Rodolfo A. Cutrera

Melatonin effects on bone: experimental facts and clinical perspectives

Abstract: Bone formation proceeds through a remodeling process that runs continuously, involving the resorption of old bone by osteoclasts, and the subsequent formation of new bone by osteoblasts. This is controlled by growth factors and cytokines produced in bone marrow microenvironment and by the action of systemic hormones, like parathyroid hormone, estradiol or growth hormone (GH). One candidate for hormonal modulation of osteoblast and osteoclast formation is melatonin. Because circulating melatonin declines with age, its possible involvement in post‐menopausal and senescence osteoporosis is considered. This review article discusses early studies on melatonin–bone relationships and recent data that suggest a direct effect of melatonin on bone. Melatonin could act as an autacoid in bone cells as it is present in high quantities in bone marrow, where precursors of bone cells are located. Melatonin dose‐dependently augmented proteins that are incorporated into the bone matrix, like procollagen type I c‐peptide. Osteoprotegerin, an osteoblastic protein that inhibits the differentiation of osteoclasts is also augmented by melatonin in vitro. Another possible target cell for melatonin is the osteoclast, which degrades bone partly by generating free radicals. Melatonin through its free radical scavenger and antioxidant properties may impair osteoclast activity and bone resorption. At least in one study melatonin was both inhibitory to osteoclastic and osteoblastic cells. Therefore, the documented bone‐protecting effect of melatonin in ovariectomized rats can depend in part on the free radical scavenging properties of melatonin. Additionally, melatonin may impair development of osteopenia associated with senescence by improving non‐rapid eye movement sleep and restoring GH secretion. Whether melatonin can be used as a novel mode of therapy for augmenting bone mass in diseases deserves to be studied.

Melatonin site and mechanism of action: Single or multiple?

ABSTRACT: By affecting the entrainment pathways of the biologic clock, melatonin has a major influence on the circadian and seasonal organization of vertebrates. In addition, a number of versatile functions that far transcend melatonin actions on photoperiodic time measurement and circadian entrainment have emerged. Melatonin is a free radical scavenger and antioxidant and it has a significant immunomodulatory activity, being presumably a major factor in an organisms defense toxic agents and invading organisms. Besides affecting specific receptors in cell membranes to exert its effects, the interaction of melatonin with nuclear receptor sites and with intracellular proteins, like calmodulin or tubulin‐associated proteins, as well as the direct antioxidant effects of melatonin, may explain many general functions of the pineal hormone.

Paraquat induces behavioral changes and cortical and striatal mitochondrial dysfunction.

Paraquat is a highly toxic quaternary nitrogen herbicide capable of increasing superoxide anion production. The aim of this research was to evaluate various behavioral changes and study cortical, hippocampal, and striatal mitochondrial function in an experimental model of paraquat toxicity in rats. Paraquat (10mg/kg ip) was administered weekly for a month. Anxiety-like behavior was evidenced in the paraquat-treated group as shown by a diminished time spent in, and fewer entries into, the open arms of an elevated-plus maze. Also, paraquat treatment induced a deficit in the sense of smell. In biochemical assays, NADH-cytochrome c reductase activity was significantly inhibited by 25 and 34% in cortical and striatal submitochondrial membranes, respectively. Striatal cytochrome oxidase activity was decreased by 24% after paraquat treatment. Also, cortical and striatal mitochondria showed 55 and 74% increased State 4 respiratory rates, respectively. Paraquat treatment decreased striatal State 3 oxygen consumption by 33%. Respiratory controls were markedly decreased in cortical and striatal mitochondria, indicating mitochondrial dysfunction after paraquat treatment, together with mitochondrial depolarization and increased hydrogen peroxide production rates. We demonstrate that paraquat induced alterations in nonmotor symptoms and cortical and striatal mitochondrial dysfunction.

