The Effect of Melatonin on Liver Superoxide Dismutase Activity, Serum Nitrate and Thyroid Hormone Levels

Japanese Journal of Physiology, 50, 149–153, 2000

Güler ÖZTÜRK, S¸ule COS¸ KUN, Denõúz ERBAS¸, and Enver HASANOGˇ LU*

Department of Physiology and *Department of Pediatric Nephrology, Faculty of Medicine, University of Gazi, Ankara, Turkey

Abstract: Melatonin is a main neurohormone levels, respectively. Blood and tissue samples of the pineal gland. The effects of melatonin on were collected. Decreased TSH, T3, T4 and ni- the level of serum thyroid-stimulating hormone trate levels were determined in the melatonin-in- (TSH), thyroxine (T4), triiodothyronine (T3), ni- jected and nighttime groups. Melatonin levels trate, melatonin and liver superoxide dismutase showed a diurnal rhythm. SOD activity increased (SOD) activity were examined in rats. Melatonin in the melatonin-treated group. The results was injected at the dose of 10 mg/kg for 7 days, demonstrate that increased SOD activity, and re- 2 h before turning the lights off. Rats were decap- duced serum TSH, T3, T4 and nitrate levels cor- itated at 10:00 a.m. and 02:00 a.m., which are the related with the serum melatonin levels. [Japa- times of the lowest and highest serum melatonin nese Journal of Physiology, 50, 149Ð153, 2000]

Key words: melatonin, nitrate, superoxide dismutase, thyroxine, triiodothyronine.

Melatonin is a neurohormone produced during the NOS is present in the pineal gland and its function ap- night by the pineal gland. Its secretion is regulated by pears to be under photoneural regulation [9]. circadian and seasonal variations in day length, trans- The lowering effect of melatonin and thyroid hor- mitted via visual projections to the suprachiasmatic mones on serum cholesterol has also been docu- nucleus, which functions as a circadian clock in mam- mented showing a parallel effect on lipid metabolism mals [1]. [10]. Melatonin is now considered an important antioxi- The aim of this study was to investigate the correla- dant [2]. It is a free-radical scavenger and general an- tion between diurnal variations of melatonin and thy- tioxidant. It possibly works via electron donation to roid hormones, antioxidant status, and nitrate produc- directly detoxify free radicals such as the highly toxic tion in plasma. hydroxyl radical [3]. Melatonin has also been reported to alter the activities of enzymes improving the total MATERIAL AND METHODS antioxidative defense capacity of the organism, i.e., nitric oxide synthase, SOD [4]. It is also implicated in Melatonin (N-acetyl-methoxy-tryptamine) was pur- the regulation of blood flow because pinealectomy chased from Sigma and dissolved in absolute ethanol, produces hypertension and exogenous melatonin re- and later in phosphate-buffered saline (PBS) with a verses this phenomenon [5]. Thyroid hormones also final ethanol concentration of less than 1%. increase the blood flow in many tissues [6]. Animals. A total of 24 male Sprague-Dawley Many types of mammalian cells produce nitric rats, age 2 months, were divided into control and oxide (NO), and it plays a major role in the regulation melatonin-treated groups. Animals were maintained at of blood pressure. NO functions as a vasodilator and 20Ϯ2°C and under 12:12-h light : dark cycle (light is synthesized by the isoforms of nitric oxide syn- from 6:00 a.m. to 6:00 p.m.). After 1 week of acclima- thases (NOS) [7]. Suppression of melatonin secretion tion, animals were randomly divided into four groups by NO has been reported [8]. The neural isoform of of six rats each and fed standard pellet food and tap

Received on June 9, 1999: accepted on December 27, 1999 Correspondence should be addressed to: Deniz Erbas¸, Department of Physiology, Faculty of Medicine, University of Gazi, 06500, Bes¸evler, Ankara, Turkey. Tel: ϩ90–312–212–9010, Fax: ϩ90–312–213–4338, E-mail: [email protected]

