Jerry Vriend

Melatonin: an ancient molecule that makes oxygen metabolically tolerable

Melatonin is remarkably functionally diverse with actions as a free radical scavenger and antioxidant, circadian rhythm regulator, anti‐inflammatory and immunoregulating molecule, and as an oncostatic agent. We hypothesize that the initial and primary function of melatonin in photosynthetic cyanobacteria, which appeared on Earth 3.5–3.2 billion years ago, was as an antioxidant. The evolution of melatonin as an antioxidant by this organism was necessary as photosynthesis is associated with the generation of toxic‐free radicals. The other secondary functions of melatonin came about much later in evolution. We also surmise that mitochondria and chloroplasts may be primary sites of melatonin synthesis in all eukaryotic cells that possess these organelles. This prediction is made on the basis that mitochondria and chloroplasts of eukaryotes developed from purple nonsulfur bacteria (which also produce melatonin) and cyanobacteria when they were engulfed by early eukaryotes. Thus, we speculate that the melatonin‐synthesizing actions of the engulfed bacteria were retained when these organelles became mitochondria and chloroplasts, respectively. That mitochondria are likely sites of melatonin formation is supported by the observation that this organelle contains high levels of melatonin that are not impacted by blood melatonin concentrations. Melatonin has a remarkable array of means by which it thwarts oxidative damage. It, as well as its metabolites, is differentially effective in scavenging a variety of reactive oxygen and reactive nitrogen species. Moreover, melatonin and its metabolites modulate a large number of antioxidative and pro‐oxidative enzymes, leading to a reduction in oxidative damage. The actions of melatonin on radical metabolizing/producing enzymes may be mediated by the Keap1‐Nrf2‐ARE pathway. Beyond its direct free radical scavenging and indirect antioxidant effects, melatonin has a variety of physiological and metabolic advantages that may enhance its ability to limit oxidative stress.

Melatonin feedback on clock genes: a theory involving the proteasome.

The expression of ‘clock’ genes occurs in all tissues, but especially in the suprachiasmatic nuclei (SCN) of the hypothalamus, groups of neurons in the brain that regulate circadian rhythms. Melatonin is secreted by the pineal gland in a circadian manner as influenced by the SCN. There is also considerable evidence that melatonin, in turn, acts on the SCN directly influencing the circadian ‘clock’ mechanisms. The most direct route by which melatonin could reach the SCN would be via the cerebrospinal fluid of the third ventricle. Melatonin could also reach the pars tuberalis (PT) of the pituitary, another melatonin‐sensitive tissue, via this route. The major ‘clock’ genes include the period genes, Per1 and Per2, the cryptochrome genes, Cry1 and Cry2, the clock (circadian locomotor output cycles kaput) gene, and the Bmal1 (aryl hydrocarbon receptor nuclear translocator‐like) gene. Clock and Bmal1 heterodimers act on E‐box components of the promoters of the Per and Cry genes to stimulate transcription. A negative feedback loop between the cryptochrome proteins and the nucleus allows the Cry and Per proteins to regulate their own transcription. A cycle of ubiquitination and deubiquitination controls the levels of CRY protein degraded by the proteasome and, hence, the amount of protein available for feedback. Thus, it provides a post‐translational component to the circadian clock mechanism. BMAL1 also stimulates transcription of REV‐ERBα and, in turn, is also partially regulated by negative feedback by REV‐ERBα. In the ‘black widow’ model of transcription, proteasomes destroy transcription factors that are needed only for a particular period of time. In the model proposed herein, the interaction of melatonin and the proteasome is required to adjust the SCN clock to changes in the environmental photoperiod. In particular, we predict that melatonin inhibition of the proteasome interferes with negative feedback loops (CRY/PER and REV‐ERBα) on Bmal1 transcription genes in both the SCN and PT. Melatonin inhibition of the proteasome would also tend to stabilize BMAL1 protein itself in the SCN, particularly at night when melatonin is naturally elevated. Melatonin inhibition of the proteasome could account for the effects of melatonin on circadian rhythms associated with molecular timing genes. The interaction of melatonin with the proteasome in the hypothalamus also provides a model for explaining the dramatic ‘time of day’ effect of melatonin injections on reproductive status of seasonal breeders. Finally, the model predicts that a proteasome inhibitor such as bortezomib would modify circadian rhythms in a manner similar to melatonin.

The Keap1-Nrf2-antioxidant response element pathway: a review of its regulation by melatonin and the proteasome.

