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Oxidation of melatonin by AAPH-derived peroxyl radicals: Evidence of a pro-oxidant effect of melatonin
Biochimica et Biophysica Acta 1790 (2009) 787–792
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g e n
Oxidation of melatonin by AAPH-derived peroxyl radicals: Evidence of a pro-oxidant effect of melatonin Valdecir F. Ximenes ⁎, Adriano S. Pessoa, Camila Z. Padovan, Daniele C. Abrantes, Fabiana Helena F. Gomes, Michele A. Maticoli, Manoel L. de Menezes Departamento de Química, Faculdade de Ciências, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Bauru, SP, Brazil
a r t i c l e
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Article history: Received 1 December 2008 Received in revised form 24 March 2009 Accepted 25 March 2009 Available online 1 April 2009 Keywords: Melatonin N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) Glutathione pro-oxidant activity
a b s t r a c t Background: Melatonin is well-established as a powerful reducing agent of oxidant generated in the cell medium. We aimed to investigate how readily melatonin is oxidized by peroxyl radicals ROO⋅ generated by the thermolysis of 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and the role of glutathione (GSH) during the reaction course. Methods: Chromatographic, mass spectroscopy, and UV–visible spectrometric techniques were used to study the oxidation of melatonin by ROO⋅ or horseradish peroxidase (HRP)/H2O2. Our focus was the characterization of products and the study of features of the reaction. Results: We found that N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and a monohydroxylated derivative of melatonin were the main products of the reaction between melatonin and ROO⋅. Higher pH or saturation of the medium with molecular oxygen increased the yield of AFMK but did not affect the reaction rate. Melatonin increased the depletion of intracellular GSH mediated by AAPH. Using the HRP/H2O2 as the oxidant system, the addition of melatonin promoted the oxidation of GSH to GSSG. Conclusions: These results show, for the first time, that melatonin radical is able to oxidize GSH. General significance: We propose that this new property of melatonin could explain or be related to the recently reported pro-oxidant activities of melatonin. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Melatonin, an endogenous secretory product, is well-established as a powerful reducing agent of oxidant species generated in the cell medium [1]. For hydroxyl radical (HO⋅), a free radical supposed to be responsible for more than 50% of the injuries resulting from deleterious oxidants [2], the reaction rates with melatonin reaches the diffusion-controlled limit [3]. Hypochlorous acid (HOCl), the microbicidal substance generated when neutrophils are stimulated, is efficiently neutralized by melatonin [4]. Indeed, melatonin is also an inhibitor of the enzyme myeloperoxidase (MPO), which catalyzes the biosynthesis of HOCl [5,6]. Melatonin reacts with peroxynitrite (ONOO−), a strong oxidant formed from the reaction ⋅ between superoxide (O−⋅ 2 ) and nitric oxide ( NO), the endotheliumderived relaxing factor [7,8]. In this case, it should be emphasized that melatonin also interacts with the radicals of peroxynitrite decay. Thus, it is difficult to distinguish between direct interaction and interaction with its radical products [7]. The ubiquitous hydrogen peroxide (H2O2), which is constantly produced in the cell medium, is efficiently reduced by melatonin through peroxidases [9,10] and cytochrome C [11] catalyzed reactions. Finally, melatonin also traps non-endogenous free radicals
such as the stable free radicals 2,2′-diphenyl-1-picrylhydrazyl (DPPH) [12], 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS⋅+) [13] and chlorpromazine radical cation (CPZ⋅+) [14]. The 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) is a water-soluble azo compound largely used as a source of peroxyl radicals (ROO⋅) [15]. Concerning the deleterious process induced by AAPH, melatonin acts as an inhibitor of the oxidative damage of DNA and hemolysis of erythrocytes [12], avoids the inactivation of the enzyme alkaline phosphatase [16], inhibits the oxidation of the fluorescence protein beta-phycoerythrin [17] and inhibits the oxidative damage to catalase [18]. These protective effects of melatonin led us to study its oxidation by AAPH-derived ROO⋅. We have investigated how readily melatonin is oxidized by this oxidant. We found that N1acetyl-N2-formyl-5-methoxykynuramine (AFMK) and monohydroxylated derivative of melatonin were the main products of the reaction between melatonin and ROO⋅. Moreover, we have obtained evidence that the melatonyl radical is able to oxidize glutathione (GSH). 2. Materials and methods 2.1. Chemicals
⁎ Corresponding author. Departamento de Química, Faculdade de Ciências, Universidade Estadual Paulista CEP 17033-360, Brazil. Tel.: +55 14 31036088; fax: +55 12 31036099. E-mail address: [email protected] (V.F. Ximenes). 0304-4165/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2009.03.021
Melatonin, horseradish peroxidase (HRP), catalase, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), GSH, glutathione oxidized (GSSG), ophthalaldehyde (OPA), AFMK, N-ethylmaleimide (NEM), metaphosphoric
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acid and AAPH were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Hydrogen peroxide (H2O2) was prepared by diluting a 30% stock solution and calculating its concentration using its absorption at 240 nm (ɛ = 43.6 M− 1 cm− 1). All the reagents used for solutions, buffers and mobile phase were of analytical grade. Melatonin stock solution was prepared by dissolving it in ethyl alcohol.
washed three times with PBS and then re-suspended using the same buffer to a 20% (v/v) cell suspension. 2.5. Determination of intracellular concentration of GSH in erythrocytes
Unless otherwise indicated, melatonin (1 mM) was incubated with 40 mM AAPH in 0.05 M sodium phosphate buffers pH 5.5 or 7.4, or phosphate buffered saline solution pH 7.4 (PBS), at 37 °C. The consumption of melatonin and the formation of products were measured by high performance liquid chromatography (HPLC) (Varian ProStar in line with UV–Vis Detector set at 254 nm and Fluorescence Detector set at 340/460 nm). HPLC analyses were carried out isocratically on a Luna C18 reversed-phase column (250 × 4.6 mm, 5 μm), with 75:25 water/acetonitrile (flow rate 1.0 mL/min) as the mobile phase. The consumption of melatonin and production of AFMK were quantified by a calibration curve constructed by the peak area plotted against the concentration of the pure standards. To measure the effect of the molecular oxygen during the reaction course, nitrogen or oxygen gas was smoothly bubbled during the course of the reaction.
