Introduction

The free-radical theory of aging has gained acceptance and matured in the 50 years since it was proposed by Harman in 1956 (1,2). In addition to free radicals, other cellular reactive byproducts of normal metabolism can lead to biomolecule damage. This had led to the formation of the “oxidative damage theory,” which suggests reactive oxygen species (ROS) are responsible for the accumulation of age-related cellular debris, and that this damage is an important contributor to aging and/or chronic diseases (3). In contrast to a century ago, when infectious diseases were a major cause of mortality, today noninfectious chronic diseases are the most likely causes of death. Disease epidemics (for example, cardiovascular diseases, type 2 diabetes, obesity, and metabolic syndrome) have become major contributors to the burden of disease and are presently emerging, or accelerating, in most developing countries (4). The baby-boomer generation is advancing in age, and the geriatric population in the developed countries will increase tremendously in the next few decades (5). More than 50% of the American public is aware of antiaging therapies, and most people currently use some form of these therapies (6). In excess of 70% of the American population uses dietary supplements daily, most commonly antioxidant vitamin and mineral supplements (7). Likewise, while the use of alternative therapies is on the rise, progress is being made in understanding aging, and the public has more access to this information (through media and the internet) than ever before.

In theory, antiaging interventions should modify the biochemical and molecular events causing aging and chronic diseases, correct physiological changes responsible for symptoms and signs of aging, and decrease the susceptibility to disease associated with aging. Vitamin A and its precursor β-carotene, vitamin C, and vitamin E are the most frequently used antioxidant vitamins. The idea that the pathophysiologic processes caused exclusively by oxygen-derived free radicals could presumably be alleviated by conventional antioxidants, such as vitamins E or C, and/or intracellular enzymatic antioxidants seems worthy of investigation. Several studies, however, have found that antioxidants offered little protection against aging or chronic diseases.

Investigators in the Physicians’ Health Study evaluated data from 83,639 U.S. male physicians, of whom 29% were taking vitamin E, vitamin C, or multivitamin supplements on a self-selected basis (8). The the study report concluded that these supplements were not associated with a significant reduction in total cardiovascular diseases or coronary heart disease mortality. Another prospective study revealed that vitamins E and C and certain carotenoids did not reduce the risk of stroke in 43,738 men 40 to 75 years old who had no cardiovascular disease or diabetes (9). The Heart Outcome Prevention Evaluation trial (10), a randomized controlled trial of patients 55 years old or older who had cardiovascular disease or diabetes, demonstrated that taking 400 IU vitamin E daily for an average of 4.5 years had no influence on cardiovascular outcomes or nephropathy. A randomized, double-blind, placebo-controlled trial of vitamin E revealed, interestingly, that vitamin E supplementation resulted in increased blood pressure in type 2 diabetic patients (11). The authors of this study concluded that the mechanism for this increase remains unknown but appears to be independent of changes in oxidative stress. A metaanalysis of a large body of published data revealed that antioxidant vitamins do not significantly reduce risk of cardiovascular death (12). Moreover, an additional metaanalysis of 11 trials revealed that high doses of vitamin E resulted in slightly but significantly higher rates of all-cause mortality compared with placebo (13). A recent systematic review and metaanalysis that included 68 randomized trials with 232,606 participants (385 publications) also showed that antioxidant vitamins may increase all-cause mortality (14) (Table 1). Oxidative stress is often an early and key event that activates numerous pathways involved in aging and development of chronic diseases. On the basis of published literature, a novel molecular approach to resolve the apparent controversy and to protect cells from oxidative stress is warranted (15).

Table 1 Summary of effects of antioxidant vitamins in chronic diseases.
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Nitrooxidative Stress: A Possible Explanation of the Controversy

A theoretical explanation for why antioxidant vitamins do not protect as expected against disease relates to the fact that oxidative stress is more complex than previously realized. This complexity was revealed by the discovery of nitric oxide (NO) and the nitric oxide synthase (NOS) family. A vital, ubiquitous molecule, NO has numerous roles in regulating physiological processes. Because of affinity of NO for the superoxide anion radical , neither enzymatic nor pharmacologic levels of conventional antioxidants successively compete with NO for and thus antioxidants do not prevent the occurrence of high peroxynitrite (ONOO−) levels (16). These findings, particularly the discovery of the involvement of ONOO− as a causative reactant, have expanded the oxidative stress theory into new areas (17).