Both melatonin and proteasome inhibitors upregulate antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GP), hemoxygenase 1 (HO-1), and NADPH:quinone oxidoreductase (NQO1). Recent evidence suggests that the antioxidant action of both melatonin and proteasome inhibitors involves the Keap1-ARE (Keap1 antioxidant response element) pathway via the upregulation of Nrf2. Melatonin and proteasome inhibitors suppress the degradation of Nrf2 and also enhance its nuclear translocation. In the nucleus Nrf2, together with a cofactor, stimulates the transcription of antioxidant enzymes and detoxifying enzymes. The ligase (E3) complex (Keap1-Cul3-Rbx1) responsible for ubiquitinating Nrf2, prior to proteasomal degradation, also ubiquitinates IkB kinase and the antiapoptotic factor Bcl-2, and possibly additional proteins. In various systems, NF-κB, which is inhibited by IkBα, is downregulated by proteasome inhibitors as well as by melatonin. Similarly in leukemic cells, Bcl-2 is down-regulated by the proteasome inhibitor, bortezomib, and also by melatonin. Thus melatonin administration modulates the activity of three separate substrates of the Keap1-Cul3-Rbx1 ubiquitin ligase. These facts could be accounted for by the hypothesis that melatonin interacts with the ubiquitin ligase complex or, more likely, by the hypothesis that melatonin acts as a proteasome inhibitor. A recent study documented that melatonin acts as a proteasome inhibitor in cancer cells as well as inhibiting chymotrypsin-like activity in cell-free systems of these cells. Further studies, however, are needed to clarify the interaction of melatonin and the ubiquitin-proteasome system as they relate to oxidative stress.

Melatonin reduces dopamine content in the neurointermediate lobe of male syrian hamsters

The effect of daily late afternoon administration of melatonin on the in situ activity of tyrosine hydroxylase (TH) was studied in the posterior pituitary (neurointermediate lobe) of the male Syrian hamster. After 3 weeks of melatonin administration, TH activity was significantly reduced in the posterior pituitary. This was associated with a significant decrease in norepinephrine (NE) content. After 5 weeks, TH activity and NE content were no longer significantly different from controls. Dopamine (DA) content of the posterior pituitary was decreased progressively by melatonin administration, with a reduction of greater than 50% after 5 weeks of treatment. These data provide evidence that melatonin has a potent inhibitory effect on the regulation of the dopaminergic system of the neurointermediate lobe--an effect that appears unrelated to changes in axonal TH.

Effects of Melatonin on Thyroid Physiology of Female Hamsters

The effects of melatonin administration on thyroid physiology of female hamsters was investigated. A protocol of 25 micrograms given daily as subcutaneous injections late in the light period was found to inhibit blood levels of thyroxin (T4), triiodothyronine (T3) and thyrotropin (TSH). The free T4 index (FT4I) and the free T3 index (FT3I) were also significantly inhibited by melatonin injections. Decreasing the photoperiod under which the hamsters were kept, from 14 h light/10 h dark (14L/10D) to 10L/14D also resulted in decreased blood levels of these hormones. A protocol of melatonin injections using 2.5 mg daily, on the other hand, did not significantly inhibit blood levels of thyroid hormones or TSH; injection of this dose every afternoon into hamsters in long photoperiod significantly augmented the blood levels of T4. Continuously available melatonin in the form of subcutaneous implants of 1 mg melatonin in beeswax did not inhibit blood levels of thyroid hormones; furthermore, such implants prevented the inhibitory effects of injections of 25 micrograms melatonin. The results are consistent with the hypothesis that melatonin interferes with neurotransmitters which influence the synthesis or release of hypothalamic thyrotropin-releasing hormone.

Effects of daily afternoon melatonin administration on monoamine accumulation in median eminence and striatum of ovariectomized hamsters receiving pargyline.

Basic fibroblast growth factor (FGF), its mRNA and the mRNA that encodes for its receptor have all been localized in the rat subfornical organ (SFO). Basic FGF is widely distributed throughout the SFO; it is present in neurons, in the vascular basement membrane of lateral blood vessels (but not those within the SFO) and in ependymal cells surrounding the SFO. Results of in situ hybridization show that the expression of basic FGF mRNA is detected throughout the organ. Similarly, the expression of flg, the gene for the putative basic FGF receptor, can also be detected in the SFO. The results all support the possibility that this growth factor may modulate the known physiological functions of the SFO.The effects of daily afternoon melatonin injections on the accumulation of monoamines were studied in extracts of median eminence, and of caudate nucleus, of intact and ovariectomized Syrian hamsters which were administered pargyline 2 h prior to sacrifice. Although no significant effect of melatonin administration on the serotonin (5HT) accumulation after pargyline could be detected, significantly increased amounts of 5HT and of the 5HT metabolite, 5-hydroxyindole acetic acid, were detected in median eminence and in caudate nucleus of melatonin-injected hamsters not treated with pargyline. In both median eminence and in posterior pituitary, dopamine (DA) concentrations were significantly reduced by melatonin administration. In the median eminence of intact hamsters, the accumulation of DA after pargyline was reduced to 22% of controls by melatonin injections; in ovariectomized hamsters, the accumulation of DA was reduced to 9% of controls by melatonin injections. The accumulation of norepinephrine after pargyline was significantly reduced by melatonin administration only in ovariectomized hamsters. No significant inhibitory effects of melatonin injections could be detected on DA accumulation in caudate nucleus. These data suggest that melatonin injections result in substantial inhibition of daytime DA synthesis in median eminence independently of its effects on gonadal steroids. Paradoxically, melatonin-induced inhibition of median eminence DA activity occurred concomitantly with suppression of pituitary and plasma prolactin (PRL). We conclude that daily afternoon melatonin injections inhibit PRL secretion and interfere with cycles of LH in spite of decreased DA activity in the median eminence.

