Evaluación del efecto de la administración de melatonina sobre la viabilidad celular, la

resistencia a estrés oxidativo inducido así como sobre la capacidad de ingestión y metabolismo

oxidativo de herofilos de tórtolas collarizas (Streptopelia risoria) jóvenes y viejas.

The decrease of melatonin production with aging contributes to the decline in immune function as organisms age. Treatment withthe exogenously administered indolamine restores the reduced immunological functions. Therefore, we investigated the effect of melatonin on viability, phagocyte ingestion capacity and free radical generation levels of heterophils from young and old ringdove (Streptopelia risoria) aged 3-4 and 11-13 years, respectively. Animals received a single oral dose of melatonin one hour before lights off for 3 consecutive days. Experiments were performed at the acrophases and nadirs of melatonin.

RESUMEN

RESUMEN

Melatonin treatment significantly increased serum melatonin levels at the acrophases but not at the nadirs of the two age groups. In both young and old animals there was increased heterophil viability at acrophases with respect to nadirs, and also increased cell resistance to oxidative stress in the old animals after the melatonin treatment. Atacrophases, index, percentage and efficiency of phagocytosis increasedsignificantly, and superoxide anion levels decreased significantly with respect to the nadir values of vehicle and melatonintreated animals, the effect being greater in young than in old ringdoves. At the nadirs, no change was observed in any parameter analyzed.

RESUMEN

In both young and old animals, phagocytosis and melatonin werepositively correlated, while superoxide anion levels and melatonin were negatively related. In conclusion, exogenous melatonin enhanced heterophil viability in old animals as well as increasingphagocytosis and free radical scavenging in both age groups during the nocturnal period, accompanied by an increase in the levels of the indolamine.

INTRODUCCION

Aging is a complex physiological process that involves a number of changes that are manifested in individual cells as well as inthe whole organism. One of these changes is a reduction of the rate of production and a significant decline in the amplitude andmean levels of melatonin – the "melatonin deficiency state"[1].

This loss of amplitude of melatonin rhythm in advanced age is both an indication as well as a cause of age-related disturbances inthe circadian pacemaker leading to chronobiological disorders [2], which is accompanied by a general deterioration of cognitive, psychological, and social functioning as well as by sleep disturbances [1,3].

INTRODUCCION

In particular, in birds there has been shown a reduction of theamplitude of the melatonin rhythm with increased age that alters their activity/rest rhythms [4], rendering the circadian pacemaker a less self-sustained, often highly damped oscillatory system [5].

Aging also affects the innate immune system [6,7]. In this case, functional impairment has been reported in neutrophils from aged individuals, finding a diminished chemotactic and phagocytic capacity, as well as disturbances in thedegree of oxidative stress [8,9].

INTRODUCCION

In recent years much attention has been devoted to the possible interaction between melatonin and the immune system [10-13].The literature suggests that there is a close connection between melatonin and immune regulation in both mammals [14 -16] and birds [17-20], showing correlations between diurnal and seasonal changes inthe immune system and the synthesis and secretion of the indolamine [21]. Melatonin may also possess significant anti-aging properties [22], acting as an ubiquitously direct free radical scavenger and a natural indirect antioxidant [22-24].

INTRODUCCION

Thus, the characteristic decline in circulating levels of the indolamine with age might somehow contribute to the diminished innate immune function typical of the age, as well as suggesting arelationship of the melatonin levels in advanced age with the total antioxidative capacity of the organism.

Previously, we have found a significant decline in nocturnal serum melatonin levels in old ringdoves (>10 years) withrespect to the concentrations observed in young animals (aged 2-3 years) [4] and that the treatment with melatonin at thedoses of 0.25 and 2.5 mg/kg b.w. in young and old animals, respectively, restored the amplitude of the serum melatonin rhythm in old doves to that of the young birds, also improving their nocturnal rest [25].