A multifactorial approach employing melatonin to accelerate resynchronization of sleep–wake cycle after a 12 time‐zone westerly transmeridian flight in elite soccer athletes

Rapid transmeridian translocation through multiple time zones has a negative impact on athletic performance. The aim of the present study was to test the timely use of three factors (melatonin treatment, exposure to light, physical exercise) to hasten the resynchronization of a group of elite sports competitors and their coaches to a westerly transmeridian flight comprising of 12 time‐zones. Twenty‐two male subjects were included in the study. They were professional soccer players and their coaches who travelled to Tokyo to play the final game of the Intercontinental Coup. The day prior to departure, urine was collected from each subject from 18:00 to 06:00 hrs to measure the melatonin metabolite 6‐sulphatoxymelatonin. Participants were asked to complete sleep log diaries from day 0 (preflight) to the day before returning to Buenos Aires (day 8). All subjects received 3 mg of melatonin p.o. daily at expected bedtime at Tokyo immediately after leaving Buenos Aires. Upon arrival at Tokyo the subjects performed a daily physical exercise routine outdoors at two restricted times of the day (from 08:00 to 11:00 hrs in the morning and from 13:00 to 16:00 hrs in the afternoon). Exposure to sunlight or physical exercise at other times of the day was avoided. Except for the number of awakenings (which increased on days 1 and 3) and sleep latency (which decreased on days 2, 6 and 8), there was an absence of significant changes in subjective sleep parameters as compared with preflight assessment. Sleep quality and morning alertness at Tokyo correlated significantly with preflight 6‐sulphatoxymelatonin excretion. Mean resynchronization rate of sleep–wake cycle to the 12 hr‐time shift was 2.13 ± 0.88 days, significantly different from the minimal resynchronization rate of 6 days expected after a 12‐time‐zones flight. The results indicate that the combination of melatonin treatment, an appropriate environmental light schedule and timely applied physical exercise can be useful to help elite athletes to overcome the consequences of jet lag.

Effect of melatonin on bone metabolism in ovariectomized rats

To assess the effect of pharmacological dose of melatonin on bone metabolism in ovariectomized rats, urinary deoxypyridinoline (a marker of bone resorption) and calcium excretion, circulating levels of calcium, phosphorus and bone alkaline phosphatase activity (a marker of bone formation), and bone mineral density (BMD), mineral content (BMC) and bone area (BA) of total body, were measured in adult rats for up to 60 days after surgery. Rats received melatonin in the drinking water (25 microg/ml water) or drinking water alone. Urinary deoxypyridinoline increased significantly after ovariectomy by 51% (30 days after surgery) and by 47% (60 days after surgery). The increase in urinary deoxypyridinoline found 30 days after ovariectomy was not observed in melatonin-treated rats. Urinary calcium concentration was similar in the 4 experimental groups studied, as was the circulating calcium concentration at every time interval examined. Fifteen days after surgery, a significant increase in serum phosphorus and bone alkaline phosphatase levels occurred in ovariectomized rats receiving melatonin as compared to their controls. Sixty days after surgery BMD, BMC and BA decreased significantly in ovariectomized rats, an effect not modified by melatonin. Serum estradiol decreased significantly by 30 days after ovariectomy to attain values close to the limit of detection of the assay by 60 days after ovariectomy. The results support the conclusion that a pharmacological amount of melatonin modifies bone remodeling after ovariectomy and that the effect may need adequate concentrations of estradiol.

Effect of Superior Cervical Ganglionectomy on 24-h Variations in Hormone Secretion from the Anterior Hypophysis and in Hypothalamic Monoamine Turnover During the Preclinical Phase of Freund's Adjuvant Arthritis in Rats