The intracellular GSH was determined by the DTNB assay, as previously described, with small modifications [19,20]. A 20% suspension of erythrocytes in PBS was added to the same volume of 25 mM AAPH solution in PBS. The reaction mixture was gently homogenized (rotary homogenizer) at 37 °C for 2.5 h. At specific intervals, 0.2 mL aliquots were removed, diluted to 1.0 mL with PBS and centrifuged at 1000 ×g for 10 min. Then, 0.7 mL of water was added to the erythrocyte pellets and the lysate was precipitated by the addition of 0.7 mL metaphosphoric acid solution [1.67 g metaphosphoric acid, 0.2 g EDTA (disodium salt), and 30 g NaCl in 100 mL water]. After 5 min, the protein precipitate was isolated from the remaining solution by centrifugation at 2000 ×g for 10 min. The supernatant (0.45 mL) was added to 0.45 mL of 300 mM Na2HPO4 and to 0.1 mL of DTNB solution (20 mg DTNB in 100 mL of 1% citrate solution). The absorbance at 412 nm was read against a blank consisting of 0.45 mL supernatant, 0.45 mL water and 0.1 mL of DTNB. A blank was prepared for each sample. The hemoglobin content (Hb) in the lysate was determined by the cyanohemoglobin method. The GSH values were expressed as μmol/g Hb.
2.3. Identification of melatonin oxidation products by LC/MS
2.6. Oxidation of GSH by AAPH and effect of melatonin
The oxidation products of melatonin were separated as described above. The outlet stream was split and 0.2 mL/min injected into the mass spectrometer (Quattro II micro, Triple Quadrupole, Micromass, Manchester, UK) equipped with an electrospray ionization source. The mass spectrometer was operated at positive and negative ionization scan modes (m/z 200–500). The capillary voltage was set at 3.0 kV and the cone voltage at 30 V in negative mode, or 3.0 kV and 25 V in positive mode. The desolvatation gas temperature was set at 300 °C and nitrogen flow rate at 600 L/h.
A solution of GSH (100 μM) was incubated with AAPH (2.5 mM) at 37 °C in PBS. Aliquots of 450 μL were removed in the indicated time and the remaining GSH was measured using the DTNB method as above. When present, melatonin was added in the beginning of the reaction. A standard curve was constructed to calculate the GSH concentration.
2.2. Oxidation of melatonin by AAPH
2.4. Preparation of erythrocyte suspensions Venous blood (10 mL) with heparin as anticoagulant was obtained from healthy volunteers after obtaining informed consent. The erythrocytes were isolated by centrifugation at 1700 ×g for 10 min,
2.7. Oxidation of GSH by HRP/H2O2/melatonin A solution of GSH (100 μM) was incubated with 50 μM H2O2 and 0.5 μM HRP at 37 °C in the presence or absence of melatonin for 15 min. The reactions were stopped by adding 10 μg/mL catalase. The supernatant (0.45 mL) was added to 0.45 mL of 300 mM Na2HPO4 and to 0.1 mL of DTNB solution (20 mg DTNB in 100 mL of 1% citrate solution). The absorbance at 412 nm was read
Fig. 1. Separation of the oxidation products of melatonin. Melatonin was oxidized by ROO⋅ and its products separated by HPLC. Complete reaction mixture was 1 mM melatonin, 40 mM AAPH, 0.05 M sodium phosphate buffer pH 7.4 at 37 °C. The supernatant was analyzed by HPLC 4 h after the reaction started. The detectors used were UV–Vis (below) and fluorescence (above).
V.F. Ximenes et al. / Biochimica et Biophysica Acta 1790 (2009) 787–792
Fig. 2. The effect of dissolved oxygen or nitrogen on ROO⋅ mediated oxidation of melatonin and production of AFMK. Complete reaction mixture was 1 mM melatonin, 40 mM AAPH, 0.05 M sodium phosphate buffer pH 7.4 at 37 °C. Loss of melatonin and formation of AFMK were measured by HPLC. Oxygen or nitrogen was bubbled during the reaction course.
against a blank consisting of 0.45 mL PBS, 0.45 mL of 300 mM Na2HPO4 and 0.1 mL of DTNB. 2.8. Production of GSSG by HRP/H2O2/melatonin A solution of GSH (100 μM) was incubated with 50 μM H2O2 and 0.5 μM HRP at 37 °C in the presence or absence of melatonin for 15 min. The reactions were stopped by adding 10 μg/mL catalase. The concentration of GSH and its oxidized form GSSG were determined by HPLC with fluorimetric detection as previously described [19]: for GSH determination, 50 μL of the reaction mixture obtained after oxidation of GSH in the presence or absence of melatonin was added to 1.0 mL of 0.1% EDTA in 0.1 M sodium hydrogenphosphate, pH 8.0. To 20 μL portion of this mixture, 300 μL of 0.1% EDTA in 0.1 M sodium hydrogenphosphate and 20 μL of 0.1% OPA in methanol, was added. The well-capped tubes were incubated at 25 °C for 15 min in dark and 20 μL injected into the HPLC system. For GSSG determination, a 200 μL aliquot of the reaction mixture was incubated at 25 °C with 200 μL of 40 mM NEM, for 25 min, in the dark, to interact with the GSH present in the sample. To this mixture, 750 μL of 0.1 M NaOH was added. A 20 μL portion of this mixture was taken for measurement of GSSG, using the procedure outlined above for GSH assay, except that 0.1 M NaOH was employed as the diluent rather than 0.1% EDTA in 0.1 M sodium hydrogenphosphate [21]. The HPLC method for the determination of GSH and GSSG is based on derivatization with OPA to form a stable, highly fluorescent derivate
Fig. 3. The pH effect on ROO⋅ mediated oxidation of melatonin and production of AFMK. Complete reaction mixture was 1 mM melatonin, 40 mM AAPH, 0.05 M sodium phosphate buffer pH 7.4 or 5.5 at 37 °C. Loss of melatonin and formation of AFMK were measured by HPLC.
