Melatonin and the Programming of Stem Cells.

Melatonin interacts with various types of stem cells, in multiple ways that comprise stimulation of proliferation, maintenance of stemness and self-renewal, protection of survival, and programming toward functionally different cell lineages. These various properties are frequently intertwined but may not be always jointly present. Melatonin typically stimulates proliferation and transition to the mature cell type. For all sufficiently studied stem or progenitor cells, melatonin's signaling pathways leading to expression of respective morphogenetic factors are discussed. The focus of this article will be laid on the aspect of programming, particularly in pluripotent cells. This is especially but not exclusively the case in neural stem cells (NSCs) and mesenchymal stem cells (MSCs). Concerning developmental bifurcations, decisions are not exclusively made by melatonin alone. In MSCs, melatonin promotes adipogenesis in a Wnt (Wingless-Integration-1)-independent mode, but chondrogenesis and osteogenesis Wnt-dependently. Melatonin upregulates Wnt, but not in the adipogenic lineage. This decision seems to depend on microenvironment and epigenetic memory. The decision for chondrogenesis instead of osteogenesis, both being Wnt-dependent, seems to involve fibroblast growth factor receptor 3. Stem cell-specific differences in melatonin and Wnt receptors, and contributions of transcription factors and noncoding RNAs are outlined, as well as possibilities and the medical importance of re-programming for transdifferentiation.

Addressing the 'hypoxia paradox' in severe COVID-19: literature review and report of four cases treated with erythropoietin analogues.

BACKGROUND: Since fall 2019, SARS-CoV-2 spread world-wide, causing a major pandemic with estimated ~ 220 million subjects affected as of September 2021. Severe COVID-19 is associated with multiple organ failure, particularly of lung and kidney, but also grave neuropsychiatric manifestations. Overall mortality reaches > 2%. Vaccine development has thrived in thus far unreached dimensions and will be one prerequisite to terminate the pandemic. Despite intensive research, however, few treatment options for modifying COVID-19 course/outcome have emerged since the pandemic outbreak. Additionally, the substantial threat of serious downstream sequelae, called 'long COVID' and 'neuroCOVID', becomes increasingly evident. Among candidates that were suggested but did not yet receive appropriate funding for clinical trials is recombinant human erythropoietin. Based on accumulating experimental and clinical evidence, erythropoietin is expected to (1) improve respiration/organ function, (2) counteract overshooting inflammation, (3) act sustainably neuroprotective/neuroregenerative. Recent counterintuitive findings of decreased serum erythropoietin levels in severe COVID-19 not only support a relative deficiency of erythropoietin in this condition, which can be therapeutically addressed, but also made us coin the term 'hypoxia paradox'. As we review here, this paradox is likely due to uncoupling of physiological hypoxia signaling circuits, mediated by detrimental gene products of SARS-CoV-2 or unfavorable host responses, including microRNAs or dysfunctional mitochondria. Substitution of erythropoietin might overcome this 'hypoxia paradox' caused by deranged signaling and improve survival/functional status of COVID-19 patients and their long-term outcome. As supporting hints, embedded in this review, we present 4 male patients with severe COVID-19 and unfavorable prognosis, including predicted high lethality, who all profoundly improved upon treatment which included erythropoietin analogues. SHORT CONCLUSION: Substitution of EPO may-among other beneficial EPO effects in severe COVID-19-circumvent downstream consequences of the 'hypoxia paradox'. A double-blind, placebo-controlled, randomized clinical trial for proof-of-concept is warranted.

Melatonin and Microglia.

Melatonin interacts in multiple ways with microglia, both directly and, via routes of crosstalk with astrocytes and neurons, indirectly. These effects of melatonin are of relevance in terms of antioxidative protection, not only concerning free-radical detoxification, but also in prevention of processes that cause, promote, or propagate oxidative stress and neurodegeneration, such as overexcitation, toxicological insults, viral and bacterial infections, and sterile inflammation of different grades. The immunological interplay in the CNS, with microglia playing a central role, is of high complexity and includes signaling toward endothelial cells and other leukocytes by cytokines, chemokines, nitric oxide, and eikosanoids. Melatonin interferes with these processes in multiple signaling routes and steps. In addition to canonical signal transduction by MT1 and MT2 melatonin receptors, secondary and tertiary signaling is of relevance and has to be considered, e.g., via the upregulation of sirtuins and the modulation of pro- and anti-inflammatory microRNAs. Many details concerning the modulation of macrophage functionality by melatonin are obviously also applicable to microglial cells. Of particular interest is the polarization toward M2 subtypes instead of M1, i.e., in favor of being anti-inflammatory at the expense of proinflammatory activities, which is well-documented in macrophages but also applies to microglia.

