The oxidation of apocynin catalyzed by myeloperoxidase: Proposal for NADPH oxidase inhibition

The oxidation of apocynin catalyzed by myeloperoxidase: Proposal for NADPH oxidase inhibition

ABB Archives of Biochemistry and Biophysics 457 (2007) 134–141 www.elsevier.com/locate/yabbi The oxidation of apocynin catalyzed by myeloperoxidase: ...

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ABB Archives of Biochemistry and Biophysics 457 (2007) 134–141 www.elsevier.com/locate/yabbi

The oxidation of apocynin catalyzed by myeloperoxidase: Proposal for NADPH oxidase inhibition Valdecir F. Ximenes b

a,b,*

, Marı´lia P.P. Kanegae b, Sandra R. Rissato a, Ma´rio S. Galhiane

a

a Departamento de Quı´mica, Faculdade de Cieˆncias, Universidade Estadual Paulista, Bauru, SP, Brazil Departamento de Ana´lises Clı´nicas, Faculdade de Cieˆncias Farmaceˆuticas, Universidade Estadual Paulista, Araraquara, SP, Brazil

Received 22 September 2006, and in revised form 10 November 2006 Available online 18 November 2006

Abstract Apocynin has been used as an efficient inhibitor of the NADPH oxidase complex and its mechanism of inhibition is linked to prior activation through the action of peroxidases. Here we studied the oxidation of apocynin catalyzed by myeloperoxidase (MPO) and activated neutrophils. We found that apocynin is easily oxidized by MPO/H2O2 or activated neutrophils and has as products dimer and trimer derivatives. Since apocynin impedes the migration of the cytosolic component p47phox to the membrane and this effect could be related to its conjugation with essential thiol groups, we studied the reactivity of apocynin and its MPO-catalyzed oxidation products with glutathione (GSH). We found that apocynin and its oxidation products do not react with GSH. However, this thiol compound was efficiently oxidized by the apocynin radical during the MPO-catalyzed oxidation. We suggest that the reactivity of apocynin radical with thiol compounds could be involved in the inhibitory effect of this methoxy-catechol on NADPH oxidase complex.  2006 Elsevier Inc. All rights reserved. Keywords: Apocynin; Myeloperoxidase; NADPH oxidase; Respiratory burst; Hypochlorous acid; Neutrophil

The generation of the superoxide anion through activation of the NADPH oxidase complex in phagocytic and non-phagocytic cells and its involvement in the pathophysiology of inflammatory and vascular diseases are widely accepted [1]. The superoxide anion is the primary reactive species generated by one-electron reduction of molecular oxygen. From this, several antimicrobial substances, such as hydrogen peroxide, hydroxyl radical, hypochlorous acid and peroxynitrite can be produced, which may also damage the tissues in which they are formed [2]. NADPH oxidase is a multienzyme complex composed of the membrane-bound cytochrome b558, three cytosolic factors (p47phox, p67phox, p40phox) and the small GTPase Rac2. When the cells are activated by stimuli such as immunocomplex, opsonized particles, arachidonic acid or phorbol myristate acetate, a cascade of events takes place, resulting in the migration of these cytosolic components to the membrane *

Corresponding author. Fax: +55 14 3103 6099. E-mail address: [email protected] (V.F. Ximenes).

0003-9861/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.11.010

and assembly of the enzyme complex, which starts producing superoxide [3]. Apocynin is a methoxy-catechol (4-hydroxy-3-methoxyacetophenone), originally extracted from the root of the medicinal herb Picrorhiza kurroa, from the Himalayas, and has shown to possess anti-inflammatory properties [4]. Apocynin has been used as an efficient inhibitor of the multienzyme complex NADPH oxidase in many experimental models involving phagocytic and non-phagocytic cells [5,6]. The mechanism of inhibition is not totally known, but involves the impairment of the translocation to the membrane of the cytosolic component p47phox of the NADPH oxidase complex [7]. A very important finding concerning this mechanism was the discovery that apocynin is a prodrug that is converted by peroxidase-mediated oxidation to a dimer, which has been shown to be more efficient than apocynin itself [8]. In neutrophils, the peroxidase (myeloperoxidase, MPO) might be responsible for the oxidation of apocynin, when these cells are activated, leading to the formation of the

