Sergio D. Paredes

Melatonin induces mitochondrial‐mediated apoptosis in human myeloid HL‐60 cells

Abstract:  The role of melatonin in the mediation of apoptotic events has recently gained attention, especially after recent studies have reported that melatonin exerts antiapoptotic actions in normal cells but may activate proapoptotic pathways in some tumor cells. Here, we have evaluated the effect of melatonin on apoptosis in the human leukemia cell line HL‐60. Melatonin treatment (1 mm) induced a significant increase in caspase‐3 and ‐9 activities. The effect of melatonin on the activation of caspases was time dependent, reaching a maximum after 12 hr of stimulation, and then decreasing to a minimum after 72 hr. Treatment with melatonin also evoked mitochondrial membrane depolarization and permeability transition pore induction, which caused loss of mitochondrial staining by calcein, and increased cell death by apoptosis/necrosis as demonstrated by propidium iodide positive‐staining of cells after 72 hr of stimulation. In addition, the exposure of cells to melatonin resulted in an activation and association of the proapoptotic proteins Bax and Bid, as well as promoting detectable increases in the expression of both proteins. We conclude that melatonin has proapoptotic and/or oncostatic effects in the human myeloid cell line HL‐60.

Protective effect of melatonin against human leukocyte apoptosis induced by intracellular calcium overload: relation with its antioxidant actions

Abstract:  Apoptosis or programmed cell death plays a critical role in both inflammatory and immune responses. Recent evidence demonstrates that control of leukocyte apoptosis is one of the most striking immune system‐related roles of melatonin. For this reason, this study evaluated the protective effects of melatonin on human leukocyte apoptosis induced by sustained cytosolic calcium increases. Such protective effects are likely mediated by melatonin’s free‐radical scavenging actions. Treatments with the specific inhibitor of cytosolic calcium re‐uptake, thapsigargin (TG), and/or the calcium‐mobilizing agonist, N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), induced intracellular reactive oxygen species (ROS) production, caspase activation as well as DNA fragmentation in human leukocytes. Also, TG‐ and/or FMLP‐induced apoptosis was dependent on both cytosolic calcium increases and calcium uptake into mitochondria, because when cells were preincubated with the cytosolic calcium chelator, dimethyl BAPTA, and the inhibitor of mitochondrial calcium uptake, Ru360, TG‐ and FMLP‐induced apoptosis was largely inhibited. Importantly, melatonin treatment substantially prevented intracellular ROS production, reversed caspase activation, and forestalled DNA fragmentation induced by TG and FMLP. Similar results were obtained by preincubating the cells with another well‐known antioxidant, i.e., N‐acetyl‐l‐cysteine. To sum up, depletion of intracellular calcium stores induced by TG and/or FMLP triggers different apoptotic events in human leukocytes that are dependent on calcium signaling. The protective effects resulting from melatonin administration on leukocyte apoptosis likely depend on melatonin’s antioxidant action because we proved that this protection is melatonin receptor independent. These findings help to understand how melatonin controls apoptosis in cells of immune/inflammatory relevance.

Jerte Valley Cherry-Enriched Diets Improve Nocturnal Rest and Increase 6-Sulfatoxymelatonin and Total Antioxidant Capacity in the Urine of Middle-Aged and Elderly Humans

Tryptophan, serotonin, and melatonin, present in Jerte Valley cherries, participate in sleep regulation and exhibit antioxidant properties. The effect of the intake of seven different Jerte Valley cherry cultivars on the sleep-wake cycle, 6-sulfatoxymelatonin levels, and urinary total antioxidant capacity in middle-aged and elderly participants was evaluated. Volunteers were subjected to actigraphic monitoring to record and display the temporal patterns of their nocturnal activity and rest. 6-sulfatoxymelatonin and total antioxidant capacity were quantified by enzyme-linked immunosorbent assay and colorimetric assay kits, respectively. The intake of each of the cherry cultivars produced beneficial effects on actual sleep time, total nocturnal activity, assumed sleep, and immobility. Also, there were significant increases in 6-sulfatoxymelatonin levels and total antioxidant capacity in urine after the intake of each cultivar. These findings suggested that the intake of Jerte Valley cherries exerted positive effect on sleep and may be seen as a potential nutraceutical tool to counteract oxidation.

