Effect of Melatonin Supplementation on Biomarkers of Oxidative Stress ...

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Effect of Melatonin Supplementation on Biomarkers of Oxidative Stress and Serum Vitamin and Mineral Concentrations in Heat-Stressed Japanese Quail K. Sahin,*,1 M. Onderci,† M. F. Gursu,‡ O. Kucuk,§ and N. Sahin† *Department of Animal Nutrition, Faculty of Veterinary Science, †Veterinary Control and Research Institute, and ‡Department of Biochemistry, School of Medicine, Firat University, 23119 Elazig, Turkey; and §Oncology and Nutrition, Barbara Ann Karmanos Cancer Institute, Wayne State University, 3990 John Road, Detroit, Michigan 48201

Primary Audience: Veterinarians, Researchers SUMMARY This study was conducted to evaluate the effects of dietary melatonin supplementation on biomarkers of oxidative stress, malondialdehyde (MDA) and homocysteine, and on serum concentrations of vitamin C, vitamin E, vitamin A, Fe, Zn, Cu, Cr, cholesterol, triglyceride, highdensity lipoprotein cholesterol, glucose, and activities of paraoxonase (PON) and arylesterase in Japanese quail (Coturnix coturnix japonica) exposed to high ambient temperature (34°C). One hundred twenty Japanese quail (10 d old) were randomly assigned to 4 treatment groups consisting of 3 replicates of 10 birds. The birds were kept in a temperature-controlled room at 22 or 34°C. Birds were fed a basal diet or the basal diet supplemented with 40 mg of melatonin/kg of diet. The experiment was terminated after 32 d. Levels of MDA in serum, liver, heart and kidney, and level of homocysteine in serum were markedly increased in heat-stressed quail, and the levels significantly decreased by melatonin supplementation (P < 0.01). Decreases in serum concentrations of vitamin C, vitamin E, vitamin A, Fe, Zn, Cu, Cr, cholesterol, triglyceride, and the activities of PON and arylesterase induced by heat stress were partially restored by melatonin supplementation (P < 0.01). Heat stress-induced increases in serum cholesterol and glucose concentrations were also partially alleviated by melatonin (P < 0.01). No interactions between melatonin and temperature were found on the measured parameters in the present study (P > 0.05). The results of the study indicate that melatonin supplementation attenuated the increase in oxidative stress and depletion in antioxidants caused by heat stress in Japanese quail. Key words: heat stress, quail, melatonin, oxidative stress, antioxidant 2004 J. Appl. Poult. Res. 13:342–348

1

To whom correspondence should be addressed: [email protected]; [email protected].

SAHIN ET AL.: EFFECT OF MELATONIN SUPPLEMENTATION

DESCRIPTION OF PROBLEM Many environmental factors, including high ambient temperature, induce oxidative stress [1, 2] and deplete antioxidants in organisms [3, 4]. Lower plasma levels for antioxidant vitamins and minerals, such as vitamin C, vitamin E, folic acid, zinc, and chromium, and increased oxidative stress were observed in stressed poultry [1, 4, 5]. Furthermore, environmental stress reduces plasma protein concentration and the activity of antioxidant enzymes, such as paraoxonase (PON) [6], and markedly increases blood glucose and cholesterol concentrations [7]. Increased oxidative stress and elevated levels of glucose and cholesterol have been associated with a high risk of chronic diseases, such as cancer, diabetes, and cardiovascular disease. These factors also affect the performance and carcass characteristics of poultry with significant economic consequences. Therefore, we have chosen the Japanese quail as a model to investigate the effects of environmental stress on oxidative stress and its consequences on the birds. To alleviate the detrimental influences of high environmental temperature on oxidative stress and performance of poultry, dietary manipulations are preferable to other methods because of their practicality and lower cost. Recent studies have shown that diets enriched with antioxidant substances, such as vitamin C, vitamin E, vitamin A, zinc, and chromium, could be used to attenuate the negative effects of environmental stress, suggesting that detrimental effects of environmental stress are largely due to induction of oxidative stress [5, 8, 9]. Environmental variables influence pineal gland in many avian species [10, 11, 12]. Arendt [13] suggested that the pineal gland acts as a photoneuroendocrine transducer that responds via melatonin release. Melatonin, the main secretory product of the pineal gland, is known to be a powerful antioxidant with its high free radical scavenging activity [14, 15, 16]. Melatonin was reported to be more efficient than vitamin E in scavenging peroxyl radicals [7]. In addition to its ability to scavenge peroxyl radicals, hydroxyl radicals, and other reactive oxygen species, melatonin also is involved in many physiological functions, such