Melatonin, Its Metabolites and Their Interference with Reactive Nitrogen Compounds.

Melatonin and several of its metabolites are interfering with reactive nitrogen. With the notion of prevailing melatonin formation in tissues that exceeds by far the quantities in blood, metabolites come into focus that are poorly found in the circulation. Apart from their antioxidant actions, both melatonin and N1-acetyl-5-methoxykynuramine (AMK) downregulate inducible and inhibit neuronal NO synthases, and additionally scavenge NO. However, the NO adduct of melatonin redonates NO, whereas AMK forms with NO a stable product. Many other melatonin metabolites formed in oxidative processes also contain nitrosylatable sites. Moreover, AMK readily scavenges products of the CO2-adduct of peroxynitrite such as carbonate radicals and NO2. Protein AMKylation seems to be involved in protective actions.

The Reserve/Maximum Capacity of Melatonin's Synthetic Function for the Potential Dimorphism of Melatonin Production and Its Biological Significance in Mammals.

In this article, we attempt to classify a potential dimorphism of melatonin production. Thus, a new concept of "reserve or maximum capacity of melatonin synthetic function" is introduced to explain the subtle dimorphism of melatonin production in mammals. Considering ASMT/ASMTL genes in the pseudoautosomal region of sex chromosomes with high prevalence of mutation in males, as well as the sex bias of the mitochondria in which melatonin is synthesized, we hypothesize the existence of a dimorphism in melatonin production to favor females, which are assumed to possess a higher reserve capacity for melatonin synthesis than males. Under physiological conditions, this subtle dimorphism is masked by the fact that cells or tissues only need baseline melatonin production, which can be accomplished without exploiting the full potential of melatonin's synthetic capacity. This capacity is believed to exceed the already remarkable nocturnal increase as observed within the circadian cycle. However, during aging or under stressful conditions, the reserve capacity of melatonin's synthetic function is required to be activated to produce sufficiently high levels of melatonin for protective purposes. Females seem to possess a higher reserve/maximum capacity for producing more melatonin than males. Thus, this dimorphism of melatonin production becomes manifest and detectable under these conditions. The biological significance of the reserve/maximum capacity of melatonin's synthetic function is to improve the recovery rate of organisms from injury, to increase resistance to pathogen infection, and even to enhance their chances of survival by maximizing melatonin production under stressful conditions. The higher reserve/maximum capacity of melatonin synthesis in females may also contribute to the dimorphism in longevity, favoring females in mammals.

Thymus-Pineal Gland Axis: Revisiting Its Role in Human Life and Ageing.

For years the thymus gland (TG) and the pineal gland (PG) have been subject of increasingly in-depth studies, but only recently a link that can associate the activities of the two organs has been identified. Considering, on the one hand, the well-known immune activity of thymus and, on the other, the increasingly emerging immunological roles of circadian oscillators and the rhythmically secreted main pineal product, melatonin, many studies aimed to analyse the possible existence of an interaction between these two systems. Moreover, data confirmed that the immune system is functionally associated with the nervous and endocrine systems determining an integrated dynamic network. In addition, recent researches showed a similar, characteristic involution process both in TG and PG. Since the second half of the 20th century, evidence led to the definition of an effectively interacting thymus-pineal axis (TG-PG axis), but much has to be done. In this sense, the aim of this review is to summarize what is actually known about this topic, focusing on the impact of the TG-PG axis on human life and ageing. We would like to give more emphasis to the implications of this dynamical interaction in a possible therapeutic strategy for human health. Moreover, we focused on all the products of TG and PG in order to collect what is known about the role of peptides other than melatonin. The results available today are often unclear and not linear. These peptides have not been well studied and defined over the years. In this review we hope to awake the interest of the scientific community in them and in their future pharmacological applications.

