Caffeine inhibits erythrocyte membrane derangement by antioxidant activity and by blocking caspase 3 activation

Biochimie 94 (2012) 393e402

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Research paper

Caffeine inhibits erythrocyte membrane derangement by antioxidant activity and by blocking caspase 3 activation Ester Tellone a, *, Silvana Ficarra a, Annamaria Russo a, Ersilia Bellocco a, Davide Barreca a, Giuseppina Laganà a, Ugo Leuzzi a, Davide Pirolli b, Maria Cristina De Rosa c, Bruno Giardina b, c, Antonio Galtieri a a b c

Organic and Biological Chemistry Department, University of Messina, V. le Ferdinando Stagno d’Alcontres 31, 98166 Messina, Italy Biochemistry and Clinical Biochemistry Institute, Catholic University, School of Medicine, L. go F. Vito n.1, I-00168 Rome, Italy C.N.R. Institute of Chemistry of Molecular Recognition, L. go F. Vito n.1, I-00168 Rome, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 March 2011 Accepted 9 August 2011 Available online 16 August 2011

The aim of this research was to investigate the effect of caffeine on band 3 (the anion exchanger protein), haemoglobin function, caspase 3 activation and glucose-6-phosphate metabolism during the oxygenationedeoxygenation cycle in human red blood cells. A particular attention has been given to the antioxidant activity by using in vitro antioxidant models. Caffeine crosses the erythrocyte membrane and interacts with the two extreme conformational states of haemoglobin (the T and the R-state within the framework of the simple two states allosteric model) with different binding affinities. By promoting the high affinity state (R-state), the caffeineehaemoglobin interaction does enhance the pentose phosphate pathway. This is of benefit for red blood cells since it leads to an increase of NADPH availability. Moreover, caffeine effect on band 3, mediated by haemoglobin, results in an extreme increase of the anion exchange, particularly in oxygenated erythrocytes. This enhances the transport of the endogenously produced CO2 thereby avoiding the production of dangerous secondary radicals (carbonate and nitrogen dioxide) which are harmful to the cellular membrane. Furthermore caffeine destabilizes the haemeeprotein interactions within the haemoglobin molecule and triggers the production of superoxide and met-haemoglobin. However this damaging effect is almost balanced by the surprising scavenger action of the alkaloid with respect to the hydroxyl radical. These experimental findings are supported by in silico docking and molecular dynamics studies and by what we may call the “caspase silence”; in fact, there is no evidence of any caspase 3 activity enhancement; this is likely due to the promotion of positive metabolic conditions which result in an increase of the cellular reducing power. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Band 3 protein Caspase 3 Haemoglobin Metabolism Erythrocytes

1. Introduction The stimulant properties of infusions prepared from coffee beans play a significant role in the worldwide popularity of such beverages and of cola drinks to which caffeine (CF), a purine alkaloid whose properties are linked to the inhibition of adenosine receptors [1], is added as a flavour enhancer. Although CF is widely consumed in beverages, little is known about its numerous biological activities and its biomedical effect. Some studies indicate that the biological effects of CF span a wide range of molecular targets [2]. Hence, it seems to contribute to cancer prevention [3]

* Corresponding author. Tel./fax: þ39 0906765442. E-mail address: [email protected] (E. Tellone). 0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2011.08.007

and to interact with adenosine receptors and phosphodiesterases being also involved in the intracellular Ca2þ release [1,4]. Moreover, CF influences eryptosis [5], intracellular ATP levels [6,7], reduces insulin sensitivity [8] and activates the erythrocyte enzyme glutathione-S-transferase [9]. The mechanisms underlying the anticarcinogenic effects of CF could be related to its effects on cell proliferation, apoptosis [10,11], angiogenesis and/or the immune system [3]. In this context we have to consider that the tumour suppressor gene p53 is a key component of the cellular emergency response which induces either cell-growth arrest or apoptosis [12,13]. Along this line He et al. [14] reported that CF induced apoptosis in JB6 C141 cells through a process involving p53 activation, the over expression of Bax protein and the activation of caspase 3, one of the key components involved in apoptosis.

