Complex as Nitric Oxide Scavenger in a Stimulated Murine

Bioconjugate Chem. 2003, 14, 1165−1170

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Dinuclear Copper(II) Complex as Nitric Oxide Scavenger in a Stimulated Murine Macrophage Model Laura Chiarantini,*,† Aurora Cerasi,† Luca Giorgi,‡ Mauro Formica,‡ Maria Francesca Ottaviani,‡ Michela Cangiotti,‡ and Vieri Fusi*,‡ Institute of Biochemistry “Giorgio Fornaini” and Istituto di Scienze Chimiche, Universita` degli Studi di Urbino “Carlo Bo”, Italy. Received April 15, 2003; Revised Manuscript Received July 20, 2003

Nitric oxide is a gaseous, short-living free radical which behaves as an important signaling molecule with pleiotropic capacities including vasodilatation, neurotransmission, and microbial and tumor cell killing, as well as in tissue damage and organ-specific autoimmune disorders. Here, a synthesized, dinuclear copper complex system in vitro obtained by the simple aza-phenolic ligand 2,6-bis{[bis-(2aminoethyl)amino]methyl}phenol (L) and Cu(II) ion has been used. The stability constants of ligand L with Cu(II) ion were determined through potentiometric measurements in aqueous solution (37.1 ( 0.1 °C, I ) 0.15 M of NaCl) to mimic the biological medium. The measurements demonstrated that [Cu2H-1L(OH)]2+ (DCu) is the predominant species present in solution at pH 7.4. The molecular structure of the ligand in this species permits the cooperation of the two copper ions in assembling the substrate, thus the complex can be used as a receptor for small molecules such as NO. As a biological model, we chose the production of NO catalyzed by inducible nitric oxide synthase obtained from RAW 264.7 murine macrophage cell line stimulated with LPS, which enabled us to prove that NO is coordinated by the DCu complex, modifying its EPR spectra. The coordination of NO with DCu reduces the level of nitrite in the culture medium of stimulated RAW 264.7 macrophages without any inhibition in the expression of iNOS.

INTRODUCTION

Nitric oxide (NO) can be synthesized by several cell types in mammalian tissues and the mechanism of its synthesis and functions have been the focus of wide investigation (1). NO plays a role as an autocrine and paracrine mediator in several physiological conditions. In particular, the effect of NO in the regulation of vascular homeostasis, as well as its involvement in neurotransmission and in host defense against infectious agents, has been demonstrated (2-4). However, it has also been reported that imbalances in intracellular NO levels may be responsible for various pathological alterations, such as septic shock, hypertension, stroke, and neurodegenerative disease (5). The production of nitric oxide is catalyzed by the enzyme nitric oxide synthase (NOS). NOS is constituted by a family of enzymes that catalyze the NADPH-dependent conversion of L-arginine to nitric oxide and L-citrulline. Three isoforms of nitric oxide synthase have been identified and cloned (6). Brain (nNOS or type I) and endothelial (eNOS or type III) enzymes are constitutively expressed, and their enzymatic activity is regulated by changes in concentration of free Ca2+ (7-9). The third member of the family is the inducible (type II) nitric oxide synthase (iNOS), which is expressed in many different cell types after induction and produces high levels of NO * Corresponding authors. Prof. Laura Chiarantini, Institute of Biochemistry “Giorgio Fornaini”, Universita` degli Studi di Urbino “Carlo Bo”, Via Saffi 2, 61029 Urbino (PU), Italy. Phone: +39 0722 305260. Fax: +39 0722 320188. E-mail: [email protected]. Prof. Vieri Fusi, Istituto di Scienze Chimiche, Universita` degli Studi di Urbino “Carlo Bo”, Piazza Rinascimento 6, Urbino (PU), Italy. † Institute of Biochemistry “Giorgio Fornaini”. ‡ Istituto di Scienze Chimiche.