Melatonin in macrophage biology: Current understanding and future perspectives.

Melatonin is a ubiquitous hormone found in various organisms and highly affects the function of immune cells. In this review, we summarize the current understanding of the significance of melatonin in macrophage biology and the beneficial effects of melatonin in macrophage-associated diseases. Enzymes associated with synthesis of melatonin, as well as membrane receptors for melatonin, are found in macrophages. Indeed, melatonin influences the phenotype polarization of macrophages. Mechanistically, the roles of melatonin in macrophages are related to several cellular signaling pathways, such as NF-κB, STATs, and NLRP3/caspase-1. Notably, miRNAs (eg, miR-155/-34a/-23a), cellular metabolic pathways (eg, α-KG, HIF-1α, and ROS), and mitochondrial dynamics and mitophagy are also involved. Thus, melatonin modulates the development and progression of various macrophage-associated diseases, such as cancer and rheumatoid arthritis. This review provides a better understanding about the importance of melatonin in macrophage biology and macrophage-associated diseases.

Aging, Melatonin, and the Pro- and Anti-Inflammatory Networks.

Aging and various age-related diseases are associated with reductions in melatonin secretion, proinflammatory changes in the immune system, a deteriorating circadian system, and reductions in sirtuin-1 (SIRT1) activity. In non-tumor cells, several effects of melatonin are abolished by inhibiting SIRT1, indicating mediation by SIRT1. Melatonin is, in addition to its circadian and antioxidant roles, an immune stimulatory agent. However, it can act as either a pro- or anti-inflammatory regulator in a context-dependent way. Melatonin can stimulate the release of proinflammatory cytokines and other mediators, but also, under different conditions, it can suppress inflammation-promoting processes such as NO release, activation of cyclooxygenase-2, inflammasome NLRP3, gasdermin D, toll-like receptor-4 and mTOR signaling, and cytokine release by SASP (senescence-associated secretory phenotype), and amyloid-ß toxicity. It also activates processes in an anti-inflammatory network, in which SIRT1 activation, upregulation of Nrf2 and downregulation of NF-κB, and release of the anti-inflammatory cytokines IL-4 and IL-10 are involved. A perhaps crucial action may be the promotion of macrophage or microglia polarization in favor of the anti-inflammatory phenotype M2. In addition, many factors of the pro- and anti-inflammatory networks are subject to regulation by microRNAs that either target mRNAs of the respective factors or upregulate them by targeting mRNAs of their inhibitor proteins.

Melatonin and inflammation-Story of a double-edged blade.

Melatonin is an immune modulator that displays both pro- and anti-inflammatory properties. Proinflammatory actions, which are well documented by many studies in isolated cells or leukocyte-derived cell lines, can be assumed to enhance the resistance against pathogens. However, they can be detrimental in autoimmune diseases. Anti-inflammatory actions are of particular medicinal interest, because they are observed in high-grade inflammation such as sepsis, ischemia/reperfusion, and brain injury, and also in low-grade inflammation during aging and in neurodegenerative diseases. The mechanisms contributing to anti-inflammatory effects are manifold and comprise various pathways of secondary signaling. These include numerous antioxidant effects, downregulation of inducible and inhibition of neuronal NO synthases, downregulation of cyclooxygenase-2, inhibition of high-mobility group box-1 signaling and toll-like receptor-4 activation, prevention of inflammasome NLRP3 activation, inhibition of NF-κB activation and upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2). These effects are also reflected by downregulation of proinflammatory and upregulation of anti-inflammatory cytokines. Proinflammatory actions of amyloid-ß peptides are reduced by enhancing α-secretase and inhibition of ß- and γ-secretases. A particular role in melatonin's actions seems to be associated with the upregulation of sirtuin-1 (SIRT1), which shares various effects known from melatonin and additionally interferes with the signaling by the mechanistic target of rapamycin (mTOR) and Notch, and reduces the expression of the proinflammatory lncRNA-CCL2. The conclusion on a partial mediation by SIRT1 is supported by repeatedly observed inhibitions of melatonin effects by sirtuin inhibitors or knockdown.