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apocynin dimer. However, it should be not discarded that other cellular oxidants are also capable of oxidizing apocynin, since this xenobiotic is an effective inhibitor of the NADPH oxidase system in endothelial cells, which do not contain MPO [8]. This study concerns the oxidation of apocynin catalyzed by MPO. We demonstrated, for the first time, that apocynin is easily oxidized by the catalytic action of MPO, the products being its dimer and trimer derivatives. The reactivity of radicals of apocynin and its dimer derivative with thiol compounds was suggested to be involved in the mechanism of inhibition of the NADPH oxidase complex. Materials and methods Chemicals Apocynin, glutathione, zymosan, taurine, catalase (EC 1.11.1.6), superoxide dismutase (SOD)1 (EC 1.15.1.1), 5,5 0 -tetramethylbenzidine (TMB) and 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Myeloperoxidase (EC 1.11.1.7) was purchased from Planta Natural Products (Vienna, Austria) and its concentration was determined from its absorption at 430 nm (e430 nm = 89,000 M1 cm1 per heme) [9]. Hydrogen peroxide was prepared by diluting a 30% stock solution purchased from Peroxidos do Brasil (Sao Paulo, SP, Brazil) and calculating its concentration using its absorption at 240 nm (240 nm = 43.6 M1 cm1) [10]. Hypochlorous acid was prepared by diluting a concentrated commercial bleach solution and calculating its concentration from its absorption at 292 nm (e = 350 M1 cm1) [9]. All the reagents used for solutions and buffers were of analytical grade.

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Identification of apocynin oxidation products by LC/MS The oxidation products of apocynin were separated as described above. The HPLC outlet stream was split and 0.2 mL/min was injected into the mass spectrometer (Quattro II Micro, Triple Quadrupole, Micromass, Manchester, UK) equipped with an electrospray ionization source. The mass spectrometer was operated with negative ionization in the full-scan mode (scan range 150–550 m/z). Spray voltage was set at 4.5 kV, the capillary temperature at 150 C and desolvation gas flow at 300 L/h.

Hypochlorous acid production by stimulated neutrophils Neutrophils (2 · 106 cells/mL) were pre-incubated at 37 C in supplemented PBS, 5 mM taurine and apocynin for 10 min. Then, the cells were stimulated by the addition of 1 mg/mL opsonized zymosan. After 30 min the reaction was stopped by adding 20 lg/mL catalase. The neutrophils were pelleted by centrifugation and the supernatant put on ice. Formation of hypochlorous acid was measured by assaying accumulated taurine chloramine (see below). The production of HOCl by the control, with apocynin omitted, was used to calculate the percentage of inhibition. The production of HOCl by activated neutrophils in the absence of apocynin was 30–40 lM.

Hypochlorous acid production by purified MPO Purified MPO (10 nM) was incubated in PBS with 5 mM taurine and the reactions were started by adding 50 lM H2O2. After 15 min, the reactions were stopped by adding 20 lg/mL catalase. The Formation of hypochlorous acid was measured by assaying the accumulated taurine chloramine (see below). The production of HOCl by the control, with apocynin omitted, was used to calculate the percentage of inhibition.

