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Keywords:

  • lipid peroxidation;
  • liposomes;
  • melatonin;
  • myeloperoxidase;
  • NO synthase;
  • xanthine oxidase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

Abstract: We have investigated the action of melatonin against lipid peroxidation in membranes including brain homogenates (BH), brain and liver microsomes (MIC), and phosphatidylcholine (PC) liposomes, as well as its effect on the activity of pro-oxidant enzymes such as constitutive neuronal nitric oxide synthase (cnNOS), xanthine oxidase (XO) and myeloperoxidase (MPO). The liposomes were reconstituted by a dialysis method, lipid peroxidation was monitored using the thiobarbituric reactive substances (TBARS) method and enzyme activities were measured spectrophotometrically. The ascorbyl and hydroxyl free radicals were generated by the reaction of ascorbic acid + FeSO4 and H2O2 + FeCl2, respectively, and peroxynitrite using a mixture of NaNO2 in an alkaline medium. Melatonin protected against lipid peroxidation induced by distinct reactive oxygen species (ROS) in all membranes tested although with different potency, in the following order BH < MIC < PC. The K0.5 for enzyme inhibition by melatonin was determined for nNOS (2.0 ± 0.1 mm), for XO (0.8 ± 0.1 mm) and for MPO (0.063 ± 0.003 mm), the latter one with high affinity. Melatonin showed a weak effect as a nitrogen monoxide (NO) scavenger in the presence of sodium nitroprusside (NO donor) and low reactivity with 1,1–diphenyl-2-picryl hydrazyl (DPPH). These results demonstrate the antioxidant action of melatonin, principally that related to the activity of pro-oxidant enzymes such as XO and MPO.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

Reactive oxygen and nitrogen species are produced in physiological conditions as a result of the normal metabolism of living organisms. However, when an imbalance in homeostasis is caused by various pathologies, the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) may increase greatly, thereby jeopardizing the affected organism. The pathologies related to oxidative stress extend from those that affect the central nervous system, such as Alzheimer's and other degenerative diseases, through to ischemia-reperfusion injury, inflammation, circulatory and other disorders of the cardiovascular system, to effects on the gastrointestinal and immune systems.

Melatonin, a pineal secretory product, whose physiological role has been thoroughly reviewed [1], is considered a potent antioxidant against stress induced by many oxidant generating systems [2, 3]. Melatonin stimulates the physiological enzymatic antioxidant defenses [4, 5], acts as a free radical scavenger and as a metal ion inactivator [6], preserves mitochondrial function and has low toxicity [7]. Indeed, melatonin protects against oxidative stress observed in some neurodegenerative diseases such as Alzheimer's, damage induced by amyloid beta-peptide [8–10] and methyl phenyl tetrapyridine (MPTP)-induced Parkinson's disease [11]. Melatonin also reduces loss of memory and brain damage induced by treatment with d-galactose [12], protects against the damage caused by oxidative stress induced by kainic acid or quinolinic acid in rat brain [13, 14] and limits dopamine auto-oxidation [15]. The same protection was observed in other diseases including diabetes induced by streptozotocin (diabetes model) and cholelithiasis [16]. Furthermore, melatonin protects biomolecules such as DNA from the oxidant ion induced by phosphine [17], lowers hydroxyl radical-induced apoptosis of cultured thymocytes in mice [18] and protects against oxidative hemolysis of red blood cells [19]. In addition, melatonin protects against paraquat-induced damage [20], reduces the levels of malondialdeyde, a product of lipid peroxidation, and nitrite/nitrate in the blood of asphyxiated newborns [21] among many others actions (for reviews see [7, 22]). Besides melatonin has been considered as a hormone, some researchers are also taken into account its antioxidant properties and classifying the molecule as an antioxidant vitamin, as it has non-receptor mediated antioxidant activity, is synthesized by the organism and is obtained from diet, similarly to vitamin D3 [23].