Melatonin and the electron transport chain.

Melatonin protects the electron transport chain (ETC) in multiple ways. It reduces levels of ·NO by downregulating inducible and inhibiting neuronal nitric oxide synthases (iNOS, nNOS), thereby preventing excessive levels of peroxynitrite. Both ·NO and peroxynitrite-derived free radicals, such as ·NO2, hydroxyl (·OH) and carbonate radicals (CO3·-) cause blockades or bottlenecks in the ETC, by ·NO binding to irons, protein nitrosation, nitration and oxidation, changes that lead to electron overflow or even backflow and, thus, increased formation of superoxide anions (O2·-). Melatonin improves the intramitochondrial antioxidative defense by enhancing reduced glutathione levels and inducing glutathione peroxidase and Mn-superoxide dismutase (Mn-SOD) in the matrix and Cu,Zn-SOD in the intermembrane space. An additional action concerns the inhibition of cardiolipin peroxidation. This oxidative change in the membrane does not only initiate apoptosis or mitophagy, as usually considered, but also seems to occur at low rate, e.g., in aging, and impairs the structural integrity of Complexes III and IV. Moreover, elevated levels of melatonin inhibit the opening of the mitochondrial permeability transition pore and shorten its duration. Additionally, high-affinity binding sites in mitochondria have been described. The assumption of direct binding to the amphipathic ramp of Complex I would require further substantiation. The mitochondrial presence of the melatonin receptor MT1 offers the possibility that melatonin acts via an inhibitory G protein, soluble adenylyl cyclase, decreased cAMP and lowered protein kinase A activity, a signaling pathway shown to reduce Complex I activity in the case of a mitochondrial cannabinoid receptor.

Inflammaging, Metabolic Syndrome and Melatonin: A Call for Treatment Studies.

The metabolic syndrome (MS) is a collection of risk factors for cardiovascular disease, including obesity, hypertension, hyperinsulinemia, glucose intolerance and dyslipidemia. MS is associated with low-grade inflammation of the white adipose tissue, which can subsequently lead to insulin resistance, impaired glucose tolerance and diabetes. Adipocytes secrete proinflammatory cytokines as well as leptin and trigger a vicious circle which leads to additional weight gain largely as fat. The imbalance between inflammatory and anti-inflammatory signals is crucial to aging. Healthy aging can benefit from melatonin, a compound known to possess direct and indirect antioxidant properties, to have a significant protective effect on mitochondrial function, to enhance circadian rhythm amplitudes, to modulate the immune system and to exhibit neuroprotective actions. Melatonin levels decrease in the course of senescence and are more strongly reduced in diseases related to insulin resistance. This short review article analyzes the multiple protective actions of melatonin that are relevant to the attenuation of inflammatory responses and progression of inflammaging and how melatonin is effective to curtail MS in animal models of hyperadiposity. The clinical data supporting the possible therapeutic use of melatonin in human MS are also reviewed. Since attention has been focused on the development of potent melatonin analogs with prolonged effects (ramelteon, agomelatine, tasimelteon, piromelatine) and in clinical trials these analogs were administered in doses considerably higher than those usually employed for melatonin, clinical trials on melatonin in the range of 50-100 mg/day are needed to further assess its therapeutic value in MS.

Melatonin and the pathologies of weakened or dysregulated circadian oscillators.