Hypochlorous acid assay Isolation of human neutrophils Neutrophils were isolated from the blood of healthy donors by Ficoll–Paque centrifugation, dextran sedimentation, and hypotonic lysis of red cells [11]. After isolation, neutrophils were resuspended in 10 mM phosphate buffer (pH 7.0) containing 10 mM potassium chloride and 140 mM sodium chloride, plus 1 mM calcium chloride, 0.5 mM magnesium chloride, and 1 mg/mL glucose (supplemented PBS). Opsonized zymosan was prepared as described by Simoes and collaborators [12]. The blood samples were taken from healthy volunteers. The study was approved by the Faculty Research Ethics Committee (Comiteˆ de E´tica em Pesquisa-FCFAR/UNESP no. 03/2006).

Oxidation of apocynin catalyzed by myeloperoxidase Apocynin (1 mM) was incubated in 50 mM phosphate buffer, pH 7.0, 25 C, with 0.1 lM MPO and various concentrations of hydrogen peroxide. The reaction was started by adding hydrogen peroxide and monitored by following the UV absorption spectrum (Hewlett Packard 8452A diode array spectrophotometer). The Products were separated in a high performance liquid chromatograph (Waters 2690 Separation Module in line with a Waters 996 UV–vis Detector set at 254 nm). HPLC analyses were carried out isocratically on a Synergi C18 reversed-phase column (250 · 4.6 mm, 4 lm), with 60:40 water/acetonitrile (flow rate 0.7 mL/min) as the mobile phase.

The accumulated taurine chloramine was assayed by adding 400 lL neutrophil supernatant to 100 lL of a solution containing 14 mM TMB dissolved in 50% dimethylformamide, 100 lM potassium iodide and 400 mM acetic acid. Under these conditions, taurine chloramine oxidizes TMB to a blue product with an absorbance maximum at 655 nm. A standard curve was generated by adding pure HOCl to PBS containing taurine [13]. The absorbance measurements were made in a SpectraMax M2 plate reader (Molecular Devices).

Oxygen uptake assays The reactions were monitored with a Clark-type oxygen electrode (Yellow Spring Instruments 5300A) coupled to an X-Y recorder (EG&G, Princeton Applied Research). The reaction was performed at 25 C, in a final volume of 3 mL, and triggered by adding hydrogen peroxide.

GSH measurement The reaction medium was composed of 1 mM apocynin, 1 mM hydrogen peroxide, 0.1 lM MPO and 1 mM GSH in 50 mM phosphate buffer, pH 7.0, at 25 C and a final volume of 1 mL. After a fixed interval, 20 lg/mL catalase was added to stop the reaction and aliquots were removed to measure the concentration of GSH by the DTNB method [14].

Results UV–vis studies

1

0

Abbreviations used: SOD, superoxide dismutase; TMB, 5,5 -tetramethylbenzidine; DTNB, 5,5-dithiobis-(2-nitrobenzoic acid); TIC, total ion chromatogram.

The oxidation of apocynin was monitored by UV–vis absorption spectrophotometry. Fig. 1 shows the difference

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products (identification below). Equimolar amounts of hydrogen peroxide and apocynin resulted in total consumption of apocynin (Fig. 2, chromatogram C). It should be mentioned that, assuming a classical peroxidase mechanism for the oxidation of phenolic compounds, this concentration of hydrogen peroxide is in excess, since the active intermediate redox compound I of peroxidases is two redox-states above the native form. For this reason, experiments were also performed with 0.5 mM hydrogen peroxide (Fig. 2, chromatogram B). In this case, apocynin was not totally oxidized. However, the relative concentration of the dimer product was higher and the trimer lower than in the experiments with an equimolar amount of hydrogen peroxide. Fig. 1. Oxidation of apocynin catalyzed by myeloperoxidase. Apocynin (1 mM) was incubated with 0.1 lM MPO and 1 mM hydrogen peroxide in 50 mM phosphate buffer, pH 7.0 at 25 C. Difference spectra against apocynin were recorded every 5 s. Arrows indicate the direction of spectral changes.

spectrum resulting when hydrogen peroxide was added to the reaction medium containing 1 mM apocynin and MPO at pH 7.0 and 25 C, blanked with the apocynin alone. The time-dependent raise in the baseline was caused by a slight increase in the turbidity of the reaction medium. The reaction was totally dependent on both hydrogen peroxide and MPO, as the lack of either component prevented the oxidation (not shown). The efficiency of MPO as a catalyst of this reaction is evident, since 1 mM of apocynin was completely oxidized in about 1 min.