Japanese Journal of Physiology Vol. 50, No. 1, 2000 149 G. ÖZTÜRK et al. water ad libitum. The rats received 7 daily subcuta- 62.5 : 37.5 (v/v)] was added to 250 ␮l of the super- neous (s.c) injections of melatonin (10 mg/kg) or PBS natant, and the solution was centrifuged at 3,000ϫg as the control 2 h before turning the lights off, and for 10 min at ϩ4°C. The aqueous upper layer was then were decapitated under ether anesthesia (under ethical collected. Subsequently 40 ␮l samples were taken consideration) at 10:00 a.m. and 02:00 a.m. from each supernatant (40 ␮l water for the negative Determination of melatonin in serum. The control), and 900 ␮l diluting buffer at 37°C was added serum was estimated by ELISA (RE 540 21 IBL- [50 mM 2-amino-2-methyl-1,3-propanediol/HCI, pHϭ Hamburg). Extraction columns were placed into glass 8.8, 0.11 mM diethylenetriaminepentaacetic acid tubes, 2ϫ1 ml of methanol (undiluted) was added, the (DTPA) as the chelating agent (Bioxytech)]. Thirty solution centrifuged at 200ϫg for 1 min, and then microliters of 1,4,6-trimethyl-2-vinylpropyl-trifluo- 2ϫ1 ml of bidistilled water was added. After column romethano-sulphonate [R2 (Bioxytech)] was then preparation, 0.5 ml of standards, controls and samples added to separate the mercaptans (RSH groups), fol- were added to the columns and centrifuged at 200ϫg lowed by energetic stirring to a vortex for 3–4 s and for 1 min. The columns were then washed with 2ϫ1 incubation in a thermal bath at 37°C (for 1 min). ml of 10% methanol in bidistilled water (v/v) and cen- Thirty microliters of 5,6,6a,11b-tetrahydro-3,9,10-tri- trifuged at 500ϫg for 1 min. Next, the columns were hydroxybenzo-fluorene [R1 (Bioxytech)], the chro- placed into clean glass tubes and 1 ml methanol was mophore which will be self oxidized as a function of added followed by centrifugation at 200ϫg for 1 min. the SOD concentration, was then added, followed by The columns were removed from tubes, the methanol stirring to a vortex (4–5 s). The samples were trans- was evaporated by nitrogen, and samples were recon- ferred to a quartz tube and the SOD activity was mea- stituted in 0.15 ml of bidistilled water. Fifty micro- sured for absorbance at 525 nm (for 1 min). The ex- liters of each extracted standard, control and sample perimental ratio Vs/Vc was obtained by reference ta- were pipetted into the appropriate wells. Fifty micro- bles which relate it to the units of activity of the en- liters of Melatonin-Biotine and 50 ␮l antiserum were zyme: Vs/Vcϭ1ϩ(SOD)/a(SOD)ϩb; Vsϭpercentage added into each well and the plate was shaken care- of sample with SOD, Vcϭmean of at least four con- fully. The plate was sealed with an adhesive foil and trols, aϭ0.073, bϭ0.93. Hence: (SOD)ϭ0.93 (Vs/Vc– incubated for 18 h at ϩ4°C. Each well was then 1)/1.073–0.073(Vs/Vc) [11]. washed with assay buffer and 150 ␮l of enzyme con- The resulting value is multiplied by the dilution jugate was pipetted into the wells followed by incuba- factor and expressed as units of activity of per milli- tion of the plate for 120 min at room temperature. liter. The detection limit of the assay was 0.2 U/ml. Each well was washed three times with assay Nitrate measurement. The amount of total ni- buffer, and 200 ␮l of substrate solution was pipetted trate in the test samples was determined by modifica- into each well. The plate was then incubated at room tion of the procedure described by Bramen and Hen- temperature for 1 h. Fifty microliters of stop solution drix [12] using the purge system of Sievers (Model was then pipetted into each well and the plate was 280 NOA). A saturated solution of VCI3 in 1 M HCI read at an optical density of 405 nm with a microtiter was prepared and filtered before use. Five milliliters plate reader. The lowest detectable level of the assay of this reagent was added to a purge vessel and it was 5 pg/ml. purged with nitrogen for 5 to 10 min before use. The Determination of thyroid hormones and purge vessel was equipped with a cold-water con- thyroid-stimulating hormone. Hormones were denser and a water jacket to permit heating of the measured by radioimmunoassay kits (Bio Bak, USA). reagent to 95°C using a circulating water bath. The The lowest detection limit of the assay was HCI vapors were removed by a gas babbler containing 0.011 ␮IU/ml for TSH, 0.2 ng/ml for T3 and 0.5 ␮g/dl 15 ml of 1 M NaOH. The gas flow rate into the chemi- for T4. luminescence detector was controlled using a needle Determination of the liver SOD activity. valve. SOD activity was estimated by the spectrophotometric Sample and standards were injected into the purge method (SOD-525, Bioxytech, France). Tissues were vessel to react with the VCI3/HCI reagent, which con- blotted on paper and weighted after washing in ice- verted nitrate and nitrite and s-nitroso compounds to cold 0.25 M sucrose. Tissues were then homogenized NO. The NO produced was detected by ozone-in- in ice-cold 0.25 M sucrose. Homogenates were cen- duced chemiluminescence in the chemiluminescence trifuged at 3,000ϫg for 10 min at ϩ4°C and super- detector. A standard curve was constructed using vari- natant was collected. Four-hundred microliters of ice- ous concentrations of NaNO3 (10–100 ␮M). The de- cold extraction reagent [ethanol–chloroform, tection limit of the assay was nanomolar to millimolar