The effect of superior cervical ganglionectomy (SCGx) on 24‐h rhythms of circulating adrenocorticotropic hormone (ACTH), growth hormone (GH), prolactin and luteinizing hormone (LH) and of hypothalamic noradrenaline content and dopamine and serotonin turnover, was assessed in rats 3 days after administering Freunds complete adjuvant. In sham‐operated rats, Freunds adjuvant injection increased serum ACTH without affecting its diurnal rhythmicity. SCGx, performed 10 days earlier, suppressed 24‐h rhythmicity and augmented mean values of circulating ACTH. A depressive effect of immunization on GH release was found in both sham‐operated and SCGx rats. GH concentrations did not exhibit diurnal rhythmicity and decreased after immunization. Time‐of‐day‐related changes in serum prolactin were significant for all examined groups, except for SCGx‐immunized rats. Freunds adjuvant administration augmented prolactin secretion. Daily changes in serum LH concentration and a decrease after immunization were found in both sham‐operated and SCGx rats. SCGx: (i) counteracted inhibition of daily variations of noradrenaline content in medial hypothalamus of Freunds adjuvant‐injected rats; (ii) decreased anterior hypothalamic dopamine turnover and augmented it in the medial hypothalamus; (iii) lowered amplitude of serotonin turnover rhythm in medial hypothalamus. The data indicate that several early changes in levels and 24‐h rhythms of circulating ACTH and prolactin, and in hypothalamic noradrenaline content and dopamine and serotonin turnover, were modified by a previous SCGx in Freunds adjuvant‐injected rats.

Effects of prenatal stress on motor performance and anxiety behavior in Swiss mice.

Stressor presence during the last weeks of gestation has been associated with behavioral disorders in later life. In this study we support further research on the long term effects of prenatal stress on Swiss mice descendants behavior. Prenatal stress procedure consisted on restraining the dams under bright light for 45 min, three times per day from the 15th day of pregnancy, until birth. After weaning, offsprings motor performance and spontaneous exploratory behavior were measured by the tight-rope and T-maze tests, respectively. We also evaluated anxiety behavior using elevated plus maze test. We found that maternal stress improves the performance of the animals in the tight rope test and that this effect was sex and age dependent: prenatal stressed males obtained the best scores during the first month of life, while in females the same was achieved at the second month. Spontaneous exploratory behavior analysis revealed that it was elevated in prenatal stressed males and that this effect persisted on time. However, we did not find significant differences on this behavioral response among both females groups. Finally, differences on anxiety behavior were found only in females: prenatally stressed animals showed a higher proportion of entries into the open arms of a plus maze (reduced anxiety) compared to the control group. Our results show that prenatal stress modifies the normal behavior of the progeny: prenatal stressed animals have a better performance in the carried out test. These notably results suggest the existence of an adaptive response to prenatal stress.

Effect of melatonin treatment on 24-h variations in responses to mitogens and lymphocyte subset populations in rat submaxillary lymph nodes.

Wistar male rats were injected s.c. with melatonin (30 μg) or vehicle, 1 h before lights off, for 11 days. Ten days after beginning melatonin treatment, rats received Freund’s complete adjuvant or its vehicle s.c., and after 2 days, they were sacrificed at six different time intervals throughout a 24‐h cycle. The mitogenic effect of lipopolysaccharide (LPS) and concanavalin A (Con A), the activity of ornithine decarboxylase (ODC) and the relative size of lymphocyte subset populations were measured in submaxillary lymph nodes. In control rats, the mitogenic effects of LPS and Con A and ODC activity peaked during the afternoon. Injection of Freund’s adjuvant induced a 10‐h shift in the diurnal rhythm of the mitogenic effect of LPS to attain maximal values at night. Melatonin pretreatment blunted the daily variations in the mitogenic activity of Con A or LPS and, when given to Freund’s adjuvant‐injected rats, augmented mesor and amplitude of diurnal rhythm in ODC activity. Maxima in B cell number occurred at night whereas those of T and B‐T cell number occurred during the afternoon. During the early phase of immunization tested, the number of B cells augmented and the amplitude of its diurnal rhythmicity increased both after immunization and following melatonin pretreatment. Maxima of 24‐h rhythms in CD4+ and CD4+/CD8+ cell populations occurred during the afternoon while those of CD8+ cells occurred at late night. Melatonin significantly augmented CD4+ cell number and decreased CD8+ cell number; it therefore augmented the CD4+:CD8+ ratio. The results suggest that pretreatment with a pharmacological dose of melatonin exerts immunomodulating effects at an early, preclinical, phase of Freund’s adjuvant‐induced arthritis in rats.