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Fig. 4. The effects of melatonin on ROO⋅ induced intracellular GSH depletion in erythrocytes. Erythrocyte suspension at 20% (v/v) was incubated in PBS at 37 °C with 25 mM AAPH in the absence (control) or presence of 100 μM melatonin. The results are mean and SD of two independent experiments using blood from two individuals and performed in duplicates.
[21,22]. The derivatives of GSH and GSSG were separated by liquid chromatography (Varian ProStar in line with Fluorescence Detector set at 350/420 nm). The analyses were carried out isocratically on a Luna C18 reversed-phase column (250 × 4.6 mm, 5 μm). The mobile phase consisted of 15% methanol in 25 mM sodium hydrogenphosphate (v/v), pH 6.0 (flow rate 0.5 mL/min) [21,22]. 3. Results As melatonin has been used as a substance able to avoid the in vitro oxidative damages triggered by AAPH-generated peroxyl radicals [12,16–18], initially, we sought to measure the reactivity and identify the main products of the reaction between melatonin and AAPH. The chromatogram depicted in Fig. 1 shows that the AFMK and monohydroxylated melatonin were the main products obtained when 1 mM melatonin was incubated with 40 mM AAPH in 0.05 M pH 7.4 phosphate buffer at 37 °C. The identification of AFMK was based on comparison with pure commercial standards and LCMS analysis: MW 264, [M-H]− 263 and [M + Na]+ 287. The peak eluting at 7 min (MW 248, [M-H]− 247 and [M + Na]+ 271 correlates with the monohydroxylated derivative of melatonin which has been identified in several oxidative processes, including the reaction with hydroxyl radicals [23], cytochrome C catalyzed oxidation [11], neutrophil mediated oxidation [6] and hypochlorous acid oxidation [4].
Fig. 5. The effect of melatonin on ROO⋅ mediated oxidation of GSH. Complete reaction mixture (control) was 100 μM GSH, 2.5 mM AAPH, PBS buffer pH 7.4 at 37 °C. GSH level was measured using the DTNB method.
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AAPH decomposes at physiological temperature (37 °C) in aqueous solutions to generate an alkyl radical (R⋅), which in the presence of molecular oxygen is converted to the corresponding ROO⋅ [15]. This radical species is responsible for the oxidation of biomolecules in several in vitro models. The involvement ROO⋅ during the oxidation of melatonin has been reinforced here, since the reaction was partially impaired when the medium was made anaerobic by bubbling nitrogen gas (Fig. 2). The thermolysis of AAPH at 37 °C is a slow reaction. The rate of radical generation is directly proportional to AAPH concentration and the total amount of radical formed can be calculated from the equation 1.36 × 10− 6 [AAPH] × t(s) [15]. Here, using this equation and 0.04 M AAPH, the production of ROO⋅ can be estimated as 196 μM in 1 h. This result correlates very well with the consumption of melatonin, which was 183 ± 36 μM during the same time interval. Hence, the production of ROO⋅ can be conceived as the rate determining step for the AAPH-promoted oxidation of melatonin. The reaction rate was not increased by bubbling molecular oxygen, which is consistent with the thermolysis been the rate controlling step. However, the yield of AFMK was increased, which is consistent with previous results regarding the oxidation of melatonin catalyzed by peroxidase [24] (Figs. 2, 3). The efficiency of production of AFMK has been also correlated with the pH when melatonin is submitted to peroxidase mediated reactions. In general, the yield of AFMK is increased when neutral to alkaline pH is used to oxidize melatonin [25]. Here, the reaction with ROO⋅ also followed this pattern (Fig. 3). The reactivity of melatonin with ROO⋅ was also studied regarding its protective effect against the depletion of intracellular GSH when erythrocytes were incubated with AAPH [26]. Here, we tested the efficacy of melatonin as an inhibitor of this oxidative process. Fig. 4 shows that melatonin was not able to avoid the depletion of intracellular GSH. Considering that melatonin reacted promptly with ROO⋅, its inability to avoid the oxidation of intracellular GSH could be an experimental evidence of a lower reactivity of melatonin with ROO⋅ if compared to the reaction between this oxidant and GSH. To test this hypothesis we measured the effect of melatonin during the ROO⋅ mediated oxidation of GSH. In these experiments, pure GSH (100 μM) was submitted to AAPH-mediated oxidation. The remaining GSH was quantified by using the DTNB method. Fig. 5 shows that, despite its reactivity with ROO⋅, melatonin was not able to inhibit the oxidation of GSH. The same result was obtained by inverting the experiment, that is, by testing the effect of GSH on melatonin oxidation (Fig. 6). In this case, GSH, as expected, inhibited the oxidation of melatonin.
Fig. 6. The effect of GSH on ROO⋅ mediated oxidation of melatonin. Complete reaction mixture (control) was 1 mM melatonin, 40 mM AAPH, 0.05 M sodium phosphate buffer pH 7.4 at 37 °C. Aliquots of the supernatant (20 μL) were injected into the HPLC in the indicated time. The concentrations of melatonin were calculated based on the peak areas and compared with pure standards.