Effects of valproate on amino acid and monoamine concentrations in striatum of audiogenic seizure-prone balb/c mice

The effects of valproate on CNS concentrations of gamma-aminobutyric acid (GABA), glulamate (GLU), glutamine (GLN); dopamine (DA), serotonin (5-HT), and metabolites were examined in tissue extracts of caudate nucleus of genetic substrains of Balb/c mice susceptible (EP) or resistant (ER) to audiogenic seizures. Generalized tonic-clonic seizures observed in EP mice were inhibited by valproate, administered 1 h prior to testing, in a dose-response fashion. Concentrations of GABA, GLU, and GLN, which were lower in EP mice than in ER mice, were significantly increased by valproate at doses of 180 and 360 mg/kg. Concentrations of homovanillic acid (HVA) and hydroxyindoleacetic acid (5-HIAA), metabolites of DA and 5-HT, were substantially increased by valproate at these doses. The in situ activity of tyrosine hydroxylase (TH) was not significantly influenced by valproate, whereas a valproate-induced increase in tryptophan hydroxylase (TPH) activity was observed in both striatum and in midbrain tegmentum. The data are consistent with the interpretation that anti-convulsive doses of valproate influences the intraneuronal metabolism of monoamines, GABA, and glutamate concurrently. Valproates influence on the metabolism of both major inhibitory (GABA) and excitatory (GLY amino acids in striatum could contribute to its anti-convulsive effects in genetically seizure prone mice, as well as to the accumulation of DA and 5-HT metabolites.

Plasma corticosterone in chicks reared under several lighting schedules

Plasma corticosterone was determined by radioimmunoassay in 6-7-week-old male broiler type chicks, reared under several carefully controlled lighting regimes. When subjects were grouped by photoperiod of rearing, chicks reared in darkness had significantly lower hormone levels than diurnal controls, or than subjects reared in continuous light. Around-the-clock sampling revealed a diurnal corticosterone rhythm, with high daytime levels and lower night-time levels. This rhythm appeared to be retained in constant light, although phase shifted or free running. Neither analysis by light intensity level nor by lights on/lights off status at the time of blood sampling revealed differences in plasma corticosterone between the experimental groups which could be attributed to these factors.

Monoamine neurotransmitters and amino acids in the cerebrum and striatum of immature rats with kaolin-induced hydrocephalus

Hydrocephalus is characterized by enlargement of the cerebral ventricles. The behavioral disturbances are, in some cases, rapidly reversible by surgical treatment suggesting that there may be a functional impairment of neurons. Hydrocephalus was induced in 3-week old rats by kaolin injection into the cisterna magna. Parietal cerebrum and striatum content of monoamine neurotransmitters and amino acids were assayed by high performance liquid chromatography (HPLC), 1, 2, or 4 weeks after induction of hydrocephalus. The ventricles exhibited progressive enlargement which was partially reversed by surgical treatment. Cerebral water content was increased at all stages. Increased levels of cerebral aspartate and glutamate suggest that there is the potential for excitatory neurotoxicity. The increase in cerebral taurine correlated negatively with the increase in water content. Cerebral concentrations of norepinephrine and serotonin, and its metabolite 5-HIAA, were increased at 1 and 2 weeks suggesting an increase in their turnover during the early stages of ventricular dilatation. Dopamine and its metabolite DOPAC were transiently diminished in the striatum at 1 and 2 weeks, respectively, suggesting that axonal projections from the brainstem may be impaired. We conclude that the effect of hydrocephalus on amino acids and monoamines varies regionally. Due to increased water content, there may be dilution effects in whole tissue, therefore, it is important to make determinations on the basis of protein content.

Melatonin as a proteasome inhibitor. Is there any clinical evidence

Proteasome inhibitors and melatonin are both intimately involved in the regulation of major signal transduction proteins including p53, cyclin p27, transcription factor NF-κB, apoptotic factors Bax and Bim, caspase 3, caspase 9, anti-apoptotic factor Bcl-2, TRAIL, NRF2 and transcription factor beta-catenin. The fact that these factors are shared targets of the proteasome inhibitor bortezomib and melatonin suggests the working hypothesis that melatonin is a proteasome inhibitor. Supporting this hypothesis is the fact that melatonin shares with bortezomib a selective pro-apoptotic action in cancer cells. Furthermore, both bortezomib and melatonin increase the sensitivity of human glioma cells to TRAIL-induced apoptosis. Direct evidence for melatonin inhibition of the proteasome was recently found in human renal cancer cells. We raise the issue whether melatonin should be investigated in combination with proteasome inhibitors to reduce toxicity, to reduce drug resistance, and to enhance efficacy. This may be particularly valid for hematological malignancies in which proteasome inhibitors have been shown to be useful. Further studies are necessary to determine whether the actions of melatonin on cellular signaling pathways are due to a direct inhibitory effect on the catalytic core of the proteasome, due to an inhibitory action on the regulatory particle of the proteasome, or due to an indirect effect of melatonin on phosphorylation of signal transducing factors.