Ewa Sewerynek

A review of the evidence supporting melatonin's role as an antioxidant

Abstract: This survey summarizes the findings, accumulated within the last 2 years, concerning melatonins role in defending against toxic free radicals. Free radicals are chemical constituents that have an unpaired electron in their outer or‐bital and, because of this feature, are highly reactive. Inspired oxygen, which sustains life, also is harmful because up to 5% of the oxygen (O2) taken in is converted to oxygen‐free radicals. The addition of a single electron to O2 produces the superoxide anion radical (O2); C2: is catalytic‐reduced by superoxide dismutase, to hydrogen peroxide (H2O2). Although H2O2 is not itself a free radical, it can be toxic at high concentrations and, more importantly, it can be reduced to the hydroxyl radical (OH). The OH is the most toxic of the oxygen‐based radicals and it wreaks havoc within cells, particularly with macromolecules. In recent in vitro studies, melatonin was shown to be a very efficient neutralizer of the OH; indeed, in the system used to test its free radical scavenging ability it was found to be significantly more effective than the well known antioxidant, glutathione (GSH), in doing so. Likewise, melatonin has been shown to stimulate glutathione peroxidase (GSH‐Px) activity in neural tissue; GSH‐PX metabolizes reduced glutathione to its oxidized form and in doing so it converts H2O2 to H2O, thereby reducing generation of the OH by eliminating its precursor. More recent studies have shown that melatonin is also a more efficient scavenger of the peroxyl radical than is vitamin E. The peroxyl radical is generated during lipid peroxidation and propagates the chain reaction that leads to massive lipid destruction in cell membranes. In vivo studies have demonstrated that melatonin is remarkably potent in protecting against free radical damage induced by a variety of means. Thus, DNA damage resulting from either the exposure of animals to the chemical carcinogen safrole or to ionizing radiation is markedly reduced when melatonin is co‐administered. Likewise, the induction of cataracts, generally accepted as being a consequence of free radical attack on lenticular macromolecules, in newborn rats injected with a GSH‐depleting drug are prevented when the animals are given daily melatonin injections. Also, paraquat‐induced lipid peroxidation in the lungs of rats is overcome when they also receive melatonin during the exposure period. Paraquat is a highly toxic herbicide that inflicts at least part of its damage by generating free radicals. Finally, bacterial endotoxin (lipopolysaccharide or LPS)‐induced free radical damage to a variety of organs is highly significantly reduced when melatonin is also administered; LPS, like paraquat, produces at least part of its damage to cells by inducing the formation of free radicals. Physiological melatonin concentrations have also been shown to inhibit the nitric oxide (NO)‐generting enzyme, nitric oxide synthase. The reduction of NO‐ production would contribute to melatonins antioxidant action since NO‐ can generate the peroxynitrite anion, which can degrade into the OH. Thus, melatonin seems to have multiple ways either to reduce free radical generation or, once produced, to neutralize them. Melatonin accomplishes these actions without membrane receptors, indicating that the indole has important metabolic functions in every cell in the organism, not only those that obviously contain membrane receptors for this molecule.

Rhythms of glutathione peroxidase and glutathione reductase in brain of chick and their inhibition by light

Melatonin was recently shown to be a component of the antioxidative defense system of organisms due to its free radical scavenging and antioxidant activities. Pharmacologically, melatonin stimulates the activity of the peroxide detoxifying enzyme glutathione peroxidase in rat brain and in several tissues of chicks. In this report, we studied the endogenous rhythm of two antioxidant enzymes, glutathione peroxidase and glutathione reductase, in five regions (hippocampus, hypothalamus, striatum, cortex and cerebellum) of chick brain and correlated them with physiological blood melatonin concentrations. Glutathione peroxidase exhibited a marked 24 h rhythm with peak activity in each brain region which had acrophases about 8 h after lights off and about 4 h after the serum melatonin peak was detected. Glutathione reductase activity exhibited similar robust rhythms with the peaks occurring roughly 2 h after those of glutathione peroxidase. We suggest that neural glutathione peroxidase increases due to the rise of nocturnal melatonin levels while glutathione reductase activity rises slightly later possibly due to an increase of its substrate, oxidized glutathione. The exposure of chicks to constant light for 6 days eliminated the melatonin rhythm as well as the peaks in both glutathione peroxidase and glutathione reductase activities. These findings suggest that the melatonin rhythm may be related to the nighttime increases in the enzyme activities, although other explanations cannot be excluded.