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Moreover recent studies have shown that CF also influences haeme ligation affinities in monomeric haemoglobin [15] and that it is able to bind into the central cavity of bovine haemoglobin [16]. In addition and in line with previous studies on human haemoglobin (Hb) binding sites for other classes of effectors (2,3diphosphoglycerate, inositol hexaphosphate, bezafibrate) [17,18], the presence of more classes of binding sites at the bovine Hb interface is also evident for CF as well as the formation of complexes with an altered structure of the Tyr and Trp residue environments (b-37 Trp, a-42 Tyr, a-140 Tyr and b-145 Tyr residues located at the a1b2 interface of bovine Hb) [16]. It is also known that Hb interacts with the cytoplasmic domain of the band 3 protein (CDB3) and that this oxygen-dependent interaction underlies a sophisticated regulatory mechanism of anion exchange and metabolism thereby playing a significant role in maintaining the structural and functional integrity of the red blood cell (RBC) [19e22]. Within this framework we have to consider that caspase 3 activation induced by oxidative stress (from pro-caspase to caspase 3) leads to the proteolytic cleavage of CDB3 [23e27], the binding sites for Hb and glycolytic enzymes. This event cancels out the oxygen-dependent modulation of RBC metabolism almost abolishing the ATP release for signal transduction in vascular epithelium [28]. In this work we report a number of experiments in order to correlate the effects of CF with band 3 anion exchange, haemoglobin-oxygen affinity, caspase 3 activation, glucose-6phosphate (G6P) metabolism and antioxidant or pro-oxidant activities. Moreover, the exact position of CF in T and R Hb is not very clear [16], so we also present the results of a computational study aimed at the identification of CF binding site of Hb using the “blind docking” approach, a powerful feature of the AUTODOCK program [29]. The dynamic behaviours of these HbeCF complexes have been also studied through a series of molecular dynamics simulations. These results provide further support to the experimental findings. 2. Methods 2.1. Materials All reagents were purchased from SigmaeAldrich (St. Louis, MO, USA). Citrate fresh human blood was obtained from informed healthy donors who declared that they had abstained from all drug treatment for at least one week prior to sample collection, in accordance with the principles outlined in the Declaration of Helsinki. 2.2. Preparation of erythrocytes Citrate blood samples were washed three times with an isoosmotic NaCl solution. During washing the white blood cells were discarded from the pellet. After washing, the RBC were resuspended (haematocrit 3%) in the incubation buffer (35 mM Na2SO4, 90 mM NaCl, 25 mM HEPES [N-(2-hydroxyethyl)-piperazine-N1-2ethanesulfonic acid], 1.5 mM MgCl2), adjusted to pH 7.4 or 7.3 and 310  20 mOsmol per kg, measured by an Osmostat OM-6020 apparatus (Daiichikagakuco, Kyoto, Japan). In experiments performed with deoxygenated erythrocytes, samples were submitted to cycles of vacuum deoxygenation and nitrogen (ultrapure) saturation at a pressure of 100 kPa. This treatment allowed us to obtain different levels of deoxygenation (from 15% up to 90%), which were checked by determining Hb saturation spectrophotometrically (Beckman DU 70 spectrophotometer) using the millimolar absorptivities reported by Zijlstra et al. [30].