Separation of oxidation products Fig. 2 shows the chromatographic profile obtained when the reaction mixture was injected into the HPLC system, five minutes after the start of reaction. This figure also shows the effect of the concentration of hydrogen peroxide on apocynin uptake and the concentration of the main

Fig. 2. Chromatographic separation of apocynin and its oxidation products. (A) Apocynin 1 mM in 50 mM phosphate buffer, pH 7.0. (B) Five minutes after addition of 0.1 lM MPO and 0.5 mM hydrogen peroxide. (C) Five minutes after addition of 0.1 lM MPO and 1 mM hydrogen peroxide. Data represent at least three experiments.

Identification of the apocynin oxidation products To identify the main oxidation products, the HPLC was coupled to a mass spectrometer equipped with an electron spray ionization source. Negative ions were generated in the full-scan mode (range 150–550 m/z). Fig. 3 shows the total ion chromatogram (TIC) and the mass spectra for apocynin and its oxidation products. Apocynin (MW 166), eluting at 6.10 min, was detected in its deprotonated form m/z 165 (MH). The peak eluting at 2.92 min (m/z 329) matches perfectly the deprotonated form of the dimer of apocynin (MW 330), which was described as one of the products of soybean peroxidase-catalyzed oxidation of apocynin [15]. The peak eluting at 11.15 min (m/z 507) matches the deprotonated hydroxylated trimer of apocynin, which was also described in the soybean peroxidase article [15]. Scheme 1 depicts the putative pathway for the oxidation of apocynin catalyzed by myeloperoxidase. Effect of glutathione on apocynin oxidation The current view is that the dimer of apocynin is responsible for the inhibitory property of apocynin [8,16]. In this regard, one possibility is that the dimer could react with essential sulfhydryl residues of p47phox, a cytosolic component of the NADPH oxidase complex. To check the reactivity of the oxidation products of apocynin with sulfhydryl compounds, we added 1 mM glutathione (GSH) after total oxidation of apocynin by MPO or activated neutrophils. The reaction medium was incubated for 10 min after addition of GSH. Both the dimer and trimer products were completely non-reactive, as checked by HPLC after the incubation (not shown). However, when GSH was added before the addition of hydrogen peroxide, the oxidation of apocynin was completely inhibited, as verified by HPLC (Fig. 4). Since GSH is a poor substrate of MPO [17], this effect cannot be explained by the competition between apocynin and GSH for the redox-active forms of MPO. A possible explanation for the non-consumption of apocynin could be the recycling of apocynin by reduction of the intermediate apocynin radical by GSH. To check this hypothesis, we

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followed the consumption of oxygen during the oxidation of apocynin in the presence or absence of GSH, as this would reveal the formation of thiyl radical [18]. The results depicted in Fig. 5 show an intense oxygen consumption when GSH was added in the reaction system. In the controls, the lack of any of the components in the reaction mixture prevented the oxygen uptake. The enzyme SOD was added to demonstrate the formation of superoxide and reinforce the involvement of GSSG anion radical during the MPO-catalyzed oxidation of apocynin in the presence of GSH, as verified for tyrosine [18]. The concomitant oxidation of GSH was confirmed using the DTNB technique (Fig. 6) [14]. We also studied the effect of GSH on subsequent oxidation of the oxidation products of apocynin. In this case, the apocynin was first oxidized and, after its total consumption and production of the dimer and trimer (checked by HPLC), a new portion of MPO and hydrogen peroxide was added, in the presence or absence of GSH. Again, a strong consumption of oxygen suggested the formation of thiyl radicals (not shown). Reaction of apocynin with stimulated neutrophils Considering the importance of the apocynin oxidation products in the mechanism of inhibition of NADPH oxidase, we performed experiments to find out whether similar products are formed when apocynin is oxidized by activated human neutrophils. In this case, isolated neutrophils were incubated with 0.1 mM apocynin for 30 min in supplemented PBS, at 37 C. The activation was performed by adding 1 mg/mL opsonized zymosan. After centrifugation, the supernatant was injected into the HPLC system. Clearly, an efficient oxidation would not be expected, since apocynin blocks the respiratory burst. However, despite the relatively inefficient reaction, the dimer of apocynin was easily identified (Fig. 7). Effect of apocynin on hypochlorous acid production by stimulated neutrophils

