Darío Acuña-Castroviejo

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.

Melatonin mitigates mitochondrial malfunction

Abstract:  Melatonin, or N‐acetyl‐5‐methoxytryptamine, is a compound derived from tryptophan that is found in all organisms from unicells to vertebrates. This indoleamine may act as a protective agent in disease conditions such as Parkinsons, Alzheimers, aging, sepsis and other disorders including ischemia/reperfusion. In addition, melatonin has been proposed as a drug for the treatment of cancer. These disorders have in common a dysfunction of the apoptotic program. Thus, while defects which reduce apoptotic processes can exaggerate cancer, neurodegenerative disorders and ischemic conditions are made worse by enhanced apoptosis. The mechanism by which melatonin controls cell death is not entirely known. Recently, mitochondria, which are implicated in the intrinsic pathway of apoptosis, have been identified as a target for melatonin actions. It is known that melatonin scavenges oxygen and nitrogen‐based reactants generated in mitochondria. This limits the loss of the intramitochondrial glutathione and lowers mitochondrial protein damage, improving electron transport chain (ETC) activity and reducing mtDNA damage. Melatonin also increases the activity of the complex I and complex IV of the ETC, thereby improving mitochondrial respiration and increasing ATP 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. 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.

Free radical-mediated molecular damage. Mechanisms for the protective actions of melatonin in the central nervous system.

Abstract: This review briefly summarizes the multiple actions by which melatonin reduces the damaging effects of free radicals and reactive oxygen and nitrogen species. It is well documented that melatonin protects macromolecules from oxidative damage in all subcellular compartments. This is consistent with the protection by melatonin of lipids and proteins, as well as both nuclear and mitochondrial DNA. Melatonin achieves this widespread protection by means of its ubiquitous actions as a direct free radical scavenger and an indirect antioxidant. Thus, melatonin directly scavenges a variety of free radicals and reactive species including the hydroxyl radical, hydrogen peroxide, singlet oxygen, nitric oxide, peroxynitrite anion, and peroxynitrous acid. Furthermore, melatonin stimulates a number of antioxidative enzymes including superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase. Additionally, melatonin experimentally enhances intracellular glutathione (another important antioxidant) levels by stimulating the rate‐limiting enzyme in its synthesis, γ‐glutamylcysteine synthase. Melatonin also inhibits the proxidative enzymes nitric oxide synthase and lipoxygenase. Finally, there is evidence that melatonin stabilizes cellular membranes, thereby probably helping them resist oxidative damage. Most recently, melatonin has been shown to increase the efficiency of the electron transport chain and, as a consequence, to reduce election leakage and the generation of free radicals. These multiple actions make melatonin a potentially useful agent in the treatment of neurological disorders that have oxidative damage as part of their etiological basis.

Melatonin, mitochondria, and cellular bioenergetics

Aerobic cells use oxygen for the production of 90–95% of the total amount of ATP that they use. This amounts to about 40 kg ATP/day in an adult human. The synthesis of ATP via the mitochondrial respiratory chain is the result of electron transport across the electron transport chain coupled to oxidative phosphorylation. Although ideally all the oxygen should be reduced to water by a four‐electron reduction reaction driven by the cytochrome oxidase, under normal conditions a small percentage of oxygen may be reduced by one, two, or three electrons only, yielding superoxide anion, hydrogen peroxide, and the hydroxyl radical, respectively. The main radical produced by mitochondria is superoxide anion and the intramitochondrial antioxidant systems should scavenge this radical to avoid oxidative damage, which leads to impaired ATP production. During aging and some neurodegenerative diseases, oxidatively damaged mitochondria are unable to maintain the energy demands of the cell leading to an increased production of free radicals. Both processes, i.e., defective ATP production and increased oxygen radicals, may induce mitochondrial‐dependent apoptotic cell death. Melatonin has been reported to exert neuroprotective effects in several experimental and clinical situations involving neurotoxicity and/or excitotoxicity. Additionally, in a series of pathologies in which high production of free radicals is the primary cause of the disease, melatonin is also protective. A common feature in these diseases is the existence of mitochondrial damage due to oxidative stress. The discoveries of new actions of melatonin in mitochondria support a novel mechanism, which explains some of the protective effects of the indoleamine on cell survival.