Fig. 7. The effect of melatonin on HRP/H2O2 mediated oxidation of GSH. Complete reaction mixture 100 μM GSH, 50 μM H2O2 and 0.5 μM HRP in PBS at 37 °C (control). The results are mean and SD of three experiments. The concentration of GSH was measured removing aliquots, adding catalase (10 μg/mL) to stop the reaction and the supernatant assayed by the DTNB method. The differences are statistically significant (p b 0.05, One Way Anova).
Another explanation for the inability of melatonin to avoid the depletion of intracellular GSH could be a direct reaction between the melatonin radical, which would be generated by the AAPH-mediated oxidation, and GSH. To test this hypothesis, we studied the effect of melatonin during the horseradish peroxidase (HRP)-catalyzed oxidation of GSH. As HRP is a poor catalyst for the oxidation of GSH by H2O2, the presence of melatonin, a better substrate to HRP, could exacerbate the oxidation of GSH through a melatonyl radical intermediate. The data depicted in Figs. 7 and 8 confirmed our hypothesis, since the oxidation of GSH and production of GSSG was significantly more efficient when melatonin was added in the reaction medium. The use of HRP/H2O2 as an oxidant system was necessary to confirm our hypothesis, since the thermolysis of AAPH is the rate controlling step during the oxidation of melatonin or GSH. As a result, this reaction would not be suitable to find a kinetic evidence of the interaction between melatonin radical and GSH. 4. Discussion Melatonin is an inhibitor of the hemolysis of erythrocytes, a feature that can be directly related to its scavenger action upon ROO⋅ [12]. By following the consumption of melatonin during AAPH thermolysis, we confirmed this chemical property of melatonin. The main products of the reaction were a hydroxylated derivative of melatonin and AFMK. These are the same products obtained when melatonin is oxidized by H2O2 in reactions catalyzed by peroxidases and cytochrome C [6,11]. The higher yield of AFMK, when oxygen was bubbled or when the pH was increased, also followed the same pattern, recently reported, for the oxidation of melatonin catalyzed by peroxidase [24,25]. Collectively, these results suggest that the mechanism of oxidation of melatonin by ROO⋅ could be conceived as an initial proton or electron abstraction of the indole ring, followed by the reaction with water or molecular oxygen. Despite its reactivity with ROO⋅, melatonin was not able to avoid the depletion of intracellular GSH in erythrocytes when these cells were incubated with AAPH. These results are in agreement with experiments where cumene hydroperoxide was used to provoke intracellular GSH depletion [27]. An explanation for these findings could be a lower reactivity of melatonin with ROO⋅, if compared with the reaction between ROO⋅ and GSH. The results here, pointed to this direction, since pure GSH inhibited the oxidation of melatonin by ROO⋅, and as expected, melatonin did not avoid the oxidation of GSH by ROO⋅. This relative low reactivity of melatonin with ROO⋅ is not unexpected. In fact, melatonin is also considered a poor lipoperoxyl radical trapping if compared with alpha-tocopherol [28,29].
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Fig. 8. The oxidation of GSH to GSSG by HRP/H2O2/melatonin. Complete reaction mixture 100 μM GSH, 50 μM H2O2 and 0.5 μM HRP in PBS at 37 °C. The supernatant was derivatized and subjected to GSH (above) and GSSG (below) analysis. (i) GSH only, (ii) complete reaction mixture and (iii) complete reaction mixture plus melatonin (100 μM).
The presence of melatonin, during the oxidation of intracellular GSH by AAPH, was not only unsuccessful in avoiding its depletion, but also exacerbated its oxidation as demonstrated in Fig. 5. For us, this was evidence that the effect of melatonin could also be explained by considering a reaction between melatonyl radicals and GSH. Our proposal is that GSH would be cycling melatonin by reducing the melatonyl radical. This GSH pro-oxidant activity has been demonstrated by many phenolic substances that have an adequate reduction potential [30–32]. As suggested by Galati et al., phenols with a reduction potential for the couple PhO⋅/PhOH higher than 850 mV (the reduction potential for the GS−/GS⋅) are able to oxidize GSH [33]. This is the case of melatonin (MLT⋅/MLTH, 950 mV) [34], thus, the reaction between melatonin radical and GSH can be considered a thermodynamically favorable reaction. The equations (1–3) are a proposal for the experimental evidence obtained here. ROS
Acknowledgments
MLTH!MLT•
ð1Þ
MLT• + GSH²MLTH + GS•
ð2Þ
2
GS•!GSSG:
In conclusion, the reactivity of melatonin radical with GSH could explain or be related to the recent findings of Albertini et al., where this indoleamine hormone caused a pro-oxidant effect through the depletion of GSH in macrophage-like U937 cells [36]. A similar effect was reported by Osseni et al. using a human liver cell line (HepG2). In this case, the use of melatonin in the range 1–10 mM also induced intracellular GSH depletion [37]. Finally, we should emphasize that the pro-oxidant activity was observed using a concentration of melatonin which is not found in the physiological medium. However, this concentration, or higher, have been used in biological and toxicological studies. For instance, such levels can be achieved when melatonin is applied topically on the skin [38,39]. Hence, in these cases, this property of melatonin should be taken into account.
ð3Þ
This in vitro pro-oxidant effect of melatonin parallels with the previous work of Tan et al., where the oxidation of oxyhemoglobin was increased by a combination of melatonin and NADH. In that case, it was demonstrated that melatonin recycles NAD radical to NADH, which produces hydrogen peroxide, during oxyhemoglobin oxidation [35].
This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) and from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). References [1] D.X. Tan, L.D. Chen, B. Poeggeler, L.C. Manchester, R.J. Reiter, Melatonin: a potent endogenous hydroxyl radical scavenger, Endocr. J. 1 (1993) 57–60. [2] P. Dziegiel, M. Podhorska-Okolow, M. Zabel, Melatonin: Adjuvant therapy of malignant tumors, Med. Sci. Monit. 14 (2008) 64–70.