The buffer used to prepare deoxygenated erythrocytes was 0.1 pH unit lower than that used for oxygenated erythrocytes, in order to compensate for the Haldane effect that occurs during deoxygenation [31]. Thus, after deoxygenation, the external pHs of oxygenated and deoxygenated samples differed by no more than 0.03 pH unit. Met-haemoglobin (met-Hb) levels and the degree of haemolysis were determined at the end of the incubation time as follows: haemolysis was determined by measuring spectrophotometrically. Hb concentration in the supernatants obtained from centrifugation at 2500  g for 5 min at 4  C; met-Hb levels were determined spectrophotometrically on lysed cells [30]. 2.3. Preparation of RBC ghosts Washed erythrocytes were lysed with ice-cold hypotonic medium containing 5 mM Tris and 5 mM KCl. After haemolysis the Hb and the intracellular contents were eliminated by centrifugation. The membranes were resealed by incubation at 37  C for 1 h with a closing buffer (20 mM HEPES, 130 mM NaCl, 1.5 mM MgCl2, pH 7.4) and used for sulphate transport determination. 2.4. Kinetic measurements Cells were incubated in the above incubation buffer at 25  C, under different experimental conditions. At several time intervals, 10 mmol of the stopping medium SITS (4-acetamido-40 -isothiocyanostilbene-2,20 -disulfonic acid) was added to each test tube containing the RBC suspension at periodic time intervals. Cells were separated from the incubation medium by centrifugation (J2-HS Centrifuge, Beckman, Palo Alto, CA, USA) and washed three times at 4  C with a sulphate-free medium to remove the sulphate trapped on the outside. After the final washing, the packed cells were lysed with perchloric acid (4%) and distilled water. Lysates were centrifuged for 10 min at 4000  g (4  C) and membranes were separated from the supernatant. Sulphate ions were precipitated from the supernatant by adding a glycerol/distilled water mixture (1:1, V/V), 4 M NaCl and 1 M HCl, 1.23 M BaCl2$2H2O to obtain a homogeneous barium sulphate precipitate. The absorbance of this suspension was measured at 350e425 nm. Sulphate concentration was determined using a calibrated standard curve, obtained by measuring the absorbance of suspensions with known amounts of sulphate [32]. The experimental data on sulphate concentration as a function of the incubation time were analyzed by best fit procedures using the following equation: c(t) ¼ cN(1  ekt) where c(t) represents sulphate concentration at time t, cN intracellular sulphate concentration at equilibrium, and k the rate constant of sulphate influx. 2.5. Measurement of oxygen dissociation curves (ODC) Erythrocytes and Hb solutions were prepared as previously described [33]. Stripped Hb was obtained by passing the haemolysate through a Sephadex G-25 column equilibrated with 0.1 M HEPES buffer, pH 7.4, containing 0.1 M NaCl. Concentrated stock solutions of 2,3-diphosphoglycerate (2,3-DPG) and CF were prepared by dissolving them in HEPES buffer. A number of O2 equilibrium experiments were performed on purified Hb in order to obtain quantitative data on oxygen affinity. The oxygen binding properties of human Hb solutions were investigated at pH 7.4 both in the absence and in presence of 100 mM CF. Values of P50 (partial pressure of the ligand at which 50% of haemes is oxygenated) and n (Hill coefficient; an empirical index of cooperativity), were determined at 37  C from absorbance changes accompanying the dioxygen molecule binding by the

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tonometric method, in the presence of the physiological allosteric effector 2,3-DPG, as well as in the presence of CF [33]. An average standard deviation of 8% for values of P50 was calculated. Met-Hb content never exceeding 2% in any of the experimental conditions. 2.6. Determination of Phosphatase PTP-1B activity Phosphatase PTP-1B activity was determined using p-nitrophenyl phosphate (p-NPP) as substrate. Briefly, membranes were suspended in 25 mM HEPES buffer of pH 7.3, containing 0.1 mM phenylmethanesulfonylfluoride (PMSF), 20 mM MgCl2 and 15 mM p-NPP, and incubated at 37  C for 30 min. After centrifugation, the release of p-nitrophenol was measured in the supernatant at 410 nm [34]. 2.7. 2,2-Diphenyl-1-picrylhydrazyl assay The free radical scavenging effect of CF was assessed by the free radical method as previously reported [35] using a stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) which forms a violet solution and reacts with antioxidants losing its colour. The colour loss and subsequent fall in absorbance is correlated to antioxidant content of the sample. Test compounds at a concentrations ranging from 1 to 100 mM, in a final volume of 3.0 ml, were mixed with 80 mM DPPH in methanol. The changes in absorbance at 517 nm were monitored over 30 min. DPPH concentration in the cuvette was chosen to give absorbance values less than 1.0. The inhibition percentage (%) of radical scavenging activity was calculated by the following equation:

Inhibitionð%Þ ¼

Ao  As  100 Ao

where Ao is absorbance of the control and As is absorbance of the sample after 30 min of incubation. 2.8. Caspase 3 assay Citrate blood samples were washed three times with an isoosmotic NaCl solution. The white blood cells were discarded from the pellet during washing. After washing, the RBC were resuspended (haematocrit 3%) in the incubation buffer (35 mM Na2SO4, 90 mM NaCl, 25 mM HEPES, 1.5 mM MgCl2), adjusted to pH 7.4 and incubated for 2 h at 37  C in the absence or in the presence of 100 mM of CF or 100 mM of tert-butyl-hydroperoxide (t-BHT). After treatment, erythrocytes were collected by centrifugation at 3000 rpm for 5 min, resuspended in HEPES buffer [100 mM HEPES pH 7.5, 20% glycerol, 5 mM DTT and 0.5 mM ethylenediaminetetraacetic acid (EDTA)] and lysed by sonication. The cell lysates were clarified by centrifugation at 15,000 rpm for 10 min. The supernatant was passed through Microcon YM 30 (Nominal Molecular Weight Limit 30,000) to obtained a partial purification of caspase 3. The cell lysates (100 ml) were incubated at 37  C for 1 h with enzyme-specific colorimetric substrates (AcDEVD-pNA 100 mM in HEPES buffer) in a final volume of 600 ml. Caspase 3 activity was analyzed with a spectrophotometer after pNA release at 405 nm, and is expressed as the n-fold value of the untreated sample compared with that obtained with purified caspase 3. 2.9. Antioxidant effects of CF on t-BHT The same experimental condition described in Caspase 3 assay was used to analyze potential antioxidant effects of CF (100 mM) against t-BHT (100 mM), incubating the erythrocyte in presence of