(6). In fact, iNOS is regulated at the transcriptional level by endotoxin and cytokines and is independent of calcium concentration in the physiological range. When activated by endotoxin, macrophages elicit the formation of inflammatory cytokines, which then activate macrophages and other cells to promote iNOS gene induction. Overproduction of NO, primarily by iNOS, has been implicated in a wide variety of disease states (10). Therefore, it is crucial to regulate the excess NO produced by iNOS when considering inflammatory reactions. Most of the currently used pharmacological inhibitors of iNOS may also affect the other two isoforms of NOS and interfere with unrelated metabolic cell pathways to some degree (2, 3). One strategy for the inhibition of NO is to scavenge or remove excess NO produced during pathological processes, avoiding inhibition of the NO metabolic pathway. For this purpose we have used a dinuclear copper complex [Cu2H-1L(OH)]2+ (DCu) as a scavenger for NO. Binuclear transition metal complexes play a central role in a variety of fields. In fact, binuclear metal complexes are successful devices for the recognition and assembly of external species of various nature such as inorganic and organic species (11-14). Many natural biological sites, such as the active centers of several metalloenzymes, are produced by two transition metal ions; thus synthetic binuclear receptors can be used to mimic the active centers (15-18). Recently, we reported the synthesis and the coordination properties of the noncyclic ligand L (Figure 1) containing a phenol moiety separating two triaza-polyamine fragments toward several M(II) metal cations (19-22). Solid-state and solution studies highlighted the ability of L to assemble two transition metal ions in close proximity. In this way, the two metals cooperate to bind a new species (OH-, Cl-, R-O-, N3-, O2, and others), which is coordinated in a

10.1021/bc030022l CCC: $25.00 © 2003 American Chemical Society Published on Web 11/04/2003

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Figure 1. Ligand L.

Figure 2. Coordination models of the [M2(H-1L)]2+ complex with a potential guest.

bridge disposition between the two metals as schematically depicted in Figure 2. The data demonstrate that the DCu species was able to lower nitrite levels (an indicator of NO presence), in the culture supernatant produced by LPS-stimulated RAW 264.7 macrophage cell line. It is also demonstrated that, through electron paramagnetic resonance (EPR) spectra analysis, the ability of the DCu species to bind the NO produced in vitro. Here it is shown that the DCu species decreases the free NO level but does not interfere with the expression of iNOS protein in macrophage cell line. MATERIAL AND METHODS.

Synthesis. Ligand 2,6-bis{[bis(2-aminoethyl)amino]methyl}phenol L and its solid copper complex [Cu2H-1L(OH)](ClO4)2 were prepared as previously described (19). An aqueous solution at pH 7.4 of this copper complex was freshly prepared by dissolving a known amount of [Cu2H-1L(OH)](ClO4)2 in water and adjusting to pH 7.4 and prompty used. Solvents and starting materials were used as purchased. EMF Measurements. Equilibrium constants for protonation and complexation reactions with L were determined by pH-metric measurements (pH ) -log [H+]) in 0.15 M NaCl at T ) 37.1 °C, using the fully automatic equipment previously described (23); the EMF data were acquired with the PASAT computer program. The combined glass electrode was calibrated as a hydrogen concentration probe by titrating known amounts of HCl with CO2-free NaOH solutions and determining the equivalent point by Gran’s method (24, 25), which gives the standard potential E° and the ionic product of water (pKw ) 13.40 (1) at T ) 37.1 °C in 0.15 M NaCl). At least three potentiometric titrations were performed for each system in the pH range 2.5-11, using different molar ratios of Cu(II)/L ranging from 1:1 to 2:1. The HYPERQUAD computer program was used to process the potentiometric data (26, 27). All titrations were treated either as single sets or as separate entities without significant variation in the values of the determined constants. UV-vis Spectroscopy. UV absorption spectra were recorded at T ) 37.1 °C on a Varian Cary-100 spectrophotometer equipped with a temperature control unit. Cell Culture. All experiments were performed on mouse macrophage cell line RAW 264.7 (kindly supplied by Prof. Umberto Benatti, Genova) cultured in DMEM

