e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 220–228

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Modulation of protein tyrosine nitration and inflammatorymediators by isoprenylhydroquinone glucoside

Ana Olmos, Rosa-Marıa Giner ∗, Marıa-Carmen Recio,Jose-Luis Rıos, Salvador ManezDepartament de Farmacologia, Facultat de Farmacia, Universitat de Valencia,Av. Vicent Andres Estelles s/n, 46100 Burjassot, Spain

a r t i c l e i n f o

Article history:

Received 23 June 2006

Received in revised form

2 November 2006

Accepted 3 November 2006

Published on line 9 November 2006

Keywords:

Prenylhidroquinone

Peroxynitrite

Nitrotyrosine

a b s t r a c t

The nitration of tyrosine caused by peroxynitrite and other reactive nitrogen species is

clearly detrimental for some physiological processes; however, its signalling role is still

open to controversy. Among the natural phenolics known for their ability to oppose free

tyrosine nitration, isoprenylhydroquinone glucoside is investigated due to its unusual

structure, which contains a simple hydroxybenzene alkylated by a hemiterpenoid moi-

ety. This hydroquinone was shown to be an effective inhibitor of peroxynitrite-induced

protein tyrosine nitration in 3T3 fibroblasts. When tested on bovine seroalbumin nitra-

tion, however, the potency was reduced by half and the effect was almost abolished in

the presence of bicarbonate. In contrast, addition of this anion had no effect on the

nitrite/hydrogen peroxide/hemin system. Isoprenylhydroquinone glucoside was also active

in the �M range on intra- and extracellular protein-bound tyrosine nitration by phor-

Hemin

Cytokines

bol 12-myristate 13-acetate-stimulated neutrophils. The effects on nitric oxide synthase

expression, interleukin-1� and tumor necrosis factor-� production by lipopolysaccharide-

stimulated macrophages were quite moderate. Thus, isoprenylhydroquinone glucoside is

an inhibitor of protein nitration in situ, but lacks effect on the generation of either nitric

oxide or inflammatory cytokines.

In the same way, the activation of p38 MAP kinase by the

1. Introduction

The physiological role of reactive oxygen species, namelysinglet oxygen, hydrogen peroxide (H2O2), superoxide (O2

−),and hydroxyl radical, began to be universally recognized atthe end of the 1960s, when McCord and Fridovich describedthe existence of a superoxide dismutase enzymatic activity(McCord and Fridovich, 1968, 1969). Reactive oxygen speciesexert oxidative, cytotoxic, and tissue-degrading functions,

and have been associated with aging and various inflam-matory conditions. More recent findings indicate, however,that they may participate in cellular signaling mechanisms

∗ Corresponding author. Tel.: +34 963543609; fax: +34 963544973.E-mail address: rmginer@uv.es (R.-M. Giner).

0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ejps.2006.11.002

© 2006 Elsevier B.V. All rights reserved.

(Hensley and Floyd, 2002). For example, enhanced H2O2 pro-duction was found to occur immediately after the activationof receptors of endogenous ligands such as platelet-derivedgrowth factor in vascular smooth muscle cells. The preven-tion of this effect with either catalase or the antioxidantcompound N-acetylcysteine was associated to a blockade inDNA synthesis and both mitogen-activated protein kinase andprotein-tyrosine kinase activities (Sundaresan et al., 1995).

pro-inflammatory cytokine interleukin-1� is mediated by theincrease of H2O2 production in astrocytes. The underlyingmechanism is the transient activation of protein phosphatase

t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 220–228 221

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ctivity, which is abolished by N-acetylcysteine and by the freeadical scavenger phenyl-tert-butyl nitrone (Robinson et al.,999).

The oxidative breakdown of the terminal imino of argi-ine by constitutive or inducible nitric oxide synthases (NOS)

eads to the formation of nitrogen monoxide (NO), a fairlytable free radical which, in combination with O2

−, forms per-xynitrite (ONOO−). Thereafter, an array of reactive nitrogenpecies (RNS) derive from both NO and ONOO− by transferringxygen atoms (Pryor and Squadrito, 1995). Recent evidenceonfers signaling relevance to the process of tyrosine nitra-ion, which is caused either by ONOO− or by nitrogen dioxide,he latter formed from nitrite, mediating hemoprotein cataly-is (Thomas et al., 2002). Pharmacological manipulation of thisind of processes include many efforts directed at the modula-ion of the overexpression of inducible NOS (Song et al., 2002),he genomic damage caused by ONOO− through the activationf poly(ADP)-ribose polymerase (PARP) (Serraino et al., 2003),he inactivation of antiproteinases (Whiteman et al., 1999),nd the nitration of aromatic aminoacids (Pannala et al., 1997).