Fig. 1. Hill plot for the binding of oxygen to Hb in the absence (closed symbols) and in the presence (open symbols) of 100 mM CF. Conditions: 1  101 M HEPES buffer plus 1  101 M NaCl and 3  103 M 2,3-DPG at pH 7.4, 37  C. For further details see “Methods” section.

the later alone or both substances. Additional control assays with the presence 100 mM of specific caspase 3 inhibitor (Z-DEVD-FMK) or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) in the samples treated with 100 mM of t-BHT were performed. 2.10. CF effects on superoxide anion generation Superoxide anions were generated using the Nishimiki method with minor modifications [36]. The reaction mix was made up of: 1 ml nitroblue tetrazolium (NBT) solution (156 mM NBT in 100 mM phosphate buffer, pH 7.4) 1 ml NADH solution (468 mM in 100 mM phosphate buffer, pH 7.4) and increasing concentrations (0e100 mM) of CF. The reaction was started by adding 100 ml of phenazine methosulphate (PMS) solution (60 mM PMS in 100 mM phosphate buffer, pH 7.4) to the mixture. The reaction mixture was incubated at 25  C for 5 min, and absorbance at 560 nm was measured against a blank sample. Decreased absorbance of the reaction mixture indicated increased superoxide anion scavenging activity. 2.11. Binding of CF to Hb Purified Hb (0.7 mg/ml) in the deoxygenated and oxygenated state was incubated for 1 h in the presence of 100 mM CF. The samples were filtered with 0.45 mm filter and unbound CF was analyzed by High Performance Liquid Chromatography (HPLC). Chromatographic analysis was performed using a Shimazu system, consisting of a LC-10AD pump system, an SPD-M10A diode array detector, a Rheodyne 7725i injector with a 20 ml sample loop and a reverse-phase Supelco C18 column (5 mm, 250  4.6 mm). The Table 1 CF binding site in R-state Hb, as calculated by AUTODOCK. Residues at a maximum distance of 4.5 Å from CF are reported. Human oxy-haemoglobin (2DN1) Binding site ID

AUTODOCK binding energy (kcal/mol)

Caffeine binding site residues

1

2.6

Leu66(a1), Leu105(a1), Leu109(a1), Leu125(a1), Leu129(a1), Ala63(a1), Phe128(a1), Val10(a1), Trp14(a1), Thr67(a1), Val70(a1)

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Table 2 CF binding sites in T-state Hb, as calculated by AUTODOCK. Residues at a maximum distance of 4.5 Å from CF are reported. Human deoxy-haemoglobin (2DN2) Binding site ID

AUTODOCK binding energy (kcal/mol)

Caffeine binding site residues

1

2.5

2

2.2

3

2.2

4

2.1

Pro95(a1), Thr137(a1), Ser138(a1), Tyr140(a1), Arg141(a1), Trp37(b2), Val1(a2) Glu39(b1), Glu43(b1), Phe42(b1), Gly46(b1), Gly47(b1), Leu48(b1), Ser49(b1) HEM(a1), Lys61(a1), Ala65(a1),Asn68(a1), Ala79(a1), Leu80(a1), Ala82(a1), Leu83(a1) HEM(b2), Arg40(b2), Phe41(b2), Glu43(b2), Ser44(b2), Leu96(b2)

mobile phase was acetonitrileewater (75e25 v/v). The flow rate was 1.0 ml/min at 25  C. CF was detected at 276 nm and determined by comparison of peak areas with a standard solution of 100 mM of CF. 2.12. Hydroxyl radical scavenging Hydroxyl radical was assayed as described by Halliwell et al. [37] with a slight modification. This assay is based on quantification of the degradation product of 2-deoxyribose by condensation with thiobarbituric acid (TBA). The reaction mixture contained, in a final volume of 1 ml: 2.8 mM 2-deoxy-2-ribose, 10 mM phosphate buffer

