Malgorzata Karbownik

Significance of Melatonin in Antioxidative Defense System: Reactions and Products

Melatonin is a potent endogenous free radical scavenger, actions that are independent of its many receptor-mediated effects. In the last several years, hundreds of publications have confirmed that melatonin is a broad-spectrum antioxidant. Melatonin has been reported to scavenge hydrogen peroxide (H2O2), hydroxyl radical (HO·), nitric oxide (NO·), peroxynitrite anion (ONOO–), hypochlorous acid (HOCl), singlet oxygen (1O2), superoxide anion (O2–·) and peroxyl radical (LOO·), although the validity of its ability to scavenge O2–· and LOO· is debatable. Regardless of the radicals scavenged, melatonin prevents oxidative damage at the level of cells, tissues, organs and organisms. The antioxidative mechanisms of melatonin seem different from classical antioxidants such as vitamin C, vitamin E and glutathione. As electron donors, classical antioxidants undergo redox cycling; thus, they have the potential to promote oxidation as well as prevent it. Melatonin, as an electron-rich molecule, may interact with free radicals via an additive reaction to form several stable end-products which are excreted in the urine. Melatonin does not undergo redox cycling and, thus, does not promote oxidation as shown under a variety of experimental conditions. From this point of view, melatonin can be considered a suicidal or terminal antioxidant which distinguishes it from the opportunistic antioxidants. Interestingly, the ability of melatonin to scavenge free radicals is not in a ratio of mole to mole. Indeed, one melatonin molecule scavenges two HO·. Also, its secondary and tertiary metabolites, for example, N1-acetyl-N2-formyl-5-methoxykynuramine, N-acetyl-5-methoxykynuramine and 6-hydroxymelatonin, which are believed to be generated when melatonin interacts with free radicals, are also regarded as effective free radical scavengers. The continuous free radical scavenging potential of the original molecule (melatonin) and its metabolites may be defined as a scavenging cascade reaction. Melatonin also synergizes with vitamin C, vitamin E and glutathione in the scavenging of free radicals. Melatonin has been detected in vegetables, fruits and a variety of herbs. In some plants, especially in flowers and seeds (the reproductive organs which are most vulnerable to oxidative insults), melatonin concentrations are several orders of magnitude higher than measured in the blood of vertebrates. Melatonin in plants not only provides an alternative exogenous source of melatonin for herbivores but also suggests that melatonin may be an important antioxidant in plants which protects them from a hostile environment that includes extreme heat, cold and pollution, all of which generate free radicals.

Effects of Melatonin Treatment in Septic Newborns

Free radicals have been implicated in the pathogenesis of neonatal sepsis and its complications. This study was conducted to determine the changes in the clinical status and the serum levels of lipid peroxidation products [malondialdehyde (MDA) and 4-hydroxylalkenals (4-HDA)] in 10 septic newborns treated with the antioxidant melatonin given within the first 12 h after diagnosis. Ten other septic newborns in a comparable state were used as “septic” controls, while 10 healthy newborns served as normal controls. A total of 20 mg melatonin was administered orally in two doses of 10 mg each, with a 1-h interval. One blood sample was collected before melatonin administration and two additional blood samples (at 1 and 4 h) were collected after melatonin administration to assess serum levels of lipid peroxidation products. Serum MDA + 4-HDA concentrations in newborns with sepsis were significantly higher than those in healthy infants without sepsis; in contrast, in septic newborns treated with melatonin there was a significant reduction (p < 0.05) of MDA + 4-HDA to the levels in the normal controls at both 1 and 4 h (p < 0.05). Melatonin also improved the clinical outcome of the septic newborns as judged by measurement of sepsis-related serum parameters after 24 and 48 h. Three of 10 septic children who were not treated with melatonin died within 72 h after diagnosis of sepsis; none of the 10 septic newborns treated with melatonin died. To our knowledge, this is the first study where melatonin was given to human newborns.