Some of the known compounds possessing inhibitoryctivity against tyrosine nitration are phenolic compoundsf plant origin that have antioxidant properties essen-ially linked to certain polyhydroxyl substitution patternsPannala et al., 1997). In this context, we have recentlyescribed the mechanism implicated in the effect of 2-

soprenylhydroquinone-1-glucoside (IHG). Despite its ratheroor antioxidant activity, this compound behaved as an effi-ient inhibitor of free tyrosine nitration induced by ONOO−

Olmos et al., 2005). As a continuation of our research project,e now report on the interaction of IHG with the nitrationf protein-bonded tyrosine in different chemical and cellu-

ar systems in order to elucidate its mode of action. To betternderstand this interaction, we have investigated the effectf IHG on tyrosine nitration in cultured fibroblasts stimu-

ated with ONOO− and in human neutrophils stimulated withhorbol 12-myristate 13-acetate (PMA). The latter producesyeloperoxidase activation and, in turn, augments tyrosine

itration. The modulation of extracellular protein nitrationas determined with the aid of bovine seroalbumin (BSA)

n the latter leukocyte model and, in cell-free systems, withither ONOO− or the combination of nitrite/heme/H2O2. Totudy a possible influence on the genesis and turnover ofO, the effect of IHG on iNOS expression and nitrite levelsas determined in cultured macrophages stimulated with

ipopolysaccharide (LPS).

. Materials and methods

.1. Chemicals and laboratory ware

nless otherwise specified, all chemicals were obtainedrom Sigma (St. Louis, MO, USA), materials for cell cultureere purchased from Sarstedt (Numbrecht, Germany), andedia and reagents were supplied by Gibco (Langley, VA,SA). The natural test compound 1-O-�-glucopyranosyl-2-

3′,3′-dimethylallyl) hydroquinone (2-isoprenylhydroquinone--glucoside, IHG) (Fig. 1A) was isolated from Phagnalon rupestreAsteraceae) and identified by ourselves (Gongora et al., 2001).otal purity of the compound was assessed by TLC and NMR.

Fig. 1 – Chemical structure of isoprenylhydroquinoneglucoside (A, IHG) and epigallocatechin gallate (B, EGCG).

Epigallocatechin gallate (EGCG, Fig. 1B) was used as a refer-ence drug. Peroxynitrite was synthesized in a quenched flowreactor as previously described (Olmos et al., 2005). Enhancedchemiluminescence (ECL) detection reagents were obtainedfrom Pierce (Rockford, IL, USA). Precision plus protein stan-dards were from BioRad (Hercules, CA, USA.). Ficoll-Paque,nitrocellulose membrane, Hyperfilm ECL, and puRE Taq Ready-To-Go PCR Beads were obtained from Amersham Biosciences(Little Chalfont, UK). EDTA-free protease inhibitor cocktailtablets were purchased from Roche (Mannheim, Germany).Polyclonal anti-3-nitrotyrosine and anti-nitric oxide synthase-2 primary antibodies were purchased from Cayman (AnnArbor, MI, USA). Mouse TNF-� and IL-1� ELISA kits werepurchased from eBioscience (San Diego, CA, USA). Rneasy®

Total RNA kit was purchased from Qiagen Ltd. (Crawley,UK); oligo (dT) primer and AMV-reverse transcriptase wereobtained from Promega (Madison, WI, USA), and IL-1� andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) primersets from R&D (Minneapolis, MN, USA).

2.2. Cell culture

Murine 3T3 fibroblasts and RAW 264.7 macrophages werecultured in Dulbecco’s modified Eagle’s medium (DMEM) sup-plemented with 10% fetal bovine serum (FBS), 100 IU/mlpenicillin, and 100 �g/ml streptomycin at 37 ◦C in a humid-ified atmosphere of 95% air and 5% CO2 in an incubator.Fibroblasts were removed from the tissue culture flask withtrypsin (5 mg/ml) and macrophages using a cell scraper. Cellswere cultured in dishes at a density of 2 × 105 cells/ml andwhen sub-confluent, the medium was substituted by PBS forfibroblasts and by DMEM supplemented with 0.5% FBS formacrophages. Then, the cells were subjected to the treatmentas indicated below.

2.3. Preparation of human neutrophils

Neutrophils were obtained from human blood buffy coats.The residual blood was mixed with an equal volume of 2%dextran in 0.9% NaCl at room temperature to allow the sed-imentation of erythrocytes. The upper leukocyte-rich layerwas centrifuged at 325 × g for 15 min at room temperature.

To remove residual erythrocytes, the pellet was resuspendedin lysis buffer (8.3 g/l NH4Cl, 1 g/l KHCO3, 37 mg/l EDTA·2H2O)and centrifuged again. Then the pellet was resuspended in10 ml Hanks’ buffered saline (HBSS) and the cells were puri-

u t i c

222 e u r o p e a n j o u r n a l o f p h a r m a c e

fied with the Ficoll-Paque gradient density method. The cellmixture was centrifuged again at 500 × g for 40 min at roomtemperature and the neutrophils were separated and resus-pended in HBSS containing 1.0 mM Ca2+ and 0.5 mM Mg2+. Cellpurity (95–98%) was established with the aid of flow cytometry(Coulter Epics XL-MCL).

2.4. Determination of cell viability

The cytotoxicity of IHG was determined with the aid of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide(MTT) assay (Mossman, 1983). Human neutrophils, murine 3T3fibroblasts, and RAW 264.7 macrophage cells were exposedto IHG at a concentration of 100 �M in a 96-microplate for1 h (neutrophils) or 24 h (fibroblasts and macrophages), afterwhich 100 �l per well of a 0.5 mg ml−1 solution of MTT wereadded. The plates were then incubated at 37 ◦C until bluedeposits were visible. This blue-colored metabolite was dis-solved in dimethylsulfoxide (DMSO) and the absorbance wasmeasured at 490 nm with a Labsystems Multiskan EX platereader. Results were expressed in absolute absorbance read-ings, with a decrease indicating a reduction in cell viability.The percentage of viability was calculated as the relation ofviable cell number/total cell number × 100.