It is presumed that initially ROS production reduced the endothelial NOS (eNOS)-derived NO within endothelial cells while activating inducible NOS (iNOS), which causes almost 1000-fold higher NO production than does eNOS under physiologic circumstances. iNOS is predominantly expressed in inflammatory cells such as macrophages, although epithelial cells from affected tissues also express the enzyme. Intensified expression of iNOS has been detected in virtually all cell types tested, including macrophages, fibroblasts, chondrocytes, osteoclasts, and epithelial cells, and results in the production of much NO in animals and patients with inflammatory diseases (18–21). The level of iNOS expression is well correlated with the degree of inflammation. A controversy arises from observations reporting both cytotoxic and cytoprotective effects of NO. In cases in which NO was found to be cytotoxic, it was questioned whether NO exerted these effects directly or indirectly, or through the formation of more reactive species such as the ONOO−. Thus, it is believed that oxygen-based free radicals are only an initial step in the pathophysiologic mechanisms of aging and chronic diseases (22). The combination of elevated NO plus excess with the formation of high levels of ONOO− is the proverbial intracellular “devil’s triangle” (Figure 1).

Figure 1
figure 1

Organization of the “devil’s triangle” within the targeted cell. UV, ionizing irradiation, smoking, hyperglycemia, dyslipidemia and chronic inflammation cause excess and NO production via several means. Normally, is readily degraded to H2O by intracellular enzymatic antioxidants. In the presence of abundant and iNOS-derived NO, the biochemically interaction of these substances inevitably produces vast amounts of ONOO− thereby reducing the degradation of by SOD. In early stages of oxidative stress, if iNOS is not fully activated, several conventional antioxidants including vitamin E and C diminish the damage via scavenging and thereby prevent the activation of the “devil’s triangle.” Unlike the classic antioxidant vitamins, melatonin is the only currently available molecule which is known to block all aspects of the “devil’s triangle” as well as to activate antioxidative enzymes at RNA expression level, thereby preventing the loss of essential cellular antioxidative ezymes.

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Changing the Nature of Oxidative Stress: The “Devil’s Triangle”

In the last two decades, NO produced in excess was found to be a toxic molecule that damages and even kills cells. In the presence of excess and abundant NO, the latter vital molecule shows its dark side. Neither nor NO is particularly toxic by itself because efficient processes exist that minimize their accumulation (16). is rapidly removed by the superoxide dismutases (SOD), with the isoenzymes of this molecule being located in the mitochondria, cytosol, and extracellular compartments. NO normally is rapidly removed by its fast diffusion through tissues into red blood cells (23), where it is quickly converted to nitrate by a reaction with oxyhemoglobin. This process limits the biological half-life of NO in vivo to less than a second. However, when both and NO are generated within a few molecular diameters of each other, they combine spontaneously to form ONOO− in a reaction that occurs at a diffusion-limited rate. Basically, every time NO and collide, they form ONOO−. NO is the only known biological molecule that reacts faster with and is produced in such high concentrations that it outcompetes endogenous SOD, hence creating the “devil’s triangle” (see Figure 1). Consequently, from a biological viewpoint, the reaction of with NO to form ONOO− is inevitable. Any of several antioxidants may have beneficial effects in acute conditions that lead to this situation (for example, acute hyperglycemia, ischemia-reperfusion, myocardial infarction). In case of chronic oxidative stress, however, total iNOS activation leads to massive ONOO− generation, against which ordinary antioxidants provide little protection. Thus, in conditions leading to chronic oxidative stress, such as chronic hyperglycemia, dyslipidemia, tobacco smoking, and prolonged drug use, which are known to produce oxidative damage, conventional antioxidants become ineffective.

How Is Peroxynitrite Harmful?

Once ONOO− forms, it can act via two distinct mechanisms. First, ONOO− has direct toxic effects leading to lipid peroxidation, protein oxidation, and DNA damage. The second mechanism involves the induction of several transcription factors, including nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1), leading to cytokine-induced chronic inflammation (Figure 2). This cascade, once activated, causes the release of proinflammatory cytokines, including tumor necrosis factor α (TNF-α) and interleukin 1β, which induce widespread inflammation. During this process, several adhesion molecules and monocyte chemoattractant proteins also become involved, widening the inflammatory response and vascular events (17,24).