Melatonin and Its Relation to the Immune System and Inflammation

Abstract: Melatonin (N‐acetyl‐5‐methoxytryptamine) was initially thought to be produced exclusively in the pineal gland. Subsequently its synthesis was demonstrated in other organs, for example, the retinas, and very high concentrations of melatonin are found at other sites, for example, bone marrow cells and bile. The origin of the high level of melatonin in these locations has not been definitively established, but it is likely not exclusively of pineal origin. Melatonin has been shown to possess anti‐inflammatory effects, among a number of actions. Melatonin reduces tissue destruction during inflammatory reactions by a number of means. Thus melatonin, by virtue of its ability to directly scavenge toxic free radicals, reduces macromolecular damage in all organs. The free radicals and reactive oxygen and nitrogen species known to be scavenged by melatonin include the highly toxic hydroxyl radical (·OH), peroxynitrite anion (ONOO−), and hypochlorous acid (HOCl), among others. These agents all contribute to the inflammatory response and associated tissue destruction. Additionally, melatonin has other means to lower the damage resulting from inflammation. Thus, it prevents the translocation of nuclear factor‐kappa B (NF‐κB) to the nucleus and its binding to DNA, thereby reducing the upregulation of a variety of proinflammatory cytokines, for example, interleukins and tumor neurosis factor‐alpha. Finally, there is indirect evidence that melatonin inhibits the production of adhesion molecules that promote the sticking of leukocytes to endothelial cells. By this means melatonin attenuates transendothelial cell migration and edema, which contribute to tissue damage.

Pharmacology and Physiology of Melatonin in the Reduction of Oxidative Stress in vivo

This brief resume summarizes the evidence which shows that melatonin is a significant free radical scavenger and antioxidant at both physiological and pharmacological concentrations in vivo. Surgical removal of the pineal gland, a procedure which lowers endogenous melatonin levels in the blood, exaggerates molecular damage due to free radicals during an oxidative challenge. Likewise, providing supplemental melatonin during periods of massive free radical production greatly lowers the resulting tissue damage and dysfunction. In the current review, these findings are considered in terms of neurodegenerative diseases, cancer, ischemia/reperfusion injury and aging. Besides being a highly effective direct free radical scavenger and indirect antioxidant, melatonin has several features that make it of clinical interest. Thus, melatonin is readily absorbed when it is administered via any route, it crosses all morphophysiological barriers, e.g., blood-brain barrier and placenta, with ease, it seems to enter all parts of every cell where it prevents oxidative damage, it preserves mitochondrial function, and it has low toxicity. While blood melatonin levels are normally low, tissue levels of the indoleamine can be considerably higher and at some sites, e.g., in bone marrow cells and bile, melatonin concentrations exceed those in the blood by several orders of magnitude. What constitutes a physiological level of melatonin must be redefined in terms of the bodily fluid, tissue and subcellular compartment being examined.

Causes of Oxidative Stress in the Pre- and Perinatal Period

Oxidative stress may be defined as an imbalance between pro-oxidant and antioxidant forces resulting in an overall pro-oxidant insult. Pregnancy is a physiological state accompanied by a high energy demand of many bodily functions and an increased oxygen requirement. Because of the increased intake and utilization of oxygen, augmented levels of oxidative stress would be expected. Arguments for a role of oxidative stress/oxidative lipid derivatives in the pathogenesis of preeclampsia are documented in many papers and evidence continues to accumulate that oxidative stress is a mediator of endothelial dysfunction and thus contributes to the cardiovascular complications of preeclampsia. Also other conditions, such as toxic substance exposure, smoking and asphyxia likewise induce oxidative stress. The oxidized lipid products generated as a consequence of these conditions are highly reactive and cause damage to cells and cell membranes. Thus, increased oxidative stress accompanied by reduced endogenous defences may play a role in the pathogenesis of a number of diseases in the newborn.