2.5. Protein-bound tyrosine nitration by ONOO− inmurine fibroblast

Cultured fibroblasts were treated with concentrations of IHGranging from 6.25 to 50 �M for 5 min at 37 ◦C and thenincubated with 125 �M ONOO− for 5 min. The reaction was ter-minated by washing with PBS and the cells were harvested in100 �l ice-cold lysis buffer (1% Triton X-100, 1% deoxycholicacid, 20 mM NaCl, 25 mM Tris–HCl, and EDTA-free proteaseinhibitor cocktail tablets). The mixture was sonicated (onecycle in 10 s) in a Branson sonifier 150 and the proteins weremeasured by means of the Bradford method with BSA as astandard. Finally, immunoblotting with anti-3-nitrotyrosineantibody was performed to determine the effect of IHG onprotein-bound tyrosine nitration (see Section 2.13).

2.6. Protein-bound tyrosine nitration in humanneutrophils stimulated with phorbol 12-myristate13-acetate (PMA)

Purified neutrophils (1 × 106/ml) were resuspended in 1 mlHBSS and incubated with 125 �M NO2

− and 1 �M PMA at 37 ◦Cfor 1 h. IHG or EGCG were tested at concentrations rangingfrom 3 to 50 �M. The cells were then harvested in 100 �l ice-cold lysis buffer (1% Triton X-100, 1% deoxycholic acid, 20 mMNaCl, 25 mM Tris–HCl, and EDTA-free protease inhibitor cock-tail tablets). The mixture was sonicated (one cycle in 10 s), theproteins were quantified with the aid of the Bradford method,and Western blot analysis was performed (see Section 2.13).

2.7. BSA nitration by human neutrophils stimulated

with PMA

In accordance with a slightly modified version of Eiserich’smethod (Eiserich et al., 1998), purified neutrophils (1 × 106/ml)

a l s c i e n c e s 3 0 ( 2 0 0 7 ) 220–228

were resuspended in 1 ml HBSS and incubated with 1 mg/mlBSA, 125 �M NO2

−, and 1 �M PMA at 37 ◦C for 1 h, either inthe absence or presence of IHG and EGCG at concentrationsranging from 3 to 50 �M. The cells were then centrifuged at11,180 × g for 15 min and the BSA present in the supernatantwas detected by means of Western blot analysis (see Section2.13).

2.8. BSA nitration of cell-free samples

2.8.1. By nitrite/heme/H2O2

BSA nitration was performed as previously described by Bianet al. (2003). In brief, 2 mg/ml BSA dissolved in 0.1 M phosphatebuffer at pH 7 were incubated with 25 �M hemin, 2 mM NO2

−,1 mM H2O2, and concentrations of IHG or EGCG ranging from 6to 100 �M for 30 min at 37 ◦C, either in the absence or presenceof bicarbonate 25 mM. The nitration of the protein was thenevaluated with the aid of Western blot analysis (see Section2.13).

2.8.2. By ONOO−

The extent of BSA nitration was monitored by means of West-ern blot analysis (see Section 2.13). In brief, 2 mg/ml BSAdissolved in 0.1 M phosphate buffer at pH 7 were vortexed for15 s with 0.5 mM ONOO− and concentrations of IHG or EGCGranging from 6 to 100 �M, either in the absence or presence ofbicarbonate 25 mM. From this point, the procedure was carriedout as described above.

2.9. Determinations related to NO turnover inmacrophages

2.9.1. Nitrite productionRAW 264.7 macrophages (1 × 106 cells/ml) were co-incubatedwith 1 �g/ml LPS at 37 ◦C for 24 h in a 96-well culture platein the presence of IHG or EGCG at concentrations rangingfrom 50 to 100 �M. Nitrite concentration in the culture super-natant was determined spectrophotometrically by means ofthe Griess reagent (Escandell et al., 2006).

2.9.2. Nitric oxide synthase-2 expressionRAW 264.7 macrophages at 1 × 106 cells/ml were co-incubatedin a 6-well culture plate with 1 �g/ml LPS at 37 ◦C for 18 hin the presence of IHG or EGCG at concentrations rangingfrom 50 to 100 �M. Cells were harvested in 100 �l ice-cold lysisbuffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl, and25 mM Tris, pH 7.4 and EDTA-free protease inhibitor cocktail).The mixture was sonicated (one cycle in 10 s), the proteinswere quantified with the Bradford method, and Western blotanalysis was performed (see Section 2.13) (Escandell et al.,2006).

2.10. Determination of tumor necrosis factor-˛ (TNF-˛)production in macrophages

RAW 264.7 macrophages were stimulated and treated as

described in the nitrite assay (Section 2.9.1). TNF-� productionin the culture supernatant was determined with the aid of aspecific enzyme immunoassay kit from eBioscience, followingthe manufacturer’s instructions.