INTRODUCCION

This was also the case when treating the animals with tryptophan, the precursor of melatonin [26]. Also, both in vivo and in vitro, the administration of low pharmacological doses of the indolamine led to an increase in the immune response [27,28]. In the present experiments, we studied the effects of oral administration of melatonin over three days to young and old ringdoves given 0.25 and 2.5 mg/kgb.w./animal/day, respectively, on the circulating levels of the indolamine and theviability, phagocytic function, and oxidative metabolism of blood heterophils at the acrophases and nadirs of themelatonin cycle. Also, correlations between the levels of the indolamine and the various immune parameters analyzed were performed in both basal, vehicle and melatonin treatment conditions.

MATERIAL Y METODOSAnimales

•Palomas hembras y machos (Streptopelia risoria) de 3-4 años de edad y 11-13 años de edad pesando 150±20 g fueron usadas en este estudio (n= 10, por cada grupo de edad). Los animales fueron criados individualmente bajo las siguientes condiciones:

22ºC; 70% humedad, fotoperiodo a 12/12 h luz/oscuridad y alimentados de forma normal. Estas especies se caracterizan por ser diurnas y monofásicas con ciclos sueño vigilia similares a los humanos, por lo que representan un buen modelo para estudiar alteraciones del ritmo circadiano por la edad incluyendo alteraciones inmunes.

MATERIALES Y METODOS

Melatonina

La solucion de Melatonina (N-acetyl-5-methoxytryptamine) fue preparada en solucion Buffer fosfato salino (PBS) , la solución base inicial de 10 mg/mL fue disuelta por agitacion, seguida por dilucion de las soluciones de trabajo (0.25 y 2.5 mg melatonin por Kg de peso).

MATERIALES Y METODOS

Experimento A: Determinación de concentraciones de melatonina en acrofase y nadir del ritmo de melatonina en cada grupo experimental A1 Animal con tratamiento

Las aves recibieron una dosis única diaria oral (0.25 mg y 2.5 mg / 0.1 mL por animal / día) de melatonina por 3 días una hora antes de apagar la luz . El grupo de animales que recibieron 0.1 mL de PBS con el mismo horario que los animales tratados con melatonina. Se uso una pipeta Pasteur (plastica) para la administración. Los niveles basales sanguíneos fueron obtenidos previo al experimento. Basado en estudios previos, la acrofase del ritmo de melatonina (tiempos en los cuales las variables alcanzan su máximo) fueron obtenidos, en los grupos que recibieron PBS , a 02:00 y 01:00 h, y el nadir (tiempos en los cuales las variables alcanzan su mínimo) a 14:00 y 13:00 h, En las aves tratadas con melatonina , las acrofases y nadir fueron respectivamente establecidos a 02:30 y 14:30 h y 03:00 y 15:00 h en aves jovenes y viejas respectivamente

MATERIALES Y METODOS

A2. Recolección de sangreMuestras sanguíneas fueron obtenidas de 10 pájaros de cada grupo de edad en acrofases y nadir, con intervalo de por lo menos una semana entre muestras . Se colecto (1 mL) sangre de la vena braquial. Las muestras fueron centrifugadas a temperatura ambiente por 15 min a 300×g. El suero fue entonces dividido en alícuotas en viales Eppendorf , y refrigeradas a -30°C hasta el momento del ensayo. Las extracciones fueron hechas antes de iniciar el tratamiento ( valores basales) y en el 3º día de tratamiento.

MATERIALES Y METODOS

A3. Ensayo de la melatonina en suero

Melatonina fue medida por medio de un kit comercial de radioinmunoensayo (IBL, Hamburg, Germany) que consiste de 125I melatonina (140 kBq), buffer ensayo, enzima, enzima-buffer, nnivel estándar melatonina, suero de conejo antimelatonina,agentes precipitantes, and control (muestras de suero liofilizado), de acuerdo a las instrucción de fabrica. Resultados fueron expresados en pg/mL.

http://www.ehu.es/biomoleculas/isotopos/ria.htm

MATERIALES Y METODOS

Experimento B: Determinación de la viabilidad de la función fagocítica y metabolismo oxidativo de heterófilos en acrofases y nadir del ritmo de melatonina .

