Myeloperoxidase-Catalyzed Oxidation of Melatonin by Activated Neutrophils

Biochemical and Biophysical Research Communications 279, 657– 662 (2000) doi:10.1006/bbrc.2000.3993, available online at http://www.idealibrary.com on

Myeloperoxidase-Catalyzed Oxidation of Melatonin by Activated Neutrophils Sueli de Oliveira Silva,* Valdecir F. Ximenes,† Luiz Henrique Catalani,† and Ana Campa* ,1 *Departamento de Ana´lises Clı´nicas e Toxicolo´gicas, Faculdade de Cieˆncias Farmaceˆuticas, and †Departamento de Quı´mica Fundamental, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo CEP 05508-900, Brazil

Received October 19, 2000

In the presence of hydrogen peroxide, horseradish peroxidase (HRP) catalyzes the production of N 1acetyl-N 2-formyl-5-methoxykynuramine from melatonin. This reaction consumes oxygen and exhibits chemiluminescence in the 440 –540 nm region. The excited cleavage product derived from the thermolysis of an intermediate dioxetane is suggested to be the emitting species. Chemiluminescence and the indole ring cleavage product were also observed when HRP/ H 2O 2 was replaced by phorbol myristate acetate or opsonized zymosan-activated neutrophils. Azide, a myeloperoxidase inhibitor, strongly suppressed melatonin oxidation. Superoxide dismutase has a strong inhibitory effect on light emission but catalase and uric acid are without effect on the emission. The oxidation of melatonin by activated neutrophils may be relevant to the in vivo functions of myeloperoxidase and melatonin. The possible biological implication of melatonin oxidation by neutrophils, especially in inflammatory conditions, is discussed. © 2000 Academic Press Key Words: chemiluminescence; melatonin; myeloperoxidase; neutrophils; peroxidase; horseradish peroxidase; reactive oxygen species; superoxide anion; indole; activated neutrophils; inflammation.

Neutrophils play an essential role in the inflammatory response. These cells are readily recruited to sites of inflammation, where they recognize and phagocyte pathogens. The killing activity of neutrophils is supported by the multienzymatic complex NADPH oxidase, that produces superoxide anion. Hydrogen peroxide is formed by dismutation of superoxide anion (1). The parallel process of enzyme degranulation completes the microbicide battery of these cells. Myeloperoxidase catalyses the formation of HOCl via a cycle Abbreviations used: HRP, horseradish peroxidase; MPO, myeloperoxidase; PMA, phorbol myristate acetate; OZ opsonized zymosan; ROS, reactive oxygen species; SOD superoxide dismutase. 1 To whom correspondence should be addressed. Fax: (55-11) 38132197. E-mail: [email protected].

involving the oxidation of MPO by H 2O 2 to compound I, the active form of the enzyme, followed by the reaction of compound I with Cl ⫺ (2). There is increasing evidence that, although the formation of HOCl is important for the microbicide activity of neutrophils, MPO also catalyses other reactions (3). Currently, it is recognized that MPO catalyzes the oxidation of common substrates through a native enzyme-compound I/compound II cycle (2) or hydroxylation involving MPO compound III (4). Furthermore, MPO appears to play a role in protein nitration, lipid peroxidation (5) and in immunomodulation (6). We recently completed a study of the chemiluminescent oxidation of a number of indole compounds by HRP at relatively high concentrations of H 2O 2 in alkaline medium (7). The intensity of chemiluminescence was found to depend basically on the indole substitution at positions 2 and 3; for 2-methyl- and 2,5-dimethylindole, a very high intensity of light emission was found. Scheme 1 shows the formation and cleavage of a dioxetane intermediate, as proposed elsewhere for indole ring cleavage reactions (8 –11). Melatonin is one of the biologically relevant indole compounds. This pineal hormone controls several essential physiological functions associated with circadian and seasonal rhythms (12). Increasing evidence also points to its participation in the immune response, with effects on leukocyte activation, cytokine and nitric oxide production and modulation of vascular permeability having been reported (13–16). The mechanism of action of this pineal hormone is completely unknown. Since melatonin is a good electron donor and reacts efficiently with hydroxyl radical (17) and peroxynitrite (18, 19), one suggestion for its biological role is its strong free radical scavenging properties. The formation of the melatonin cation radical and further reaction of this radical to form the corresponding indole ring cleavage product have been proposed in studies on melatonin oxidation (17–19). Here we focus on the oxidation of melatonin by HRP/ H 2O 2, the resultant chemiluminescence and the possi-