Dynamic aspects of melatonin's actions merit increasing future attention. This concerns particularly entirely different effects in senescent, weakened oscillators and in dysregulated oscillators of cancer cells that may be epigenetically blocked. This is especially obvious in the case of sirtuin 1, which is upregulated by melatonin in aged tissues, but strongly downregulated in several cancer cells. These findings are not at all controversial, but are explained on the basis of divergent changes in weakened and dysregulated oscillators. Similar findings can be expected to occur in other accessory oscillator components that are modulated by melatonin, among them several transcription factors and metabolic sensors. Another cause of opposite effects concerns differences between nocturnally active laboratory rodents and the diurnally active human. This should be more thoroughly considered in the field of metabolic syndrome and related pathologies, especially with regard to type 2 diabetes and other aspects of insulin resistance. Melatonin was reported to impair glucose tolerance in humans, especially in carriers of the risk allele of the MT2 receptor gene, MTNR1B, that contains the SNP rs10830963. These findings contrast with numerous reports on improvements of glucose tolerance in preclinical studies. However, the relationship between melatonin and insulin may be more complex, as indicated by loss-of-function mutants of the MT2 receptor that are also prodiabetic, by the age-dependent time course of risk allele overexpression, by progressive reduction in circadian amplitudes and melatonin secretion, which are aggravated in diabetes. By supporting high-amplitude rhythms, melatonin may be beneficial in preventing or delaying diabetes.

Taxon- and Site-Specific Melatonin Catabolism.

Melatonin is catabolized both enzymatically and nonenzymatically. Nonenzymatic processes mediated by free radicals, singlet oxygen, other reactive intermediates such as HOCl and peroxynitrite, or pseudoenzymatic mechanisms are not species- or tissue-specific, but vary considerably in their extent. Higher rates of nonenzymatic melatonin metabolism can be expected upon UV exposure, e.g., in plants and in the human skin. Additionally, melatonin is more strongly nonenzymatically degraded at sites of inflammation. Typical products are several hydroxylated derivatives of melatonin and N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK). Most of these products are also formed by enzymatic catalysis. Considerable taxon- and site-specific differences are observed in the main enzymatic routes of catabolism. Formation of 6-hydroxymelatonin by cytochrome P450 subforms are prevailing in vertebrates, predominantly in the liver, but also in the brain. In pineal gland and non-mammalian retina, deacetylation to 5-methoxytryptamine (5-MT) plays a certain role. This pathway is quantitatively prevalent in dinoflagellates, in which 5-MT induces cyst formation and is further converted to 5-methoxyindole-3-acetic acid, an end product released to the water. In plants, the major route is catalyzed by melatonin 2-hydroxylase, whose product is tautomerized to 3-acetamidoethyl-3-hydroxy-5-methoxyindolin-2-one (AMIO), which exceeds the levels of melatonin. Formation and properties of various secondary products are discussed.

On the significance of an alternate pathway of melatonin synthesis via 5-methoxytryptamine: comparisons across species.

Melatonin is a phylogenetically ancient molecule. It is ubiquitously present in almost all organisms from primitive photosynthetic bacteria to humans. Its original primary function is presumable to be that of an antioxidant with other functions of this molecule having been acquired during evolution. The synthetic pathway of melatonin in vertebrates has been extensively studied. It is common knowledge that serotonin is acetylated to form N-acetylserotonin by arylalkylamine N-acetyltransferase (AANAT) or arylamine N-acetyltransferase (SNAT or NAT) and N-acetylserotonin is, subsequently, methylated to melatonin by N-acetylserotonin O-methyltransferase (ASMT; also known as hydroxyindole-O-methyltransferase, HIOMT). This is referred to as a classic melatonin synthetic pathway. Based on new evidence, we feel that this classic melatonin pathway is not generally the prevailing route of melatonin production. An alternate pathway is known to exist, in which serotonin is first O-methylated to 5-methoxytryptamine (5-MT) and, thereafter, 5-MT is N-acetylated to melatonin. Here, we hypothesize that the alternate melatonin synthetic pathway may be more important in certain organisms and under certain conditions. Evidence strongly supports that this alternate pathway prevails in some plants, bacteria, and, perhaps, yeast and may also occur in animals.

Stunted Growth. Proceedings of the 23rd Aschauer Soiree, Held at Aschauhof, Germany, November 7th 2015.

Twenty-four scientists met at Aschauhof, Altenhof, Germany, to discuss the associations between child growth and development, and nutrition, health, environment and psychology. Meta-analyses of body height, height variability and household inequality, in historic and modern growth studies published since 1794, highlighting the enormously flexible patterns of child and adolescent height and weight increments throughout history which do not only depend on genetics, prenatal development, nutrition, health, and economic circumstances, but reflect social interactions. A Quality of Life in Short Stature Youth Questionnaire was presented to cross-culturally assess health-related quality of life in children. Changes of child body proportions in recent history, the relation between height and longevity in historic Dutch samples and also measures of body height in skeletal remains belonged to the topics of this meeting. Bayesian approaches and Monte Carlo simulations offer new statistical tools for the study of human growth.

