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Oral Administration of Melatonin to Old Ring Doves (Streptopelia risoria) Increases Plasma Levels of Melatonin and Heterophil Phagocytic Activity

Department of Animal Physiology, Faculty of Science, University of Extremadura, Badajoz, Spain.

Address reprint requests to: M^a del Pilar Terrón Sánchez, Department of Physiology, Faculty of Science, University of Extremadura, Avda Elvas s/n. 06071- Badajoz, Spain. E-mail: pilarts{at}unex.es

	Abstract

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We analyzed the effect of oral melatonin (23 µg/0.1 ml/animal/d; 1 h before dark on 12 consecutive days) in old birds, in natural photoperiods, on the hormone's plasma levels, and phagocytosis. Blood collections were performed daily at 2:00 AM and 4:00 PM until 5 days after the treatment. From day 1, the melatonin levels were significantly higher than basal levels at both times. Values at 2:00 AM were significantly higher than the 4:00 PM values. After treatment, the melatonin levels declined, returning from day 14 to basal values at both hours. At 2:00 AM, phagocytosis was significantly greater than that obtained at 4:00 PM and greater than basal values. The 4:00 PM values were only significantly greater than basal on days 6 and 8, parallel to a decline in superoxide anion levels, which were lowest at 2:00 AM. Melatonin administered to old ring doves increases the differences between nocturnal and diurnal plasma levels, and, in parallel, increases phagocytosis and reduces superoxide radical levels in heterophils.

Perhaps the condition, in which the stimulatory effects of melatonin on the immune system are best demonstrated occur in those situations in which the immune system is depressed. The many models of immunosuppression include aging, physical stress, infectious diseases, and treatment with corticoids and antitumoral or adrenergic drugs (2). There is a growing literature to indicate that there are some important links between the 24-hour rhythm of melatonin and age-related changes in physiology and behavior. Because melatonin acts by providing information to the organism about its overall temporal organization, this indolamine would seem to have the potential to be an important pharmacological agent for attenuating age-related changes in circadian organization, immune system, sleep, and other disorders that accompany aging (5). Previously, we had found a significant decline in both nocturnal and diurnal plasma melatonin levels in old ring doves (>8 years) with respect to the concentrations observed in mature (adult, aged 3–5 years) and young animals (aged 1–3 years); also, there is an absence of a circadian melatonin rhythm (the levels remaining more or less constant throughout the 24 hours) in these birds (6). Our results thus corroborated previous reports that melatonin is synthesized and secreted during the dark period of the light/dark cycle, and that plasma concentrations decline in advanced age (7). We also reported previously an enhancing dose-dependent effect of melatonin, at both physiological and pharmacological concentrations, on phagocytic function in the dove. Likewise, melatonin limits oxidative stress, which results from the function of the immune system (6,8,9).

In the present experiments, we studied the effect in old ring doves of oral administration of melatonin at the pharmacological dose of 23 µg/animal/day on the plasma levels of melatonin during a 12-day treatment period and in the 5 subsequent days; furthermore, we examined the effect of this treatment on the phagocytic function and oxidative metabolism of blood heterophils. Plasma determinations were carried out at two specific times (2:00 AM and 4:00 PM), with the heterophil phagocytic activity being evaluated at these times on alternate days from the beginning until the end of the treatment.

	METHODS

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Animals
Male and female ring doves (Streptopelia risoria) aged 8–10 years (old for this species) were used in the study (n = 8). The birds were housed isolated in cages measuring 40 x 40 x 45 cm, and fed ad libitum in a room with an outside window (natural lighting) and indirect ventilation. The study was conducted from April to June, when the natural photoperiod was approximately 14 hours light and 10 hours dark (dark period from 9:30 PM ± 30 minutes to 7:30 AM ± 30 minutes). The temperature was constant and maintained at 22 ± 2°C. Some birds were randomly chosen for a blood sample. The study was approved by the Ethical Committee of the University of Extremadura (Badajoz, Spain) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Melatonin
N-acetyl-5-methoxytryptamine (melatonin; Sigma, St. Louis, MO) was prepared in phosphate-buffered saline (PBS) solution, starting from a base solution of 10 mg/ml, which was dissolved by stirring, and used at the pharmacological concentration of 230 µg/ml (a dose 10 times higher than that used in our in vitro experiments, considering the volume of each animal's blood to be approximately 10 ml).