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g e n
Oxidation of melatonin by AAPH-derived peroxyl radicals: Evidence of a pro-oxidant effect of melatonin Valdecir F. Ximenes ⁎, Adriano S. Pessoa, Camila Z. Padovan, Daniele C. Abrantes, Fabiana Helena F. Gomes, Michele A. Maticoli, Manoel L. de Menezes Departamento de Química, Faculdade de Ciências, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Bauru, SP, Brazil
a r t i c l e
i n f o
Article history: Received 1 December 2008 Received in revised form 24 March 2009 Accepted 25 March 2009 Available online 1 April 2009 Keywords: Melatonin N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) Glutathione pro-oxidant activity
a b s t r a c t Background: Melatonin is well-established as a powerful reducing agent of oxidant generated in the cell medium. We aimed to investigate how readily melatonin is oxidized by peroxyl radicals ROO⋅ generated by the thermolysis of 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and the role of glutathione (GSH) during the reaction course. Methods: Chromatographic, mass spectroscopy, and UV–visible spectrometric techniques were used to study the oxidation of melatonin by ROO⋅ or horseradish peroxidase (HRP)/H2O2. Our focus was the characterization of products and the study of features of the reaction. Results: We found that N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and a monohydroxylated derivative of melatonin were the main products of the reaction between melatonin and ROO⋅. Higher pH or saturation of the medium with molecular oxygen increased the yield of AFMK but did not affect the reaction rate. Melatonin increased the depletion of intracellular GSH mediated by AAPH. Using the HRP/H2O2 as the oxidant system, the addition of melatonin promoted the oxidation of GSH to GSSG. Conclusions: These results show, for the first time, that melatonin radical is able to oxidize GSH. General significance: We propose that this new property of melatonin could explain or be related to the recently reported pro-oxidant activities of melatonin. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Melatonin, an endogenous secretory product, is well-established as a powerful reducing agent of oxidant species generated in the cell medium [1]. For hydroxyl radical (HO⋅), a free radical supposed to be responsible for more than 50% of the injuries resulting from deleterious oxidants [2], the reaction rates with melatonin reaches the diffusion-controlled limit [3]. Hypochlorous acid (HOCl), the microbicidal substance generated when neutrophils are stimulated, is efficiently neutralized by melatonin [4]. Indeed, melatonin is also an inhibitor of the enzyme myeloperoxidase (MPO), which catalyzes the biosynthesis of HOCl [5,6]. Melatonin reacts with peroxynitrite (ONOO−), a strong oxidant formed from the reaction ⋅ between superoxide (O−⋅ 2 ) and nitric oxide ( NO), the endotheliumderived relaxing factor [7,8]. In this case, it should be emphasized that melatonin also interacts with the radicals of peroxynitrite decay. Thus, it is difficult to distinguish between direct interaction and interaction with its radical products [7]. The ubiquitous hydrogen peroxide (H2O2), which is constantly produced in the cell medium, is efficiently reduced by melatonin through peroxidases [9,10] and cytochrome C [11] catalyzed reactions. Finally, melatonin also traps non-endogenous free radicals
such as the stable free radicals 2,2′-diphenyl-1-picrylhydrazyl (DPPH) [12], 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS⋅+) [13] and chlorpromazine radical cation (CPZ⋅+) [14]. The 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) is a water-soluble azo compound largely used as a source of peroxyl radicals (ROO⋅) [15]. Concerning the deleterious process induced by AAPH, melatonin acts as an inhibitor of the oxidative damage of DNA and hemolysis of erythrocytes [12], avoids the inactivation of the enzyme alkaline phosphatase [16], inhibits the oxidation of the fluorescence protein beta-phycoerythrin [17] and inhibits the oxidative damage to catalase [18]. These protective effects of melatonin led us to study its oxidation by AAPH-derived ROO⋅. We have investigated how readily melatonin is oxidized by this oxidant. We found that N1acetyl-N2-formyl-5-methoxykynuramine (AFMK) and monohydroxylated derivative of melatonin were the main products of the reaction between melatonin and ROO⋅. Moreover, we have obtained evidence that the melatonyl radical is able to oxidize glutathione (GSH). 2. Materials and methods 2.1. Chemicals
⁎ Corresponding author. Departamento de Química, Faculdade de Ciências, Universidade Estadual Paulista CEP 17033-360, Brazil. Tel.: +55 14 31036088; fax: +55 12 31036099. E-mail address: [email protected] (V.F. Ximenes). 0304-4165/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2009.03.021
Melatonin, horseradish peroxidase (HRP), catalase, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), GSH, glutathione oxidized (GSSG), ophthalaldehyde (OPA), AFMK, N-ethylmaleimide (NEM), metaphosphoric
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acid and AAPH were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Hydrogen peroxide (H2O2) was prepared by diluting a 30% stock solution and calculating its concentration using its absorption at 240 nm (ɛ = 43.6 M− 1 cm− 1). All the reagents used for solutions, buffers and mobile phase were of analytical grade. Melatonin stock solution was prepared by dissolving it in ethyl alcohol.
washed three times with PBS and then re-suspended using the same buffer to a 20% (v/v) cell suspension. 2.5. Determination of intracellular concentration of GSH in erythrocytes
Unless otherwise indicated, melatonin (1 mM) was incubated with 40 mM AAPH in 0.05 M sodium phosphate buffers pH 5.5 or 7.4, or phosphate buffered saline solution pH 7.4 (PBS), at 37 °C. The consumption of melatonin and the formation of products were measured by high performance liquid chromatography (HPLC) (Varian ProStar in line with UV–Vis Detector set at 254 nm and Fluorescence Detector set at 340/460 nm). HPLC analyses were carried out isocratically on a Luna C18 reversed-phase column (250 × 4.6 mm, 5 μm), with 75:25 water/acetonitrile (flow rate 1.0 mL/min) as the mobile phase. The consumption of melatonin and production of AFMK were quantified by a calibration curve constructed by the peak area plotted against the concentration of the pure standards. To measure the effect of the molecular oxygen during the reaction course, nitrogen or oxygen gas was smoothly bubbled during the course of the reaction.