B1. Animales TratadosLas aves recibieron una única dosis diaria (0.25 mg y 2.5 mg / 0.1 mL por animal / día) de melatonina por 3 días consecutivos una horaantes de apagar la luz. Animales que recibieron PBS 0.1 mL con el mismo horario de las aves tratadas con melatonina, usando una pipeta Pasteur (plástica). Los niveles basales fueron tomados antes del tratamiento.

MATERIALES Y METODOS

B2. Aislamiento de leucocitos heterófilos Leucocitos heterófilos fueron obtenidos inmediatamente después de colectar 1 mL de sangre (en acrofase y nadir de la melatonina en cada grupo experimental) de la vena braquial al cual fueron añadidos 0.5 mL de (PBS) y 0.5 mL de heparina litio, seguido por centrifugación a 600×g por 15 min en una gradiente usando Histopaque (1 mL of 1119, 1 mL of 1077; Sigma, St. Louis, MO, USA).

MATERIALES Y METODOS

B3. Viabilidad celular bajo estrés oxidativo Viabilidad celular fue evaluada usando calcein como un fluorescent probe, following a technique previously described [29]. Para cargar calceina las células fueron incubadas por 45 min con 1 μM acetoxymethyl (calcein AM) a 37 ºC, centrifugado 10 min a 2100 rpm, resuspendido el precipitado en in buffer fresco. Fluorescencia fue registrada de alícuotas de 2 ml usando un espectrofotomet ro Muestras fueron excitads a 494 nm y la fluorescencia resultante fue medida a 520 nm. Despues incubada con 1 μM, 10 μM 100 μM 100 o 1 mM peroxido de hidrogeno (H2O2) por 30 o 60 min at 37 ºC, células fueron centrifugadas y resuspendidas en buffer fresco La calceina fluorescente que quedo en las celulas despues de la incubacion fue registrada.

MATERIALES Y METODOSB4. Fagocitosis de particulas de latexpLos ensayos de fagocitosis de particulas inertes (latex beads) were performed according to a technique previously described [30]. Alicuotas de 200 μl de la suspension de fagocitos (5×105 cells/mL) fueron colocadas en the wells of plastic macrophage migration-inhibition factor (MIF) type plates, and despues de after 30 min deincubacion a 37 ºC en un horno con atmósfera con CO2 al 5%, la monocapa adherida fue lavada con PBS a 37 ºC. Luego 20 μl de partículas de latex (1.09-mm diameter particle size, dilluted to 1% in PBS; Sigma, St. Louis, MO, USA) y 200 μl PBS fueron añadidos, seguidos mpor otros 30 -min de incubación bajo iguales condiciones Finalmente, las muestras fueron fijadas y teñidast con Diff-Quick (Dade Behring, Liederbach, Germany) conteniendo methanol (5 min), eosin (five passes), and haematoxylin (five passes). Las placas fueron lavadas con agua y secadas , seguidas por conteo en microscopio de inmersión a una magnificación de 100×. El número de particulas ingeridas por 100 heterófilos expresó el indice de fagocitosis de partículas de latex (PI). El porcentaje de células que fagocitaron por lo menos 1 paticula de latex expresaron el porcentaje de fagocitosis (PP). La relacion PI:PP dió la eficiencia de fagocitosis (PE).

MATERIALES Y METODOS

B5. Quantitative nitroblue tetrazolium(NBT) testThe method used was that described by our group [28]. una alícuota de 250 μl de suspensión de heterófilos (1×106 cells/mL) fue incubado por 60 minutos con un volumen igual de NBT (Sigma, St. Louis, MO,USA, 1mg/mL in PBS solution) en la presencia de 50 μl de partículas de latex (S, stimulated samples) (1.09 mm, diluted to 1% in PBS).Alicuotas de suspensión de heterófilos incubados en la ausencia de partículas de latex fueron usadas como muestras no estimuladas (NS). En todos los casos, despu'es de shaking incubation a 37 ºC, la reacción fue detenida 2.5 mL of 0.5 N hydrochloric acid. Los tubos fueron centrifugadospor 30 min at 600×g, y la absorbancia del sobrenadante fue determinada en un espectrofotómetro a 525 nm usando dioxano como control blanco. La estimulación de NBT reduction fue entonces determinado como un porcentaje relativo a la absorbancia obten8ida en los tubos sin partículas de latex