Experimental Design
The birds received a single daily oral dose (23 µg/0.1 ml/animal/d) of melatonin for 12 consecutive days, 1 hour before dark (at about 8:30 PM). The administration of melatonin was carried out using a plastic Pasteur pipette. Control animals received 0.1 ml of PBS with the same schedule as the melatonin-treated animals. Basal animals are animals studied before treatment that have not been given PBS or melatonin. Blood collections were performed at 2:00 AM under dim red light and again at 4:00 PM. The extractions were continued for 5 days following the end of the treatment (until day 17). The blood collection schedule was based on the following: (i) 2:00 AM is halfway through the acrophases of both the young and mature animals; (ii) 4:00 PM is the mesor (midline estimating statistic of rhythm) of the young and mature animals; and (iii) in old animals, no significant differences had been observed in the melatonin levels at 4:00 PM and 2:00 AM (6).

Plasma Collection
Blood drawn from the brachial vein was transferred unheparinized to a preprepared tube containing EDTA, and centrifuged at room temperature for 15 minutes at 300 x g. The plasma was then divided into aliquots in Eppendorf vials, and kept frozen at –30°C until use.

Measurement of Melatonin in Plasma
Melatonin levels were determined by means of a commercial radioimmunoassay kit (IBL, Hamburg, Germany) that consisted of ¹²⁵I-melatonin (<140 kBq), assay buffer, enzyme, enzyme buffer, melatonin standards, rabbit antimelatonin antiserum, precipitating agent, and controls (lyophilized plasma samples), according to the manufacturers instructions. Results are expressed in pg/ml.

Studies of Phagocytosis and Oxidative Metabolism
The studies of phagocytosis and oxidative metabolism (nitroblue tetrazolium test, NBT) were carried out on alternate days from the beginning until the end of the treatment (days 2, 4, 6, 8, 10, and 12).

Isolation of Heterophil Leukocytes
Heterophil leukocytes were obtained from 1 ml of blood drawn from the brachial vein to which 0.5 ml of PBS solution and 0.5 ml of lithium heparin were added; this was followed by centrifugation at 600 x g for 15 minutes through a Histopaque gradient (1 ml of 1119, 1 ml of 1077; Sigma, St. Louis, MO). The heterophils were then washed in PBS and adjusted according to each trial (5 x 10⁵ cells/ml for phagocytosis and 1 x 10⁶ cells/ml for NBT).

Phagocytosis of Latex Beads
The phagocytosis of inert particles (latex beads) was performed according to the technique described in Rodríguez and Lea (10). Aliquots of 200 µl of the phagocyte suspension were put into the wells of plastic macrophage migration inhibition factor (MIF)-type plaques, and, after 30 minutes incubation at 37°C in an oven with a 5% CO₂ atmosphere, the adhered monolayer was washed with PBS at 37°C. Then, 20 µl of latex beads (1.09 mm, diluted to 1% in PBS) and 200 µl PBS were added, followed by another 30 minutes of incubation under the same conditions as previously. Finally, the samples were fixed and stained with Diff-Quick (Dade Behring, Liederbach, Germany) containing methanol (5 min), eosin (five passes), and hematoxylin (five passes). The plaques were rinsed with tap water and dried, followed by counting under oil-immersion phase-contrast microscopy at 100x. The number of particles ingested per 100 heterophils was expressed as the latex-bead phagocytosis index (PI). The percentage of cells that had phagocytosed at least 1 latex bead was expressed as the phagocytosis percentage (PP). The ratio PI:PP was calculated, giving the phagocytosis efficiency (PE).

Quantitative NBT Test
The method described by Terrón and colleagues (6) was used. An aliquot of 250 µl of heterophil suspension (1 x 10⁶ cells/ml) was incubated for 60 minutes with an equal volume of NBT (Sigma, St. Louis, MO, 1 mg/ml in PBS solution) in the presence of 50 µl of latex beads (S, stimulated samples) (1.09 mm, diluted to 1% in PBS). Aliquots of the heterophil suspension incubated in the absence of latex beads were used as nonstimulated samples (NS). In all cases, after shaking incubation at 37°C, the reaction was stopped with 2.5 ml of 0.5 N hydrochloric acid.