150 Japanese Journal of Physiology Vol. 50, No. 1, 2000 Melatonin Is an Antioxidant

.(Table 1. Effect of melatonin on liver SOD, serum nitrate and thyroid hormones levels (mean؎SD

Control group Melatonin-treated group

10:00 a.m. (nϭ6) 02:00 a.m (nϭ6) 10:00 a.m. (nϭ6) 02:00 a.m. (nϭ6)

Melatonin (pg/ml) 10.28Ϯ1.81a 74.05Ϯ13.22b 56.42Ϯ7.17c 122.2Ϯ36.2d Nitrate (␮M) 3.18Ϯ1.60e 1.29Ϯ0.43f 2.70Ϯ1.56g 1.12Ϯ0.37h SOD (U/ml) 10.85Ϯ3.23i 21.96Ϯ10.40j 27.37Ϯ6.59k 39.41Ϯ12.12l m n o p T3 (ng/ml) 0.74Ϯ0.05 0.52Ϯ0.06 0.65Ϯ0.06 0.50Ϯ0.07 r s t u T4 (mg/dl) 5.12Ϯ0.13 3.60Ϯ0.53 4.81Ϯ0.47 3.11Ϯ0.11 TSH (␮IU/ml) 2.01Ϯ0.53v 0.62Ϯ0.30w 1.26Ϯ0.11x 0.24Ϯ0.09y a–b, a–c, a–d, c–d, i–k, i–l, m–n, m–p, r–s, r–u, t–u, v–w, v–y, w–x, x–ypϽ0.001. e–f, e–h, v–x, n–o, o–ppϽ0.01. c–d, j–l, s–t, w–ypϽ0.02. b–d, f–g, g–h, i–j, k–l, m–o, s–upϽ0.05.

Table 2. Groups and levels (Group factor has two levels: control and melatonin treated; and time factor has two levels: 10:00 a.m. and 02:00 a.m.).