The intracellular GSH was determined by the DTNB assay, as previously described, with small modifications [19,20]. A 20% suspension of erythrocytes in PBS was added to the same volume of 25 mM AAPH solution in PBS. The reaction mixture was gently homogenized (rotary homogenizer) at 37 °C for 2.5 h. At specific intervals, 0.2 mL aliquots were removed, diluted to 1.0 mL with PBS and centrifuged at 1000 ×g for 10 min. Then, 0.7 mL of water was added to the erythrocyte pellets and the lysate was precipitated by the addition of 0.7 mL metaphosphoric acid solution [1.67 g metaphosphoric acid, 0.2 g EDTA (disodium salt), and 30 g NaCl in 100 mL water]. After 5 min, the protein precipitate was isolated from the remaining solution by centrifugation at 2000 ×g for 10 min. The supernatant (0.45 mL) was added to 0.45 mL of 300 mM Na2HPO4 and to 0.1 mL of DTNB solution (20 mg DTNB in 100 mL of 1% citrate solution). The absorbance at 412 nm was read against a blank consisting of 0.45 mL supernatant, 0.45 mL water and 0.1 mL of DTNB. A blank was prepared for each sample. The hemoglobin content (Hb) in the lysate was determined by the cyanohemoglobin method. The GSH values were expressed as μmol/g Hb.
2.3. Identification of melatonin oxidation products by LC/MS
2.6. Oxidation of GSH by AAPH and effect of melatonin
The oxidation products of melatonin were separated as described above. The outlet stream was split and 0.2 mL/min injected into the mass spectrometer (Quattro II micro, Triple Quadrupole, Micromass, Manchester, UK) equipped with an electrospray ionization source. The mass spectrometer was operated at positive and negative ionization scan modes (m/z 200–500). The capillary voltage was set at 3.0 kV and the cone voltage at 30 V in negative mode, or 3.0 kV and 25 V in positive mode. The desolvatation gas temperature was set at 300 °C and nitrogen flow rate at 600 L/h.
A solution of GSH (100 μM) was incubated with AAPH (2.5 mM) at 37 °C in PBS. Aliquots of 450 μL were removed in the indicated time and the remaining GSH was measured using the DTNB method as above. When present, melatonin was added in the beginning of the reaction. A standard curve was constructed to calculate the GSH concentration.
2.2. Oxidation of melatonin by AAPH
2.4. Preparation of erythrocyte suspensions Venous blood (10 mL) with heparin as anticoagulant was obtained from healthy volunteers after obtaining informed consent. The erythrocytes were isolated by centrifugation at 1700 ×g for 10 min,
2.7. Oxidation of GSH by HRP/H2O2/melatonin A solution of GSH (100 μM) was incubated with 50 μM H2O2 and 0.5 μM HRP at 37 °C in the presence or absence of melatonin for 15 min. The reactions were stopped by adding 10 μg/mL catalase. The supernatant (0.45 mL) was added to 0.45 mL of 300 mM Na2HPO4 and to 0.1 mL of DTNB solution (20 mg DTNB in 100 mL of 1% citrate solution). The absorbance at 412 nm was read
Fig. 1. Separation of the oxidation products of melatonin. Melatonin was oxidized by ROO⋅ and its products separated by HPLC. Complete reaction mixture was 1 mM melatonin, 40 mM AAPH, 0.05 M sodium phosphate buffer pH 7.4 at 37 °C. The supernatant was analyzed by HPLC 4 h after the reaction started. The detectors used were UV–Vis (below) and fluorescence (above).
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Fig. 2. The effect of dissolved oxygen or nitrogen on ROO⋅ mediated oxidation of melatonin and production of AFMK. Complete reaction mixture was 1 mM melatonin, 40 mM AAPH, 0.05 M sodium phosphate buffer pH 7.4 at 37 °C. Loss of melatonin and formation of AFMK were measured by HPLC. Oxygen or nitrogen was bubbled during the reaction course.
against a blank consisting of 0.45 mL PBS, 0.45 mL of 300 mM Na2HPO4 and 0.1 mL of DTNB. 2.8. Production of GSSG by HRP/H2O2/melatonin A solution of GSH (100 μM) was incubated with 50 μM H2O2 and 0.5 μM HRP at 37 °C in the presence or absence of melatonin for 15 min. The reactions were stopped by adding 10 μg/mL catalase. The concentration of GSH and its oxidized form GSSG were determined by HPLC with fluorimetric detection as previously described [19]: for GSH determination, 50 μL of the reaction mixture obtained after oxidation of GSH in the presence or absence of melatonin was added to 1.0 mL of 0.1% EDTA in 0.1 M sodium hydrogenphosphate, pH 8.0. To 20 μL portion of this mixture, 300 μL of 0.1% EDTA in 0.1 M sodium hydrogenphosphate and 20 μL of 0.1% OPA in methanol, was added. The well-capped tubes were incubated at 25 °C for 15 min in dark and 20 μL injected into the HPLC system. For GSSG determination, a 200 μL aliquot of the reaction mixture was incubated at 25 °C with 200 μL of 40 mM NEM, for 25 min, in the dark, to interact with the GSH present in the sample. To this mixture, 750 μL of 0.1 M NaOH was added. A 20 μL portion of this mixture was taken for measurement of GSSG, using the procedure outlined above for GSH assay, except that 0.1 M NaOH was employed as the diluent rather than 0.1% EDTA in 0.1 M sodium hydrogenphosphate [21]. The HPLC method for the determination of GSH and GSSG is based on derivatization with OPA to form a stable, highly fluorescent derivate
Fig. 3. The pH effect on ROO⋅ mediated oxidation of melatonin and production of AFMK. Complete reaction mixture was 1 mM melatonin, 40 mM AAPH, 0.05 M sodium phosphate buffer pH 7.4 or 5.5 at 37 °C. Loss of melatonin and formation of AFMK were measured by HPLC.