The tubes were centrifuged for 30 minutes at 600 x g and 4°C, the supernatant was discarded, and the reduced NBT (blue formazan) was extracted from the cell pellet with 1 ml of dioxan. The tubes were then centrifuged for 30 minutes at 600 x g, and the absorbance of the supernatant was determined in a spectrophotometer at 525 nm using dioxin as the blank control. The stimulation of NBT reduction was then determined as a percentage relative to the absorbance obtained in the tubes without latex beads.

Statistical Analysis
Data are expressed as mean (X) ± standard error (SE) of the number of determinations carried out in duplicate. The variables were tested for normality, and the different groups were compared using the Scheffe analysis of variance (ANOVA) parametric F test, with p <.05 taken as the level of significance between groups.

Correlations by multiple regression of the different capacities with the melatonin values at both hours studied were taken as significant if r² > 0.5.

	RESULTS

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The results shown in Figure 1 indicate that, from the first day of melatonin administration (23 µg/animal/d), plasma levels of the indolamine were generally higher than the corresponding basal levels at both 2:00 AM and 4:00 PM. Also, the values were significantly higher (p <.05) at 2:00 AM than at 4:00 PM. In particular, at 2:00 AM: (i) there was a significant increase (p <.05) over the basal values on every day studied during treatment as well as on day 13, the first day following the last treatment, and (ii) at 4:00 PM there was a significant increase (p <.05) over the basal values from days 3 to 9 (inclusive). At the end of treatment, the melatonin levels began to decline steadily at both times of day, reaching levels close to the basal values by day 14, 2 days following the melatonin treatment. No significant changes were observed in the control animals after the administration of PBS, the vehicle used for melatonin administration (results not shown).

View larger version (59K): [in this window] [in a new window]   Figure 1. Changes in plasma levels of melatonin in old birds during 12 days of treatment with indolamine (23 µg/ml) and for the 5 subsequent days. Each value represents the mean ± SE (standard error) of eight determinations carried out in duplicate. a = p <.05 with respect to the corresponding basal value at 2:00 AM; b = p <.05 with respect to the corresponding basal value at 4:00 PM; * = p <.05 with respect to the value obtained at 4:00 PM on the same day. B = basal, animals before treatment

View larger version (31K): [in this window] [in a new window]   Figure 2. A: The phagocytosis index (number of latex beads phagocytosed per 100 heterophils); B: phagocytosis percentage (% of 100 cells that have phagocytosed at least 1 latex bead); and C: phagocytosis efficiency (phagocytosis index/phagocytosis percentage) in old birds given melatonin (23 µg/ml). Each value represents the mean ± SE (standard error) of eight determinations performed in duplicate. a = p <.05 with respect to the corresponding basal value at 2:00 AM; b = p <.05 with respect to the corresponding basal value at 4:00 PM; * = p <.05 with respect to the value obtained at 4:00 PM on the same day; Basal, animals before treatment

View larger version (20K): [in this window] [in a new window]   Figure 3. Results of the correlation study between the plasma levels of melatonin (pg/ml) and the values of the phagocytosis index at 4:00 PM (A) and at 2:00 AM (B) in old birds treated with melatonin (23 µg/ml)

View larger version (17K): [in this window] [in a new window]   Figure 4. Results of the correlation study between the relative increment (RI) in the melatonin concentrations (2:00 AM vs 4:00 PM) and the relative increment of the phagocytosis index (2:00 AM vs 4:00 PM) in old birds treated with melatonin (23 µg/ml). (RI = value at 2:00 AM minus value at 4:00 PM, divided by the value at 4:00 PM)

View larger version (39K): [in this window] [in a new window]   Figure 5. The percentage stimulation of the reduction of nitroblue tetrazolium (NBT) in heterophils incubated in the presence of latex beads in old birds treated with melatonin (23 µg/ml). S = samples stimulated with latex beads; NS = nonstimulated samples. Each value represents the mean ± SE (standard error) of eight determinations carried out in duplicate. a = p <.05 with respect to the corresponding basal value at 2:00 AM; b = p <.05 with respect to the corresponding basal value at 4:00 PM; * = p <.05 with respect to the value obtained at 4:00 PM on the same day. Basal, animals before treatment