Melatonin but not vitamins C and E maintains glutathione homeostasis in t-butyl hydroperoxide-induced mitochondrial oxidative stress

Mitochondria do not contain catalase and are therefore largely dependent on reduced glutathione (GSH) and glutathione peroxidases for its antioxidant protection. When GSH levels are greatly decreased, hydrogen peroxide accumulates leading to extensive mitochondrial damage. Melatonin has antioxidant properties and prevents toxic effects of reactive oxygen species by maintaining cellular GSH homeostasis. Thus, we examined the influence of melatonin and other classical antioxidants such as vitamins C and E on GSH content and the activity of the GSH‐related enzymes (glutathione peroxidase and reductase) in isolated rat liver and brain mitochondria treated with t‐butyl hydroperoxide (i‐BHP). In control mitochondria melatonin (100 nM) significantly increases GSH content and glutathione peroxidase and reductase activities. After incubation with 100 μM i‐BHP, the mitochondrial hydroperoxides level increased, 90% of mitochondrial GSH was oxidized to GSSH, and the activities of GSH‐related enzymes were almost totally inhibited. Melatonin (100 nM) counteracted the changes in GSH, GSH‐related enzymes and hydroperoxides induced by i‐BHP in cultured mitochondria. In the presence of 100 nM melatonin, the activity of the respiratory chain complexes I and IV, measured in submitochondrial particles prepared from rat liver and brain mitochondria, increased significantly. Vitamin C was virtually without effect, and only 1 mM vitamin E increased GSH and reduced hydroperoxide mitochondrial contents. Our results suggest that melatonin, but not vitamins C and E, prevents the toxic effects of hydroperoxides on mitochondria by regenerating their GSH content.

Extrapineal melatonin: sources, regulation, and potential functions

Endogenous melatonin is synthesized from tryptophan via 5-hydroxytryptamine. It is considered an indoleamine from a biochemical point of view because the melatonin molecule contains a substituted indolic ring with an amino group. The circadian production of melatonin by the pineal gland explains its chronobiotic influence on organismal activity, including the endocrine and non-endocrine rhythms. Other functions of melatonin, including its antioxidant and anti-inflammatory properties, its genomic effects, and its capacity to modulate mitochondrial homeostasis, are linked to the redox status of cells and tissues. With the aid of specific melatonin antibodies, the presence of melatonin has been detected in multiple extrapineal tissues including the brain, retina, lens, cochlea, Harderian gland, airway epithelium, skin, gastrointestinal tract, liver, kidney, thyroid, pancreas, thymus, spleen, immune system cells, carotid body, reproductive tract, and endothelial cells. In most of these tissues, the melatonin-synthesizing enzymes have been identified. Melatonin is present in essentially all biological fluids including cerebrospinal fluid, saliva, bile, synovial fluid, amniotic fluid, and breast milk. In several of these fluids, melatonin concentrations exceed those in the blood. The importance of the continual availability of melatonin at the cellular level is important for its physiological regulation of cell homeostasis, and may be relevant to its therapeutic applications. Because of this, it is essential to compile information related to its peripheral production and regulation of this ubiquitously acting indoleamine. Thus, this review emphasizes the presence of melatonin in extrapineal organs, tissues, and fluids of mammals including humans.

Extrapineal melatonin: analysis of its subcellular distribution and daily fluctuations

Abstract:  We studied the subcellular levels of melatonin in cerebral cortex and liver of rats under several conditions. The results show that melatonin levels in the cell membrane, cytosol, nucleus, and mitochondrion vary over a 24‐hr cycle, although these variations do not exhibit circadian rhythms. The cell membrane has the highest concentration of melatonin followed by mitochondria, nucleus, and cytosol. Pinealectomy significantly increased the content of melatonin in all subcellular compartments, whereas luzindole treatment had little effect on melatonin levels. Administration of 10 mg/kg bw melatonin to sham‐pinealectomized, pinealectomized, or continuous light‐exposed rats increased the content of melatonin in all subcellular compartments. Melatonin in doses ranging from 40 to 200 mg/kg bw increased in a dose‐dependent manner the accumulation of melatonin on cell membrane and cytosol, although the accumulations were 10 times greater in the former than in the latter. Melatonin levels in the nucleus and mitochondria reached saturation with a dose of 40 mg/kg bw; higher doses of injected melatonin did not further cause additional accumulation of melatonin in these organelles. The results suggest some control of extrapineal accumulation or extrapineal production of melatonin and support the existence of regulatory mechanisms in cellular organelles, which prevent the intracellular equilibration of the indolamine. Seemingly, different concentrations of melatonin can be maintained in different subcellular compartments. The data also seem to support a requirement of high doses of melatonin to obtain therapeutic effects. Together, these results add information that assists in explaining the physiology and pharmacology of melatonin.