Fig. 3. Analysis of MPO-catalyzed oxidation of apocynin using liquid chromatography with mass spectrometry. Apocynin (1 mM) was incubated for 5 min at 25 C in 50 mM phosphate buffer, pH 7.0, containing 0.1 lM MPO and 0.5 mM hydrogen peroxide for five minutes. (A) total ion current (TIC) of the reaction mixture. (B–D) the mass spectra of peaks eluting at 2.92 (dimer), 6.10 (apocynin) and 11.15 min (hydroxylated trimer), respectively. The mass spectra were obtained in the negative ion mode (MH ion).

If apocynin is a substrate for MPO and also inhibits the NADPH oxidase complex, then the production of hypochlorous acid by stimulated neutrophils is likely to be affected. Therefore, we studied the effect of apocynin on both MPO-catalyzed and extracellular production of hypochlorous acid by activated neutrophil. Fig. 8A shows that apocynin was limited as an inhibitor of MPO-catalyzed production of hypochlorous acid. In fact, whereas 5 lM was able to inhibit about 50–60% of the chlorination activity, concentration as high as 1000 lM did not reach total inhibition. However, apocynin was an effective inhibitor of the generation of this microbicidal compound, with an IC50 of 19 lM, when stimulated neutrophils were used as a source of hypochlorous acid (Fig. 8B). As a control, we performed experiments to decide whether a direct scavenging effect of apocynin on hypochlorous acid could account for the observed inhibition. For that purpose, pure

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Scheme 1. Pathway for the oxidation of apocynin catalyzed by myeloperoxidase or activated neutrophils.

Fig. 4. The effect of glutathione on MPO-catalyzed oxidation of apocynin. Apocynin (1 mM) was incubated during 2 min at 25 C in 50 mM phosphate buffer, pH 7.0, containing 0.1 lM MPO, 0.5 mM hydrogen peroxide (control); in the absence of MPO (MPO); in the presence of 1 mM GSH (+GSH). The reaction was started by adding hydrogen peroxide. The supernatant was injected in the HPLC (see Material and methods). Data represent at least three experiments.

hypochlorous acid (30 lM) was added to the reaction mixture of PBS, taurine and apocynin (10–100 lM) and the taurine–chloramine produced was measured as above. The lack of any effect shows that, at least at the concentrations tested here, apocynin was unable to compete with taurine for the hypochlorous acid and also did not react with taurine–chloramine (not shown). Discussion The use of apocynin as an inhibitor of the activation of the NADPH oxidase complex is based on the inhibition of the assembly process, as the migration of the p47phox com-

Fig. 5. Time course of oxygen uptake during oxidation of apocynin. Apocynin (1 mM) was incubated at 25 C in 50 mM phosphate buffer, pH 7.0, containing 0.1 lM MPO, 0.5 mM hydrogen peroxide and 1 mM GSH (line 1, complete system); plus 20 lg/mL SOD (line 2); no GSH (line 3); no apocynin (line 4); no MPO (line 5). The reaction was started by addition of the hydrogen peroxide. The buffer solution was initially saturated with air (200 mM) by stirring the opened container for 5 min. Data represent at least three experiments.