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.11. Determination of interleukin-1ˇ (IL-1ˇ)roduction in macrophages

AW 264.7 macrophages were stimulated and treated asescribed in the nitrite assay (Section 2.9.1). IL-1� production

n macrophage cell lysates obtained with lysis buffer (10 mMEPES pH 7.6, 10 mM KCl, 1.5 mM MgCl2, 0.8% Triton, 1 mMitiothreitol, 2 mM phenylmethylsulfonyl fluoride, and EDTA-ree protease inhibitor cocktail) was determined with the aidf a specific enzyme immunoassay kit from eBioscience, fol-

owing the manufacturer’s instructions.

.12. Dihydrorhodamine (DHR) 123 oxidation inacrophages

acrophages (0.5 × 106 cells/ml) were co-incubated in six-wellulture plate with 1 �M PMA at 37 ◦C for 4 h in the presencef IHG (50 and 100 �M), EGCG (3.12–12.5 �M) or vehicle. Thirtyinutes before harvesting the cells, DHR 123 (0.2 mg/ml) was

dded to the medium. Then cells were washed, resuspendedn PBS and analyzed immediately by flow cytometry using FL-1etector (485 nm excitation/530 nm emission) (Walrand et al.,003).

.13. Western blot analysis

qual amounts of proteins were loaded on 10% SDS-PAGEnd transferred onto a nitrocellulose membrane for 90 mint 125 mA. The membrane was subsequently blocked inhosphate buffered saline (PBS) containing 0.5% BSA, 1%olyvinylpyrrolidone-10, 1% PEG, 0.2% Tween 20, and 10 mMaF at room temperature for 2 h and then incubated with thenti-3-nitrotyrosine primary antibody at 1:3000 dilution in theame blocking buffer at 4 ◦C overnight. The resulting blotsere washed and incubated with horseradish peroxidase-

onjugated goat anti-rabbit immunoglobulin G at 1:12,000ilution in blocking buffer. After washing, immunoreactiveroteins were visualized with the aid of an enhanced chemi-

uminesce system (ECL plus kit), after which the membranesere exposed to Hyperfilm ECL. The intensities of the bandsere quantified with a Scion Image for Windows Program. Theasal nitration value (blank) was then subtracted from that ofach treated sample. For NOS-2, the membranes were blockedn PBS-Tween 20 containing 5% (w/v) low fat milk and thenncubated with anti-NOS-2 polyclonal antibody (1:1000 dilu-ion). For �-actin, which was used as an internal control toormalize for differences in protein amounts among differentell samples, the membranes were incubated with anti �-actin1:10,000 dilution).

.14. Reverse transcriptase-polymerase chain reactionRT-PCR)

AW 264.7 macrophages were cultured in six-well platest 2 × 106 cells/well. When sub-confluent, the medium waseplaced with DMEM without FBS and after 24 h, stimuli were

dded. The plates were washed with PBS and the total cellularNA was extracted with the Rneasy® Total RNA kit in accor-ance with the manufacturer’s protocol. The concentrationf the extracted RNA was determined spectrophotometrically

l s c i e n c e s 3 0 ( 2 0 0 7 ) 220–228 223

at a wavelength of 260 nm. cDNA was synthesized by incu-bating l �g of total RNA with a reaction mixture containingoligo (dT) primer and AMV-reverse transcriptase. The result-ing cDNA samples were then amplified by means of a PCR, inwhich puRE Taq Ready-To-Go PCR beads with IL-1� and GAPDHprimer sets were used. Semi-quantitative PCR was carriedout on an Eppendorf Mastercycler under the following condi-tions: initial denaturing temperature of 94 ◦C for 4 min; cyclingparameters at 94 ◦C for 45 s, 55 ◦C for 45 s, and 72 ◦C for 45 s for28 cycles; and finally, an elongation temperature of 72 ◦C for10 min. Following the reaction, the amplified products wererun on 2% agarose gel in TBE buffer. Densitometry analysiswas performed with a Scion Image for Windows Program. Thelevel of cytokine mRNA was then normalized against those ofGAPDH mRNA.

2.15. Statistical analysis

Data were expressed as mean ± S.E.M. Statistical analysis wasperformed with a one-way analysis of variance (ANOVA),followed by Dunnett’s t-test for multiple comparisons. In com-parisons with the control group, values of P less than 0.05 wereconsidered to be statistically significant. Inhibition percent-ages (%I) were calculated from the differences between thetest compound-treated group and the control group. Inhibitoryconcentration 50% (IC50) values were calculated from thedose/response linear regression plots. When the IC50 valueswere calculated from Western blotting images, they should beconsidered apparent.

3. Results

The effect of IHG on cellular protein nitration was determinedafter studying two different pathways that affect protein-bound tyrosine. The presence of 3-nitrotyrosine (3-NT), anindicator of the RNS formation, was established in both casesby means of Western blot analysis with a 3-NT antibody. In thefibroblast protocol, tyrosine nitration is induced by ONOO−,which is formed from the reaction of O2

− and NO and pro-duces nitration either via NO2

• or a CO2 adduct (Augusto etal., 2002). In the second experimental design, myeloperoxidase(MPO) from human neutrophils produces tyrosine nitration,either via oxidation of NO2

− in the presence of H2O2 to formNO2

•, or via the reaction of NO2− with the main MPO product,

HOCl, to form NO2Cl as a nitrating species. Tyrosine nitra-tion in the cell lysate was reduced by exposure of cells to IHGin a concentration dependent manner (Fig. 2). IHG and EGCGwere very active in both systems; their IC50 values were 12 and7 �M, respectively, in fibroblast protein-bound tyrosine nitra-tion stimulated with ONOO−, while their IC50 values were 11and 7 �M, respectively (Fig. 3), in the case of human neutrophilprotein-bound tyrosine nitration stimulated with PMA. Thetest compounds at 100 �M did not exhibit any cytotoxicity oneach cell types used and cellular viability was higher than 95%in the MTT test (data not shown).