Individual and synergistic antioxidative actions of melatonin: studies with vitamin E, vitamin C, glutathione and desferrrioxamine (desferoxamine) in rat liver homogenates

The pharmacological effects of melatonin, vitamin E, vitamin C, glutathione and desferrioxamine (desferoxamine) alone and in combination on iron‐induced membrane lipid damage in rat liver homogenates were examined by estimating levels of malondialdehyde and 4‐hydroxyalkenals (MDA + 4‐HDA). Individually, melatonin (2.5–1600 μM), vitamin E (0.5–50 μM), glutathione (100–7000 μM) and desferrioxamine (1–8 μM) inhibited lipid peroxidation in a concentration‐dependent manner. Vitamin C had both a pro‐oxidative (25–2000 μM) and an antioxidative (2600–5000 μM) effect. The IC50 (concentration that reduces damage by 50%) values were 4, 10, 426, 2290 and 4325 μM for vitamin E, desferrioxamine, melatonin, glutathione and vitamin C, respectively. The synergistic actions of melatonin with vitamin C, vitamin E, and glutathione were systematically investigated. When melatonin was combined with vitamin E, glutathione, or vitamin C, the protective effects against iron‐induced lipid peroxidation were dramatically enhanced. Even though melatonin was added at very low concentrations, it still showed synergistic effects with other antioxidants at certain concentrations. Furthermore, melatonin not only reversed the pro‐oxidative effects of vitamin C, but its efficacy in reducing lipid peroxidation was improved when it was combined with prooxidative concentrations of vitamin C. The results provide new information in terms of the possible pharmacological use of the combination of melatonin and classical antioxidants to treat free radical‐related conditions.

DNA oxidatively damaged by chromium(III) and H2O2 is protected by the antioxidants melatonin, N1-acetyl-N2-formyl-5-methoxykynuramine, resveratrol and uric acid

Chromium (Cr) compounds are widely used industrial chemicals and well known carcinogens. Cr(III) was earlier found to induce oxidative damage as documented by examining the levels of 8-hydroxydeoxyguanosine (8-OH-dG), an index for DNA damage, in isolated calf thymus DNA incubated with CrCl(3) and H(2)O(2). In the present in vitro study, we compared the ability of the free radical scavengers melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), resveratrol and uric acid to reduce DNA damage induced by Cr(III). Each of these scavengers markedly reduced the DNA damage in a concentration-dependent manner. The concentrations that reduced 8-OH-dG formation by 50% (IC(50)) were 0.10 microM for both resveratrol and melatonin, and 0.27 microM for AFMK. However, the efficacy of the fourth endogenous antioxidant, i.e. uric acid, in terms of its inhibition of DNA damage in the same in vitro system was about 60--150 times less effective than the other scavengers; the IC(50) for uric acid was 15.24 microM. These findings suggest that three of the four antioxidants tested in these studies may have utility in protecting against the environmental pollutant Cr and that the protective effects of these free radical scavengers against Cr(III)-induced carcinogenesis may relate to their direct hydroxyl radical scavenging ability. In the present study, the formation of 8-OH-dG was likely due to a Cr(III)-mediated Fenton-type reaction that generates hydroxyl radicals, which in turn damage DNA. Once formed, 8-OH-dG can mutate eventually leading to cancer; thus the implication is that these antioxidants may reduce the incidence of Cr-related cancers.

Anticarcinogenic actions of melatonin which involve antioxidative processes: comparison with other antioxidants

The complex processes of carcinogenesis often involve oxidative stress. Numerous indicators of oxidative damage are enhanced as the result of the action of carcinogens. Several antioxidants, with different efficacies, protect against oxidative abuse caused by carcinogens. Recently, melatonin (N-acetyl-5-methoxytryptamine) and related indoleamines have attracted attention because of their high antioxidant and anticarcinogenic activity. Some antioxidants, e.g. ascorbic acid, play an ambivalent role in antioxidative defense, since, under specific conditions, they are strongly prooxidant. Among known antioxidants, melatonin has been an often investigated experimental agent in reducing cancer initiation and inhibiting the growth of established tumors. The indoleamine has been shown to protect macromolecules from oxidative mutilation induced by carcinogens. In these studies, a variety of in vitro and in vivo models were used and numerous indices of oxidative damage were evaluated. The protective effects of melatonin and several other indoleamine antioxidants against cellular damage caused by carcinogens make them potential supplements in the treatment or co-treatment at several stages of cancer.