pH 7.4, 25 mM FeCl3; 100 mM EDTA; 2.8 mM H2O2; 100 mM ascorbic acid and varying concentrations (0e100 mM) of the test sample. The sample was incubated for 1 h at 37  0.5  C in a water bath. At the end of the incubation period, 1 ml of 1% (w/v) TBA was added to each mixture followed by the addition of 1 ml of 2.8% (w/v) trichloroacetic acid (TCA). The solutions were heated in a water bath at 100  C for 15 min to develop the pink coloured malondialdehydeethiobarbituric acid adduct. After cooling, the absorbance was measured at 532 nm against an appropriate blank solution. All tests were performed three times. Trolox was used as a positive control. The inhibition (%) of test compounds was calculated using the following formula:

Inhibitionð%Þ ¼

Ao  As  100 Ao

2.13. CF docking to T and R Hb Visualization and molecular graphics were done using Discovery Studio (Accelrys Inc.) on a HP XW8600 workstation running Red Hat Enterprise Linux 5. Ligand binding studies were carried out using AUTODOCK (version 4.2) suite of programs [29]. A Lamarckian genetic algorithm (LGA) searches the conformational space of the ligand in the vicinity of the macromolecule and ranks the docked molecules on the basis of its binding energy. Its graphical front-end, AUTODOCKTOOLS was used to add polar hydrogens and partial charges for proteins and ligands using the

Fig. 2. View of the binding modes of CF on (A) R-state and (B) T-state Hb suggested by molecular docking studies. Each cluster position is defined in Tables 1 and 2. Also illustrated for the purpose of comparison are (C) the corresponding region in the T-state and (D) the R-state. a chains (red) and b chains (yellow) of Hb are shown in ribbon representation. Haeme (green) and CF (blue) are displayed in stick representation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Fluctuations of CFefree (black) and CFebound (red) Hbs. Shown is root mean square fluctuations (RMSF) of the Ca atoms relative to the average MD structures of oxy- (A) and deoxyHb (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Kollman United Atom and Gasteiger charges, respectively; AUTOGRID pre-calculates a three-dimensional grid of interaction energy based on molecular coordinates; and AUTODOCK performs docking simulations. For CF, the coordinates were taken from the ZINC database, entry 1084; for T and R states of Hb, the crystallographic structures of deoxygenated and oxygenated human haemoglobin (deoxyHb and oxyHb) were obtained from the Protein Data Bank, PDB ID: codes: 2DN2 and 2DN1, respectively [38]. The default settings of AUTODOCK were used, with the exception of the docking runs, which were set to 200. The docking process was performed in two steps. In the first, the docking procedure was applied to the whole protein target, without imposing the binding site (“blind docking”). The grid field was a 76 Å cube, with grid points separated by 0.6 Å, centred on the Hb molecule. In the second step, we docked the ligands in each of the binding sites found in the first step (“refined docking”). This time, the grid field was a 25 Å cube with grid points separated by 0.2 Å centred on the best scored conformation obtained in the first step. 2.14. Molecular dynamics simulations The lowest energy HbeCF conformations obtained from the docking simulations were submitted to aqueous-phase molecular dynamics (MD) simulations. For the purpose of comparison, MD simulations on the CFefree deoxyHb and oxyHb structures were also performed. The MD simulations and the analysis of the trajectories were carried out using GROMACS 4.0.7 [39] and the GROMOS96 43a1 force field [40]. All starting structures were immersed in a cubic box with periodic boundary conditions and were solvated with explicit SPC water molecules. Each system was then neutralized by 6 Naþ counter ions that were added at random positions to the bulk solvent. The box dimensions (9.0 nm  9.0 nm  9.0 nm) were set to allow at least 1.0 nm between the protein and the box faces on each side. Following steepest descents minimization, the systems were equilibrated under NVT conditions for 100 ps at 300 K, followed by 100 ps under NPT conditions, applying position restraints to the protein and ligand atoms. Finally, all restraints were removed and the molecular dynamics were run for 5 ns at 300 K. van der Waals interactions were modelled using 6-12 Lennard-Jones potentials with a 1.4 nm cutoff. Long-range electrostatic interactions

