Melatonin, a major hormone produced by the pineal gland, plays an important role in various physiological processes, including regulation of circadian and endocrine rhythms, aging, stimulation of immune functions and it also has neuro-protective and cardio-protective effects. Melatonin synthesis in the pineal gland is rather low during daytime as compared to night-time. Night-time melatonin production is significantly attenuated with aging.

Anti-oxidants may have a beneficial effect on many age-related diseases. In the free radical theory of aging, it is suggested that accumulated free radical damage may be responsible for degenerative process during aging. In addition, free radical scavengers such as reduced glutathione (GSH) are defense systems in the aging process. Free radicals can attack a wide variety of cellular components, including DNA, proteins, and membrane lipids. Lipid peroxidation plays an important role in tissue injury. Malondialdehyde (MDA), a stable metabolite of the free radical-mediated lipid peroxidation cascade, is widely used as marker for oxidative stress. GSH is an important endogenous anti-oxidant which levels are affected by oxidative stress.

Melatonin is known to be a potent free radical scavenger and anti-oxidant. Total anti-oxidant capacity of human serum positively correlates with its melatonin concentration. Thus, reduction of melatonin production with increasing age may be a factor in increased oxidative damage in the elderly. Reiter et al. (1999) showed that reduction of endogenous melatonin levels due to pinealectomy leads to oxidative damage as animals’ age. Previous observations demonstrated that the physiological levels of melatonin are important in protecting against oxidative stress-induced damage. Pinealectomy aggravates and melatonin administration attenuates oxidative injury. As far as we know, effects of short-term administration of exogenous melatonin on GSH levels in liver tissue in relation to pinealectomy have not been well documented yet. The present study was designed to explore the effects of pinealectomy (and thus the role of endogenous melatonin) and exogenous melatonin on rat liver tissue in vivo.

Male Wistar rats, weighing 150–200 g, were placed in a quiet and temperature- (21±2 °C) and humidity-(60±5%) controlled room in which a 12–12 h light-dark cycle was maintained. Two months before melatonin injections, pinealectomy was performed. All experiments were performed between 9.00 and 17.00 h. The rats were divided into three groups of 10 rats each: control (non-Px), Px+vehicle and Px+melatonin. Melatonin or vehicle was administrated daily intraperitoneally by injection for 10 days. Twenty-four hours after the last injection, rats were sacrified, livers were quickly removed and divided into two parts. One part was put in a formaldehyde solution for routine histopathological examination by light microscopy. The other part was put in liquid nitrogen and stored at ?70 °C until assayed for MDA and GSH levels.

Melatonin (Sigma, St. Louis, MO, USA) was dissolved in ethanol and then diluted in saline (0.9% NaCl wt/vol). All protocols in the present study were approved by the local Ethics Committee of the Medical School of the Firat University, Elazig, Turkey.

Pinealectomy was performed as described previously. Rats were anesthetized with ketamine hydrocloride (75 mg/kg) and xylazine (8 mg/kg) before operation. The entire procedure was completed within 15 min. Px was confirmed by the histological evaluation of the gland for each animal.

Tissues were homogenized with ice-cold 150 mM KCl for determination of the levels of MDA and GSH. MDA levels in the homogenates were determinated spectrophotometrically by measuring the presence of thiobarbituric acid reactive substances. GSH levels were determined by the spectrophotometric method, which is based on the use of Elman’s reagent. The results were expressed as nmol/g tissue.

Liver-tissue samples were fixed in 10% neutral buffered formalin and then processed for paraffin embedding. Sections of the blocks were stained with hematoxylin–eosin. Light microscopic examinations were performed in a blinded fashion. Histological findings with respect to portal tract lesions, dilatation and congestion of sinusoids were expressed semi-quantitatively (0, absent; +, slightly present; ++, moderately present; +++, severely present) according to the dissemination of damage.

Data are expressed as means±SEM; when p<0.05, the difference was considered to be statistically significant. Multiple comparisons between experimental groups were analyzed by one-way ANOVA with a Tukey post hoc test.

MDA levels are summarized in Table 1. Px caused a significant increase in MDA levels as compared with the control (non-Px) group. Melatonin given to Px rats significantly reduced MDA levels in liver tissue.

Values are means±SEM (n=10 in each group) for sham-operated rats (Non-Px), Px rats treated with vehicle (Px+vehicle), and Px rats treated with melatonin (Px+melatonin).

^a Significantly different from Non-Px group (p<0.005).
^b Significantly different from Px+melatonin group (p<0.001).
^c Significantly different from Non-Px group (p<0.01).

^d Significantly different from Px+melatonin group (p<0.005).

Liver GSH levels were significantly lower in the Px group than in the control group. Melatonin administration significantly increased GSH levels.

Semi-quantitative light microscopical results are shown in Table 2. Light microscopical examination of sections of control animals showed normal liver tissue characteristics. It was found that various grades of sinusoidal dilatation developed in all Px rats, which was reduced by melatonin treatment. Severity of mononuclear cell infiltration and sinusoidal congestion were significantly lower in the Px+melatonin group than in the Px+vehicle group.

In the present study, we found that Px causes significant liver damage (oxidative damage and structural changes) as compared with control (sham-operated) rats and tissue damage in Px rats is prevented by administration of exogenous melatonin. This protection was manifested by reduced levels of lipid peroxidation products, increased levels of GSH and less tissue damage in the livers of rats.

It is important to clarify whether reduction of circulating melatonin has a critical role in liver tissue damage. Pinealectomy is known to remove the nocturnal elevation in serum melatonin levels and decreases melatonin concentrations. In the present study, we investigated the role of endogenous melatonin and the effects of short-term administration of exogenous melatonin in liver tissue after Px. It has been reported that Px may cause hypertension up to 60 days, and therefore, we used rats that were pinealectomized at 2 months before melatonin administration to eliminate any possible effect of Px-induced hypertension on tissue damage. Indeed, our previous studies indicated that either mean arterial pressure and heart rate values were not significantly different between both experimental groups.