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SCHEME 1. Proposed route for indole ring cleavage products.

bility of oxidation of melatonin by MPO. Besides a high MPO content, activated neutrophils also produce large amounts of reactive oxygen species; these cells are thus equipped with the chemical machinery necessary for the oxidation of biological substrates. Hence, we evaluated whether the oxidation of melatonin could occur during neutrophil activation. Finally, the possible biological implications of melatonin oxidation by activated neutrophils are discussed.

Biological Oxygen Monitor. The reaction mixtures were lyophilized, resuspended in tetrahydrofuran and analyzed by high performance liquid chromatography using a SHIMADZU LC-10A system coupled to SPD-10A UV-Vis and RF535 fluorescence detectors, respectively. The chromatographic analyses were carried out on a Zorbaz 4.6 mm ⫻ 25 cm Silica column (P.N. 880952-701) using 1:1 acetonitrile: THF as the mobile phase in the isocratic mode at a flow rate of 0.4 mL/min. The absorbance was measured at 254 nm and fluorescence emission at 450 nm with 340 nm excitation. The mass spectra were obtained employing a Hewlett–Packard 5988 quadrupole mass spectrometer attached to a 5890 gas chromatograph using a HP1 column (12 m ⫻ 0.25 mm ⫻ 0.25 ␮m).

MATERIALS AND METHODS

Experimental conditions. Unless otherwise stated, the standard reaction mixture for the HRP catalyzed reaction was 1 ␮mol/L HRP, 0.5 mmol/L H 2O 2, 1 mmol/L melatonin in phosphate buffer 17.5 mmol/L, pH 7.4. Final volumes were 0.3 mL for the luminometer and 3 mL for the photon-counter and oxygen monitor. All measurements were made at 37°C. For the oxidation of melatonin by stimulated neutrophils, the standard reaction mixture was 1 mmol/L melatonin and 1 ⫻ 10 6 neutrophils/assay. Phorbol myristate acetate (16 ng/ assay) or opsonized zymosan (1 ⫻ 10 7 particles/assay) were used as stimuli.

Catalase (EC 1.11.1.6; from bovine liver), superoxide dismutase (SOD; EC 1.15.1.1; from bovine erythrocytes), horseradish peroxidase (HRP; EC 1.11.1.7; type VI), myeloperoxidase (MPO, EC 1.11.1.7, from human leukocytes), melatonin, Histopaque, dextran, DMSO, PMA, zymosan, glucose and uric acid were from Sigma Chemical Co. (St. Louis, MO). H 2O 2, sodium azide, Na 2HPO 4, KH 2PO 4, KCl, CaCl 2, MgCl 2, ethanol, acetonitrile and tetrahydrofuran were from Merck (Darmstadt, Germany and Rio de Janeiro). Stock solutions of PMA were prepared in DMSO (100 ␮mol/mL) and stored at ⫺20°C. Zymosan solutions (10 mg/mL) were prepared by dissolution in saline-phosphate buffer 10 mmol/L, pH 7.4, followed by sonication, and centrifuged and stored at ⫺20°C. Aliquots of the zymosan stock solution were opsonized by incubation for 60 min at 37°C with human fresh serum. Opsonized zymosan was maintained frozen at ⫺10°C and used within one week. Melatonin stock solutions (10 mM) were prepared in 30% ethanol and used for at most one week. Dextran was prepared in 0.9% NaCl. All other stock solutions were prepared as aqueous solutions. Neutrophils were isolated from the blood of healthy volunteer donors essentially by the method of Bo¨yum (20). Blood (10 ml), collected into heparin (50 IU/ml blood), was diluted with an equal volume of saline–phosphate buffer 10 mmol/L, pH 7.4, and carefully layered on to 10 ml of a commercial gradient of Ficoll–Hypaque (Histopaque; d ⫽ 1.077). The tube was centrifuged at 2500 rpm at room temperature for 20 min. The supernatant, rich in mononuclear cells, was discarded and 20 mL of 5% dextran added to the pellet. The tube was homogenized and maintained for 45 min at room temperature to allow erythrocyte sedimentation. The resulting supernatant, rich in granulocytes, was recovered, washed with salinephosphate buffer and the pellet submitted to hypotonic treatment with 10 mL of distilled water to promote lysis of contaminated erythrocytes. After one minute, the isotonicity was restored by the addition of 5 mL of 2.7% NaCl and phosphate buffer. The sample was centrifuged at 2500 rpm at room temperature for 5 min, the supernatant discarded and 1 mL of saline-phosphate buffer added to the pellet. The density of cells was counted in a Neubauer chamber. To follow the time course of chemiluminescence, we used either an EG&G Berthold LB96V Microplate Luminometer or a “photoncounting” system composed of an EG&G PAR 1121A Amplifierdiscriminator and a Thorn EMI 9658AM photomultiplier cooled to ⫺12°C by a Thorn EMI FACT-50 MKIII thermoelectric cooler. The last system offers the possibility of using cut-off filters in front of the PMT tube. Oxygen consumption was monitored with a YSI-5300