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Fig. 4. The effects of melatonin on ROO⋅ induced intracellular GSH depletion in erythrocytes. Erythrocyte suspension at 20% (v/v) was incubated in PBS at 37 °C with 25 mM AAPH in the absence (control) or presence of 100 μM melatonin. The results are mean and SD of two independent experiments using blood from two individuals and performed in duplicates.
[21,22]. The derivatives of GSH and GSSG were separated by liquid chromatography (Varian ProStar in line with Fluorescence Detector set at 350/420 nm). The analyses were carried out isocratically on a Luna C18 reversed-phase column (250 × 4.6 mm, 5 μm). The mobile phase consisted of 15% methanol in 25 mM sodium hydrogenphosphate (v/v), pH 6.0 (flow rate 0.5 mL/min) [21,22]. 3. Results As melatonin has been used as a substance able to avoid the in vitro oxidative damages triggered by AAPH-generated peroxyl radicals [12,16–18], initially, we sought to measure the reactivity and identify the main products of the reaction between melatonin and AAPH. The chromatogram depicted in Fig. 1 shows that the AFMK and monohydroxylated melatonin were the main products obtained when 1 mM melatonin was incubated with 40 mM AAPH in 0.05 M pH 7.4 phosphate buffer at 37 °C. The identification of AFMK was based on comparison with pure commercial standards and LCMS analysis: MW 264, [M-H]− 263 and [M + Na]+ 287. The peak eluting at 7 min (MW 248, [M-H]− 247 and [M + Na]+ 271 correlates with the monohydroxylated derivative of melatonin which has been identified in several oxidative processes, including the reaction with hydroxyl radicals [23], cytochrome C catalyzed oxidation [11], neutrophil mediated oxidation [6] and hypochlorous acid oxidation [4].
Fig. 5. The effect of melatonin on ROO⋅ mediated oxidation of GSH. Complete reaction mixture (control) was 100 μM GSH, 2.5 mM AAPH, PBS buffer pH 7.4 at 37 °C. GSH level was measured using the DTNB method.
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AAPH decomposes at physiological temperature (37 °C) in aqueous solutions to generate an alkyl radical (R⋅), which in the presence of molecular oxygen is converted to the corresponding ROO⋅ [15]. This radical species is responsible for the oxidation of biomolecules in several in vitro models. The involvement ROO⋅ during the oxidation of melatonin has been reinforced here, since the reaction was partially impaired when the medium was made anaerobic by bubbling nitrogen gas (Fig. 2). The thermolysis of AAPH at 37 °C is a slow reaction. The rate of radical generation is directly proportional to AAPH concentration and the total amount of radical formed can be calculated from the equation 1.36 × 10− 6 [AAPH] × t(s) [15]. Here, using this equation and 0.04 M AAPH, the production of ROO⋅ can be estimated as 196 μM in 1 h. This result correlates very well with the consumption of melatonin, which was 183 ± 36 μM during the same time interval. Hence, the production of ROO⋅ can be conceived as the rate determining step for the AAPH-promoted oxidation of melatonin. The reaction rate was not increased by bubbling molecular oxygen, which is consistent with the thermolysis been the rate controlling step. However, the yield of AFMK was increased, which is consistent with previous results regarding the oxidation of melatonin catalyzed by peroxidase [24] (Figs. 2, 3). The efficiency of production of AFMK has been also correlated with the pH when melatonin is submitted to peroxidase mediated reactions. In general, the yield of AFMK is increased when neutral to alkaline pH is used to oxidize melatonin [25]. Here, the reaction with ROO⋅ also followed this pattern (Fig. 3). The reactivity of melatonin with ROO⋅ was also studied regarding its protective effect against the depletion of intracellular GSH when erythrocytes were incubated with AAPH [26]. Here, we tested the efficacy of melatonin as an inhibitor of this oxidative process. Fig. 4 shows that melatonin was not able to avoid the depletion of intracellular GSH. Considering that melatonin reacted promptly with ROO⋅, its inability to avoid the oxidation of intracellular GSH could be an experimental evidence of a lower reactivity of melatonin with ROO⋅ if compared to the reaction between this oxidant and GSH. To test this hypothesis we measured the effect of melatonin during the ROO⋅ mediated oxidation of GSH. In these experiments, pure GSH (100 μM) was submitted to AAPH-mediated oxidation. The remaining GSH was quantified by using the DTNB method. Fig. 5 shows that, despite its reactivity with ROO⋅, melatonin was not able to inhibit the oxidation of GSH. The same result was obtained by inverting the experiment, that is, by testing the effect of GSH on melatonin oxidation (Fig. 6). In this case, GSH, as expected, inhibited the oxidation of melatonin.
Fig. 6. The effect of GSH on ROO⋅ mediated oxidation of melatonin. Complete reaction mixture (control) was 1 mM melatonin, 40 mM AAPH, 0.05 M sodium phosphate buffer pH 7.4 at 37 °C. Aliquots of the supernatant (20 μL) were injected into the HPLC in the indicated time. The concentrations of melatonin were calculated based on the peak areas and compared with pure standards.