Control group Melatonin-treated group 10:00 a.m. 02:00 a.m. n XϮSD XϮSD XϮSD XϮSD

Melatonin 12 42.16Ϯ34.49a 89.20Ϯ42.44b 33.28Ϯ24.5c 98.09Ϯ9.9d Nitrate 12 2.23Ϯ1.47e 1.91Ϯ1.36f 2.94Ϯ1.50g 1.21Ϯ0.39h SOD 12 16.39Ϯ9.35i 33.39Ϯ11.22j 19.08Ϯ9.90k 30.70Ϯ14.00l m n o p T3 12 0.63Ϯ0.12 0.57Ϯ0.10 0.69Ϯ0.07 0.51Ϯ0.07 r s t u T4 12 4.36Ϯ0.87 3.96Ϯ 0.94 4.96Ϯ0.3 3.36Ϯ0.44 TSH 12 1.32Ϯ0.83v 0.75Ϯ 0.54w 1.63Ϯ0.53x 0.43Ϯ0.29z a–b, c–b, i–j, o–p, t–u, x–zpϽ0.001. v–wpϽ0.01. g–h, k–l, m–npϽ0.05. and assay sensitivity was ϳ1pM. els of the control and melatonin-treated groups during Statistical analysis. All data are expressed as in daytime and nighttime (pϽ0.05) (Tables 1 and 2). the meanϮstandard deviation (SD). A two-way factor- ial analysis of variance (ANOVA) test was applied. DISCUSSION There were two levels of group factor: control and melatonin-treated. Two time factors were also in- The action of melatonin is highly pleiotropic. It in- volved: 02:00 a.m. and 10:00 a.m. (Table 2). volves: firstly, direct effects via spesific binding sites in various peripheral tissues and cells of vertebrates, RESULTS including immunomodulation; secondly, systemic in- fluence on the cytoskeleton and nitric oxide forma- Nighttime serum melatonin levels were found to be tion, mediated by calmodulin; and thirdly, antioxida- significantly higher than those of daytime levels in tive protection [13]. control (pϽ0.001) and melatonin-treated groups In vitro studies have demonstrated that melatonin is (pϽ0.02) (Tables 1 and 2). a scavenger of oxyradicals and peroxynitrite, and an The SOD activity in the liver was increased at inhibitor of NO [14]. Melatonin can inhibit NO pro- nighttime in control and melatonin-treated groups duction by reducing iNOS expression in leucocytes (pϽ0.001) (Tables 1 and 2). and inhibit catalytic activity of NOS in rat cerebellum Serum nitrate levels were decreased in the mela- and hypothalamus by a calmodulin- mediated mecha- tonin-treated and nighttime groups. This was a singifi- nism [15–17]. Recent studies indicated that NO is in- cant difference between daytime and nighttime levels volved in ischemic brain damage. In addition to the in the control and melatonin-injected groups (pϽ0.01 inhibition of neuronal NOS activity, the cytoprotective and pϽ0.05 respectively) (Tables 1 and 2). effect of melatonin during ischemia may be due to its T3, T4 and TSH levels during nighttime were found ability to directly scavenge free radicals [18]. It is sug- to be significantly lower than those during daytime for gested that the inhibitory effect of melatonin on NO the control and melatonin-injected groups (pϽ0.001). production may be responsible for the protective effect There were also significant difference between the lev- of the hormone on neuronal structures [18]. NO and

Japanese Journal of Physiology Vol. 50, No. 1, 2000 151 G. ÖZTÜRK et al. cGMP rhythms in the frontal cortex of the chick are This work was supported by the Turkish Prime Ministry inversely related to the melatonin rhythm. When State Planning Organization and Gazi University Research melatonin levels increase (at night or after melatonin Center. injection) NO and cGMP levels decrease [18, 19]. In our study, serum nitrate levels decreased inversely re- REFERENCES lated to the serum melatonin levels, and this is in 1. Delegrange P and Guardiola-Lemaitre B: Melatonin, its agreement with previous studies [14, 18]. receptors, and relationships with biological rhythm dis- Thyroid hormones increase the basal rate of oxygen orders. Clin Neuropharmacol 20: 482–510, 1997 consumption in all tissues except the brain, gonads 2. 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