RESULTS Attention was focused specifically on the oxidation of melatonin by two peroxidases; HRP and MPO. The action of MPO was investigated in both a cell-free reaction system and in the presence of neutrophils. Oxidation of melatonin by HRP/H 2O 2. Melatonin is oxidized by HRP/H 2O 2, forming one main product isolated by HPLC and identified by CG-MS as N 1-acetylN 2-formyl-5-methoxykynuramine [MS (m/z): 264(7), 176(69), 160(100), 150(24), 117(13)]. Under the conditions used, neither HRP nor H 2O 2 alone caused any modification of melatonin. Figure 1 shows a typical chromatogram observed for the melatonin/HRP/H 2O 2 reaction. After 60 min of reaction, melatonin was completely consumed and a new peak with a retention time higher than melatonin and showing fluorescence appeared. The excitation and emission spectra of this product are shown in Fig. 2. Chemiluminescent light emission during the reaction of melatonin/HRP/H 2O 2 (Fig. 3) lasts several minutes and is absolutely dependent on the addition of all reactants. None of the controls, i.e., melatonin/HRP, melatonin/H 2O 2 or HRP/H 2O 2, triggered light emission under the conditions assayed. Although the intensity of light emission observed during melatonin oxidation by HRP/H 2O 2 is insufficient to allow unambiguous assign-

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FIG. 1. HPLC profiles of the melatonin (1 mmol/L)/HRP (1 ␮mol/ L)/H 2O 2 (0.5 mmol/L) system after 60 min of reaction in phosphate buffer 17.5 mmol/L, pH 7.4, 37° C. The signals corresponds to absorbance at 254 nm (left) and fluorescence at 450 nm (␭ exc 340 nm) (right). Melatonin (M) is completely consumed and a new peak, assigned as K, that has a longer retention time than melatonin (M) and is fluorescent appeared. This chromatogram is representative of seven experiments.

FIG. 3. Light emission of the melatonin/HRP/H 2O 2 system (■). The reaction conditions are the same as those in Fig. 1. The control HRP/H 2O 2 (F) is also shown The controls melatonin/HRP and melatonin/H 2O 2 do not induce any observable chemiluminescence. The addition of SOD (10 ␮g/reaction) causes a partial decrease in light emission (Œ). This is one representative experiment out of three.

ment of the emissive species, excited N 1-acetyl-N 2formyl-5-methoxykynuramine formed by the cleavage of a dioxetane intermediate is a likely candidate. The use of cut-off filters indicated that the spectral region of light emission is 440 –540 nm (Table 1). Light intensity and kinetics are strongly dependent on H 2O 2 concentration in the range of 0.016 to 5 mmol/L (Fig. 4). At lower H 2O 2 concentrations, the light emission is brief and more intense, while at higher concentrations the emission persists longer and the maximum intensity is lower. In both cases, the chemiluminescence kinetics are marked by an abrupt drop. At the lower concentrations of H 2O 2 (0.016 – 0.1mM), light emission could be partially restored by readdition of H 2O 2. At H 2O 2 concentrations higher than 1mmol/L, the light emission decreased and could be partially restored by the subsequent addition of HRP, but not H 2O 2 or melatonin. As expected, this indicates that, at high H 2O 2 concentration, inactivation of HRP takes place. A H 2O 2-dependent effect on chemiluminescence was also observed in a set of exper-