Melatonin in plants and other phototrophs: advances and gaps concerning the diversity of functions.

Melatonin is synthesized in Alphaproteobacteria, Cyanobacteria, Dinoflagellata, Euglenoidea, Rhodophyta, Phae ophyta, and Viridiplantae. The biosynthetic pathways have been identified in dinoflagellates and plants. Other than in dinoflagellates and animals, tryptophan is not 5-hydroxylated in plants but is first decarboxylated. Serotonin is formed by 5-hydroxylation of tryptamine. Serotonin N-acetyltransferase is localized in plastids and lacks homology to the vertebrate aralkylamine N-acetyltransferase. Melatonin content varies considerably among species, from a few picograms to several micrograms per gram, a strong hint for different actions of this indoleamine. At elevated levels, the common and presumably ancient property as an antioxidant may prevail. Although melatonin exhibits nocturnal maxima in some phototrophs, it is not generally a mediator of the signal 'darkness'. In various plants, its formation is upregulated by visible and/or UV light. Increases are often induced by high or low temperature and several other stressors including drought, salinity, and chemical toxins. In Arabidopsis, melatonin induces cold- and stress-responsive genes. It has been shown to support cold resistance and to delay experimental leaf senescence. Transcriptome data from Arabidopsis indicate upregulation of genes related to ethylene, abscisic acid, jasmonic acid, and salicylic acid. Auxin-like actions have been reported concerning root growth and inhibition, and hypocotyl or coleoptile lengthening, but effects caused by melatonin and auxins can be dissected. Assumptions on roles in flower morphogenesis and fruit ripening are based mainly on concentration changes. Whether or not melatonin will find a place in the phytohormone network depends especially on the identification of molecular signals regulating its synthesis, high-affinity binding sites, and signal transduction pathways.

Melatonin, noncoding RNAs, messenger RNA stability and epigenetics--evidence, hints, gaps and perspectives.

Melatonin is a highly pleiotropic regulator molecule, which influences numerous functions in almost every organ and, thus, up- or down-regulates many genes, frequently in a circadian manner. Our understanding of the mechanisms controlling gene expression is actually now expanding to a previously unforeseen extent. In addition to classic actions of transcription factors, gene expression is induced, suppressed or modulated by a number of RNAs and proteins, such as miRNAs, lncRNAs, piRNAs, antisense transcripts, deadenylases, DNA methyltransferases, histone methylation complexes, histone demethylases, histone acetyltransferases and histone deacetylases. Direct or indirect evidence for involvement of melatonin in this network of players has originated in different fields, including studies on central and peripheral circadian oscillators, shift work, cancer, inflammation, oxidative stress, aging, energy expenditure/obesity, diabetes type 2, neuropsychiatric disorders, and neurogenesis. Some of the novel modulators have also been shown to participate in the control of melatonin biosynthesis and melatonin receptor expression. Future work will need to augment the body of evidence on direct epigenetic actions of melatonin and to systematically investigate its role within the network of oscillating epigenetic factors. Moreover, it will be necessary to discriminate between effects observed under conditions of well-operating and deregulated circadian clocks, and to explore the possibilities of correcting epigenetic malprogramming by melatonin.

Melatonin and the theories of aging: a critical appraisal of melatonin's role in antiaging mechanisms.