View larger version (22K): [in this window] [in a new window]   Figure 6. Results of the correlation study between the plasma levels of melatonin (pg/ml) and the values of the nitroblue tetrazolium (NBT) reduction at 4:00 PM (A) and at 2:00 AM (B) in old birds treated with melatonin (23 µg/ml)

View larger version (16K): [in this window] [in a new window]   Figure 7. Results of the correlation study between the relative increment (RI) of the plasma concentration of melatonin (2:00 AM vs 4:00 PM) and the relative decrement (RD) of the nitroblue tetrazolium (NBT) reduction (2:00 AM vs 4:00 PM) in old birds treated with melatonin (23 µg/ml). RI = value at 2:00 AM minus value at 4:00 PM, divided by the value at 4:00 PM; RD = value at 4:00 PM minus value at 2:00 AM, divided by the value at 2:00 AM)

	DISCUSSION

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Recently, we have confirmed that old ring doves exhibit a decline in their innate immune function, which could be due, at least in part, to the absence of the circadian rhythm of melatonin (14). Here, the focus has been on the age-related changes in melatonin levels and in the possible effect of administering the indolamine on the phagocytic activity (phagocytosis and oxidative metabolism) during aging. In general, from the first day of administration, the plasma levels were greater than the corresponding basal levels both at night and in the late afternoon, with the nocturnal values being greater than the daytime values. A progressive increase in nocturnal melatonin levels was observed until day 5 of treatment, as a consequence of exogenous melatonin administration. Similar results were obtained in diurnal plasma levels. Besides, a sharp drop in diurnal plasma melatonin levels was observed after 10 days of treatment despite the exogenous treatment of melatonin. As a whole, these results show that, in old ring doves after 10 days of treatment, the homeostasis of melatonin gives rise to similar diurnal and nocturnal values to those observed in young birds (6). After treatment, melatonin declined steadily at both time-points studied, reaching levels similar to the basal values by day 14, two days after the last administration of melatonin. It is interesting that, once melatonin administration was finished, plasma levels of the hormone decreased back to basal values, which indicates that treatment does not reduce the endogenous secretion of melatonin by negative feedback; this is greatly important in relation to the therapeutic use of melatonin. In addition, the phagocytosis indices at 2:00 AM were significantly higher than both the basal values and the 4:00 PM values throughout the treatment period. The nocturnal increased phagocytosis seemed in general to be due to both phagocytosis percentage (number of activated heterophils) and the phagocytic efficiency (amount of antigen ingested by each activated phagocyte). Phagocytosis at 4:00 PM was only greater than the basal level on days 6 and 8 (days with highest melatonin levels both diurnal and nocturnal, and with the same number of activated heterophils (phagocytic percentage).

Also, with the melatonin administration, there was a clear positive correlation between the levels of this agent and the phagocytosis index at both time points studied. This indicates that, as the plasma melatonin levels of old birds rise, there is a concomitant increase in the capacity of their blood heterophils to phagocytose latex beads. This coincides with earlier findings of a positive melatonin–phagocytosis correlation in young ring doves (15), and with the enhancement of the phagocytic activity of heterophils from mature and old animals after in vitro incubation with either physiological or pharmacological concentrations of melatonin (6,9). Finally, the correlation study between the relative increments of the phagocytosis values and plasma melatonin levels (2:00 AM vs 4:00 PM) also showed that the greater the day–night difference in melatonin concentration, the greater the difference observed in phagocytosis values.

The increase in both the plasma melatonin levels and phagocytic activity of the heterophils in these old animals is of particular interest because the melatonin deficiency of old age is related to suppressed immunocompetence (16). In this sense, Cardinali and colleagues (17) found that in vivo treatment with melatonin not only restored circadian immune rhythms in the old animals, but also found that pharmacological levels of indolamine could hyperstimulate the immune system and exacerbate autoimmune processes. It is known that in both mammals and birds, a phase–response curve on the effect of melatonin on circadian rhythms is present (3,23). In this sense, the oral treatment with melatonin could induce a phase shift in the circadian system, which would affect the light–dark values of phagocytosis. Melatonin, in addition to its direct effect on immune cells, augments the amplitude, and perhaps delays or advances the phase, of the underlying central oscillator, thus indirectly affecting the immune response (18).