Nitric oxide (NO) is a free radical gaseous molecule that is a mediator of vital physiologic functions. However, when the production of NO by the nitric oxide synthases (NOS) is exacerbated by an oxidative stress or by a pathological condition, its production must be controlled. The reaction of nitrogen monoxide and superoxide anion generates peroxynitrite (ONOO). The overproduction of ONOO contributes to a very important feature of tissue damaging mechanisms during pathological processes, in addition to decreasing the availability of NO produced in physiological conditions. Since ONOO is a highly reactive molecule, it reacts with cellular components such as membrane lipids and proteins, thereby disturbing their function and, consequently, cellular homeostasis [24–26]. The literature shows that melatonin possesses potent antioxidant potential, also against RNS. In this context melatonin inhibits nitric oxide synthase activity [27] and decreases NO-induced lipid peroxidation in brain homogenates (BH) [28]. Melatonin also possesses, although weak vasoconstrictor activity [29], perhaps by acting as a nitrogen monoxide scavenger, and decreases NO-induced lipid peroxidation in rat retinal homogenates [30]. However paradoxically, it was shown that melatonin protects against the free radical-induced impairment of nitrogen monoxide production in the human umbilical artery, probably by its ability to scavenge hydroxyl radicals [31]. In addition, melatonin attenuates neuronal NADPH-d/nitric oxide synthase expression in the hypoglossal nucleus of adult rats following peripheral nerve injury [32].

Myeloperoxidase (MPO) plays a central role in infection and inflammation, where its role is to convert hydrogen peroxide and chloride to hypochlorous acid (HOCl). Although HOCl has an important role in killing microorganisms it has high reactivity and the ability to damage biomolecules by oxidation, both directly and by decomposing them to form chlorine gas (Cl2) [33].

In this study we investigated the in vitro antioxidant potential of melatonin on lipid peroxidation induced by the ascorbyl and hydroxyl radicals and by peroxynitrine in different models of biological membranes including BH, microsomes (MIC) and phosphatidylcholine (PC) liposomes. We report also the reactivity of melatonin with 1,1–diphenyl-2-picryl hydrazyl (DPPH), an inhibitory effect on xanthine oxidase (XO), constitutive brain nitric oxide synthase (cnNOS) and MPO activities, and a weak effect as a nitrogen monoxide scavenger.

Drugs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

Melatonin, PC, tricine, succinic acid, myoglobin and thiobarbituric acid were purchased from Sigma Chemical Company® (St Louis, MO, USA). The other reagents were purchased from Merck® AG (Darmstadt, Germany). Melatonin, freshly prepared, was dissolved in a minimum volume of absolute ethanol and diluted in water depending on the assay.

Animals

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

Wistar male albino rats (160–190 g) were used. They were maintained in the animal facility of the Universidade Federal de Santa Catarina and housed in an air-conditioned room (approximately 24°C) with controlled lighting (lights on from 07:00 to 19:00 hr). All the animals were maintained with pelleted food (Nuvital, Nuvilab CR1, Curitiba, PR, Brazil) and tap water available ad libitum. Fasted animals were deprived of food for at least 16 hr but allowed free access to water. All the animals were maintained in accordance with ethical recommendations of the Brazilian Veterinary Medicine Council and the Brazilian College of Animal Experimentation.

DPPH reactivity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

The radical DPPH is considered a stable radical and its maximal absorption occurs at 517 nm. It is used as a tool to study the free radical scavenging action of molecules, an approach that is independent of metal or enzymatic activity. The assay is based on the incubation of all reagents for 1 hr at 37°C in an ethanolic solution of 200 μm DPPH and the optical density is measured afterwards at 517 nm. The percentage inhibition in the sample was calculated based on a control with 100% DPPH in the absence of the molecule being tested [34].

Brain homogenates

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

Rats were killed by ether inhalation and their brains removed. The brains were then homogenized in a proportion of 1:5 (w/v) in a cold buffer containing 20 mm KH2PO4 pH 7.4, 150 mm NaCl and 0.1% Triton X-100. The sample was then centrifuged and an aliquot of the supernatant was incubated for 20 min at 37°C in a buffer containing 0.1 m Tris-HCl pH 7.4 plus the lipid peroxidation inducers.

Brain MIC preparation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

Microsomes were obtained by differential centrifugation with calcium aggregation, according to Schenkman [35]. The fractions obtained were immediately placed in a freezer at −70°C for the later determination of antioxidant activity. The protein concentration was determined according to Lowry et al. [36].

Liposomes preparation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

Bilayer liposomes were prepared by cholate dialysis as described previously [37]. Briefly, the method consists of the solubilization of the phospholipids (PC) at 50 mg/mL in a buffer containing 10 mm tricine, 20 g/L sodium cholate, 10 g/L deoxicholate at pH 8.0 followed by a dialysis procedure at 30°C for 5 hr.

Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

Lipid peroxidation was induced by the addition of 25 μm FeSO4 and 500 μm ascorbate in a reaction medium containing 2 mg microsomal protein/mL or BH or liposomes (lipids at 2.5 mg/mL), and 0.1 m Tris-HCl, pH 7.4. The samples were incubated for 30 min at 37°C and the extent of lipid peroxidation was determined by the thiobarbituric acid method [38]. The amount of thiobarbituric reactive species (TBARS) was calculated using an extinction coefficient of 1.56 × 105/m/cm.

Lipid peroxidation induced by hydroxyl radical

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

This method was adapted from Gutteridge and Halliwell [39]. Hydroxyl radical in this experiment was produced from different variations of the Fenton reaction that generates the hydroxyl radical from the reaction of hydrogen peroxide and FeCl3 + ascorbate. Lipid peroxidation was induced by the addition of 25 μm FeCl3 and 100 μm ascorbate in a reaction medium containing MIC (protein 1 mg/mL) or liposomes (lipids at 2.5 mg/mL) and 10 mm KH2PO4, pH 7.4. The lipid peroxidation was determined by the TBARS method as described above.

Assay for nitric oxide synthase activity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

The NOS activity was determined by the method based on myoglobin oxidation by NO produced in the reaction medium. The myoglobin oxidation was monitored spectrophotometrically at 405 nm. The NOS activity was calculated using the slope of the curve after 10 min of reaction. The reaction medium contained 50 mm KH2PO4, pH 7.2, 1.2 mm, MgCl2, 0.25 mm CaCl2, 20 μml-arginine, 60 mml-valine, 1.2 mml-citruline, 1 mmdithiothreitol (DTT), 4 μm flavin adenine dinucleotide (FAD), 5 μm flavin mononucleotide (FMN), 10 μm tetrahydrobiopterin (TBH4), 120 μm NADPH and 0.1 nmol mioglobin. A control assay was carried out in the presence of 1 mm ethylene glycol tetraacetic acid (EGTA) to characterize the calcium independent NOS activity. A calibration curve of myoglobin oxidation was obtained where the protein was totally oxidized by potassium ferrocyanide [41].

Assay for nitric oxide

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

Nitric oxide was released by the incubation of 40 mm sodium nitroprusside in a medium containing 20 mm NaH2 PO4, pH 7.2 at room temperature, in the presence or absence of melatonin at different concentrations. NO was measured indirectly through nitrite formation. Nitrite was monitored spectrophotometrically at 540 nm by the Griess reagent (N-1-naphthyl-ethylenediamine, 0.1% w/v and sulfanilamide, 1% w/v in H3PO4 5% v/v) [42]. The results were obtained using a standard curve for NaNO2.

Assay for XO activity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

Xanthine oxidase activity was evaluated spectrophotometrically at 295 nm, through the formation of uric acid derived from xanthine. The reactions were carried out at 25°C, for 10 min, in a medium containing 100 μm xanthine, 0.1 m phosphate buffer, pH 7.8 and 0.04 U/mL XO [43].

Assay for MPO activity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

Lungs of rats were homogenized in an ice-cold 0.1 m phosphate buffer at pH 7.4, containing 0.5% cetyl trimethylammonium bromide as previously described [44] and freeze-thawed three times. The samples were centrifuged at 12,000 g at 4°C for 20 min. The supernatant was assayed in a reaction medium containing 50 mm phosphate buffer, pH 6.0 at 25°C, o-dianisidine-2HCl (0.167 mg/mL) and H2O2 (0.005 %). The enzyme activity was determined by the slope of the absorption curve set at 450 nm. A standard curve of MPO activity was obtained previously with a commercial enzyme batch (Sigma).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

The antioxidant potential of melatonin was first analyzed by observing the reactivity with DPPH, a violet-colored stable radical that absorbs strongly at 517 nm. As the electron becomes paired off in the presence of a free radical scavenger, the absorption decreases and the resulting decoloration is stoichiometrically related to the number of electrons captured. Melatonin showed low reactivity with this radical. At a concentration of 5.5 mm the compound decreases DPPH absorption by only 40% (results not shown). This assay has been proposed in the literature as a first screen for the antioxidant potential of new molecules. In our laboratory we have observed that some molecules do not show reactivity with DPPH while at the same time they are very potent hydroxyl radical scavengers and have great potential against lipid peroxidation. In fact, melatonin prevented lipid peroxidation, although with different potencies, in all lipid membranes examined in this work. Fig. 1 shows the inhibition of lipid peroxidation induced by the ascorbyl radical in BH. In this figure it is also possible to observe that melatonin promotes recovery from the basal lipid oxidation, which is probably caused by molecular oxygen during experimental handling. This observation might explain the results obtained with lipid peroxidation assays where the protection by melatonin decreased TBARS to values under the control.

image

Figure 1. Effect of melatonin on lipid peroxidation induced by ascorbyl radical in brain homogenates. Ascorbyl was generated by the reaction of FeSO4 + ascorbate and lipid peroxidation was measured by the TBARS method. Basal oxidation indicates the amount of oxidation not induced by the radical, in vitro. (○) Basal oxidation; (•) Ascorbyl; (▴) Ascorbyl – basal oxidation. The results are represented by the mean ± S.E.M., n = 3.