The classic theories of aging such as the free radical theory, including its mitochondria-related versions, have largely focused on a few specific processes of senescence. Meanwhile, numerous interconnections have become apparent between age-dependent changes previously thought to proceed more or less independently. Increased damage by free radicals is not only linked to impairments of mitochondrial function, but also to inflammaging as it occurs during immune remodeling and by release of proinflammatory cytokines from mitotically arrested, DNA-damaged cells that exhibit the senescence-associated secretory phenotype (SASP). Among other effects, SASP can cause mutations in stem cells that reduce the capacity for tissue regeneration or, in worst case, lead to cancer stem cells. Oxidative stress has also been shown to promote telomere attrition. Moreover, damage by free radicals is connected to impaired circadian rhythmicity. Another nexus exists between cellular oscillators and metabolic sensing, in particular to the aging-suppressor SIRT1, which acts as an accessory clock protein. Melatonin, being a highly pleiotropic regulator molecule, interacts directly or indirectly with all the processes mentioned. These influences are critically reviewed, with emphasis on data from aged organisms and senescence-accelerated animals. The sometimes-controversial findings obtained either in a nongerontological context or in comparisons of tumor with nontumor cells are discussed in light of evidence obtained in senescent organisms. Although, in mammals, lifetime extension by melatonin has been rarely documented in a fully conclusive way, a support of healthy aging has been observed in rodents and is highly likely in humans.

Chronobiology of Melatonin beyond the Feedback to the Suprachiasmatic Nucleus-Consequences to Melatonin Dysfunction.

The mammalian circadian system is composed of numerous oscillators, which gradually differ with regard to their dependence on the pacemaker, the suprachiasmatic nucleus (SCN). Actions of melatonin on extra-SCN oscillators represent an emerging field. Melatonin receptors are widely expressed in numerous peripheral and central nervous tissues. Therefore, the circadian rhythm of circulating, pineal-derived melatonin can have profound consequences for the temporal organization of almost all organs, without necessarily involving the melatonin feedback to the suprachiasmatic nucleus. Experiments with melatonin-deficient mouse strains, pinealectomized animals and melatonin receptor knockouts, as well as phase-shifting experiments with explants, reveal a chronobiological role of melatonin in various tissues. In addition to directly steering melatonin-regulated gene expression, the pineal hormone is required for the rhythmic expression of circadian oscillator genes in peripheral organs and to enhance the coupling of parallel oscillators within the same tissue. It exerts additional effects by modulating the secretion of other hormones. The importance of melatonin for numerous organs is underlined by the association of various diseases with gene polymorphisms concerning melatonin receptors and the melatonin biosynthetic pathway. The possibilities and limits of melatonergic treatment are discussed with regard to reductions of melatonin during aging and in various diseases.

A Comparison of B16 Melanoma Cells and 3T3 Fibroblasts Concerning Cell Viability and ROS Production in the Presence of Melatonin, Tested Over a Wide Range of Concentrations.

Melatonin is a pleiotropic molecule with many cellular and systemic actions, including chronobiotic effects. Beneficial effects are widely documented concerning the treatment of neoplastic diseases in vivo as well as reductions in viability of cultured cells from melanoma, one of the most aggressive cancers in humans. However, studies of its effects on non-tumor cells in vitro have not focused on viability, except for experiments aiming to protect against oxidotoxicity or other toxicological insults. Furthermore, there is no agreement on the range of effective melatonin concentrations in vitro, and the mechanisms that reduce cell viability have remained unclear. Tumor cell-specific increases in the production of reactive oxygen and nitrogen species (ROS/RNS) may provide a possible explanation. Our aim was to analyze the potential inhibition of tumor (B16 melanoma 4A5) and non-tumor cell (3T3 Swiss albino) viability using a wide range of melatonin concentrations (10-11-10-2 M), and to determine whether intracellular ROS enhancement was involved in this process. In the absence of fetal bovine serum (FBS), low melatonin concentrations (10-9-10-5 M) reduced the proliferation of melanoma cells with no effect in fibroblasts, whereas, in the presence of FBS, they had no effect or even increased the proliferation of both fibroblast and melanoma cells. Melatonin concentrations in the upper millimolar range increased ROS levels and reduced the viability of both cell types, but more markedly so in non-tumor cells. Thus, low melatonin concentrations reduce proliferation in this specific melanoma cell line, whereas high concentrations affect the viability of both tumor (B16 4A5 melanoma) and non-tumor (3T3 fibroblasts) cells. Increased ROS levels in both lines indicate a role for ROS production in the reduction of cell viability at high-but not low-melatonin concentrations, although the mechanism of action still remains to be elucidated.