Figure 2
figure 2

Consequences of the “devil’s triangle” (92). ONOO− exerts its harmful effects directly and indirectly. It causes activation of transcriptional factors leading to proinflammatory gene expression. As a result, nitrooxidative stress becomes an inflammatory process. Interactions between transcriptional factors and proinflammatory products induce a vicious circle. These cytokines spread the inflammatory message through the circulation. Unless excess and iNOS-derived NO production are terminated (for example, through normalizing of blood glucose level, cessation of smoking), this mechanism continues to create damage within cells (for example, endothelial dysfunction). Moreover, ONOO− directly damages all macromolecules including lipids, proteins, and DNA. It is well known that both oxygen and nitrogen-based reactants directly damage mitochondria. The majority of NAD+ consumed by PARP also slows the rate of glycolysis and mitochondrial respiration, and eventually leads to cellular dysfunction. Antioxidant vitamins have no effect on ONOO−-induced subsequent cellular damage. Melatonin, however, is a unique molecule which possesses beneficial effects in virtually all steps of the pathophysiologic process. Melatonin blocks proinflammatory cytokine production by reducing NF-κB and AP-1 translocation into the nucleus and directly inhibits iNOS and inflammatory enzyme COX-2. These features break the vicious circle and alleviate the inflammation. Melatonin preserves cellular energy by several means and has been shown to prevent all types of nitrooxidative damage at the mitochondrial level.

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A direct toxic effect of ONOO− at the site of its production involves an intriguing process that decides the fate of cells. ONOO− is per se not a radical but is a powerful nitrosating agent. ONOO− interacts with and covalently modifies all major types of biomolecules including membrane lipids, thiols, proteins, and DNA (17,25). In addition, ONOO− can yield the hydroxyl (OH•) and nitrogen dioxide radicals (less than 30% yield). Although this is a minor process in biology, OH• is a potent reactant and oxidizes relevant targets, including amino acids (tyrosine, phenylalanine, histidine), sugars, and lipids (26,27). The generation of ONOO− also decreases the availability of NO for G-protein stimulation and vasodilatation, thus further contributing to endothelial dysfunction and leading to elevated blood pressure. In addition, ONOO− can inhibit SOD as well as other antioxidant molecules and systems, a process that leads to positive feedback cycles of intracellular oxidant generation and increased radical damage (28). ONOO− activates matrix metalloproteinases (MMPs) (29,30) and triggers the expression of selectins and cellular adhesion molecules via enhancement of NF-κB activation, thereby promoting proinflammatory responses.

The mutagenic properties of ONOO− induced modified products have also been determined (31,32). Several studies have shown that NO itself does not induce DNA single-strand breaks in vitro in plasmid DNA (33,34), whereas exposure of plasmid DNA to preformed ONOO− (35) or NO plus generated concurrently induces such strand breaks (36). Single-strand breakage can be induced by treatment with very low concentrations of ONOO−, indicating that this agent is a potent inducer of this type of damage to DNA (37). DNA cleavage caused by ONOO− was observed at almost every nucleotide, with a small preference for guanine residues. Furthermore, it has been reported that ONOO− inactivates several enzymes that are critically involved in the repair of DNA damage. These observations suggest additional pathways by which ONOO− may be associated with not only elevated DNA damage but also impairment of DNA repair capacity (38).

Poly(ADP-ribose) Polymerase Activation

ONOO− induces both apoptosis and necrosis of cells. More highly elevated exposure of this agent is associated with necrosis rather than apoptosis (28,39). In this mechanism, activation of the DNA repair enzyme poly(ADP ribose)polymerase-1 (PARP-1), a member of PARP enzyme family, mediates ONOO−-induced necrosis. PARP-1 detects and signals DNA strand breaks induced by a variety of genotoxic insults, including ionizing radiation, alkylating agents, oxidants (essentially OH* and ONOO−), and free radicals (40–44). When strand breaks occur at the time of binding to DNA, PARP transfers ADP-ribose units from the respiratory coenzyme nicotinamide adenine dinucleotide (NAD+) to various nuclear proteins. From a physiological viewpoint, PARP-1 activity and poly(ADP-ribosyl)ation reactions are implicated in DNA repair, genomic stability maintenance, gene-transcription regulation, and DNA replication. An important function of PARP-1 is to allow DNA repair and cell recovery under conditions associated with a low level of DNA damage. In case of severe DNA injury, over-activation of PARP-1 depletes the cellular stores of NAD+, an essential cofactor in principal energy production mechanisms including the glycolytic pathway, the tricarboxylic acid cycle, and the mitochondrial electron transport chain (ETC). As a result, the loss of NAD+ leads to a marked reduction in the cellular pools of ATP, resulting in cellular dysfunction and death via the necrotic pathway (28,39). This scenario is known as the “suicide hypothesis” of PARP activation and seems to be a regulatory mechanism to eliminate cells after irreversible DNA injury. A vast amount of experimental evidence has established that the PARP-1 pathway of cell death plays a pivotal role in tissue injury and organ dysfunction in numerous disease processes (41,45,46).