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Figure 2 shows the results of lipid peroxidation induced by the ascorbyl and hydroxyl radicals in brain MIC and PC liposomes. The IC50s for hydroxyl radical were 0.43 ± 0.03 mm, and 0.30 ± 0.02 mm, respectively, while those for the ascorbyl radical were 1.10 ± 0.03 mm and 0.40 ± 0.02 mm, respectively. All the IC50s related to lipid peroxidation are summarized in Table 1, including the results with brain homogenate and results using peroxynitrite as the lipoperoxidation inducer. It is interesting to note that melatonin protected against the action of ROS and RNS on three kinds of lipid membrane preparation: brain homogenates, natural membranes with endogenous antioxidant defenses, MIC, in which the major element is endoplasmic reticulum membrane containing phosphatidylcholine (PC), cholesterol, sphingomyelin (SM), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), and PC liposomes in which the phospholipid used was 1,2-diacyl-sn-glycero-3-phosphocholine, a major structural phospholipid in brain, besides other highly polyunsaturated fatty-acid side-chains being important targets of oxidative stress.

image

Figure 2. Comparison of the effect of melatonin on lipid peroxidation induced by ascorbyl, hydroxyl radicals and by peroxynitrite in brain homogenate, brain microsomes and in PC liposomes. Ascorbyl was generated by the reaction of FeSO4 + ascorbate (500 μm); hydroxyl by FeCl3 + ascorbate (100 μm) + H2O2; peroxynitrite was previously synthesized and used at 2.7 mm, the lipid peroxidation was measured by the TBARS method. (▮) Brain microsomes; (□) PC liposomes; (▵) Brain homogenate. The results are represented by the mean ± S.E.M., n = 3.

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Table 1.  Effect of melatonin against lipid peroxidation in different lipid bilayers, induced by ascorbyl and hydroxyl radicals and by peroxynitrite
MembraneIC50 (mm)
AscorbylHydroxylPeroxynitrite
  1. The lipid peroxidation was evaluated by levels of TBARS, as described in Material and methods. The assays were performed in triplicate and the results are represented by the media ± S.E.M., n = 3.

Brain homogenate0.27 ± 0.030.6 ± 0.050.9 ± 0.10
Brain microsomes1.1 ± 0.100.43 ± 0.050.25 ± 0.04
Phosphatidylcholine liposomes0.4 ± 0.060.3 ± 0.040.8 ± 0.09

The major function of XO is to catalyze the oxidation of hypoxanthine and xanthine to uric acid. As a consequence it produces also the superoxide anion radical. Inhibitors of this enzyme activity are seen as antioxidants because they prevent the production of very deleterious molecules. Uric acid is found in the organism as urate and, although it has antioxidant properties [45], its low solubility in water leads to its crystallization out of solution provoking joint inflammation and the painful characteristics of gout. Although melatonin was not able to scavenge directly superoxide radical (Oinline image) (data not shown) it was able to decrease the radical formation through the inhibition of XO activity. However, this reduction occurred with low affinity as the K0.5 obtained was 0.8 ± 0.1 mm (Fig. 3). Inhibition of XO activity added to a peroxynitrite scavenger action [46] furnishes molecules such as melatonin with the potential to be very well adapted also in the pathogenesis of ischemic injury and atherosclerosis, which is characterized by an overproduction of the superoxide anion; indeed, in atherosclerosis it has been confirmed that peroxynitrite is also present.

image

Figure 3. Inhibition of xanthine oxidase activity by melatonin. The enzyme activity was measured by the spectrophotometric detection of uric acid at 295 m, 100% of enzyme activity was 10.5 ± 0.9 mmol uric acid/min. The results are represented by the mean ± S.E.M., n = 4.