MATERIALES Y METODOS

Statistical analysisDatos fueron expresados como media (X) ± desviación standard del número de determinaciones llevado a cabo en duplicado. Los resultados fueron analizados usando test no paramétricos: the ANOVA two-way test by addition of Friedman ranges (paired samples) and the ANOVA one-way test by addition ofKruskal-Wallis ranges (unpaired samples) for multiple comparisons.correlaciones por regresión múltiple de diferentes capacidades para los valores de melatonina en las horas estudiadas fueron tomados como significativas R2>0.05.

RESULTADOS

Experiment A: Determinación de las concentraciones de melatonina en acrofase y nadir del ciclo de melatoninaEn cada grupo experimental jovenes y viejos) Como no se observaron diferencias significativas were observed entre los resultados obtenidos en los valores basales (avesantes del tratamiento) y niveles de aves tratadas solo con PBS , los valores basales fueron omitidos de los resultados. Figura 1 muestra las concentraciones séricas de melatonina en acrofase y nadir en aves jovenes y viejas tratados o no tratados con 0.25 and 2.5 mg/kg po kg de peso de melatonina respectivamente

RESULTADOS

Administración de melatonina incrementó significativamente (p<0.05) los niveles circulantes de indolamina en el periodo nocturno en ambos grupos de edad ; no hubieron diferencias entre los niveles alcanzados en sus acrofases Sin embargo en los pájaros tratados con PBS los niveles séricos de melatonina en la acrofase en los animales jovenes fueron significativamente más altos (p<0.05) que los valores medidos en los pájaros viejos.No fueron observados cambios durente el día en cualquiera de los grupos de edad (valores nadir ). Table 1 muestra el efecto de incrementadas concentraciones H2O2 (desde 1 μM a 1 mM) sobre la viabilidad celular de los heterófilos. Los resultados indican que H2O2 induce una disminución dosis-dependiente en la viabilidad de heterófilos . Sin embargo, este efecto inhibitorio fue estadisticamente significativ (p<0.05) con respecto a los valores de control H2O2-free medium) sólo a la dosis de 1 mM, para ambos grupos de edad y en ambos tiempos de incubación , Ambos grupos, los tratados con PBS o los tratados con melatonina , y en ambos nadir (periodo diurno) y en acrofase (periodo nocturno)

RESULTADOS

Las reducciones en el periodo nocturno fueron significativamente más bajas (p<0.05) que aquellos en el periodo diurno, excepto heterófilos de aves trratadas con melatonina incubados con H2O2 por 30 min durante el periodo nocturno , en cuyo caso no fuero notadas diferencias significativas con respecto a sus grupos control. Hubo también una estadisticamente significativa disminución co 100 μM en aves no tratadas jovenes y viejos a 60 min de incubación con H2O2. Las diferencias entre los dos grupos de edad fueron sólo significativas (p<0.05) en las aves no tratadas durante el periodo nocturno a 60 min deincubación con H2O2. Dentro del mismo grupo de edadY periodo de estudio (acrofase o nadir), hubo significativas diferencias (p<0.05) en el caso de los valores de acrofase obtenidos en aves tratadas con PBS versus melatonina en ambos tiempos de incubación en las aves viejas

RESULTADOSFigure 1. Serum levels of melatonin at their corresponding hours of acrophase and nadir in vehicle and after 3 days of treatment one hour before lights off with 0.25 and 2.5 mg/kg b.w. of melatonin in young and old ringdoves, respectively. Each value represents the mean ± standard deviation of 10 determinations performed in duplicate. (a) p<0.05 with respect to their corresponding values in vehicle group in the same group of age; (b) p<0.05 with respect to their corresponding values in young animals.

RESULTADOS

Experiment B: Determination of viability, phagocytic function, and oxidative metabolism of heterophils at acrophases and nadirs of melatonin rhythm in each experimental group (young vs old)

Figure 2A shows the variations in the capacity of the heterophils to ingest latex beads (PI) in vehicle conditions and after 3 days of melatonin treament for both age groups. At the acrophases, in both young and old animals, the treatment with melatonin significantly raised (p<0.05) the PI with respect to the corresponding values in the vehicle group. However, no significant differences were observed at the nadirs.