Melatonin reduces body weight gain and increases nocturnal activity in male Wistar rats

AIM This study evaluated the effect of the administration of melatonin, the chief secretory product of the pineal gland, on the body weight in male Wistar rats. MAIN METHODS The animals were housed for 4months in cages equipped to log horizontal activity within a thermostatically-controlled chamber, under a 12h/12h light/dark photoperiod (lights on at 08:00h). After acclimatization, the animals were divided into two groups: (1) control animals, and (2) melatonin-treated animals. Melatonin was administered in tap water (20μg/ml), and fresh drinking fluid was changed twice weekly. Rats were fed a standard diet ad libitum. KEY FINDINGS Food and water intake, body weight, the amplitude of the activity/rest rhythm (motor activity), and blood melatonin and glucose concentrations were measured. The administration of melatonin did not influence either food or water intake or glucose levels relative to those found in the control animals. However, melatonin administration reduced body weight gain and increased nocturnal locomotor activity. The peak concentration of melatonin was found at night coinciding with the increase in nocturnal activity. SIGNIFICANCE The results show that exogenous melatonin reduces body weight gain without having marked effects on metabolism. This may be due in part to the increased nocturnal activity shown by the animals treated with the indoleamine.

Melatonin enhances hydrogen peroxide-induced apoptosis in human promyelocytic leukaemia HL-60 cells

Melatonin is an indoleamine secreted by the pineal gland that shows multiple tasks. This ubiquitously acting free radical scavenger has recently been shown to stimulate the production of reactive oxygen species (ROS) in tumour cells, making them undergo apoptosis, whilst it prevents apoptosis in healthy cells. The mechanisms by which melatonin exerts these dual actions are, however, not yet clearly understood. Thus, the aim of this study was to further investigate how melatonin can enhance oxidative stress-induced apoptosis in a leukaemia cell line. The results show that melatonin increased the apoptotic effects of H2O2 in human myeloid HL-60 cells as assessed by cellular viability, mitochondrial permeability transition induction, mitochondrial membrane depolarization, ROS generation, caspases 3, 8 and 9 activity, phosphatidylserine externalization, and DNA fragmentation techniques. When healthy leucocytes were exposed to H2O2, melatonin increased the viability of the cells. Taken together, the findings indicate that melatonin is a potential physiological tool capable of protecting healthy cells from chemotherapy-induced ROS production as well as inducing tumour cell death. Because cancer cells manifest increased oxidative stress as a result of their elevated metabolism, the use of melatonin may be useful in impairing their ROS buffering capacity.

Melatonin and Tryptophan Affect the Activity–Rest Rhythm, Core and Peripheral Temperatures, and Interleukin Levels in the Ringdove: Changes With Age

Aging is known to alter the circadian rhythms of melatonin, serotonin, thermoregulatory responses, cytokine production, and sleep/wakefulness which affect sleep quality. We tested the possible palliative effects of a 3-day administration of melatonin (0.25 or 2.5 mg/kg of body weight [b.w.] to young and old ringdoves, respectively) or tryptophan (300 mg/kg of b.w. to old ringdoves) on these rhythms. Doves are a monophasic, diurnal species; these characteristics are similar in humans. Old animals presented lower melatonin and serotonin levels; higher interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha values; and reductions in the Midline-Estimating Statistic of Rhythm and amplitude of activity-rest rhythm and in the amplitude of the core temperature rhythm. Melatonin raised serum melatonin levels; tryptophan increased both melatonin and serotonin levels. Melatonin and tryptophan lowered nocturnal activity, core temperature, and cytokine levels and increased peripheral temperature in both groups. Melatonin or tryptophan may limit or reverse some of the changes that occur in sleep-wake rhythms and temperature due to age.

Circadian levels of serotonin in plasma and brain after oral administration of tryptophan in rats.

