Assessment the Role of Melatonin in Iron Toxicity in Male Rats

 

 

 

 

 

INTRODUCTION

Iron is critical to normal cellular function and plays an important role in metabolic processes including O₂ transport, electron transport, oxidative phosphorylation and energy production, xenobiotic metabolism, DNA synthesis, cell growth, apoptosis, gene regulation and inflammation (1). It is also a necessary cofactor for the synthesis of neurotransmitters, dopamine, nor epinephrine and serotonin (2).  In spite of its requirement in the body, a misbalance in iron homeostasis can be devastating; accumulation of iron has been associated with hemochromatosis and production of free radicals that potentially leads to neuronal damage (3). Increased levels of iron in brain regions have been reported in neurodegenerative disorders (4). In addition, iron induces lipid peroxidation, protein carbonyl expression and depletion of lipid soluble antioxidants in testicular tissue results in disruption of spermatogenesis (5, 6), impairment in testicular activity and affects the androgenicity of male rats (7).

Melatonin (N-acetyl-5-methoxytryptamine), the main product of the pineal gland, which is outside of the blood–brain barrier, acts as an endocrine hormone since it is released into the blood(8). Melatonin has potent antioxidant activities (9). Because of its highly lipophilic character, it can easily cross cell membranes(10), and the blood–brain barrier (11). Several studies reported that melatonin and its metabolites are excellent scavengers of reactive oxygen and nitrogen species (12). Others demonstrated that melatonin prevents the brain oxidative damage induced by traumatic brain injury in the immature rats (13). In addition, MLT displayed a marked role in the protection of testicular toxicity induced by radiation and different mutagenic and clastogenic agents (14, 15).The aim of the present study was to evaluate the possible protective effect of melatonin on iron overload and toxicity on brain & testes of male albino rats.

METHODS

Animals and animal’s treatment:Adult maleWistar albino rats, with an average body weight of 100-120g, were kept under good ventilation; adequate stable diet and water were allowed ad libitum. They were maintained on normal light/dark cycle.  Rats were divided into four groups comprising of six rats in each. The first group served as a control group, where animals were given normal diet, and received nothing of treatment throughout the period of the study. Second group served as melatonin treated group, where animals were injected intraperitonealy (i.p) with freshly prepared melatonin at dose of 15 mg/kg body weight day after day for 8 weeks. Third group served as iron group, where animals were received ferrous sulphate dissolved in distilled water orally at dose of 1g/kg body weight day after day for eight weeks. Forth group served as melatonin and iron treated group, where animals were injected i.p. with freshly prepared melatonin at dose of 15 mg/kg body weight and then received ferrous sulphate dissolved in distilled water orally at dose of 1g/kg body weight day after day for 8 weeks. All injections were administered in the volume of 0.5ml/ injection.

Biochemical analyses:All experimental protocols were approved by the Animal Ethics Committee of Mansoura University. At the end of the experimental period, overnight fasted rats were sacrificed. Blood samples were collected in clean non-heparinized centrifuge tubes and only few droplets were placed in clean heparinized tubes for hematological parameters then the tubes were let to stand for 15 min at 30^◦C after which the non-heparinized tubes were centrifuged at 3000 G for 15 min. Blood sera were carefully separated and were kept at – 20^◦C for subsequent analysis.

Hematological parameters including the count of red blood corpuscles (RBCs), white blood cells (WBCs), hemoglobin (Hb), platelets count and hematocrit percent were conducted using hematological analyzer (Sysnex Ts-21) Japan (16). Serum ferritin was determined according to the method of Young (17). Serum transferrin was estimated according to the method of Yamanishi and Iyama, 2003 (18). Iron concentration in serum, brain and testis were measured according to the method of Dreux (19), zinc level according to the method of Hayakawa (20), and gamma-glutamyle transaminase (γ-GT) activity according to the method of Szasz (21).

Testis specimens were removed, weighed and then homogenized in phosphate buffer (50 mM, pH 7.0) to form about 10% (w/v) homogenate.The brain was carefully removed, weighed and then quickly frozen on dry ice until biochemical assay. Thereafter, brain of each animal was divided longitudinally into two parts: One was weighed, homogenized in 75% high performance liquid chromatography (HPLC) methanol 10% (W/V) for HPLC determination of monoamines content. The other part was weighed and homogenized in phosphate buffer pH 7.0 to form 10% (w/v) homogenate for other biochemical assay.  The homogenates were subjected to the following biochemical analysis: the lipid peroxidation product, malondialdehyde (MDA), was measured by the thiobarbituric acid assay, according to the method of Ohkawa (22). The reduced glutathione (GSH) content was measured according to the method of Prins and Loose (23). Catalase (CAT) activity was assayed according to Bock (24).  The superoxide dismutase (SOD) activity was assayed according to the procedure of Nishikimi (25).  Brain neurotransmitter concentrations were determined by HPLC method Pagel (26).