RESULTADOS

On the other hand, at both acrophases and nadirs, the PI in youngvehicle and melatonin-treated ringdoves were significantly higher (p<0.05) than thecorresponding values in old animals. Figure 2B shows the results for the PP (percentage of activated heterophils). At the acrophases, the melatonin-treated percentages were significantly greater(p<0.05) than the vehicle values in both young and old ringdoves. At the nadirs, however, there were no significant differences between the groups. Similarly to the PI, in this parameter there were also significant differences (p<0.05) between the untreated and treated birds, except inthe case of the phagocytosis percentages of untreated animals obtained at the nadir.

RESULTADOS

Similar results to those of the PI and PP were found in the number of latex beads ingested by each activated heterophil (PE, Figure 2C), but only during the nocturnal period. Thus, at the acrophases, the PEincreased significantly (p<0.05) in both young and old treated animals in comparsion with their respective vehicle values. Also, the nocturnal values were significantly higher (p<0.05) in the vehicle and melatonin-treated young groups than in the old groups. During the diurnal period, there were no differences between the different experimental groups.

RESULTADOS

Figure 3 shows the correlations between the serum melatonin levels (pg/mL) and the vehicle PI, PP, and PE from vehicletreated birds at the acrophases and nadirs in young and old animals. There was a positive correlation in both age groups. In the case of the PE, the results also showed positive, but moderate, correlations in both old and young animals. The relationship between serum melatonin levels (pg/mL) and the PI, PP, and PE obtained at the acrophases and nadirs in old and youngmelatonin-treated animals is shown in Figure 4. Here, as in vehicle groupparameters, there is also a positive correlation in all the measurements, with their correlation coefficients being higher than those obtained in the vehicle conditions.

RESULTADOSFigure 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 vehicle conditions and after 3 days of treatment in young and old birds given melatonin (0.25 and 2.5 mg/kg b.w. one hour before lights off, respectively), at the corresponding hours of acrophase and nadir. Each value represents the mean ± standard deviation of 10 determinations performed in duplicate. (a) p<0.05 with respect to their corresponding values in vehicle group in the same group of age; (b) p<0.05 with respect to their corresponding values in young animals.

RESULTADOS

RESULTADOSFigure 3 (A, B, C, D, E, F). Results of the correlation study between the vehicle serum levelsof melatonin (pg/mL) and the vehicle values of the phagocytosis index, phagocytosis percentage and phagocytosis efficiency obtained in young and old ringdoves at their acrophases and nadirs.

RESULTADOS

RESULTADOSFigure 4 (A, B, C, D, E, F). Results of the correlation study between the serum levels of melatonin (pg/mL) and the values of the phagocytosis index, phagocytosis percentage and phagocytosis efficiency obtained at acrophases and nadirs after 3 days in young and old ringdoves treated one hour before lights off with 0.25 and 2.5 mg/kg b.w. of melatonin, respectively.

RESULTADOS

RESULTADOS

Figure 5 shows the variations in oxidative metabolism (NBT reduction) in vehicle conditions and after 3 days of melatonin treament in both age groups. At the acrophases, the melatonin treatment significantly decreased (p<0.05) the levels of NBT reduced by heterophils in both age groups. Thus, the NBT values in the youngvehicle-treated animals were lower than in the corresponding old groups. However, there were no significant differences between young and old-treated animals. During the diurnal period, there were nodifferences between the values obtained at the nadirs of the different experimental groups.

RESULTADOSFigure 5. The percentage stimulation of the reduction of nitroblue tetrazolium (NBT) in heterophils incubated in the presence of latex beads in vehicle conditions and after 3 days of treatment in young and old birds given 0.25 and 2.5 mg melatonin per kg b.w. Respectively. Each value represents the mean ± standard deviation of 10 determinations performed in duplicate. (a) p<0.05 with respect to their corresponding values in the vehicle group in the same group of age; (b) p<0.05 with respect to their corresponding values in young animals.