iments carried out with commercial purified MPO (Fig. 5). A partial effect of SOD on the light emission of the melatonin/HRP/H 2O 2 system is observed. SOD (5 ␮g/ assay) only partially inhibits the light emission (36 ⫾ 9%) (Fig. 3). Increasing SOD 10-fold does not result in greater inhibition. Although the melatonin/HRP/H 2O 2 reaction consumes oxygen, the consumption cannot be easily measured since, at the concentrations of H 2O 2 assayed, the HRP/H 2O 2 reaction itself produces oxygen. Considering that air-equilibrated water contains 0.2 mmol/L of O 2, we have estimated the approximate amount of oxygen consumed from the difference between the HRP/ H 2O 2 and melatonin/HRP/H 2O 2 reactions (Table 2). Oxidation of melatonin by activated-neutrophils. Neutrophils activated with PMA or opsonized zymosan generate a large amount of reactive oxygen species that can be used for MPO-catalyzed melatonin oxidation. PMA or OZ-activated neutrophils, but not resting cells, oxidized melatonin, forming a product with the same HPLC retention time and spectral characteristics as that produced by the oxidation of melatonin by HRP/ H 2O 2. Melatonin is only partially consumed (22% after

TABLE 1

Effect of Cutoff Filters on the Light Emission of the Melatonin (1 mmol/L)/HRP (1 ␮mol/L)/H 2O 2 (0.5 mmol/L) Reaction

FIG. 2. Spectral features of the product isolated from the melatonin/HRP/H 2O 2 reaction labeled as K in Fig. 1 and identified by CG-MS as N 1-acetyl-N 2-formyl-5-methoxykynuramine. The excitation spectrum (A) was recorded with a fixed emission wavelength of 440 nm and the emission spectra (B) with a fixed excitation wavelength of 340 nm. 659

Cutoff filters (nm)

Emission (%)

370 400 440 480 540 600

100 100 84 57 3 1

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Oxygen Uptake by the Melatonin (1 mmol/L)/HRP (1 ␮mol/L)/H 2O 2 (0.5 mmol/L) System

FIG. 4. Effect of H 2O 2 concentration on the light emission of the melatonin/HRP/H 2O 2 system. The conditions are the same as in Fig. 1 except for the variation of the H 2O 2 concentration: 5.0 (■); 1.0 (F); 0.5 (Œ); and 0.3 mmol/L () and, in the inset, 0.1 (■); 0.05 (F); 0.033 (Œ) and 0.016 mmol/L (}).

one hour of reaction). The addition of azide, an MPO inhibitor, completely eliminates the formation of the indole cleavage product. The light emission profile of the activated neutrophil/melatonin reaction depends on the stimulus utilized (Fig. 6). Thus, when neutrophils were stimulated with PMA, the light emission was shorter than when opsonized zymosan was used. Indeed, these profiles bear a resemblance to the oxidative burst monitored by luminol-enhanced chemiluminescence (21). These data indicate that the reactive oxygen species formed during the oxidative burst define the rate of melatonin oxidation. For both stimuli, the light emission depends on cell density. Both maximal intensity and total integrated intensity increase in the range of 0.25 to 1.0 ⫻ 10 6 cells/assay (data not shown) and the reaction is markedly inhibited by the addition of SOD and azide, but unaffected by catalase or uric acid (Table 3). DISCUSSION The oxidation of melatonin by HRP/H 2O 2 produces N 1-acetyl-N 2-formyl-5-methoxykynuramine. During

FIG. 5. Effect of H 2O 2 concentration on the light emission of the melatonin/MPO/H 2O 2 system. The conditions are the same as in Fig. 1 but with 0.3 units of MPO/assay and H 2O 2 concentration of: 5.0 (■); 1.66 (Œ); 0.5 (F); and 0.16 mmol/L (), and in the inset, 0.05 (■); 0.033 (Œ); and 0.016 mmol/L (F).