Serotonin, one of the most important neurotransmitters in the central nervous system, is synthesized by the amino acid, tryptophan. Given that this essential amino acid is consumed in the diet, the aim of this study was to evaluate the effect of orally administered L-tryptophan (125 mg/kg) on circadian variations in the levels of serotonin in brain and plasma. We used male Wistar rats of 14 +/- 2 weeks of age (n = 240), maintained under conditions of a 12-hr light:dark cycle, and food and water ad libitum. Tryptophan administration was by gavage in a daily single dose at 7 p.m. for 7 days. The serotonin levels were measured by ELISA every hour at night (8 p.m. to 8 a.m.) and every 4 hr during daytime (8 a.m. to 8 p.m.). The results show that in both the tryptophan-treated and untreated groups the highest values appeared during the beginning of the darkness with a peak at 9, 10 and 11 p.m. in controls, and at 9 p.m. in the tryptophan-treated group. After tryptophan administration, the levels of serotonin were significantly higher in the plasma and all the brain regions analysed than in the control group. This increase of serotonin levels was greater in the pineal gland than in other brain regions, and the least in plasma. In conclusion, oral administration of tryptophan during 7 days enhances serotonin levels over a 24-hr period, and produces an advance in the peak of serotonin in both plasma and different brain regions.

Melatonin is able to delay endoplasmic reticulum stress-induced apoptosis in leukocytes from elderly humans.

The mechanisms regulating neutrophil apoptosis are basically unaffected by the aging process. However, a significant impairment of cell survival occurs in elderly individuals following neutrophil challenge with pro-inflammatory stimuli, such as granulocyte-macrophage colony-stimulating factor (GM-CSF). The goal of the present study was to prove the effects of melatonin supplementation on apoptosis induced by calcium signaling in human leukocytes from elderly volunteers. Treatments with the specific inhibitor of cytosolic calcium re-uptake, thapsigargin, and/or the calcium mobilizing agonist, N-formyl-methionyl-leucyl-phenylalanine (fMLP), induced mitochondrial membrane depolarization, caspase activation, phosphatidylserine (PS) externalization, and DNA fragmentation in leukocytes from both young and elderly volunteers, although such effects were much more evident in aged leukocytes. Importantly, melatonin treatment substantially preserved mitochondrial membrane potential, reversed caspase activation, reduced PS exposure and forestalled DNA fragmentation in leukocytes from both age groups. In conclusion, melatonin is able to delay endoplasmic reticulum stress-induced apoptosis in aged leukocytes and may counteract, at the cellular level, age-related degenerative phenomena linked to oxidative stress.

Circadian variations of serotonin in plasma and different brain regions of rats

Most of the physiological processes that take place in the organism follow a circadian rhythm. Serotonin is one of the most important neurotransmitters in our nervous system, and has been strongly implicated in the regulation on the mammalian circadian clock, located in the suprachiasmatic nuclei (SCN). The present study analysed the levels of serotonin over a period of 24 h in the plasma and in different brain regions. The model used was of male Wistar rats, 14 ± 2 weeks of age (n = 120), maintained under conditions of 12 h light and 12 h dark, and food and water ad libitum. The serotonin levels were measured by ELISA every hour at night (20:00–08:00 h) and every 4 h during the daytime (08:00–20:00 h). Ours results show that the maximum levels of serotonin in plasma were obtained at 09:00 and 22:00 and a minor peak at 01:00 h. In hypothalamus there was a significant peak at 22:00 and two minor peaks at 17:00 and 02:00 h; the same occurred in hippocampus with a significant peak at 21:00, and two secondary peaks at 24:00 and 05:00 h; in cerebellum there were two peaks at 21:00 and 02:00 h, while in striatum and pineal there were peaks at 21:00 h and 23:00, respectively. In conclusion, the higher levels of serotonin were during the phase of darkness, which varies depending on the region in which it is measured.

Hydrogen peroxide increases the phagocytic function of human neutrophils by calcium mobilisation

We have studied the effect of exogenous administration of hydrogen peroxide (H2O2) on phagocytic activity of human neutrophils. The treatment of cells with increasing concentrations of H2O2 evoke a significant elevation of phagocytic function assayed as phagocytic index, percentage and efficiency; and was similar to that induced by the calcium mobilising agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This stimulatory effect was reduced by pre-treatment of neutrophils with catalase and abolished in neutrophils loaded with the intracellular calcium quelator dimethyl BAPTA. In the absence of extracellular calcium, treatment of cells with H2O2 resulted in a increase in [Ca2+]i, indicating the release of calcium from intracellular stores. H2O2 abolished the typical calcium release stimulated by the physiological agonist fMLP, while depletion of agonist-sensitive calcium pools by fMLP was able to prevent H2O2-induced calcium release. We conclude that H2O2 induces calcium release from agonist-sensitive stores and consequently increase the phagocytosis process.