ponent to the membrane is impeded in its presence [7]. It is also known that the oxidation of apocynin plays an important role in its inhibitory effect. In fact, the dimeric oxidation product of apocynin decreases the non-inhibitory lag phase normally observed when apocynin is used as an inhibitor of NADPH oxidase in endothelial cells [8]. Furthermore, the inhibitory potency of this drug is augmented when neutrophils are activated by opsonized zymosan instead of phorbol ester, since the former provokes an intense release of MPO [19]. Here, we have demonstrated, for the first time, that MPO is a very efficient catalyst for

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Fig. 6. Oxidation of GSH during MPO-catalyzed oxidation of apocynin. Apocynin (1 mM) was incubated during 30 s at 25 C in 50 mM phosphate buffer, pH 7.0, containing 0.1 lM MPO, 0.5 mM hydrogen peroxide and 1 mM GSH (control). The concentration of GSH was measured removing aliquots, adding catalase (20 lg/mL) to stop the reaction and the supernatant assayed by the DTNB method [14].

the oxidation of apocynin and that the main products of oxidation are its dimer and trimer derivatives. The experiments with various concentrations of hydrogen peroxide demonstrated clearly that the trimer compound is formed by oxidation of the dimer. Obviously, higher oligomers may also be expected at lower concentrations and this might explain the slight turbidity of the reaction medium. We also demonstrated that the dimer was produced when activated neutrophils were incubated with apocynin, a finding that reinforces the reported experimental evidence that this product could play an important role in the mechanism of NADPH oxidase inhibition in neutrophils [19]. Although the trimer compound is formed by oxidation of the dimer, as shown in the studies with pure MPO, the absence of this product in the neutrophil assay may be explained by assuming that insufficient hydrogen peroxide was generated in this system. Apocynin is a strong inhibitor of the production of hypochlorous acid by stimulated neutrophils. However, its action on purified MPO was limited, since total inhibition was not verified for concentration as high as

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1000 lM. This result is not totally unexpected since the inhibition of the chlorination activity is usually caused by xenobiotics that are excellent substrates for the redox intermediate compound I and poor substrate for compound II of MPO, as a consequence, these xenobiotics promote the accumulation of compound II, which is inactive for the production of hypochlorous acid [20,21]. As we have demonstrated, this is not the case of apocynin which is an excellent substrate of MPO. Thus, we concluded that the strong inhibitor effect of apocynin on the production of hypochlorous acid by stimulated neutrophils might be the result of an additive effect of NADPH oxidase inhibition and, to a lesser extent in less extension, due to competition with chloride for the catalytic active site of MPO. This is a further evidence of the importance of apocynin as an anti-inflammatory drug. Apocynin is not the only compound that inhibits the assembly of the NADPH-oxidase system. The same property has been reported for N-ethylmaleimide and oxidation products of 1-naphthol. The explanation for the effects of these compounds is based on their conjugation with essential thiol residues present in the cytosolic component p47phox, which could impair its activation and consequent migration to the cell membrane [22,23]. For apocynin itself, the relationship between the inhibition of the translocation of p47phox and conjugation with the thiols has not been verified and the mechanism by which apocynin inhibits NADPH oxidase remains unknown. Here we verified that neither apocynin nor the dimer and trimer derivatives were able to conjugate with GSH, which is a common representative of thiol compounds. However, we obtained strong evidence that GSH is able to react with apocynin radical and/or its dimer radical, which are formed during MPO catalyzed oxidation. In fact, it is well known that GSH is able to react with phenolic compounds, such as tyrosine, during peroxidase-catalyzed oxidation. The following reactions describe the suggested pathway for oxygen consumption and thiyl radical formation during horseradish peroxidase-catalyzed oxidation of tyrosine [18].