The effect on BSA tyrosine nitration induced bynitrite/heme/H2O2 was determined in a cell-free system.IHG at 100 �M produced a moderate reduction which wasunaffected by 25 mM bicarbonate (37 and 38% inhibition in

224 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 220–228

Fig. 2 – Effects of IHG (A) and EGCG (B) on protein-boundtyrosine nitration by peroxynitrite in murine fibroblast. Arepresentative Western blot of 3-nitrotyrosine (3-NT) andthe associated quantitative analysis are shown.3-Nitrotyrosine formation is normalized to �-actin contentfor each sample and expressed as the mean of theratio ± S.E.M of at least three separate experiments. B:

** *

Fig. 3 – Effects of IHG (A) and EGCG (B) on protein-boundtyrosine nitration in human neutrophils stimulated withPMA. A representative Western blot of the experiment isshown. 3-Nitrotyrosine (3-NT) formation is determined bydensitometry and normalized to �-actin content for eachsample. Each bar chart shows the mean of the ratio ± S.E.Mof at least four different experiments. B: blank; C: control.

blank; C: control. P < 0.01, P < 0.05 as compared withcontrol group (Dunnett’s t test).

the absence or presence of bicarbonate, respectively). EGCG at100 �M exhibited the same behavior, albeit with higher activ-ity (78% versus 88% inhibition) (Fig. 4). However, when IHGwas assayed at 50 �M on nitrite/PMA-induced BSA nitrationby human neutrophils, it produced a significant inhibition of58%. The IC50 value was 28 �M, in the same range as that ofEGCG (IC50 = 32 �M) (Fig. 5).

Regarding BSA nitration induced by ONOO−, the effect ofIHG was similar to that of EGCG (IC50 values of 28 and 48 �M,respectively) and both compounds, at 100 �M, abolished the

reaction. In contrast, inhibitory effect of 100 �M IHG droppedin the presence of bicarbonate to a 25% inhibition, whereasthe activity of EGCG was only moderately reduced to 52%(Fig. 6).

**P < 0.01 as compared with control group (Dunnett’s t test).

In LPS-stimulated macrophages incubated with 100 �MIHG, iNOS expression was reduced and the subsequent pro-duction of nitrite was slightly inhibited. Moreover, exposureof EGCG-treated macrophages to LPS clearly reduced nitritelevels and iNOS expression (Table 1). As shown in Fig. 7,IHG did not affect DHR 123 oxidation in PMA-stimulatedmacrophages, whereas EGCG showed an antioxidant effect ina dose-dependent manner.

In LPS-stimulated macrophages, IHG moderately reducedthe levels of IL-1� in cell lysates although failed to affectIL-1� mRNA expression (Fig. 8). EGCG reduced in a higherextent both IL-1� production and IL-1� mRNA expression with

e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 220–228 225

Fig. 4 – Effects of IHG (A) and EGCG (B) on nitration of BSAinduced by nitrite/heme/H2O2 in absence and presence ofbi

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Fig. 5 – Effects of IHG and EGCG on nitration of BSA byhuman neutrophils stimulated with PMA. A representativeWestern blot is shown. Bar charts in the lower graph showthe band densitometric intensity of 3-nitrotyrosine. Dataare obtained from three independent experiments and arepresented as the mean ± S.E.M. B: blank; C: control.

icarbonate (25 mM). A representative Western blot of fourndependent experiments is shown. B: blank; C: control.

espect to the control (Fig. 8). TNF-� production was onlylightly affected by EGCG (Table 1).

. Discussion

great amount of evidence confirms that structural mod-fications of proteins and nucleotides made by ONOO− orO2

• accompany the progression of both acute and chronicnflammatory diseases (Szabo, 2003; Dedon and Tannenbaum,004). Such chemical modifications can be readily observed inoth cell cultures and tissue samples obtained from in vivoxperiments. Probably the major and best-known chemicalanifestation of so-called nitrative stress is the nitration of

yrosine residues, produced via three main pathways. Therst pathway is induced by ONOO− formed from the reactionetween NO and O2

−; the second one is mediated by peroxi-ases to form NO2

• in the presence of NO2−; and in the last,

O trapping of the tyrosyl radical leads first to nitrosotyro-ine and then, through the formation of an iminoxyl radical,o nitrotyrosine (Gunther et al., 1997).