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as immune response, energy metabolism, and temperature regulation. Furthermore, melatonin stimulates the activity of several enzymes related to the antioxidant defense system [17, 18]. The influence of dietary melatonin supplementation on the components of the antioxidant defense system of heat-stressed poultry can be evaluated by measuring biomarkers of oxidative stress, the serum levels of some antioxidant vitamins and minerals, and activities of antioxidant enzymes. The objective of this study was, therefore, to investigate how dietary melatonin supplementation would influence serum and tissue levels of malondialdehyde (MDA), serum levels of homocysteine, vitamin C, vitamin E, vitamin A, minerals related to the antioxidant defense system (Fe, Zn, Cu, and Cr), and activities of PON and arylesterase in Japanese quail reared under high ambient temperature (34°C).

MATERIALS AND METHODS Birds, Diets, Experimental Design, and Data Collection One hundred twenty, 10-d-old Japanese quail (Coturnix coturnix japonica) [19] were used in the study. The experiment was in accordance with animal welfare and was conducted at Veterinary Control and Research Institute of Elazig, Turkey. The birds were randomly assigned, according to a 2 × 2 factorial design, to 4 treatment groups consisting of 3 replicates of 10 birds in each group. The first factor was the temperature. The birds were kept in an environmentally controlled room at 22°C with a relative humidity of 64.3 + 3.3% (thermoneutral groups) or 34°C with a relative humidity of 43.8 + 2.5% (for 8 h/d; 0900 to 1700 h; heat stress groups). The second factor was melatonin [20] supplementation to the diet at level of 0 or 40 mg/kg of diet. At both temperatures, birds were fed either a basal (control) diet or the basal diet supplemented with 40 mg of melatonin/kg of diet. The birds were fed a starter diet until 21 d of age followed by a maintenance diet from 21 to 42 d. The diets and fresh water were offered ad libitum. Light was provided continuously (24 h) throughout the experiment. Ingredients and chemical composition of the basal diet are shown in Table 1. The basal diets

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344 TABLE 1. Ingredients and chemical analyses of the starter and grower diets fed to Japanese quail (g/100 g) Ingredient

Starter

Grower

Soybean meal Ground corn Wheat Animal-vegetable fat Dicalcium phosphate Salt Limestone, ground DL-Methionine Vitamin and mineral premixesA ME, MJ/kg

37.42 45.83 8.00 4.64 1.20 0.31 1.68 0.17 0.75 12.9

29.10 43.70 18.80 4.50 1.02 0.31 1.76 0.09 0.75 12.9

Chemical analysis CP Calcium, % Available phosphorus, % Zinc, mg/kg Copper, mg/kg Iron, mg/kg Chromium, µg/kg

23.00 1.0 0.46 45 60 27 1.5

20.21 0.95 0.40 40 5.6 26 1.3

A The vitamin premix provided the following per kilogram: all-trans-retinyl acetate, 1.8 mg; cholecalciferol, 0.025 mg; all-rac-α-tocopherol acetate, 1.25 mg; menadione (menadione sodium bisulfate), 1.1 mg; riboflavin, 4, 4 mg; thiamine (thiamine mononitrate), 1.1 mg; vitamin B6, 2.2 mg; niacin, 35 mg; Ca-pantothenate, 10 mg; vitamin B12, 0.02 mg; folic acid, 0.55 mg; D-biotin, 0.1 mg. The mineral premix provided the following per kilogram: manganese (from manganese oxide), 40 mg; iron (from iron sulfate), 12.5 mg; zinc (from zinc oxide), 25 mg; copper (from copper sulfate), 3.5 mg; Iodine (from potassium iodide), 0.3 mg; selenium (from sodium selenite), 0.15 mg; choline chloride, 175 mg.

were formulated using NRC [21] guidelines and contained 23 to 20% (starter-grower) protein and 12.9 MJ/kg ME. Laboratory Analyses At the end of study, blood samples were collected from 9 birds (3 per replicate) randomly chosen from each treatment. Blood samples were kept on ice and protected from light until they were processed to prevent any artifactual oxidation during the experiments. Blood samples centrifuged at 3,000 × g for 10 min, sera were collected, and serum MDA, homocysteine, vitamin C, vitamin E, vitamin A, PON, arylesterase, glucose, and cholesterol concentrations were determined. Procedures for other measurements are described elsewhere [22].