Reaction time (minutes)

Oxygen consumed (mmol/L)

0 5 10 20 30

0 0.06 0.09 0.13 0.16

this reaction, oxygen is consumed and chemiluminescence is observed. The route for N 1-acetyl-N 2-formyl-5methoxykynuramine formation could involve the melatonyl radical formed by the native enzyme/compound I/compound II cycle. However, the eventual participation of compound III cannot be excluded. Several routes might form compound III from HRP or MPO (3, 22, 23). The similarity between compound III and the catalytically active form of the enzyme indole 2,3dioxygenase have already been pointed out by Kettle and Winterbourn (4). Indoleamine 2,3-dioxygenase is one of the enzymes responsible for indole catabolism via the formation of a product of indole ring opening (24). Although this work provides no direct evidence as to the nature of the peroxidase cycle involved in melatonin oxidation, the possibility of involvement of both a native/compound I/compound II and a native/ compound III cycle could explain the partial inhibition by SOD observed in the melatonin/HRP/H 2O 2 system (Fig. 3). The fact that, in activated neutrophils, compound III is the predominant form of myeloperoxidase (25) could explain the higher inhibition promoted by SOD (Table 3). The amount of oxygen consumed during melatonin oxidation catalyzed by HRP/H 2 O 2 can only be roughly estimated since, at the hydrogen peroxide concentrations needed to observe N 1 -acetyl-N 2 -

FIG. 6. Light emission during the reaction of melatonin (1mmol/ L)/activated neutrophils (1.0 ⫻ 10 6 cells/assay). Neutrophils were stimulated with PMA (16 ng/assay) (solid curve) or opsonized zymosan (1.0 ⫻ 10 7 particles/assay) (dotted curve). The reactions were carried out in phosphate buffer 17.5 mmol/L, pH 7.4, 37°C. This is a representative example out of 15 experiments with different neutrophil preparations.

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ACKNOWLEDGMENTS

TABLE 3

Effect of Oxygen Radical Scavengers on the Chemiluminescence of Opsonized Zymosan-Activated Neutrophils in the Presence of Melatonin Reaction system

% of the control

n

Complete system ⫺ neutrophils ⫺ opsonized zymosan ⫺ melatonin ⫹ catalase (10 ␮g/assay) ⫹ SOD (10 ␮g/assay) ⫹ azide (1 mmol/L) ⫹ uric acid (1 mmol/L)

100 11 ⫾ 3 3⫾1 10 ⫾ 2 94 ⫾ 1 15 ⫾ 5* 7 ⫾ 2* 99 ⫾ 18

15 3 7 3 11 11 11 9

Note. The data are means ⫾ SD for n experiments. *P ⱕ 0.001.

formyl-5-methoxykynuramine formation and chemiluminescence, a significant amount of oxygen is produced due to the catalase-like activity of HRP. The estimated value of 0.16 mmol/L of oxygen consumed in 30 min (Table 2) might still be low since additional O 2 may be generated by the reaction of the putative intermediate melatonin radical with H 2 O 2 , in a similar fashion to that described for chlorpromazine (26). That MPO participates in the formation of N 1 acetyl-N 2 -formyl-5-methoxykynuramine and chemiluminescence in activated neutrophils is clearly deduced from the strong inhibitory effect of azide (a MPO inhibitor). Although several different reactive oxygen species are formed during the neutrophil oxidative burst, the participation of species other than O 2⫺ seems less probable since catalase and uric acid have no effect (Table 3). The biological relevance of melatonin oxidation by activated neutrophils has to be considered in light of the following data: (i) neutrophils and monocytes possess receptors for melatonin (27–29); (ii) inflammatory cells can synthesize melatonin (30); and (iii) some tissues have high concentrations of melatonin (31). One additional important point to be considered is that, during the inflammatory process, neutrophils are primed by cytokines and other inflammatory factors, resulting in an increment in ROS production by these cells (32). Hence, it is reasonable to suppose that the oxidation of melatonin can occur in vivo, especially under conditions of neutrophil activation. Despite the fact that the electronic excited species formed during melatonin oxidation could not be unambiguously identified, there is no doubt that an excited state is formed. It is thus intriguing to speculate that an excited species might play a role in melatonin action. This is the spirit of Cilento’s proposal that excited states formed in vivo may be able to modulate biologically important responses (33, 34).

The authors thank the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP, Sa˜o Paulo) and the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq, Brası´lia) for grant support.

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