Statistical analysis:The results were evaluated by One Way ANOVA (analysis of variance) test and post comparison was carried out with LSD test. Group results were then expressed as mean ± the standard error of the mean (SEM). The values of P≤ 0.05 were considered statistically significant.

RESULTS

In the current study, the administration of melatonin (15 mg/kg) did not change any of hematological parameters except hematocrit percent (Ht %) that observed a significant increase compared to the iron intoxicated rats. On the other hand, administration of ferrous sulphate (1g/kg body weight) caused significant decrease in white blood cells count and in hematocrit percent but no significant changes was observed in other hematological parameters comparing to control group (Table 1).

As shown in Tables 2 and 3, the administration of ferrous sulphate produced significant increase in the brain and testicular iron and in serum levels of ferritin, iron, and γ-GT but transferrin levels and zinc concentration was decreased as compared with control group. The administration of MLT against iron intoxication resulted in a significant reduction in the brain and testicular iron and in serum ferritin and iron levels as well as γ-GT activity, while significant increase was observed in serum transferrin level compared to iron intoxicated rats.

The data in Tables 4 shows the effect of various treatments on lipid peroxidation and antioxidant levels in the brain and the testis. In rats received iron alone, the values of thiobarbituric acid-reactive substances (TBARS) were significantly higher in the brain and testicular tissues than in the control group. While the activity of GSH, SOD, and CAT were significantly decreased in these tissues when compared to the control group. The administration of MLT alone caused significant decreases in TBARS in the brain tissue, while produced significant increase in SOD activity in testicular tissue as compared with control group. On the other hand, the MLT administration as a protective agent to the rats intoxicated with iron had significantly increased in the measured antioxidant parameters including GSH, SOD, and CAT compared to iron intoxicated group.

Administration of MLT alone caused an increase in dopamine content. The iron intoxicated rats showed a significant decrease in the brain neurotransmitters as compared to the control group. On the other hand, MLT administration against iron intoxication caused a significant increase in dopamine and serotonin levels while no significant changes were observed in norepinephrine levels, all as compared with iron intoxicated animal group (Table 5).

DISCUSSION

Iron is an essential metal involved in multiple physiological and biochemical processes throughout the body, including brain. In spite of its requirement in the body, accumulation of iron has been associated with hemochromatosis and production of free radicals that potentially leads to neuronal damage (4). Excess iron is toxic due  to oxidative metabolism, that free iron assists in the generation of the most devastatingly toxic hydroxyl radical (^•OH) in what is referred to as the Haber-Weiss and Fenton reactions (27).

In the present study, the administration of ferrous sulphate to rats elevated serum iron levels, ferritin, and iron concentration in brain and testis homogenate but decreased serum transferrin, a situation permissive of iron overload. Increasing serum ferritin levels may be one of the first clinical signs of iron overload that within cells, iron is stored as ferritin. Supporting to these results,Abd El-Baky (28) demonstrated that the administration of 0.5 or 1.0 gm of ferrous sulfate/kg B wt caused significant increase in serum iron and ferritin levels.

Testicular iron content demonstrated significant increase, which in agreement with Lucesoli (5) and El-Seweidy (7) who considered testicular tissues as secondary target for accumulated iron. It considers that testes produce their own transferrin, and participates in iron shuttle system which fulfills the requirements of this metal for spermatogenesis (29).

The current data showed no significant changes in blood Hb level, red blood corpuscles’, and platelets’ content in iron group which in line with the study of Barbosa (30). While there were significant decrease in white blood corpuscles’ and Ht% in iron group as compared with the control group due to the accumulation of iron, which leads to suppression of bone marrow resulting in reduction of total and differential leucocytes counts (31).The present results show a significant decrease in serum zinc concentration in iron group and in melatonin iron treated group as compared with the control group. Supporting these results, zinc levels in beta thalassemia major patients were significantly decreased in most of the studies as compared to the controls. This may be due to the release of zinc from hemolysed red cells (32).  On the contrary, the present results disagree with those obtained by Elseweidy and Abd El-Baky (33) who demonstrated that serum zinc increased significantly after intake of iron-fortified diet.