RESULTADOS

Figure 6 shows the correlations between the serum melatonin levels (pg/mL) and the superoxide anion levels (percentage of NBT reduction) at the acrophases and nadirs in young and old animals in vehicletreatedbirds. There was a clear negative correlation in young animals. In the case of the old animals, the correlation coefficient was lower than that in the young animals, although the correlation was still moderately negative. The relationship between the serum melatonin levels (pg/mL) and superoxide anion levels (percentage of NBT reduction) obtained atthe acrophases and nadirs in old and young melatonin-treated animals is shown in Figure 7. In this case, in contrast to levels of the indolamine and the reduced NBT values in vehicle-treated birds, in both young and old animals, there were clear negative correlations, with coefficientsbeing greater than the correlations found with the vehicle group values.

DISCUSION

Aging is associated with the deterioration of numerous physiological functions, which leads to age-related pathologies. Among them is the deterioration of the 24- hour rhythm of melatonin production due to changes in the morphology, physiology, and biochemistry of the pineal gland, which results in a significant reduction of nocturnal melatonin levels [31,32]. Since melatonin appears to act as a synchronizer of the circadian clock, giving a timerelated signal to a number of bodilyfunctions, one of which is the circadian organization of the organism’s defence [33], the age-related reduction of circulating levels of the pineal product may alter the circadian structure of the immune response.

DISCUSIONIn previous studies, we found a reduction in the serum nocturnal melatonin levels of old ringdoves with respect to the values found in young animals [4]. The results of the present study confirmed this, documenting a significant decline in melatonin when comparing the vehicle nocturnal (acrophase) levels of the indole in old and young ringdoves. Phagocytosis is an important element in non-specific immunity and is fundamental for the host’s defence against infection [34]. In birds, heterophils represent the first line of defence against invading agents [35] since their ability to ingest and kill different antigens plays a central role in host defence mechanisms. The phagocytosis experiments performed herein showed decreases in latex ingestion capacity (PI) and in the number of activated cells (PP), which resulted in a reduction of the PE, when comparing the old vehicle group nocturnal values to those of the young; in both groups they were higher than their respective diurnal values.

DISCUSIONFigure 6 (A, B). Results of the correlation study between the serum vehicle levels ofmelatonin (pg/mL) and the vehicle values of the nitroblue tetrazolium (NBT) reduction obtained in young and old ringdoves at their acrophases and nadirs.

DISCUSION

Having ingested an antigen, phagocytic cells trigger a metabolic burst, giving rise to the production of free radicals includingthe superoxide anion (O2 .-). In the present work, we used the NBT reduction test to evaluate the respiratory burst as indicatedby the levels of O2 .- in blood heterophils after inert particle phagocytosis. In vehicle-treated animals, lower levels of O2 .- at the acrophase (when melatonin levels were highest) than during the nadir were found, with the values being higher in the case of the aged individuals.

DISCUSION

Since age-related changes in pineal function appear to be related, at least partially, to immune-system efficiency [36], the observed impairment of immune function in old animals during the nocturnal period compared to the young birds could be due to the reduction of the circulating levels of the indolamine. Given that a number of studies support the immunoregulatory action of melatonin on the body’s innate immunity [10,12], we focused on the possible effects of exogenous melatonin on the phagocytic process and free-radical scavenging. Previous studies in our animal model have shown an enhancement of the phagocytic activity of heterophils from mature and old animals after in vitro incubation with either physiological or pharmacologicalconcentrations of melatonin [27,37]. Also, the oral administration of the indole to aged birds increased heterophil phagocytic activity with an increase in plasma levels of the indolamine [28].

DISCUSION

In the present study it was found not only in the aged animals but also in the young birds, treatment with the indolamine produced a significant nocturnal rise in the acrophase of circulating melatonin levels as well as augmented phagocytosis parameters, with the latter being significantly greater than those in the old animals; this was not the case at the melatonin nadir. Also, in both young and old animals, melatoninphagocytic correlations were obtained in both vehicle and melatonin-treated individuals. This indicates that as serum melatonin levels rise so does the latex bead phagocytic capacity of the heterophils.This is consistent with earlier findings demonstrating that melatonin has a general immuno-enhancing effect in many animals including birds [19,20], mammals [12,13,38] and humans [39].