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supplemented with 10% heat-inactivated foetal calf serum (FCS), 2.0 mM glutamine, and 1% antibiotics. Cultures were maintained in exponential growth at 37 °C in a humidified atmosphere of 95% air-5% CO2. Cell Activation. RAW 264.7 cells were incubated for 24 h in FCS-free DMEM culture medium at 37 °C at 80% of confluence. Cell stimulation to promote iNOS synthesis in intact murine macrophages was achieved by adding 1 µg/mL lipopolysaccharide (LPS) from Escherichia coli (serotype 0111:B4, Sigma, St. Louis, MO) for 30 min, and then the cells were washed and maintained in the serum free (without phenol red) DMEM medium with several concentrations (0.001-10.0 µM) of DCu or ligand L or Cu(ClO4)2 for 18 h to elicit the expression of iNOS and the production of NO. Cellular Toxicity. RAW 264.7 cells were seeded in 96-well plates (Greiner, International PBI, Italy) at a concentration of 1 × 104 cells/mL (100 µL/well) and treated with several concentrations of DCu or ligand L or Cu(ClO4)2 (0.1-10.0 µM) for 24 h. The in vitro toxicity of RAW 264.7 cells in the presence of several compounds was tested by MTT assay (28, 29). Briefly, 5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) in PBS was sterilized by filtration. At the time indicated, 10 µL of MTT solution was added, and plates were incubated at 37 °C for 4 h. Then 100 µL of 0.4% of 0.04 N HCl in 2-propanol was added and thoroughly mixed to dissolve the dark blue crystals. The plates were then read on a Microplate Reader (Benchmark, Bio-Rad), using a test wavelength of 570 nm and a reference wavelength of 630 nm. The experiments were run in quadruplicate. Measurement of Nitrate/Nitrite. Total nitrate and nitrite was measured by enzymatic reduction of nitrate to nitrite analysis using the Griess assay (30) which provides a measure of total NO produced by the RAW 264.7 cells. Nitrate was converted to nitrite by the action of nitrate reductase from Aspergillus niger (Sigma, Milan, Italy). Briefly, after RAW 264.7 cells were treated with several concentrations of DCu, ligand L, or Cu(ClO4)2 in the presence of LPS, the supernatants were immediately centrifuged at 750g in an Eppendorf microcentrifuge to remove cells in suspension. Then 100 µL aliquots of culture supernatants were incubated with 50 µM NADPH, 5 µM FAD, and 0.01 U of nitrate reductase for 20 min at 37 °C. After this incubation, the medium was mixed with 100 µL of Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthylethylenediamine dihydrochloride in 5% H3PO4) and incubated at room temperature for 10 min. The absorbance at 540 nm was measured with a spectrophotometer. Nitrite levels were determined using NaNO2 as a standard. Detection of iNOS by Western Blot Analysis. iNOS in the RAW 264.7 macrophage cell line was detected by Western blot as described previously (31). Briefly, after cells (1 × 106 cells/dishes) (Φ 35 mm, Sarstedt, Italy) were stimulated with LPS and treated with tested compounds, they were scraped and lysed with a lysis buffer containing 50 mM Tris-HCl, pH 7.6, 0.25 M sucrose, 2% (w/v) SDS, 2 µg/mL leupeptin, 1 µg/mL pepstatin, 2 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM EDTA, and 5 mM N-ethylenemaleimide. Cellular extracts were immediately boiled for 5 min, sonicated to shear the DNA, and centrifuged at 10 000g in an Eppendorf microcentrifuge to remove insoluble debris. Protein content was assayed by Lowry’s method (32). Equal amounts of protein extracts (20 µg) were resolved on 8% SDS-polyacrilamide gels, transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, UK), and then detected with an

Dinuclear Cu(II) Complex as Nitric Oxide Scavenger

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Table 1. Protonation Constants (log K) of L Determined by Means of Potentiometric Measurements in 0.15 M NaCl Aqueous Solution at 37.0 °C reaction

log K

L + H+ ) HL+ HL+ + H+ ) H2L2+ H2L2+ + H+ ) H3L3+ H3L3+ + H+ ) H4L4+ H4L4+ + H+ ) H5L5+

10.47(1)a 9.40(1) 9.02(1) 7.52(2) 2.59(4)

a Values in parentheses are the standard deviations of the last significant figure.

Table 2. Logarithm of the Equilibrium Constants Determined in 0.15 M NaCl Aqueous Solution at 37.0 °C for the Complexation Reactions of L with Cu(II) Ion reaction

log K

Cu2+ + L ) CuL2+ Cu2+ + L + H+ ) CuLH3+ Cu2+ + L + 2H+ ) CuLH24+ Cu2+ + L + 3H+ ) CuLH35+ 2Cu2+ + L ) Cu2L4+ 2Cu2+ + L ) Cu2H-1L3+ + H+ 2Cu2+ + L + H2O ) Cu2H-1L(OH)2+ + 2H+

20.90(2) 29.72(3) 33.13(4) 36.29(7) 29.59(2) 25.03(2) 19.09(5)

a Values in parentheses are the standard deviations of the last significant figure.