Apart from an examination of inflammatory cytokineelease, the focus of the present paper concerns the ability ofHG to affect the nitration of tyrosine residues in proteins. Tohis end, different experimental designs have been applied. In

Table 1 – Effects of IHG and EGCG on the assays of macrophage

iNOS Nitrite (�M)

Mean ± S.E.M. %I Mean ± S.E.M. %I

Control 0.69 ± 0.09 – 18.60 ± 0.53 –IHG 0.51 ± 0.12 26 16.18 ± 0.41* 13EGCG 0.17 ± 0.06* 75 11.16 ± 0.14** 40

Values are expressed as the mean of inducible nitric oxide synthase (iNproduction of three independent experiments. Inhibition percentages (%Icompared with control group (Dunnett’s t test).

**P < 0.01, *P < 0.05 as compared with control group(Dunnett’s t test).

some cases ONOO− has been added directly to BSA or culturedfibroblasts; in others, nascent NO2

• has been generated in situeither by combining nitrite/heme/H2O2 or by exploiting theperoxidase machinery of human blood neutrophils. In addi-tion, the possible influence on the synthesis of NO has beenstudied by examining the expression of macrophage-iNOS andthe levels of waste nitrite.

Taken together, the results indicate that IHG is most effec-tive in the inhibition of intracellular protein nitration, eitherthat caused in fibroblasts after exposure to ONOO−, or thatcaused in neutrophils after treatment with PMA. This agent isan activator of several isoforms of protein kinase C and therebyupregulates RNS in different cells by means of NOS induction(Paul et al., 1995; Tepperman et al., 2000) and the production of

O2

− and H2O2 (Steinbrenner et al., 2005; Benna et al., 1997). Thefact that only slightly lower potencies were observed when IHGwas tested for its protective activity against the nitration of

function upon stimulation by lipopolysaccharide

IL-1� (pg/ml) TNF-� (ng/ml)

Mean ± S.E.M. %I Mean ± S.E.M. %I

79.07 ± 0.38 – 77.59 ± 1.31 –49.81 ± 0.22** 37 69.05 ± 1.88* 1134.01 ± 0.48** 57 53.54 ± 0.99** 31

OS)/�-actin band densitometric intensity, nitrite, IL-1� and TNF-�

) were obtained for test compounds at 100 �M. **P < 0.01, *P < 0.05 as

226 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 220–228

Fig. 6 – Effects of IHG (A) and EGCG (B) on nitration of BSAinduced by peroxynitrite in absence or presence ofbicarbonate (25 mM). A representative Western blot isshown. Bar charts show the band densitometric intensityof 3-nitrotyrosine. Data are obtained from threeindependent experiments and are presented as the

** *

Fig. 7 – Effect of IHG (A) and EGCG (B) on dihydrorhodamine123 oxidation in macrophages stimulated with PMA. Arepresentative histogram of the experiment is shown. Thetable indicates the mean of fluorescence intensity ofrhodamine 123 ± S.E.M. of three different experiments.

mean ± S.E.M. B: blank; C: control. P < 0.01, P < 0.05 ascompared with control group (Dunnett’s t test).

BSA, again induced by either ONOO− or PMA, suggests that theinhibitory effect depends neither on the direct participation

of ONOO− nor on whether the protein target is intracellu-lar or extracellular. Our previous results obtained for IHG onONOO− reactivity discourage the hypothesis that the effect isdue to the antioxidant properties associated with its phenolic

*P < 0.05 as compared with control group (Dunnett’s t test).

e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a

Fig. 8 – Effect of IHG and EGCG on macrophage IL-1� geneexpression. RAW 264.7 macrophages were incubated for 6 hin presence of IHG or EGCG (50 and 100 �M). B: blank; C:control. Total RNA was then extracted and RT-PCRperformed for IL-1� and GAPDH gene expression.Electrophoretogram of a representative experiment iss

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r

hown.

haracter and instead point to a genuine and more selectiveompetition for the nitrating species (Olmos et al., 2005). Theddition of bicarbonate makes such a competition much moreifficult because of the great avidity of carbon dioxide – inquilibrium with bicarbonate – for ONOO−. The capture ofhis anion does not lead to its inactivation, but rather to annhancement of its nitrating and oxidizing power upon liber-tion of NO2

• and carbonate radicals. It is important to notehat carbonate radical can extract one electron from tyrosineo give tyrosine radical, which is the substrate for nitrationith NO2 (Squadrito and Pryor, 2002).

In contrast to the strong inhibitory effects shown in thebove experiments, IHG exhibited only weak action whenhe protein nitration was induced by hemin/H2O2. Theoreti-ally, this method is based on the oxidation of nitrite to NO2

•

nd involves the transient formation of an oxoferryl species;t is thus a mechanism very similar to that of peroxidasenzymes, and therefore to that subjacent in the nitra-ion induced by PMA in neutrophils (Herold and Rehmann,003):

e(III)porphyrin + H2O2 + NO2−

→ O = Fe(IV)porphyrin + H2O + NO2•

espite this similarity, only 37% inhibition against BSA nitra-ion was observed for IHG at 100 �M. As we had formerlyroved that IHG does not inhibit human leukocyte MPO at all

Gongora et al., 2002), it can be concluded that IHG does notnteract directly with the heme group.