DISCUSION

Melatonin has been demonstrated to be a potent scavenger of damaging free radicals [40]. In vitro and in vivo studies have reported that, at both physiological and pharmacological concentrations, the pineal indole protects against free radical damage [41,42]. In the current study, melatonin administration enhanced the reduction inO2 .- levels observed in vehicle-treated groups in both young and old animals. This is likely due to the rise of the circulating serum levels of melatonin produced due to the exogenous administration of the indole. Also, negative correlations were found in both vehicle and melatonin-treated groups between serum melatonin circulating levels at acrophases and nadirs and their respective NBT values obtained at those times. This indicates that rising serum levels of melatonin are accompanied by a decline in the O2 .- levels produced by heterophils. This confirms previous studies that have shown melatonin to be anantioxidant, under both in vitro and in vivo conditions [42].

DISCUSION

H2O2 is a reactive oxygen intermediate which is formed after oxygen is reduced by two electrons. H2O2 is the immediate precursor of the highly toxic hydroxil radical (·OH). In itself, H2O2 is not toxic atphysiological concentrations but, through the Fenton or Haber-Weiss reactions, it is converted to ·OH which, once generated, attacks any macromolecule within its diffusion distance [43]. Herein, the use ofH2O2 induced a dose-dependent effect in both young and old dove heterophil viability; this effect of H2O2 was significant when the cells were incubated with the higher concentrations. It is particularly interesting that, with the concentration of 100 μM, cells from treated animals incubated for 60 min in the presence of H2O2 did not exhibit asignificant reduction in their viability in young or old birds. This contrasts with the cells subjected to the same conditions but taken from non-melatonin treated birds.

DISCUSION

Also, although the concentration of 1 mM reduced cell survival in both young and old ringdoves, the reductions reached at the acrophases were significantly lower than those at the nadirs. Similarly, it is important to note that, as stated above, circulating levels of melatonin were significantly higher during the acrophases with respect to the nadirs. Although melatonin treatment did not influence heterophil survival in young animals, in the old birds the decline in viability was lower in treated animals at both 30 and 60 min of incubation with H2O2. A variety of studies have reported a scavenging action of melatonin against hydrogen peroxide [24,44-46]. As heterophils form important components of the avian innate immune system, the stimulatory action of melatonin shown in the present study reflects a significant immuno-enhancing property.

DISCUSION

Also, the indole enhanced the neutralization of the O2 .- derived from the immune function, while at the same time contributing to increased heterophil survival against H2O2 in both age groups. This is especially important in the case of aged organisms, since it is known that many hormones that are associated with maintenance of immune function (such as melatonin) also decline with advancing age, and the interrelationship between the endocrine system and the immune systemis regarded as of crucial importance in normal physiology and in mediating ageassociated degenerative diseases [47-50].The current results will hopefully stimulatefuture studies of the application of melatonin as “replacement therapy” to limit or reverse some of the effects of the changes that occur during immunosenescence.

DISCUSIONFigure 7 (A, B). Results of the correlation study between the serum levels of melatonin (pg/mL) and the values of the nitroblue tetrazolium (NBT) reduction obtained in young and old ringdoves at their acrophases and nadirs after 3 days treated one hour before lights off with 2.5 and 0.25 mg/kg b.w. of melatonin, respectively.

DISCUSIONTable 1. Effect of H2O2 on cell viability of heterophils in non-treated animals (NT) and after 3 days of melatonin treatment (T) in young and old birds given melatonin (0.25 and 2.5mg/kg b.w. one hour before lights off, respectively), at their corresponding hours of acrophase and nadir.

Each value represents the mean ± SD of 10 determinations. a) p<0.05 with respect to thevalues obtained in the controls (incubated in a free-H2O2 medium); b) p<0.05 with respect to their respective diurnal values; c) p<0.05 with respect to their respective values in the young animals; d) p<0.05 with respect to their respective values in the non-treated animals (at the basal conditions).