antibody specific against iNOS (M-19, Santa Cruz Biotechnology Inc., Santa Cruz, CA). The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG and immune complexes were visualized with the ECL detection kit (Amersham Life Science, UK) according to the manufacturer’s instructions. Membranes were stripped and reprobed with an antibody against actin (Sigma, Milan, Italy). Immunoreactive bands were quantitated by laser densitometry and iNOS levels were normalized to actin. EPR Analysis: Samples and Instrumentation. For EPR analyses, the media from culture supernatant was collected in 2 mm glass tubes sealed at both sides and stored in liquid nitrogen before the measurements. EPR spectra were recorded by means of a EMX-Bruker spectrometer operating at X band (9.5 GHz) and interfaced to a IBM PC computer (Bruker software) for data acquisition and handling. The temperature was controlled with a Bruker ST3000 variable-temperature assembly. The spectra were considered valid only on condition of reproducibility of the spectral line shape. Radical survival was nevertheless controlled by reproducibility of the EPR signal intensity. No variation of the EPR spectra was found after 1 day, after storing the samples in liquid nitrogen. RESULTS

Chemical and UV Analysis. Table 1 and Table 2 report the protonation constant and the stability constants of ligand L with Cu(II), respectively, for both equilibrium reactions determined potentiometrically in aqueous 0.15 M NaCl solution at 37.0 °C. The constants values are similar to those determined in 0.15 M N(CH3)4Cl solution at 25.0 °C previously discussed (19). The experimental condition was chosen to mimic the same culture medium condition of the dinuclear system used in the in vitro studies. The distribution diagram of the species of the L/Cu(II) system as a function of pH is reported in Figure 3. The diagram shows that in the culture medium at pH 7.4, the species [Cu2H-1L(OH)]2+ (DCu) is prevalent in solution an thus can be considered the active species for the interaction with nitric oxide.

Figure 3. Distribution diagrams of the species as a function of pH in 0.15 M NaCl, aqueous solution at 37.0 °C for the system L/Cu(II): [Cu2+] ) 2 × 10-3 M; [L] ) 1 × 10-3 M.

The crystal structure of the DCu species was previously reported (19), and it can be depicted as in Figure 2; in this case, the guest is OH- anion. To understand whether the environment of the two copper ions was modified in the presence of the biological medium, UV spectra were recorded in aqueous solutions containing L and Cu(II) in a 1:2 molar ratio at pH 7.4 using either NaCl ionic strength of the potentiometric measurements or culture medium at 37 °C. Dinuclear complex dissolved in the culture medium was used in the 0.5-100 mM range. The spectrum recorded at pH 7.4 in 0.15 M NaCl aqueous solution, where the DCu species is prevalent in solution, shows two bands at λmax 243 ( ) 9300 cm-1 mol-1 dm3) and 288 ( ) 4900 cm-1 mol-1 dm3) nm, due to the phenolate moiety engaged in the coordination of the metals; in the visible region, two more bands showing λmax 408 nm ( ) 450 cm-1 mol-1 dm3) attributed to phenolate metal charge transfer and a large band showing λmax 725 nm ( ) 420 cm-1 mol-1 dm3), due to the d-d electron transfer, are also observed. These four bands, having approximately the same profiles and , are also present in the culture medium, suggesting that the DCu species are preserved. Effects of DCu on Nitrite Level. Nitrite production was measured in the culture medium of RAW 264.7 murine macrophage cell line treated with DCu species or ligand L or Cu(ClO4)2 (0.001-10.0 µM) as described in Materials and Methods. Nitrite levels in stimulated macrophages, whether treated or not with ligand L or Cu(ClO4)2 at the several concentrations tested, did not show any differences (Figure 4). On the contrary, stimulated macrophages treated with DCu showed a reduced level of nitrite. The percentage of inhibition of nitrite level is shown in Figure 4. All the compounds used were tested to exclude any toxic effect on the cell line. Data are shown in Figure 5. Effects of DCu on iNOS Expression. To examine the effect of DCu, ligand L, or Cu(ClO4)2 on iNOS protein expression, RAW 264.7 cells were stimulated with 1 µg/mL of LPS for 30 min and subsequently treated with the above-mentioned compounds for 18 h. The cellular extracts were prepared and tested for iNOS expression by western blot analysis as described in Materials and Methods. The evaluation of iNOS levels by laser densitometry of the immunoreactive band normalized to actin is reported (Figure 6). Immunoblot analysis of unstimulated macrophage cellular extracts revealed no iNOS expression (Figure 6, lane 1). LPS-activated cells treated with DCu at several