Characterization of the protective effects of melatonin and related indoles against α‐naphthylisothiocyanate‐induced liver injury in rats

The protective effect of melatonin, 6‐hydroxymelatonin and N‐acetylserotonin against α‐naphthylisothiocyanate (ANIT)‐induced liver injury was investigated and compared in rats injected once with the hepatotoxicant (75 mg/kg body weight). In rats injected with ANIT alone, liver injury with cholestasis developed within 24 h, as indicated by both serum levels of alanine aminotransferase (SGPT) and aspartic acid aminotransferase (SGOT) activities and serum total bilirubin concentration. The administration of melatonin or 6‐hydroxymelatonin (10 mg/kg body weight) to ANIT‐injected rats reduced significantly the serum levels of both SGPT and SGOT and the serum total bilirubin concentration. For all hepatic biochemical markers, melatonin was more effective that 6‐hydroxymelatonin. By comparison, the administration of N‐acetylserotonin (10 mg/kg body weight) to ANIT‐injected rats did not reduce the serum levels of either hepatic enzymes or the serum total bilirubin concentration. In ANIT‐injected rats, hepatic lipid peroxidation (LPO) was significantly higher than in control animals and this increase was significantly reduced by either melatonin, 6‐hydroxymelatonin or N‐acetylserotonin. Furthermore, ANIT treatment caused a significant reduction in liver microsomal membrane fluidity and this reduction was completely reversed by the three indoles. The liver from ANIT‐injected rats showed several histopathological alterations; above all there was an acute infiltration of polymorphonuclear neutrophils and an increase in the number of apparent apoptotic hepatocytes. The concurrent administration of melatonin reduced the severity of all morphological alterations, specially the neutrophil infiltration and the number of presumed apoptotic cells. On the contrary, the administration of 6‐hydroxymelatonin or N‐acetylserotonin did not provide any protective effect in terms of the histopathological alterations. These results indicate that melatonin protects against ANIT‐induced liver injury with cholestasis in rats, and suggests that this protective effect is likely due to its antioxidant properties and above all to its capacity to inhibit liver neutrophil infiltration, a critical factor in the pathogenesis of ANIT‐induced liver injury. 6‐hydroxymelatonin, although able to provide partial protection against the ANIT‐induced hepatic injury, probably through its antioxidant properties by mechanisms that are unclear, was unable to reduce neutrophil infiltration. Finally, N‐acetylserotonin in the experimental conditions of this study, only exhibited some antioxidant protection but had no protective effect against ANIT‐induced hepatic damage. J. Cell. Biochem. 80:461–470, 2001.

Induction of lipid peroxidation in hamster organs by the carcinogen cadmium: Amelioration by melatonin

Cadmium is a well-known human carcinogen. Lipid peroxidation is involved in cadmium-related toxicity and carcinogenesis. Melatonin is an effective antioxidant and free radical scavenger. The potential protective effects of melatonin against cadmium-induced lipid peroxidation in hamster brain, heart, kidney, testes, lung, and liver were examined. Lipid peroxidation was induced by intraperitoneal injection of cadmium chloride [single dose of 1 mg/kg body weight (bw)]. To test whether melatonin would protect against the toxicity of the carcinogen, the melatonin was injected peritoneally at a dose of either 15 mg/kg bw or 5 mg/kg bw, 0.5 h before cadmium treatment and thereafter at 8 h intervals during the day in the 48 h interval following the cadmium injection. One group of hamsters received only a single melatonin injection (a dose of 15 mg/kg bw, 30 min prior to cadmium). Forty-eight hours after cadmium injection, lipid peroxidation increased in brain, heart, kidney, testes, and lung. Either multiple injections of melatonin at both the 5 and 15 mg/kg bw doses, or a single injection of 15 mg/kg bw, prevented the cadmium-related increases in lipid peroxidation in brain, heart and lung. Cadmium-induced lipid peroxidation in kidney was prevented by melatonin when it was given as a single dose of 15 mg/kg bw. Melatonin slightly, but not significantly, reduced cadmium-induced lipid peroxidation in testes. It is concluded that cadmium toxicity, at least with regard to the resulting lipid peroxidation, is reduced by administering melatonin.