The involvement of the NO/nitrosotyrosine pathway in theode of action of IHG could be formally considered. However,

aking into account the fact that neutrophils, unlike othernflammatory cells such as activated macrophages, producenly moderate amounts of NO, the artificial addition of NO2

−

o neutrophils should minimize the trapping of NO and the

onsequent formation of nitrosotyrosine by diverting the pro-ess in such a way that considerable amounts of NO2

• areormed. This would mean that the effect of IHG could dependnstead on the consumption of intracellular NO2

•, a process

l s c i e n c e s 3 0 ( 2 0 0 7 ) 220–228 227

that is not feasible under the experimental conditions of thecell-free assay of BSA nitration.

Determining the effects on both the expression of iNOSin macrophages and on the levels of nitrite in the culturemedium indicates in a straightforward manner the influ-ence on the production of NO. In this context, and giventhe low inhibition percentages at 100 �M, we must state thatin this case, IHG exerts physiologically negligible activity. Itspotential pharmacological benefits thus do not derive fromits interaction with any early biosynthetic step, but ratherfrom the blockade of the preformed nitrating species, as hasbeen demonstrated. Its slight effect on the production of IL-1�

and its even weaker effect on TNF-� in response to bac-terial LPS corroborate this hypothesis. These two cytokinesare among the most prominent mediators for a number ofinflammatory signals, including the induction of NOS throughtranscriptional activation (Dinarello, 2000). Moreover, IL-1�

can be regarded as a toll-like activator because of the homologyof its receptor with the cytosolic domain of toll-like recep-tors, transmembrane proteins that mount an inflammatorydefense against different exogenous stimuli, including LPS(Takeda and Akira, 2004). It is thus evident that, at phar-macologically significant concentrations, IHG is not able tomodulate NOS activation by restricting cytokine production,nor by any other mechanism.

As a concluding remark, it should be stated that the naturalhydroquinone IHG clearly inhibits the protein tyrosine nitra-tion induced by either peroxynitrite or nitrite/heme in cellularand chemical systems. The relevance of these results is basedon the fact that, unlike many other phenolics, IHG is devoidof strong antioxidant or peroxidase-inhibitory activity. Thus,not only is this selectivity towards tyrosine nitration interest-ing from a mechanistic point of view, but it may also provide amanner in which to further determine the importance of thistype of post-translational amino acid modification in experi-mental models of inflammation.

Acknowledgements

This work was supported by the Spanish Ministry of Sci-ence and Technology (Project SAF 2002-00723). Ana Olmoswas a recipient of a grant from Generalitat Valenciana(CTBPRA/2002/56). We are indebted to the Centre de Trans-fusions de la Comunitat Valenciana and the Departament deFisiologia de la Universitat de Valencia (Valencia, Spain) forgenerous supply of human blood and cultured 3T3 fibroblasts,respectively. We also thank Dr. Rosario Gil-Benso (Departa-ment de Patologia, Universitat de Valencia) for her assistancein cell cultures.

e f e r e n c e s

Augusto, O., Bonini, M.G., Amanso, A.M., Linares, E., Santos, C.C.,De Menezes, S.L., 2002. Nitrogen dioxide and carbonate

radical anion: two emerging radicals in biology. Free Radic.Biol. Med. 32, 841–859.

Benna, J.E., Dang, P.M., Gaudry, M., Fay, M., Morel, F., Hakim, J.,Gougerot-Pocidalo, M.A., 1997. Phosphorylation of therespiratory burst oxidase subunit p67(phox) during human

u t i c

228 e u r o p e a n j o u r n a l o f p h a r m a c e

neutrophil activation. Regulation by protein kinaseC-dependent and independent pathways. J. Biol. Chem. 272,17204–17208.

Bian, K., Gao, Z., Weisbrodt, N., Murad, F., 2003. The nature ofheme/iron-induced protein tyrosine nitration. Proc. Natl.Acad. Sci. U.S.A. 100, 5712–5717.

Dedon, P.C., Tannenbaum, S.R., 2004. Reactive nitrogen species inthe chemical biology of inflammation. Arch. Biochem.Biophys. 423, 12–22.

Dinarello, C.A., 2000. Proinflammatory cytokines. Chest 118,503–508.

Eiserich, J.P., Hristova, M., Cross, C.E., Jones, A.D., Freeman, B.A.,Halliwell, B., van der Vliet, A., 1998. Formation of nitricoxide-derived inflammatory oxidants by myeloperoxidase inneutrophils. Nature 391, 393–397.

Escandell, J.M., Recio, M.C., Manez, S., Giner, R.M., Cerda-Nicolas,M., Rıos, J.L., 2006. Dihydrocucurbitacin B, isolated fromCayaponia tayuya, reduces damage in adjuvant-inducedarthritis. Eur. J. Pharmacol. 532, 145–154.

Gongora, L., Giner, R.M., Manez, S., Recio, M.C., Rıos, J.L., 2001.New prenylhydroquinone glycosides from Phagnalon rupestre.J. Nat. Prod. 64, 1111–1113.

Gongora, L., Giner, R.M., Manez, S., Recio, M.C., Rıos, J.L., 2002.Effects of caffeoyl conjugates of isoprenyl-hidroquinoneglucoside and quinic acid on leukocyte function. Life Sci. 71,2995–3004.

Gunther, M.R., His, L.C., Curtis, J.F., Gierse, J.K., Marnett, L.J., Eling,T.E., Mason, R.P., 1997. Nitric oxide trapping of the tyrosylradical of prostaglandin H synthase-2 leads to tyrosineiminoxyl radical and nitrotyrosine formation. J. Biol. Chem.272, 17086–17090.

