catabolised to form water and O2 via CAT or to water via GSH-Px, can be metabolised to the extremely toxic hydroxyl radical (•OH). As can be seen in the figure, this free radical can induce DNA damage, lipid peroxidation or do damage to proteins.
Melatonin can be converted into a metabolite cyclic 3-hydroxymelatonin when it reacts with two hydroxyl radicals, which has been found to be excreted in the urine.1,9
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An AFMK pathway (figure 5)1 also exists that is apparent capable of scavenging up to 10 ROS/RNS.6
Figure 5 Free radical scavenging and AFMK pathway for melatonin and melatonin metabolites
Pandi-Perumal et al. (2006)1
AFMK and AMK are potent free radical scavengers and protectors against oxidative stress, but also has anti-inflammatory and immunoregulatory effects by inhibiting tumor necrosis factor-alpha (TNFα) and interleukin-8 formation (IL-8) and synthesis of prostaglandins.6
The main metabolite of melatonin, 6-hydroxymelatonin, can also be generated in melatonin's reaction with ROS/RNS, as well as another melatonin metabolite, 2-hydroxymelatonin.6
Melatonin and its chronobiotic effects
Melatonin is a major regulator of the circadian rhythm and acts via the suprachiasmatic nuclei (SCN) in the hypothalamus. Melatonin acts through MT1 and MT2 receptors in the SCN and MT1 is associated with inhibitory effects of electrical activity in SCN neurons, whereas melatonin's actions on MT2 are involved with the regulation of the circadian rhythm.5
Melatonin synthesis peaks during the dark phase and is inhibited by light. Exogenous melatonin administration at certain time periods results in shifts in the circadian rhythm. It is therefore considered to be useful in the treatment of jet lag and in promotion of restful sleep, although melatonin is probably not a direct hypnotic.7
Melatonin is a compound with so many diverse functions in the body, for example its regulatory role in circadian rhythm, antioxidant and oncostatic properties. This compound is of pharmacological significance, but is also sold 'over the counter' as a food supplement and is resultantly not patentable. This restricts pharmacological interest to a certain extent, although the development of melatonin analogs that are more efficient in its capability to exert these functions, for example increased half-life with retension of effect, may be of significant value for researchers and the pharmaceutical industry.
Melatonin is also involved in the protection against neurodegenerative diseases, depression and immune function.
There is no doubt as to how important melatonin is with regards to physiological functioning is in living organisms. It is critical for our perception of time and date and is protects us from hazardous environmental agents as well as from ourselves (eg, the free radicals produced via natural oxygen metabolism with which we cannot exist without).
More research into the mechanisms by which melatonin exerts its antioxidant effects and how extrapineal melatonin can be induced or inhibited as well as drug-drug interactions would be of great benefit when developing therapeutic strategies in the combat of diseases, such as Alzheimer's Disease, Parkinson's Disease, insomnia, diseases of the immune system, cancer and many more.