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Although melatonin showed a weak direct reaction with nitrogen monoxide (results not shown) in a medium where sodium nitroprusside, an NO donor, releases the molecule in a certain period of time, as described in Material and methods, the pineal hormone indole inhibited cnNOS, although with a low affinity of K0.5 = 2.0 ± 0.1 mm (Fig. 4). This result is in accordance with that obtained by Pozo et al. [27], and partially with Noda et al. [47] as they argue that melatonin is an NO scavenger. The decrease in NO synthesis is a positive event when the organism is in an oxidative stress state because an overproduction of this radical provokes the production of peroxynitrite. According to Escames et al. [28] melatonin was able to prevent lipid peroxidation induced by NO. Taken together, these observations, suggest that melatonin exerts a double protection against the deleterious actions of NO as it inhibits NOS activity and scavenges peroxynitrite.

image

Figure 4. Inhibition of neuronal nitric oxide synthase by melatonin. The enzyme activity was measured by the spectrophotometric detection of myoglobin oxidation at 405 nm, 100% of enzyme activity was 25 ± 5 pmol NO/min/mg protein. The results are represented by the mean ± S.E.M., n = 3.

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Myeloperoxidase is an enzyme present in activated neutrophils and plays a central role in infection and inflammation. The physiological action of this enzyme is to convert hydrogen peroxide and chloride to HOCl, although it is also able to degrade hydrogen peroxide to oxygen and water [33], about 5% of the hydrogen peroxide consumed by the enzyme is used to produce tyrosyl radicals [48, 49]. Melatonin inhibited the enzyme activity strongly, as can be seen in Fig. 5, which shows the kinetics of enzyme inhibition, while the inset of Fig. 5 shows the results re-plotted to obtain the K0.5 which was 0.063 mm. Although this enzyme can be considered as antioxidant because it dismutates ROS as mentioned above, it is also a pro-oxidant enzyme because of the production of HOCl acid. Inhibition of this enzyme in a stress oxidative condition has beneficial effects.

image

Figure 5. Inhibition of myeloperoxidase activity by melatonin. The enzyme activity was measured spectrophotometrically at 450 nm, in the presence of H2O2 and o-dianisidine-HCl, 100% of enzyme activity was 4.0 ± 1.0 U/min/mg protein. The results are represented by the mean ± S.E.M., n = 4.

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Based on the results shown in this work and the data described in the literature we conclude that melatonin has many antioxidant properties; it protects against lipid peroxidation induced in distinct lipid bilayers by different reactive species, acts as a ROS and RNS scavenger, and stimulates a number of endogenous anti-oxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GRd), and catalase (CAT) [6, 50]. Melatonin also inhibits the pro-oxidant enzymes nitric oxide synthase, cyclooxygenase and lipoxygenase [27, 51]. In this work we also observed that melatonin inhibits the neuronal nitric oxide synthase activity, besides XO and MPO activities. In the case of the last two enzymes this is the first demonstration of their direct inhibition.

It is known that the physiological concentration of melatonin is dependent on which body compartment is being considered. Some authors assume that melatonin physiological concentrations are those determined in the blood plasma (picomolar and low nanomolar range). However it has been observed that in bile and in the cerebrospinal fluid of the third ventricle melatonin concentrations are orders of magnitude higher than in the blood ([52] and references therein). It is also known that melatonin is effective against oxidative stress at physiological and pharmacological concentrations [53, 54]. Despite this discussion, in this work the majority of antioxidant indexes for melatonin obtained was at millimolar range and we suggest that this pineal product could be co-administered with the traditional treatments in several pathologies, including AIDS where the oxidative stress induced by nitric oxide and apoptosis were found to be strongly associated [55], or in procedures strongly linked to oxidative stress such as organ reperfusion in the case of transplantation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References

This work was supported by a grant and fellowships from CNPq (Conselho Nacional de Desenvolvimento Tecnológico), and a grant from FUNCITEC (Fundação de Ciência e Tecnologia de Santa Catarina). Adriana Teixeira is a master's degree student in neuroscience. The authors also would like to thank Gareth P. Cuttle for assistance with the English correction of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Drugs
  6. Animals
  7. DPPH reactivity
  8. Brain homogenates
  9. Brain MIC preparation
  10. Liposomes preparation
  11. Lipid peroxidation induced by ascorbyl radical (iron-ascorbate)
  12. Lipid peroxidation induced by hydroxyl radical
  13. Lipid peroxidation induced by peroxynitrite
  14. Assay for nitric oxide synthase activity
  15. Assay for nitric oxide
  16. Assay for XO activity
  17. Assay for MPO activity
  18. Results and discussion
  19. Acknowledgments
  20. References
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