Fig. 7. Oxidation of apocynin by stimulated neutrophils. Apocynin (1 mM) was incubated at 37 C in 10 mM PBS buffer in the presence of neutrophils (2 · 106 cells/mL) for 30 min. The cells were stimulated with 1 mg/mL opsonized zymosan, and subsequently the reactions were stopped by adding 20 lg/ mL catalase. The neutrophils were pelleted by centrifugation and the supernatant injected into the HPLC. Data represent at least three experiments.

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Fig. 8. Effect of apocynin on hypochlorous acid production by (A) purified MPO and (B) stimulated neutrophils. Purified MPO (10 nM) was incubated in PBS with 5 mM taurine and the reactions were started by adding 50 lM H2O2. The results are means and SD of triplicates. Neutrophils (2 · 106 cells/mL) were incubated at 37 C in supplemented PBS, with 5 mM taurine and the reaction triggered by 1 mg/mL opsonized zymosan. Formation of hypochlorous acid was measured by assaying accumulated taurine chloramine. See methods section for further details.

HRP

2 Tyr þ H2 O2 ! 2Tyr þ 2H2 O

ð1Þ

Try þ GSH Try þ GS GS þ GS GSSG

ð2Þ ð3Þ

GSSG þ O2 GSSG þ O 2

ð4Þ

We used this chemical property of the thiyl radical to demonstrate its involvement during MPO-catalyzed oxidation of apocynin in the presence of GSH. The increase in the rate of oxygen consumption when SOD was present is an evidence of the intermediacy of GSSG anion radical, since the dismutation of superoxide may shift the equilibrium to the right (reaction 4). Moreover, SOD will prevent superoxide from reacting with native MPO, thus inhibiting the enzyme through formation of inactive compound III [25]. We suggest that the reactivity of the apocynin radical with thiol compounds, represented here by GSH, could be a pathway by which this substance could interact with thiol residues of the cytosolic component of the NADPH oxidase leading to the deactivation. Corroborating this hypothesis, apocynin markedly decreases the intracellular reduced/oxidized glutathione ratio (GSH/GSSG) in stimulated monocytes and supplementation of the culture medium with GSH impairs the action of apocynin [7]. Additionally, a pro-oxidant effect of apocynin was also described in non-phagocytic cells and this effect was abolished by the use of superoxide-specific scavengers tiron and SOD [16]. Indeed, a consequence of the oxygen consumption caused by reaction of some phenol radicals with GSH or other thiol compounds is the production of superoxide [18], as verified here. Scheme 2 depicts the putative mechanism for the involvement of GSH during MPOcatalyzed oxidation of apocynin. As stated above, the assumption that the metabolism of apocynin is important for its inhibitory function is based in the found that its dimer is more efficient than apocynin itself in phagocytic and non-phagocytic cells [8,16]. Here, we are not against this property of apocynin. Indeed, the dimer was also able to react with GSH during its MPO-catalyzed oxidation. This is really expected, as this product must be more reactive with MPO than apocynin itself. In conclusion, the property of apocynin as an inhibitor of NADPH oxidase and its dependence on MPO-catalyzed oxidation might be linked to the reaction of apocynin

Scheme 2. Proposal for the involvement of GSH during MPO-catalyzed oxidation of apocynin (Apo-OH). See above-mentioned references for further information.

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Scheme 3. Proposal for apocynin/MPO-mediated inhibition of the cytosolic NADPH oxidase p47phox.

radical and/or its dimer radical with intracellular GSH or directly with essential thiols of the cytosolic factor p47phox. Scheme 3 depicts the putative pathways by which apocynin and its dimer could inhibit the activation of the cytosolic p47phox and the importance of MPO in this process. Acknowledgment This study was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Brazil. References [1] L.C. Azevedo, M. Janiszewski, F.G. Soriano, F.R. Laurindo, Endocr. Metab. Immune Disord. Drug Targets 6 (2006) 159–164. [2] J. Schiller, B. Fuchs, J. Arnhold, K. Arnold, Curr. Med. Chem. 10 (2003) 2123–2145.

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