Hensley, K., Floyd, R.A., 2002. Reactive oxygen species andprotein oxidation in aging: a look back, a look ahead. Arch.Biochem. Biophys. 397, 377–383.

Herold, S., Rehmann, F.J., 2003. Kinetics of the reactions ofnitrogen monoxide and nitrite with ferryl hemoglobin. FreeRadic. Biol. Med. 34, 531–545.

McCord, J.M., Fridovich, I., 1969. Superoxide dismutase: anenzymatic function for erythrocuprein (hemocuprein). J. Biol.Chem. 244, 6049–6055.

McCord, J.M., Fridovich, I., 1968. The reduction of cytochrome c bymilk xanthine oxidase. J. Biol. Chem. 243, 5753–5760.

Mossman, T., 1983. Rapid colorimetric assay for the cellulargrowth and survival: application to proliferation andcytotoxicity assays. J. Immunol. Methods 65, 55–63.

Olmos, A., Manez, S., Giner, R.M., Recio, M.C., Rıos, J.L., 2005.Isoprenylhydroquinone glucoside: a new non-antioxidantinhibitor of peroxynitrite-mediated tyrosine nitration. Nitric

Oxide 12, 54–60.

Pannala, A., Rice-Evans, C.A., Halliwell, B., Singh, S., 1997.Inhibition of peroxynitrite-mediated tyrosine nitration bycatechin polyphenols. Biochem. Biophys. Res. Commun. 232,164–168.

a l s c i e n c e s 3 0 ( 2 0 0 7 ) 220–228

Paul, A., Pendreigh, R.H., Plevin, R., 1995. Protein kinase C andtyrosine kinase pathways regulatelipopolysaccharide-induced nitric oxide synthase activity inRAW 264.7 murine macrophages. Br. J. Pharmacol. 114,482–488.

Pryor, W.A., Squadrito, G.L., 1995. The chemistry of peroxynitrite:a product from the reaction of nitric oxide with superoxide.Am. J. Physiol. 268, L699–L722.

Robinson, K.A., Stewart, C.A., Pye, Q.N., Nguyen, X., Kenney, L.,Salzman, S., Floyd, R.A., Hensley, K., 1999. Redox-sensitiveprotein phosphatase activity regulates the phosphorylationstate of p38 protein kinase in primary astrocyte culture. J.Neurosci. Res. 55, 724–732.

Serraino, I., Dugo, L., Dugo, P., Mondello, L., Mazzon, E., Dugo, G.,Caputi, A.P., Cuzzocrea, S., 2003. Protective effects ofcyanidin-3-O-glucoside from blackberry extract againstperoxynitrite-induced endothelial dysfunction and vascularfailure. Life Sci. 73, 1097–1114.

Song, Y.S., Park, E.H., Hur, G.M., Ryu, Y.S., Lee, Y.S., Lee, J.Y., Kim,Y.M., Jin, C., 2002. Caffeic acid phenethyl ester inhibits nitricoxide synthase gene expression and enzyme activity. CancerLett. 175, 53–61.

Squadrito, G.L., Pryor, W.A., 2002. Mapping the reaction ofperoxynitrite with CO2: energetics, reactive species, andbiological implications. Chem. Res. Toxicol. 15, 885–895.

Steinbrenner, H., Ramos, M.C., Stuhlmann, D., Mitic, D., Sies, H.,Brenneisen, P., 2005. Tumor promoter PMA stimulates MMP-9secretion from human keratinocytes by activation ofsuperoxide-producing NADPH oxidase. Free Radic. Res. 39,245–253.

Sundaresan, M., Yu, Z.X., Ferrans, V.J., Irani, K., Finkel, T., 1995.Requirement for generation of H2O2 for platelet-derivedgrowth factor signal transduction. Science 270, 296–299.

Szabo, C., 2003. Multiple pathways of peroxynitrite cytotoxicity.Toxicol. Lett. 140/141, 105–112.

Takeda, K., Akira, S., 2004. TLR signaling pathways. Semin.Immunol. 16, 3–9.

Tepperman, B.L., Chang, Q., Soper, B.D., 2000. Protein kinase Cmediates lipopolysaccharide- and phorbol-inducednitric-oxide synthase activity and cellular injury in the ratcolon. J. Pharmacol. Exp. Ther. 295, 1249–1257.

Thomas, D.D., Espey, M.G., Vitek, M.P., Miranda, K.M., Wink, D.A.,2002. Protein nitration is mediated by heme and free metalsthrough Fenton-type chemistry: an alternative to the NO/O2

−

reaction. Proc. Natl. Acad. Sci. U.S.A. 99, 12691–12696.Walrand, S., Valeix, S., Rodriguez, C., Ligot, P., Chassagne, J.,

Vasson, M.P., 2003. Flow cytometry study ofpolymorphonuclear neutrophil oxidative burst: a comparison

of three fluorescent probes. Clin. Chim. Acta 331, 103–110.

Whiteman, M., Szabo, C., Halliwell, B., 1999. Modulation ofperoxynitrite- and hypochlorous acid-induced inactivation of�1-antiproteinase by mercaptoethylguanidine. Br. J.Pharmacol. 126, 1646–1652.