Based on the proposed mechanisms, several steps may be selected as potential pharmacological targets to block the hazards of free radicals: reduce excess ROS production; prevent intracellular enzymatic antioxidant depletion; stimulate intracellular enzymatic antioxidants; inhibit iNOS overactivation; increase eNOS-produced NO bioavailability; detoxify nitrogen-based species; inhibit (normalize) NF-κB and AP-1 activation and/or lower NF-κB binding to DNA; reduce proinflammatory cytokines such as TNF-α; limit adhesion molecule production; prevent lipid peroxidation and/or repair damaged lipids; limit protein oxidation and nitration; block PARP-1 activation, thereby preserving NAD+ and cellular energy; and reduce DNA damage and/or promote its repair. Identification of these pharmacological targets suggests the need for a multifunctional agent with antioxidant, iNOS-inhibitory, and ONOO−-scavenging properties. An endogenously produced indolamine, melatonin, is the one molecule that has these characteristics. This indolamine has a variety of means by which it influences the physiology of organisms; some of these actions are receptor-mediated whereas others are receptor-independent (47,48).

Melatonin: A Versatile Antioxidant

A very large body of evidence indicates that melatonin is a major scavenger of both oxygen- and nitrogen-based reactive molecules (49–53), including ONOO− (54–56). Melatonin has scavenging actions at both physiologic and pharmacologic concentrations. Not only melatonin but also several of its metabolites can detoxify free radicals and their derivatives (57–59). Studies also reveal that melatonin eliminates the decomposition products of ONOO−, including OH•, , and the carbonate radical in the presence of physiological carbon dioxide concentrations (60–62). Melatonin also supports several intracellular enzymatic antioxidant enzymes, including SOD and glutathione peroxidase (GSH-Px) (63,64). Moreover, melatonin induces the activity of γ-glutamylcysteine synthetase, thereby stimulating the production of another intracellular antioxidant, glutathione (GSH) (65). A number of studies have shown that melatonin is significantly better than the classic antioxidants in resisting free-radical–based molecular destruction. In these in vivo studies, melatonin was more effective than vitamin E (66–68), β-carotene (69), and vitamin C (69–71), and superior to garlic oil (72). Beneficial antioxidant effects of melatonin have been recently shown in clinical settings for several chronic diseases, including patients with rheumatoid arthritis (73), elderly patients with primary essential hypertension (74), and females with infertility (75).

Several antioxidants reportedly preserve the activities of SOD and/or GSH-Px. These effects are indirect, however, owing to their ability to scavenge free radicals and protect the protein from damage. Melatonin, on the other hand, possesses genomic actions and regulates the expression of several genes, including those for SOD and GSH-Px. Melatonin influences both antioxidant enzyme activity and cellular mRNA levels for these enzymes under physiological conditions and during elevated oxidative stress (63), possibly through epigenetic mechanisms (76–78). The occurrence of these two features in a single molecule is unique for an antioxidant, and both actions protect against pathologically-generated free radicals.

Of particular interest is the possible role of melatonin as a bioenergetic agent that can improve and maintain mitochondrial function. The majority of molecular oxygen (O2) inhaled and eventually taken up by cells is processed in the mitochondrial ETC, where it is converted to water after its four-electron reduction. During this reductive process, however, partially reduced species of O2 produce ROS and RNS. A specific isoform of NOS, mitochondrial NOS, is a constitutively expressed enzyme proposed to exist in mitochondria, where it produces NO within mitochondria. NO is a physiological regulator of respiration and also the rate of ATP synthesis. Elevation of NO levels higher than the physiological concentrations may inhibit complexes I, III, and IV. In this eventuality, mitochondrial electron transfer reactions are compromised and electron leakage is exaggerated, leading to increased formation of and all downstream oxidants (79). We recently reviewed the mechanisms by which melatonin improves mitochondrial respiration and increases ATP synthesis under physiological and poisonous conditions (80). Consequently, the antioxidant and free-radical-scavenging capacities of melatonin protect proteins of the ETC and mtDNA from ROS/RNS-induced oxidative damage. This protective effect limits the loss of intramitochondrial GSH, improves ETC activity, and reduces mtDNA damage. Melatonin’s actions at the mtDNA level also increase the expression of complex IV and the activity of complex I and complex IV of the ETC (81).