Melatonin inhibits expression of the inducible NO synthase II in liver and lung and prevents endotoxemia in lipopolysaccharide-induced multiple organ dysfunction syndrome in rats

We evaluated the role of melatonin in endotoxemia caused by lipopolysaccharide (LPS) in unanesthetized rats. The expression of inducible isoform of nitric oxide synthase (iNOS) and the increase in the oxidative stress seem to be responsible for the failure of lungs, liver, and kidneys in endotoxemia. Bacterial LPS (10 mg/kg b.w) was i.v. injected 6 h before rats were killed and melatonin (10–60 mg/kg b.w.) was i.p. injected before and/or after LPS. Endotoxemia was associated with a significant rise in the serum levels of aspartate and alanine aminotransferases, γ‐glutamyl‐transferase, alkaline phosphatase, creatinine, urea, and uric acid, and hence liver and renal dysfunction. LPS also increased serum levels of cholesterol and triglycerides and reduced glucose levels. Melatonin administration counteracted these organ and metabolic alterations at doses ranging between 20 and 60 mg/kg b.w. Melatonin significantly decreased lung lipid peroxidation and counteracted the LPS‐induced NO levels in lungs and liver. Our results also show an inhibition of iNOS activity in rat lungs by melatonin in a dose‐dependent manner. Expression of iNOS mRNA in lungs and liver was significantly decreased by melatonin (60 mg/kg b.w., 58–65%). We conclude that melatonin inhibits NO production mainly by inhibition of iNOS expression. The inhibition of NO levels may account for the protection of the indoleamine against LPS‐induced endotoxemia in rats.—Crespo, E., Macías, M., Pozo, D., Escames, G., Martín, M., Vives, F., Guerrero, J. M., Acuña‐Castroviejo, D. Melatonin inhibits expression of the inducible NO synthase II in liver and lung and prevents endotoxemia in lipopolysaccharide‐induced multiple organ dysfunction syndrome in rats. FASEB J. 13, 1537–1546 (1999)

Melatonin-induced increased activity of the respiratory chain complexes I and IV can prevent mitochondrial damage induced by ruthenium red in vivo

Melatonin displays antioxidant and free radical scavenger properties. Due to its ability with which it enters cells, these protective effects are manifested in all subcellular compartments. Recent studies suggest a role for melatonin in mitochondrial metabolism. To study the effects of melatonin on this organelle we used ruthenium red to induce mitochondrial damage and oxidative stress. The results show that melatonin (10 mg/kg i.p.) can increase the activity of the mitochondrial respiratory complexes I and IV after its administration in vivo in a time‐dependent manner; these changes correlate well with the half‐life of the indole in plasma. Melatonin administration also prevented the decrease in the activity of complexes I and IV due to ruthenium red (60 μg/kg i.p.) administration. At this dose, ruthenium red did not induce lipid peroxidation but it significantly reduced the activity of the antioxidative enzyme glutathione peroxidase, an effect also counteracted by melatonin. These results suggest that melatonin modulates mitochondrial respiratory activity, an effect that may account for some of the protective properties of the indoleamine. The mitochondria‐modulating role of melatonin may be of physiological significance since it seems that the indoleamine is concentrated into normal mitochondria. The data also support a pharmacological use of melatonin in drug‐induced mitochondrial damage in vivo.

Mitochondria and chloroplasts as the original sites of melatonin synthesis: a hypothesis related to melatonin's primary function and evolution in eukaryotes

Mitochondria and chloroplasts are major sources of free radical generation in living organisms. Because of this, these organelles require strong protection from free radicals and associated oxidative stress. Melatonin is a potent free radical scavenger and antioxidant. It meets the criteria as a mitochondrial and chloroplast antioxidant. Evidence has emerged to show that both mitochondria and chloroplasts may have the capacity to synthesize and metabolize melatonin. The activity of arylalkylamine N‐acetyltransferase (AANAT), the reported rate‐limiting enzyme in melatonin synthesis, has been identified in mitochondria, and high levels of melatonin have also been found in this organelle. From an evolutionary point of view, the precursor of mitochondria probably is the purple nonsulfur bacterium, particularly, Rhodospirillum rubrum, and chloroplasts are probably the descendents of cyanobacteria. These bacterial species were endosymbionts of host proto‐eukaryotes and gradually transformed into cellular organelles, that is, mitochondria and chloroplasts, respectively, thereby giving rise to eukaryotic cells. Of special importance, both purple nonsulfur bacteria (R. rubrum) and cyanobacteria synthesize melatonin. The enzyme activities required for melatonin synthesis have also been detected in these primitive species. It is our hypothesis that mitochondria and chloroplasts are the original sites of melatonin synthesis in the early stage of endosymbiotic organisms; this synthetic capacity was carried into host eukaryotes by the above‐mentioned bacteria. Moreover, their melatonin biosynthetic capacities have been preserved during evolution. In most, if not in all cells, mitochondria and chloroplasts may continue to be the primary sites of melatonin generation. Melatonin production in other cellular compartments may have derived from mitochondria and chloroplasts. On the basis of this hypothesis, it is also possible to explain why plants typically have higher melatonin levels than do animals. In plants, both chloroplasts and mitochondria likely synthesize melatonin, while animal cells contain only mitochondria. The high levels of melatonin produced by mitochondria and chloroplasts are used to protect these important cellular organelles against oxidative stress and preserve their physiological functions. The superior beneficial effects of melatonin in both mitochondria and chloroplasts have been frequently reported.