The claims for melatonin as an anti-aging agent are also based on studies indicating that it can have immunomodulatory and tumor-suppressive effects by acting as a potent free-radical scavenger. Indeed, various in vivo and in vitro studies have shown that melatonin exhibits immunoenhancing properties and might modulate certain immune functions at the same time that it attenuates oxidation reactions (3,6,8,9,19–21,23).

In contrast to phagocytosis, melatonin administration caused a decrease in superoxide anion levels at 2:00 AM, which were also lower than at 4:00 PM. Indeed, the 4:00 PM values were only lower than the basal values on day 6 (one of the days in which the melatonin levels were the highest at that hour). Also, as we observed previously in young animals that have a clear circadian rhythm of melatonin with high nocturnal levels (15), in old animals there was a clear negative correlation between the levels of superoxide anion and plasma melatonin following its administration. This indicates that increasing plasma levels of melatonin are accompanied by a clear decline in superoxide anion levels in the blood heterophils of Streptopelia risoria; this is consistent with a large amount of data documenting the free-radical scavenging and antioxidant activities of melatonin (24,25).

The finding that reduction in melatonin levels with age may contribute to aging and the onset of age-related diseases is supported by the recent observations that melatonin is a potent scavenger of damaging free radicals (26). Indeed, numerous in vitro and in vivo studies have documented the capacity of both physiological and pharmacological melatonin concentrations to protect against free-radical mutilation of essential molecules (27). Melatonin also promotes the activity of antioxidative enzymes, thereby further reducing oxidative damage (25). In the ring dove, we have shown that, in vitro, melatonin reduces superoxide anion levels by modulating the activity of the antioxidant enzyme superoxide dismutase (23), and that it suppresses both basal and antigen-induced lipid peroxidation in heterophils at both pharmacological (28) and physiological (29) concentrations. Furthermore, clinical tests with melatonin have proven highly successful; as a result, an increase in the use of melatonin in disease states and processes where free-radical damage is involved seems reasonable (27,30).

Considering all results obtained to date, our research has confirmed in this bird species a dose-dependent enhancing effect of melatonin on the phagocytic function, while at the same time neutralizing the superoxide anion radical derived from immune function. Recently, Terron and colleagues (14), in a comparative study of heterophil phagocytic function in young and old ring doves and of its relationship with melatonin levels at different times of day, found in the young animals an enhancing effect of melatonin on the heterophils in combination with its free-radical scavenging action, but they did not find this to be the case in the old animals. As a result, it was concluded that the difference could be due, at least in part, to the fact that old birds probably lacked a circadian rhythm of melatonin.

Summary
While aging is a multifactorial process, the age-related decline in melatonin secretion seems to be a contributing factor to the functional decline. Indeed, sleep inefficiency, circadian rhythm dysregulation, reduced antioxidant protection, depressed immune function, and so forth, may be direct consequences of the loss of melatonin with age (16). The possible therapeutic and physiopathological implications of the immunoenhancing properties of melatonin have only been sparingly investigated. In general, further work is required for there to be a consensus on the "replacement therapy" use of melatonin to limit or ameliorate some of the effects of age-associated changes.

	Acknowledgments

 
This investigation was supported by a research grant from the Ministerio de Ciencia y Tecnología (BFI2002-04583-C02-01). During the performance of this work, M.P.T. and S.D.P. were supported by a personal researcher training grant from the Consejería de Educación, Ciencia y Tecnología / Fondo Social Europeo (Junta de Extremadura, FIC00B013 and FIC02A049, respectively). The authors would like to express their thanks to Ms Elena Circujano Vadillo for her technical assistance.

	Footnotes

 
Decision Editor: James R. Smith, PhD

Received May 5, 2004

Accepted August 1, 2004

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Terrón, M. d. P. Articles by Rodríguez, A. B. Search for Related Content PubMed PubMed Citation Articles by Terrón, M. d. P. Articles by Rodríguez, A. B.