Melatonin against iNOS and ONOO−

In many inflammatory processes, ONOO− rather than other reactive molecules is the predominant molecule that determines the fate of cells. Once formed by the coupling of NO and , ONOO− cannot be removed or scavenged by vitamins E or C, or by other conventional antioxidants. As a multifunctional antioxidant, however, melatonin and its metabolites have unique features not shared by the usual antioxidants, including iNOS-inhibitory (82–86) and ONOO−-scavenging (49,54,62,87,88) properties. These features of melatonin, apart from direct antioxidative effects, have been documented in chemical-induced hyperglycemia (89–91) and other circumstances such as colitis (84), liver and lung damage (83), and alkylating-agent toxicity (55,56,92,93). Thus, melatonin is the only currently available molecule known to block all aspects of the “devil’s triangle.”

Melatonin has been shown to ameliorate inflammation by blocking transcriptional factors and TNF-α (94,95). A large body of evidence confirms that these cytokines induce formation of free radicals and promote iNOS activity and transcriptional factor activation within cells. These events inevitably induce a vicious cycle of cellular damage. In the case of ONOO−-induced DNA damage, PARP overactivates in a genome-repair process and consumes NAD+ as a substrate, causing an energy crisis within cells, leading to their eventual necrosis. Preservation of NAD+ and cellular energy production may facilitate PARP to repair the DNA damage rather than blocking PARP; melatonin preserves cellular energy production (96–98) and protects against DNA damage (99).

Under physiologic conditions in resting cells, eNOS-derived NO suppresses both iNOS and cyclooxygenase-2 (COX-2) expression by reducing NF-κB translocation into the nucleus. However, it is well documented that NO derived from iNOS during inflammatory processes further potentiates COX-2 activity through the NF-κB pathway (100), thereby exaggerating the inflammatory process. This effect is not unexpected given that ONOO− directly activates COX-2 as well (101). In the case of chronic inflammation, inhibition of COX-2 and iNOS (rather than COX-2 only) would be beneficial in reducing the severity of inflammation. A recent, intriguing report (102) suggests that neither tryptophan nor serotonin, but only melatonin, inhibits COX-2 and iNOS transcriptional activation.

Another advantage of melatonin over classical antioxidants is its lack of prooxidative actions. All classical antioxidants are potential electron donors and they exhibit both reduced and oxidized forms. Once they donate an electron to neutralize a free radical, they are transformed from a reduced to an oxidized state. Usually, the oxidized form will be regenerated to the reduced state through the mechanism known as redox reaction or recycling. In this pathway, the recycling of vitamin C or vitamin E occurs at the expense of GSH. In many cases, however, GSH is a better antioxidant than either vitamin C or vitamin E (103). Because these antioxidants are electron donors and exhibit redox reactions, their oxidized forms also can oxidize other molecules. Therefore, classical antioxidants are prooxidants. Melatonin sacrifices itself and does not participate in redox cycling after scavenging free radicals. As previously mentioned, melatonin not only does not consume cellular GSH, it also preserves or even increases the content of GSH in tissues. Thus, melatonin is classified as a suicidal or terminal antioxidant (104).

Concluding Remarks

Melatonin has been administered in both physiological and pharmacological amounts to humans and animals, and there is widespread agreement that it is a nontoxic molecule (105). In pregnant rats, the maternal lowest-no-observed-effect level was found to be 200 mg/kg/day, and the developmental no-observed-adverse-effect level was ≥200 mg/kg/day (106). Melatonin is easily synthesized in a pharmacologically pure form and is inexpensive and affordable; thus, because of its versatility in protecting against nitrooxidative stress and reducing inflammation, melatonin could have significant potential to improve public health (107).

Disclosure

We declare that the authors have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.