Evidence for local synthesis of melatonin in golden hamster retina.

Daily variations in melatonin content of retinas of pinealectomized and sham-operated golden hamsters were studied. Melatonin content showed significant daily variations with maximal values at night (i.e. early in the night in pinealectomized hamsters and late at night in sham-operated animals). Moreover, mean retinal melatonin levels augmented significantly after pinealectomy. In vitro the augmented melatonin levels found in retinas incubated in darkness for 8 h was suppressed by exposure to light, indicating the ability of hamster retina to regulate melatonin synthesis in isolated conditions. Taken together, the in vivo and in vitro results support daily variations of melatonin content of exclusive retinal origin.

Experimental allergic encephalomyelitis in male Lewis rats subjected to calorie restriction

This work analyzes the effect of calorie restriction on the development of experimental allergic encephalomyelitis (EAE) in Lewis rats. Plasma levels of ACTH, corticosterone, prolactin and growth hormone (GH) and mitogenic responses in submaxillary lymph nodes were measured. Male Lewis rats (6 weeks-old) were submitted to a calorie restriction equivalent to 66% of food restriction or to a normal diet. Fifteen days later, rats were injected with complete Freund’s adjuvant plus spinal chord homogenate (SCH) or with complete Freund’s adjuvant alone. Rats were monitored daily for clinical signs of EAE and were killed on day 15 after immunization. Only rats subjected to normal diet exhibited clinical signs of the disease. The increase in plasma ACTH and corticosterone found after SCH immunization in controls was not detectable in calorie restricted rats. Likewise, the correlation between circulating ACTH and corticosterone was no longer found after calorie restriction. Generally, calorie restriction by itself augmented plasma ACTH or corticosterone and this increase was not further amplified by SCH immunization. Only calorie restricted rats exhibited augmented plasma prolactin levels after SCH immunization, and decreased plasma GH levels regardless of immunization. Calorie restriction depressed the mitogenic response of lymphoid cells to concanavalin A but not to lipopolysaccharide. Calorie restricted rats did not exhibit augmented mitogenic response to concanavalin A following SCH immunization as those found in controls. The results are compatible with the view that the course of EAE can be significantly modified by caloric restriction.ResumenEn este trabajo se analiza el efecto de la restricción calórica en el desarrollo de encefalitis alérgica experimental (EAE) en ratas de la cepa Lewis. Se evaluaron los niveles plasmáticos de ACTH, corticosterona, prolactina y hormona de crecimiento, y las respuestas mitogénicas en ganglios linfáticos submaxilares. Se utilizaron ratas macho de 6 semanas de vida sometidas a restricción calórica del 66% o a dieta normal. Tras 15 días, se les inyectó adyuvante completo de Freund emulsionado con homogenado de médula espinal (HME) o sólo el adyuvante. Se evaluaron diariamente los signos clínicos de EAE, sacrificándose las ratas a los 15 días de la inmunización. Sólo los animales mantenidos con dieta normal mostraron signos clínicos de la enfermedad. Las ratas con dieta restringida no exhibieron los aumentos significativos en ACTH y corticosterona detectados en ratas inmunizadas con HME, ni tampoco se obtuvo la correlación significativa entre los niveles circulantes de ACTH y corticosterona observada en los animales sometidos a dieta normal. De forma general, la restricción calórica aumentó los niveles de ACTH y corticosterona y este aumento no se afectó por la inmunización con HME. Sólo en las ratas sometidas a restricción calórica se obtuvo aumento en la secreción de prolactina tras la inmunización con HME. Los niveles de hormona de crecimiento disminuyeron en los animales con dieta restringida. La restricción calórica redujo la respuesta mitogénica de los ganglios linfáticos ante la concana-valina A, pero no ante el lipopolisacárido. La inmunización con HME provocó un aumento de la respuesta mitogénica ante concanavalina A sólo en ratas con dieta normal. Estos resultados indican que la restricción calórica puede afectar significativamente la evolución de la encefalitis alérgica experimental.