Statistical Analyses Sample size was calculated based on a power of 85% and a P value of 0.05. We considered a 10% improvement as satisfactory. Given that assumption, a sample size of 10 per treatment arm was calculated. The data were analyzed for the effects of melatonin and temperature using the general linear model procedure of SAS software [34]. We considered P < 0.05 to be statistically significant.

RESULTS The effects of supplemental melatonin during heat stress on MDA, homocysteine, and vitamins C, E, and A are shown in Tables 2 to 4. Malondialdehyde levels in serum, liver, heart and kidney, and homocysteine level in serum were markedly increased in heatstressed quail, and these were prevented by melatonin supplementation (P < 0.01). Decreases in serum concentrations in vitamin C, vitamin E, vitamin A, Fe, Zn, Cu, Cr, and the activities of PON and arylesterase by heat stress were partially restored by melatonin supplementation (P < 0.01). Heat stress-induced increase in serum cholesterol and glucose concentrations were also partially alleviated by melatonin (P < 0.01). No interactions between melatonin and temperature were found on the measured parameters in the present study (P > 0.05).

DISCUSSION Stressful environmental conditions cause oxidative stress and diminished antioxidant status in vivo. In the present study, we evaluated the effect of dietary melatonin supplementation on biomarkers related to the antioxidant defense system in Japanese quail reared at high ambient temperature. It has been reported that environmental stress, including high temperature-induced stress, causes oxidative stress with increased production of MDA [1, 2] and decreased concentrations of vitamins E, C, and A [3, 5, 35] in serum. In the present study, high ambient temperature induced increased serum MDA and homocysteine concentrations and lowered vitamins C, E, and A concentrations, which were reversed when melatonin was supplemented in the diet. This protective

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TABLE 2. Effects of melatonin (M) on the antioxidant status of quail reared under heat stressA (34°C) Thermoneutral Item B

Serum MDA, nmol/mL Liver MDA, nmol/g Heart MDA, nmol/g Kidney MDA, nmol/g Homocysteine, µmol/L Vitamin C, µmol/L Vitamin E, µmol/L Vitamin A, µmol/L

High temperature

P-value

Control

Melatonin

Control

Melatonin

SEM

T

M

T×M

0.95 3.2 1.5 1.8 17 43.5 2.0 1.55

0.91 2.8 1.3 1.5 15 45.8 2.1 1.54

2.3 4.8 2.6 2.5 24 26.1 1.3 1.10

1.5 3.9 2.1 2.0 20 34.9 1.8 1.28

0.4 0.5 0.2 0.1 1.3 6.2 0.3 0.05

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

0.01 0.01 0.01 0.05 0.01 0.01 0.01 0.01

0.83 0.76 0.92 0.55 0.90 0.61 0.59 0.79

Values are means n = 9. MDA = malondialdehyde.

A B

action of melatonin confirms previously reported findings of other investigators [15, 36, 37]. Melatonin, the main secretory product of the pineal gland, is known to be a powerful antioxidant with its high free radicals scavenging activity [14, 15, 16]. Melatonin exerts antioxidant activity by directly scavenging free radicals and inhibiting the production of lipid peroxidation [36]. Melatonin has been reported to be more effective than vitamin E as a free radical scavenger. Physicochemical properties of the melatonin molecule give it high diffusion ability; thus, it can easily diffuse through subcellular compartments, such as cytosol, nucleus, cellular membranes, and mitochondria [17, 38]. In addition to its ability to scavenge peroxyl radicals, hydroxyl radicals, and other reactive oxygen species, melatonin also is involved in many physiological functions, such as immune response, energy metabolism, and temperature regulation [15, 39]. Furthermore,

melatonin stimulates the activity of several enzymes related to the antioxidative defense system [17, 18]. By reducing the concentrations of reactive species, the antioxidant enzymes counteract the processes of carcinogenesis [40]. For example, Mn-superoxide dismutase has been suggested to function as a tumor preventive factor [41], and melatonin increases tissue mRNA levels for superoxide dismutase [42]. In addition, melatonin augments production and regeneration of glutathione, one of the major intracellular antioxidant molecules. Melatonin also increases the activity of catalase [43, 44]. Another antioxidant activity of melatonin is inhibition of the activity of nitric oxide synthase, which catalyzes the formation of NO [36, 44]. Similar to the results of the present study, the activities of antioxidant enzymes, such as PON, decreased when animals were exposed to high and low ambient temperatures [6]. Melatonin supplementation, in the

TABLE 3. Effects of melatonin (M) on serum metabolites in quail reared under heat stressA (34°C) Temperature (T) Thermoneutral Item Basal paraoxonase, U/L NaCI-stimulated paraoxonase, U/L Arylesterase, U/L Cholesterol, mmol/L HDL-cholesterol, mmol/LB Triglyceride, mmol/L Glucose, mmol/L Values are means n = 9. HDL = High-density lipoprotein.