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Figure 4. Effects of DCu on nitrite level. Cells (1 × 106 cells) were incubated for 30 min with LPS (1 µg/mL) and further incubated for 18 h in DMEM (without phenol red) culture medium with several concentrations of DCu (column black), ligand L (column gray) or Cu(ClO4)2 (column white). Nitrite levels in the supernatants of culture medium were measured using Griess reagent as described in Materials and Methods. The values are the mean ( SD of three different experiments. For all samples: p e 10-3 (vs LPS stimulated).

Figure 5. MTT assay in RAW 264.7 cells. Murine macrophage cell line (104/well) was incubated for 24 h with different concentrations (0.1-10 µM) of DCu (column black), ligand L (column gray), or Cu(ClO4)2 (column white). Each data point represents the mean ( SD of quadruplicate samples from two different experiments.

Figure 6. iNOS expression in stimulated RAW 264.7 cells. Immunoblot analysis of iNOS levels in murine macrophages not stimulated (lane 1) and LPS stimulated (lane 2-11). DCu was present at the concentration of 0.001 µM (lane 3); 0.01 µM (lane 4); 0.1 µM (lane 5); 0.5 µM (lane 6); 1.0 µM (lane 7); 2.0 µM (lane 8); 4.0 µM (lane 9); 8.0 µM (lane 10); and 10.0 µM (lane 11). Blots were probed with anti iNOS and actin antibodies. More details on the stimulation protocol are described in Materials and Methods.

concentrations (0.001-10.0 µM) showed no significant effect on the iNOS protein level (Figure 6, lanes 2-11). Even when ligand L or Cu(ClO4)2 were used, the iNOS expression was not affected (data not shown). EPR Analysis. EPR experiments were performed on DCu or ligand L dissolved in NaCl 0.15 M aqueous solution at pH 7.4 or in culture medium. Murine macrophages stimulated with LPS or unstimulated were grown in the presence or absence of DCu or ligand L, and on these media, EPR spectra were performed. Spectra

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Figure 7. EPR spectra at -196 °C of DCu (a) and of murine macrophages stimulated with LPS, grown in the absence (b) and in the presence of DCu (c).

obtained from culture medium alone were also recorded as negative control. All the EPR spectra were recorded at different temperatures, 37 °C, -103 °C, and at -196 °C. Spectra of DCu or ligand L in 0.15 M NaCl aqueous solution in the culture medium or in media obtained from unstimulated macrophages in the presence or absence of the two compounds appeared EPR silent at all temperatures. This is expected since dinuclear copper complexes in solution show a detectable signal only at temperatures low enough to provide a powder spectrum, due to spinexchange effect, ferro- or ferri-magnetic properties correlated to fast relaxation (33-38). On the contrary, DCu in the presence of NO generated by LPS-stimulated macrophages showed a detectable EPR signal. The EPR signals, obtained at -196 °C for stimulated cells in the absence and in the presence of DCu, and for DCu alone, are shown in Figure 7. The broad line, centered at g ) 2.004 (g values were measured compared to diphenylpicryl hydrazide-DPPH (g ) 2.0036) added as an external reference) is attributed, on the basis of similar spectra reported in the literature, to the perpendicular adsorption of mononuclear copper complexes and shows a partial resolution of the hyperfine coupling between the unpaired electron and the nuclear spins of nitrogen nuclei (aii ) 65 G). The parallel adsorptions, centered at g|, approximately 2.21, are poorly resolved and not shown. No further EPR lines were found in different field ranges. The spectrum at 37 °C is a very noisy broad signal (not shown) centered at g, approximately 2.1. The signals shown in Figure 7 address the purpose of the present study: a significant spectral variation occurs because of the linking of NO, produced by stimulated cells, to the dinuclear Cu(II) complex. The spectrum resulting from mononuclear Cu(II), in the presence of NO, indicates that the NO coordination influences the environment of copper ions in DCu. This can occur through the formation of a structure similar to that reported in the literature for an equivalent DCu complex coordinating NO- (39), and probably an electron transfer also occurs between NO and Cu(II). DISCUSSION