The current study also demonstrated a significant increase in serum γ-GT activity after iron overload exposure. γ-GTis a key enzyme in the catabolism of GSH (34). It has been shown that the extracellular cleavage of GSH by γ-GT induces the production of ROS, suggesting that γ-GT used as a marker of oxidative stress (35). Therefore, the present results suggested that serum γ-GT might be one of the enzymes related to oxidative stress induced by iron overload. This confirmed by decreased GSH level in iron overload animals. Meister (36) reported that the GSH works as an antioxidant due to the presence of numerous–SH groups, which can react with the free radicals and products of lipid peroxidation such as lipid peroxides and aldehydes protecting in this way against development of oxidative stress (37).

Thus, it is supposed that the reduction in GSH might result from the utilization of –SH groups to scavenge free radicals formed (38). Malondialdehyde (MDA) is a secondary product of lipid peroxidation and is released because of the toxic effect ofROS (39). The major forms of cellular damage induced by iron overload is lipid peroxidation, The present study showed a significant increase TBARS concentration in rat brain and testis homogenates as compared to control levels, indicating high level of oxidative stress, which markedly enhanced with increasing iron toxicity. Despite the fact that brain is well protected by the blood brain barrier, it cannot provide full protection against circulating inflammatory agents that can generate radicals in the brain (40). Supporting our results, Maharaj (41) stated that intrahippocampal injection of Fe (II) resulted in a significant increase in lipid peroxidation levels in comparison to control levels.

In addition, El-Seweidy, and El-Baky(33) reported that testicular contents of MDA in rats received biscuits enriched with iron demonstrated significant increase while GSH showed significant decrease.The current data showed significant decrease in SOD and CAT activities in brain and testicular tissues in iron group as compared with the control group. These results agreed with Lucesoli and Fraga (5) who indicated that the activities of SOD and CAT in testis were significantly decreased in iron overload animals. Jagetia (42) demonstrated that GSH depletion, reduced activities of antioxidant enzymes like SOD and CAT in testicular tissue after iron treatment which confirmed by the present data.The present study showed that in iron treated group brain dopamine and serotonin decreased significantly as compared with the control group. These results were in accordance with the previous studies of Elseweidy and Abd El-Baky (33) who demonstrated that intake of iron-fortified diet significantly decreased brain serotonin and dopamine.

Logroscino (43) also reported that Iron not only induces oxidative stress but damages proteins, membranes, and nucleic acids and eventually leads dopaminergic neuron death in the substantianigra. Supporting these data, JimnezDeL Rio (44) have reported that serotonin-binding proteins (SBP), located in brain extract are involved in storage, protection and transport of serotonin as well as catecholamines. Fe (II) only increases such binding, it is approved that Fe (II) binds first to SH- group SBP. In addition, Monoamines form coordination bonds with trapped iron leading to potential change in SBP functions. Other studies in animal models of Parkinson disease (PD) also suggested that a high iron level in the brain is associated with loss of dopaminergicsubstantianigra pars compacta (SNc) neurons (45, 46).

The current study demonstrated that melatonin has a protective action on brain and testis oxidative injury secondary to experimental iron overload. It is hypothesized that if oxidative stress is involved in the origin of a disease, then successful antioxidant treatment should delay or prevent the onset of that disease (47).Melatonin, the chief product of the pineal gland, is an effective direct free radical scavenger and indirect antioxidant. It detoxifies both ROS and reactive nitrogen species (RNS), both groups of which are abundantly produced in the brain (11). The present results suggest that melatonin might decrease the production of destructive free radical induced by iron toxicity. This is attributed, in apart, to the ability of melatonin to quench hydroxyl radicals and scavenge H₂O₂ generated from a Fenton reaction. Melatonin possesses an electron-rich aromatic indole ring and functions as an electron donor, thereby reducing and repairing electrophilic radicals (48). Besides, melatonin as well as its metabolites reportedly possesses iron-binding properties (49, 50) and have the ability to participate in maintaining iron pool at appropriate level resulting in control of iron homeostasis.

In other studies, Melatonin controlled iron levels after lead intoxication (51, 52). In addition, melatonin controls oxidative stress and modulated the levels of iron and iron binding proteins during adriamycin treatment (53).In the present study, the data showed that melatonin did not affect the levels of zinc in serum confirmed the findings by Othman (53). Moreover, it did bind several other heavy metals (49).Taken together, the chelation of iron ions by melatonin is an important factor in its anti-free radical effects subsequently, preventing or at least decreasing iron mediated tissue oxidative stress (53). The current investigation demonstrated marked normalization of serum iron, ferritin and transferrin levels in melatonin iron treated group as compared with iron group. These findings are consistence with Othman (53) who suggested that melatonin could prevent oxidation of iron binding proteins. Because of small size of melatonin enough to have direct access to the iron core of ferritin through the pores of the protein shell, inhibits redox cycling and generation of toxic ROS.