Melatonin reduces both basal and bacterial lipopolysaccharide-induced lipid peroxidation in vitro

The protective effect of melatonin against lipopolysaccharide (LPS)-induced oxidative damage was examined in vitro. Lung, liver, and brain malonaldehyde (MDA) plus 4-hydroxyalkenals (4-HDA) concentrations were measured as indices of induced membrane peroxidative damage. Homogenates of brain, lung, and liver were incubated with LPS at concentrations of either 1, 10, 50, 200, or 400 micrograms/ml for 1 h and, in another study, LPS at a concentration of 400 micrograms/ml for either 0, 15, 30, or 60 min. Melatonin at increasing concentrations from 0.01-3 mM either alone or together with LPS (400 micrograms/ml) was used. Liver, brain, and lung MDA + 4-HDA levels increased after LPS at concentrations of 10, 50, 200 or 400 micrograms/ml; this effect was concentration-dependent. The highest levels of lipid peroxidation products were observed after tissues were incubated with an LPS concentration of 400 micrograms/ml for 60 min; in liver and lung this effect was totally suppressed by melatonin and partially suppressed in brain in a concentration-dependent manner. In addition, melatonin alone was effective in brain at concentrations of 0.1 to 3 mM, in lung at 2 to 3 mM, and in liver at 0.1 to 3 mM; in all cases, the inhibitory effects of melatonin on lipid peroxidation were always directly correlated with the concentration of melatonin in the medium. The results show that the direct effect of LPS on the lipid peroxidation following endotoxin exposure is markedly reduced by melatonin.

Paraquat toxicity and oxidative damage: Reduction by Melatonin

The ability of melatonin to protect against paraquat-induced oxidative damage in rat lung, liver, and serum was examined. Changes in the levels of malondialdehyde (MDA) plus 4-hydroxyalkenals (4-HDA) and reduced and oxidized glutathione concentrations were measured. Paraquat (50 mg/kg) was injected i.p. into either Sprague-Dawley or Wistar rats with or without the co-administration of 5 mg/kg melatonin. Paraquat alone increased MDA + 4-HDA levels in serum and lungs of both rat strains, with these increases being abolished by melatonin co-treatment. Paraquat also decreased reduced glutathione levels and increased oxidized glutathione concentrations in lung and liver; these changes were negated by melatonin. The effect of melatonin on paraquat-induced mortality was also studied. Paraquat at a dose of 79 mg/kg was lethal for 50% of animals within 24 hr; when administered together with melatonin, the LD50 for paraquat increased to 251 mg/kg.

Lipopolysaccharide-induced hepatotoxicity is inhibited by the antioxidant melatonin

Oxidative damage to the liver of lipopolysaccharide-treated rats was evaluated using four parameters: level of lipid peroxidation, changes in total GSH and GSSG concentrations and hepatic morphology. Bacterial lipopolysaccharide (10 mg/kg b.w.) was injected i.p. either at 6, 16 or 24 h before animals were killed. Lipopolysaccharide increased lipid peroxidation most dramatically when it is injected 6 h before killing. Hepatic total GSH increased after lipopolysaccharide in a time-dependent manner. The highest level of GSSG and largest GSSG/total GSH ratio were also observed in the group of animals injected with lipopolysaccharide 6 h before tissue collection. In a second study, lipopolysaccharide was injected 6 h before the animals were killed, with or without 1 mg/kg b.w. melatonin. Melatonin totally abolished lipopolysaccharide-induced increase in lipid peroxidation, exaggerated the rise in total GSH and reversed the increase in GSSG concentration. The liver showed obvious histological degenerative changes after lipopolysaccharide, effects that were counteracted by melatonin administration. The protection conferred by melatonin is presumably due to its antioxidant activity.

Melatonin counteracts lipid peroxidation induced by carbon tetrachloride but does not restore glucose-6 phosphatase activity

Abstract: Carbon tetrachloride (CC14) exerts its toxic effects by the generation of free radicals. In this study we investigated whether melatonin, a potent free radical scavenger, could prevent the deleterious effects of CC14. Liver homogenates and liver microsomes were incubated with CCI4 in the presence of melatonin and lipid peroxidation and glucose‐6 phosphatase (G6Pase) activity were determined. All doses of CC14 (1, 0.5, 0.1 raM) produced significantly high levels of lipid peroxidation, as reflected by increased levels of malonaldehyde and 4‐hydroxyalkenals, in both liver homogenates and liver microsomes. These doses of CC14 concommitantly reduced the activity of microsomal G6Pase. Co‐incubation with melatonin dose‐dependently (2, 1, 0.5 raM) inhibited the production of lipid peroxidation, but it was unable to restore the activity of G6Pase. In in vivo studies, rats were also treated with melatonin (10 mg/kg, i.p.), given 30 min before and 60 min after the administration of CC14 (5 ml/kg, i.p.). Significantly elevated levels of lipid peroxidation were measured in the liver and kidney. Melatonin prevented the CCl4‐induced lipid peroxidation in the kidney, but not in the liver. These data suggest that melatonin may provide protection against some of the damaging effects of CCI4, possibly due to its ability to scavenge toxic free radicals.