A B

High temperature

P-value

Control

Melatonin

Control

Melatonin

SEM

T

M

T×M

686 760 63 4.1 0.9 1.8 10.8

705 781 62 3.9 0.7 1.7 10.5

450 536 38 5.7 1.3 2.3 12.6

518 630 50 5.0 1.1 2.0 11.4

29 50 8 0.5 0.03 0.1 0.3

0.01 0.01 0.01 0.01 0.01 0.01 0.01

0.01 0.01 0.05 0.05 0.01 0.01 0.01

0.79 0.86 0.45 0.67 0.90 0.52 0.54

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TABLE 4. Effects of melatonin (M) on serum concentrations of Fe, Zn, Cu, and Cr in quail reared under heat stressA (34°C; µg/dL) Temperature (T) Thermoneutral

High temperature

P-value

Item

Control

Melatonin

Control

Melatonin

SEM

T

M

T×M

Iron Zinc Copper Chromium

392 130 25 0.029

402 136 23 0.030

295 103 16 0.015

350 114 20 0.022

32 5.6 2.3 0.002

0.01 0.01 0.01 0.01

0.01 0.01 0.01 0.01

0.75 0.60 0.43 0.66

Values are means n = 9.

A

present study, resulted in an increase in the activity of PON and arylesterase, 2 members of the antioxidant defense system. It is reported that PON inhibits oxidation of low-density lipoproteins, which is an important stage in the early development of atherosclerosis. In addition, PON can destroy lipids in oxidized lowdensity lipoproteins [45]. Homocysteine, a sulfur-containing amino acid, is formed largely from methionine, and it is remethylated to methionine primarily by methionine synthase, which depends on folate and cobalamin. This conversion is the main way of maintaining a low plasma homocysteine level. Any condition, including stress, which impairs folate and cobalamin metabolism may cause increased serum homocysteine levels [4, 46]. Hyperhomocysteinemia is a significant risk factor for cardiovascular disease and has recently been suggested as a tumor marker [47]. Baydas et al. [37] reported that melatonin decreased plasma homocysteine levels while causing an increase in total glutathione level, which is in agreement with the results of the present study. It is also reported that plasma homocysteine levels increase in pinealectomized rats, and melatonin administration reduces these levels [37, 43] The effect of melatonin supplementation on serum homocysteine level reported in the present study is of interest, and it may direct further studies through the role of melatonin in cancer prevention. In the present study, elevated serum glucose, triglycerides, and cholesterol concentra-

tions were decreased with dietary melatonin supplementation. Decreases of glucose and cholesterol concentrations in the current study may be attributed to a decline in glucocorticoid secretion, which increases glucogenesis. Similar effects of different antioxidants on the glucose and lipid metabolism have been reported [5, 48]. Melatonin exerts significant effects on lipid metabolism. Plasma level of total cholesterol is reduced by melatonin, which was also observed in the present study. This effect of melatonin might be due to enhanced clearance of endogenous cholesterol [49, 50]. Mineral excretion is increased under stressful conditions [51]. El-Husseiny and Creger [52] reported a low retention rate for Ca, Cu, Fe, K, Mg, Mn, Na, P, and Zn in birds subjected to a high ambient temperature. Ozturk et al. [53] reported a relation between Zn and melatonin at the stage of absorption of Zn. Zinc protects the red cell membrane against cellular changes caused by lipid peroxidation, and melatonin reduces peroxidative damage on cellular membrane by enhancing the absorption of Zn. Copper and zinc are essential trace elements for the activities of Cu and Zn-superoxide dismutase, respectively [54]. Chromium is essential for activating certain enzymes and is postulated to function as an antioxidant [55, 56]. Therefore, restoration of deficiencies of these minerals, as observed in the present study, would increase the activities of these antioxidant enzymes and thus alleviate oxidative stress.

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CONCLUSIONS AND APPLICATIONS 1. Melatonin supplementation partially restored the heat stress-induced impairment in antioxidant status. 2. As melatonin supplementation significantly decreased the concentration of homocysteine, which has been suggested as an independent risk factor for coronary heart disease and cancer, the results of this study may also be applicable to prevention of cancer and cardiovascular diseases.

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Acknowledgment The authors thank the Veterinary Control and Research Institute of Elazig for providing the experimental facility.