In the present data, treatment with melatonin resulted in a significant increase in haematocrit percent, which in agreement with the study of Ozmerdivenli (54) but no significance changes were observed in other hematological parameters. On the other hand, γ-GT is inversely associated with antioxidants (55), while melatonin has the ability to protect macromolecules against oxidative stress and increase the activity of antioxidant enzymes (56, 9). Accordingly, melatonin might inhibit serum γ-GT activity to exert its antioxidant effect. This might be in agreement with Ogeturk (57) who stated that the elevations in γ-GT and total iron levels induced by CCl₄ injections were significantly reduced by melatonin. El-Missiry (58) found that the treatment with melatonin before irradiation displayed significant amelioration in the elevation of serum γ-GT activity.

The present result demonstrates that melatonin has a protective action against oxidative stress in brain and testis induced by iron overload, evidenced by a significant decrease in MDA levels in brain and testis homogenate of melatonin iron treated group as compared to iron group. These results were in accordance with Maharaj (41) who reported that the injection of melatonin or6-hydroxymelatonin (6-OHM) in combination with Fe²⁺ significantly inhibited lipid damage in the hippocampus induced by the neurotoxin. In addition, Chen (59) reported that melatonin able to protect against Fe²⁺ induced lipid peroxidation in vivo. Furthermore, melatonin reportedly suppressed lipid peroxidation induced by iron (60).

Melatonin not only does not consume cellular GSH, it also preserves or even increases the content of GSH in tissues (61). According to this view, melatonin cause significant increase in brain and testis GSH levels in melatonin treated group as compared with iron group, and hence to inhibit formation of extracellular and intracellular ROS (62). This result is in agreement with those obtained by El-Sawi (63) that when melatonin was given to animals injected with Delta-aminolevulinic acid, the decrease in GSH levels in rat brain was significantly improved. In addition, Feng(64) demonstrated that early melatonin treatment inAlzheimer’s disease is able to reduce TBARS levels, increase GSH content, SOD activity and prevent those processes that lead to cell death in a amyloid precursor protein (APP) transgenic mouse model.

It is commonly accepted that SOD is considered as a major antioxidant enzyme, because it dismutases the superoxide anion (O₂·‾) radical to hydrogen peroxide (H₂O₂) and reduces the formation of peroxynitrite.  Antolin (65) indicated that melatonin increased the tissue mRNA levels for both manganese and copper SOD levels in animals treated with melatonin.CAT is an inducible enzyme, decomposes H₂O₂, and is involved in the antioxidant defense mechanisms of mammalian cells. Thus, it is an index of increased H₂O₂ production (66). Melatonin was found also to stimulate the activity of CAT, which is also involved in reducing the H₂O₂ and thus reduce the generation of hydroxyl radicals (67).

In the current study, both CAT and SOD activity improved by melatonin treatment , melatonin administration was able to neutralize a number of toxic reactants including ROS and free radicals (68) such combination of actions indicating in reduced accumulation of lipid peroxidation hence prevent cascade generation of other harmful species. This might due to the important role of melatonin in activating antioxidant defenses such as SOD and CAT (56). This in agreement with Okatani (69) who demonstrated that administration of melatonin to pregnant rats increased the activities of SOD in fetal brain tissues.

A significant increase in both neurotransmitter, dopamine and serotonin activity were observed in melatonin iron treatment group as compared with iron group.  Cabrera (70) found that6-hydroxymelatonin (6-OHM), like melatonin, is able to protect the hippocampus against lipid peroxidation and neuronal damage caused by the neurotoxin Fe²⁺.  Furthermore, Melatonin as well as its metabolites are multi-faceted free radical scavengers and are effective against both oxygen and nitrogen-based reactants and might attenuate neural damage resulting from craniocerebral trauma (71, 72). The administration of melatonin ameliorates a hemi-Parkinson condition in rats caused by intranigral application of the catecholaminergic neurotoxin 6-OHDA.

CONCLUSION

In conclusion, MLT is effective in the prevention and amelioration of the toxic effects of iron-overload that can potentially cause oxidative stress in addition to counteracting the deleterious effects of iron overload in both testis and brain.