Melatonin prevents increases in neural nitric oxide and cyclic GMP production after transient brain ischemia and reperfusion in the Mongolian gerbil (Meriones Unguiculatus)

ABSTRACT: While nitric oxide (NO) has been implicated as a mediator of glutamate excitotoxicity after cerebral ischemia/reperfusion, melatonin has been reported to inhibit brain NO production by suppressing nitric oxide synthase. The purpose of the present studies was to determine the effect of exogenous melatonin administration on NO‐induced changes during brain ischemia/reperfusion. Indicators of cerebral cortical and cerebellar NO production [nitrite/nitrate levels and cyclic guanosine monophosphate(cGMP)] were used to estimate neural changes after transient bilateral carotid artery ligation followed by reperfusion in adult Mongolian gerbils (Meriones unguiculatus). Results show for the first time that melatonin prevents the increases in NO and cGMP production after transient ischemia/reperfusion in frontal cerebral cortex and cerebellum of Mongolian gerbils. The inhibitory effect of melatonin on NO production and its ability to scavenge free radicals and the peroxynitrite anion may be responsible for the protective effect of melatonin on neuronal structures during transient ischemia followed by reperfusion.

Melatonin reduces H2O2-induced lipid peroxidation in homogenates of different rat brain regions.

Abstract: The ability of melatonin to modify H2O2‐induced lipid peroxidation in brain homogenates was determined. The concentrations of brain malonaldehyde (MDA) and 4‐hydroxyalkenals (4‐HDA) were assayed as an index of induced membrane oxidative damage. Homogenates from five different regions of the brain (cerebral cortex, cerebellum, hippocampus, hypothalamus, and corpus striatum) derived from two different strains of rats, Sprague‐Dawley and Wistar, were incubated with either H2O2 (5 mM) alone or H2O2 together with melatonin at increasing concentrations ranging from 0.1 to 4 mM. The basal level of lipid peroxidation was strain‐dependent and about 100% higher in homogenates from the brain of Wistar rats than those measured in Sprague‐Dawley rats. MDA + 4‐HDA levels increased after H2O2 treatment in homogenates obtained from each region of the brain in both rat strains but the sensitivity of the homogenates from Sprague‐Dawley rats was greater than that for the homogenates from Wistar rats (increases after H2O2from 45 to 165% compared 20 to 40% for Sprague‐Dawley and Wistar rats, respectively). Melatonin co‐treatment reduced H202‐induced lipid peroxidation in brain homogenates in a concentration‐dependent manner; the degree of protection against lipid peroxidation was similar in all brain regions.

Suppressive effect of melatonin administration on ethanol-induced gastroduodenal injury in rats in vivo

Melatonin protection against ethanol‐induced gastroduodenal injury was investigated in duodenum‐ligated rats. Melatonin, injected i.p. 30 min before administration of 1 ml of absolute ethanol, given by gavage, significantly decreased ethanol‐induced macroscopic, histological and biochemical changes in the gastroduodenal mucosa. Ethanol‐induced lesions were detectable as haemorrhagic streaks. Ethanol administration damaged 36% and 25% of the total gastric and duodenal surface, respectively. Melatonin treatment reduced ethanol‐induced gastric and duodenal damage to 14% and 8%, respectively. When indomethacin was given together with ethanol, the gastric damaged area was 44% of the total surface, while the duodenal damaged area was 35%; melatonin administration reduced the damage to only 13% of the total gastric surface and to 12% of total duodenal surface. Both stomach and duodenum of ethanol‐treated animals showed polymorphonuclear leukocyte (PMN) infiltration. The number of PMN increased more than 600 and 200 times in stomach and duodenum, respectively, following ethanol administration. Melatonin treatment reduced ethanol‐induced PMN infiltration by 38% in the stomach and 20% in the duodenum. In indomethacin‐ethanol‐treated rats, the number of PMN increased by 875% compared to control group in the stomach and by 264% in duodenum. Melatonin administration reduced the indomethacin‐ethanol‐induced PMN rise by 57% in the stomach and 40% in the duodenum. Gastroduodenal total glutathione (tGSH) concentration and glutathione reductase (GSSG‐Rd) activity were significantly reduced following ethanol and indomethacin‐ethanol administration. Melatonin ameliorated both the decrease in tGSH concentration as well as the reduction of GSSG‐Rd activity elicited by ethanol both in the stomach and duodenum; melatonin was effective against indomethacin‐ethanol‐induced damage only in the stomach. Ethanol‐induced gastroduodenal damage is believed to be mediated by the generation of free radicals. Recently, a number of in vivo and in vitro experiments have shown melatonin to be an effective antioxidant and free radical scavenger; thus, we conclude that the protection by melatonin against ethanol‐induced gastroduodenal injury is due, at least in part, to its radical scavenging activity.

H2O2 induced lipid peroxidation in rat brain homogenates is greatly reduced by melatonin

Melatonin protection against H2O2-induced lipid peroxidation in brain homogenates was measured in vitro. The level of malonaldehyde (MDA) plus 4-hydroxyalkenals (4-HDA) was assayed in brain homogenates as an index of induced membrane oxidative damage. Brain homogenates were co-incubated with H2O2 alone or in combination with melatonin. Brain MDA + 4-HDA increased after H2O2 (0.1-5 mM) with the effect being both concentration- and time-dependent. Melatonin (0.1-5 mM) protected against H2O2-induced lipid peroxidation of brain homogenates in a concentration-dependent manner.