Fig. 7. The effect of melatonin on HRP/H2O2 mediated oxidation of GSH. Complete reaction mixture 100 μM GSH, 50 μM H2O2 and 0.5 μM HRP in PBS at 37 °C (control). The results are mean and SD of three experiments. The concentration of GSH was measured removing aliquots, adding catalase (10 μg/mL) to stop the reaction and the supernatant assayed by the DTNB method. The differences are statistically significant (p b 0.05, One Way Anova).
Another explanation for the inability of melatonin to avoid the depletion of intracellular GSH could be a direct reaction between the melatonin radical, which would be generated by the AAPH-mediated oxidation, and GSH. To test this hypothesis, we studied the effect of melatonin during the horseradish peroxidase (HRP)-catalyzed oxidation of GSH. As HRP is a poor catalyst for the oxidation of GSH by H2O2, the presence of melatonin, a better substrate to HRP, could exacerbate the oxidation of GSH through a melatonyl radical intermediate. The data depicted in Figs. 7 and 8 confirmed our hypothesis, since the oxidation of GSH and production of GSSG was significantly more efficient when melatonin was added in the reaction medium. The use of HRP/H2O2 as an oxidant system was necessary to confirm our hypothesis, since the thermolysis of AAPH is the rate controlling step during the oxidation of melatonin or GSH. As a result, this reaction would not be suitable to find a kinetic evidence of the interaction between melatonin radical and GSH. 4. Discussion Melatonin is an inhibitor of the hemolysis of erythrocytes, a feature that can be directly related to its scavenger action upon ROO⋅ [12]. By following the consumption of melatonin during AAPH thermolysis, we confirmed this chemical property of melatonin. The main products of the reaction were a hydroxylated derivative of melatonin and AFMK. These are the same products obtained when melatonin is oxidized by H2O2 in reactions catalyzed by peroxidases and cytochrome C [6,11]. The higher yield of AFMK, when oxygen was bubbled or when the pH was increased, also followed the same pattern, recently reported, for the oxidation of melatonin catalyzed by peroxidase [24,25]. Collectively, these results suggest that the mechanism of oxidation of melatonin by ROO⋅ could be conceived as an initial proton or electron abstraction of the indole ring, followed by the reaction with water or molecular oxygen. Despite its reactivity with ROO⋅, melatonin was not able to avoid the depletion of intracellular GSH in erythrocytes when these cells were incubated with AAPH. These results are in agreement with experiments where cumene hydroperoxide was used to provoke intracellular GSH depletion [27]. An explanation for these findings could be a lower reactivity of melatonin with ROO⋅, if compared with the reaction between ROO⋅ and GSH. The results here, pointed to this direction, since pure GSH inhibited the oxidation of melatonin by ROO⋅, and as expected, melatonin did not avoid the oxidation of GSH by ROO⋅. This relative low reactivity of melatonin with ROO⋅ is not unexpected. In fact, melatonin is also considered a poor lipoperoxyl radical trapping if compared with alpha-tocopherol [28,29].
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Fig. 8. The oxidation of GSH to GSSG by HRP/H2O2/melatonin. Complete reaction mixture 100 μM GSH, 50 μM H2O2 and 0.5 μM HRP in PBS at 37 °C. The supernatant was derivatized and subjected to GSH (above) and GSSG (below) analysis. (i) GSH only, (ii) complete reaction mixture and (iii) complete reaction mixture plus melatonin (100 μM).
The presence of melatonin, during the oxidation of intracellular GSH by AAPH, was not only unsuccessful in avoiding its depletion, but also exacerbated its oxidation as demonstrated in Fig. 5. For us, this was evidence that the effect of melatonin could also be explained by considering a reaction between melatonyl radicals and GSH. Our proposal is that GSH would be cycling melatonin by reducing the melatonyl radical. This GSH pro-oxidant activity has been demonstrated by many phenolic substances that have an adequate reduction potential [30–32]. As suggested by Galati et al., phenols with a reduction potential for the couple PhO⋅/PhOH higher than 850 mV (the reduction potential for the GS−/GS⋅) are able to oxidize GSH [33]. This is the case of melatonin (MLT⋅/MLTH, 950 mV) [34], thus, the reaction between melatonin radical and GSH can be considered a thermodynamically favorable reaction. The equations (1–3) are a proposal for the experimental evidence obtained here. ROS
Acknowledgments
MLTH!MLT•
ð1Þ
MLT• + GSH²MLTH + GS•
ð2Þ
2
GS•!GSSG:
In conclusion, the reactivity of melatonin radical with GSH could explain or be related to the recent findings of Albertini et al., where this indoleamine hormone caused a pro-oxidant effect through the depletion of GSH in macrophage-like U937 cells [36]. A similar effect was reported by Osseni et al. using a human liver cell line (HepG2). In this case, the use of melatonin in the range 1–10 mM also induced intracellular GSH depletion [37]. Finally, we should emphasize that the pro-oxidant activity was observed using a concentration of melatonin which is not found in the physiological medium. However, this concentration, or higher, have been used in biological and toxicological studies. For instance, such levels can be achieved when melatonin is applied topically on the skin [38,39]. Hence, in these cases, this property of melatonin should be taken into account.
ð3Þ
This in vitro pro-oxidant effect of melatonin parallels with the previous work of Tan et al., where the oxidation of oxyhemoglobin was increased by a combination of melatonin and NADH. In that case, it was demonstrated that melatonin recycles NAD radical to NADH, which produces hydrogen peroxide, during oxyhemoglobin oxidation [35].
This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) and from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). References [1] D.X. Tan, L.D. Chen, B. Poeggeler, L.C. Manchester, R.J. Reiter, Melatonin: a potent endogenous hydroxyl radical scavenger, Endocr. J. 1 (1993) 57–60. [2] P. Dziegiel, M. Podhorska-Okolow, M. Zabel, Melatonin: Adjuvant therapy of malignant tumors, Med. Sci. Monit. 14 (2008) 64–70.
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