 

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Table 1: Hematological parameters in control and different treated animal groups

Marker	Control MLT Iron MLT + Iron
RBCS(106/mm3)   WBCS (103/mm3)   Hb  (g/dl)   Platelets (103/mm3)   Hematocrit %	6.7±0.2           7+ 0.1               6.7+0.3                  7.0+0.1   7.9+0.9             6.0+ 0.7               4.5+0.2a 6.1+0.7   12.8+0.4           13.7+0.1            13.2+0.5               14.1+ 0.2   447+32.9         371.3+18.1         353.8+45.6           353.6+27.2   34.1+0.5          35.8+0.4              31.2+1a               35.1+0.3b

Red blood corpuscles (RBCs), white blood cells (WBCs), and hemoglobin (Hb).All values are expressed as mean +SEM of six independent replicates,  ^a Significant (P<0.05) as compared with the control group, ^ъ Significant (P<0.05) as compared with the iron group

 

 

 

 

Table 2: Serum ferritin, transferrin, iron, and zinc concentrations and γ-GT activities in control and different treated animal groups

Marker	Control                MLT              Iron                    MLT+ Iron
Ferritin (ng/ml)   Transferrin(mg/dl)   Iron (μmol/l)      Zinc (μg/dl)     γ-GT(U/L)	0.30+0.09            0.26+ 0.05          0.76+0.1a          0.39+0.1b   398+5.3              387+ 5                348+1.3a          380+7a,b   68.8+2.8             68.3+2                84.14+1a       76.42+ 1.7a,b   43.04+4              35.08+1.1            28.6+0.4a         33.34+1.7a   17.4+0.8             14.4+0.4              25.8+1.6a         20.2+1.1b

 

Gamma-glutamyle transaminase (γ-GT) All values are expressed as mean ± SEM of six independent replicates, ^a Significant (P<0.05) when compared with the control group,

^ъ Significant (P<0.05) when compared with the iron group

 

 

Table 3: Brain and testicular iron (Fe) in control and different treated animal groups

 

Tissue	Control MLT Iron MLT+ Iron
Brain iron(μg/g)   Testicular iron(μg/g)	15.49+0.4        15.41+0.2        20.24+0.4a           17.92+0.2a,b   14.80+ 0.5       14.76+ 0.1       19.4+ 0.5a             15.5+0.3b

All values are expressed as mean +SEM of six independent replicates, ^a Significant (P<0:05) ascompared to control group, ^ъ Significant (P<0:05) as compared to iron group

 

 

 

 

 

Table 4: The activity of SOD, CAT, GSH and TBARS in brain and testis in control and different treated animal groups

Tissue	Control           MLT               Iron                MLT+Iron
Brain	TBARS (nmol/g)  270+2              207+ 5a        374+6a  253+10b   GSH (mg/g)0.27+0.01       0.28+0.04     0.13+0.01a        0.24+ 0.02b   SOD (U/g)                        186+0.8          192+0.8         166+2a              185+3b   CAT (µmolH2O2/sec/g)0.39+0.06       0.43+0.1       0.09+0.02a0.36+0.08b
Testis	TBARS(nmol/g)146+4               142+ 3              274+9a                229+2a,b   GSH(mg/g)  0.3+0.02          0.36+0.03       0.16+0.01a         0.26+ 0.05b   SOD(U/g)                   187+1.4            195+0.5a           183+1.6a           188+0.9b   CAT(µmolH2O2/sec/g) 0.28+0.04     0.31+0.03      0.07+0.02a       0.24+0.02b

 

Thiobarbituric acid-reactive substances (TBARS), malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT).  All values are expressed as mean +SEMof six independent replicates, ^a Significant (P<0:05) when compared with the control group, ^ъ Significant (P<0:05) when compared with the iron group

 

 

 

 

 

 

Table 5:  Brain dopamine, serotonin, and norepinephrine levels in control and different treated animal groups

Neurotransmitter	Control           MLT                 Iron                 MLT+Iron
Dopamine (µg/g)   Serotonin (µg/g)   Norepinephrine  (μg/g)	8.23+0.4        9.61+ 0.8a5.89+0.3a7.1+0.2a,b   3.9+0.3         3.8+0.08          2.8+0.1a            3.5+ 0.1b   16.5+0.9       16.48+0.13     14.99+0.02a15.22+0.04a

All values are expressed as mean ± SEM of six independent replicates, ^aSignificant (P<0.05) when compared with the control group, ^ъ Significant (P<0.05) when compared with the iron group