In this in vitro biological model, exposure of macrophages to the proinflammatory bacteria LPS induces the expression of iNOS and the consequent production of a large amount of NO responsible for tissue damage and

Dinuclear Cu(II) Complex as Nitric Oxide Scavenger

autoimmune diseases. Therefore, the reduction of NO concentration without interfering with NOS enzyme is desirable. Recent attention has been directed toward the development of synthetic metal receptors, formed by transition metal ions capable of coordinating NO molecules (40). Many of the synthetic metal receptors present copper ion as the coordinating center (41, 42). Synthetic dinuclear complexes, in which the two metal ions are placed in close proximity, are good metal receptors for small molecules; the binding center is formed by both metal ions and they cooperate to bind the molecule in a stable, coordinated bridge disposition between the two metal ions (19, 21, 43). This knowledge prompted us to synthesize a dinuclear copper complex (DCu) having the two Cu(II) ions placed close to each other and to investigate whether DCu acts as a NO scavenger, forming a stable complex. The selectivity of this scavenger for the nitric oxide responsible for pathological effects is not based on specificity for any particular enzyme, but rather on compartmental localization and rate of reaction with NO. To prove that DCu in a physiological environment, such as the culture medium, is the active species for NO coordination, potentiometric and UV/Vis measurements were performed; the formation constants of the complexed species for the system L/Cu(II) calculated by potentiometric measurements in 0.15 M NaCl aqueous solution at 37 °C revealed the presence of a stable dinuclear complex (DCu) in solution (Table 2 and Figure 3), representing the only species present at pH 7.4. Moreover, UV/Vis spectra on L/Cu(II) in aqueous solution or in culture medium proved that the DCu complex is preserved in the biological medium and therefore can be considered the active species interacting with NO. MTT experiments on DCu showed no toxicity on RAW 264.7 in the concentration range tested. The biological experiments show that it is the DCu complex and not the ligand L or Cu(ClO4)2 that was able to reduce nitrite accumulation in the culture medium of the LPS-stimulated RAW 264.7 macrophage cell line (Figure 4) without inhibiting iNOS protein expression as shown in Figure 6. To determine if the DCu complex is involved in NO reduction, EPR experiments were performed to exploit the magnetic properties of both DCu and NO species. As expected, the DCu complex is EPR silent in all media used in absence of NO; this property is due to the closeness of the two Cu(II) ions which, in solution, produce a spin-exchange effect correlated to fast relaxation. As well, the medium containing NO alone is also EPR silent. At the same time, the EPR spectra recorded in the presence of both NO and DCu show detectable signals below -103 °C that could be generated by the presence of a stable DCu-NO complex. These spectra are due to the mononuclear copper complex, and they can be obtained by a spatial separation of the two copper ions in DCu which can be achieved by the coordination of a molecule of NO in a bridge disposition as observed in similar studies (39). However, redox processes involving coordinated NO and Cu(II) ions cannot be excluded although they should represent a minority since the oxidation products of NO (nitrite or nitrate) decrease in the experiments. Certainly, the EPR signals are due to Cu(II) ion when NO binding occurs. This could be explained by the separation of the two Cu(II) ions in the complexed species which behave, for EPR experiments, as a mononuclear complex. In this case, it results that NO acts on DCu complex generating an EPR signal and probably facilitates the electron transfer between the

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metal ions and the ligand. Currently, inorganic chemical studies on the NO-DCu system in aqueous solution are in progress to elucidate these aspects. In fact, all the experiments performed in biological conditions are not suitable to unequivocally identify the stoichiometry of the complex DCu-NO formed, mainly due to the low concentration of the solutions used. However, previous studies (19, 21) on the coordination properties of DCu indicate that it binds external species through cooperation of both metal ions; thus it is possible to hypothesize that DCu binds only one NO molecule in a bridge disposition as shown in Figure 2, preserving the oxidation state of the two copper ions. Our model provides unequivocal evidence that DCu reduces nitrite accumulations in RAW 264.7-stimulated cell line by scavenging NO produced while not inhibiting iNOS expression. The low toxicity of DCu, together with its activity in the cells studied, indicates a potential use in biological models where the removal of NO is advisable. Moreover, the success of DCu as a NO scavenger in vitro points out the potential of the synthesis and investigation of new dinuclear copper complexes suitable for the detection of NO produced in vitro/vivo. ACKNOWLEDGMENT

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