were calculated using Particle Mesh Ewald method, with a cutoff for the real space term of 0.9 nm. Covalent bonds were constrained using the LINCS algorithm. The time step employed was 2 fs and the coordinates were saved every 2 ps for analysis which was carried out using the standard GROMACS tools. 2.15. Statistical analysis Data are presented as mean  standard deviation (S.D.). The data were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s HSD. A P < 0.05 was regarded as indicating a significant difference. 3. Results and discussion HPLC experiments show that CFeHb binding is oxygendependent being related to the TeR transition of Hb. In particular oxyHb presents a greater affinity towards CF than the deoxyHb, as indicated by the amount of free CF measured in the oxygenated state (60 mM) compared to deoxygenated one (77 mM) and by the partial pressure of oxygen expressed as log P50 that goes from 1.119 (control value) to 1.004 in the presence of 100 mM CF (Fig. 1) supporting a significant stabilization of the high affinity state of Hb. Based on these evidences we performed the analysis of CF docking to T and R Hb states: the resulting docked conformations were clustered into families of similar binding modes, with a root mean square deviation (RMSD) clustering tolerance of 2 Å. We should recall that a small number of clusters indicate strong and

Table 3 Effect of CF concentrations on the rates of sulphate transport in RBCs at high (HOS) and low (LOS) oxygen saturation and in ghosts. Statistical analysis were performed by one-way ANOVA, followed by Tukey’s HSD. Each samples shows a significant difference at P < 0.05.

HOS LOS Ghosts

Rate constant (min1)

Rate constant (min1)

Control

Plus 100 mM CF

0.012  0.0025 (n ¼ 6) 0.0052  0.0004 (n ¼ 5) 0.034  0.0025 (n ¼ 5)

0.034  0.0054 (n ¼ 4) 0.0062  0.0006 (n ¼ 5) 0.046  0.005 (n ¼ 4)

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Fig. 4. Effects of additives on erythrocytic caspase 3 activity. RBCs were incubated and treated as described in “Methods” to analyze caspase 3 activity in the absence (a) or in the presence of: 100 mM of CF in HOS (b) and LOS (c); 100 mM of t-BHT (e); 100 mM of t-BHT plus 100 mM of CF (f); 100 mM of t-BHT plus 100 mM of trolox (g); 100 mM of t-BHT plus 100 mM of Z-DEVD-FMK (h). The assay was also performed with 7.5 Unit of purified caspase 3 (d). Values are the means  standard deviation of at least three experiments (n ¼ 3). The differences between control and compounds-treated erythrocytes were analyzed by one-way ANOVA, followed by Tukey’s HSD. Different number of asterisk indicates significant differences at P < 0.05.

specific binding, while a large number of clusters indicates a weaker or lower specificity of binding as the solutions sort into many different binding conformations or orientations. Due to the symmetry of the haemoglobin molecule, redundant orientations were eliminated. Analysis of the “blind docking” step, i.e. without prior specification of the binding site, shows that AUTODOCK predicted in R Hb only one binding cluster with significantly lower binding energy compared with the other ones. Differently from the R-state, molecular docking reveals 4 significant clusters of sites for CF on T-state Hb. Tables 1 and 2 list energetically favourable CF binding models at the end of the “refined docking” step when ligands were docked in each of the binding sites previously found and the use of an improved grid resolution allowed better evaluation of the proteineligand interactions. Residues identified as being no more than 4.5 Å from the docked CF are reported in the tables. When CF molecule was docked into R-state Hb, it was found to be embedded in a hydrophobic cavity of the alpha subunit. Helices A, B and E constitute the “wall” of the cavity, whereas the “floor” is represented by helices G and H (Fig. 2A). In the calculated model, the carbonyl oxygen atom in 2-position of CF forms a hydrogen bond with the OG1 atom of Thr67 (E16) of R-state Hb (Fig. 2A). A number of hydrophobic interactions involving Trp14 (A14), Ala63 (E12), Leu125 (H8), Phe128 (H11), Leu129 (H12) further stabilize CF molecule (Table 1). In the case of T-state Hb the models fall broadly into four clusters, which we have designated 1, 2, 3 and 4 (Table 2). Our computational determinations indicate that CF molecule occupies only surface binding sites which are highlighted in Fig. 2B and listed in Table 2. The lowest energy orientation found (cluster 1) is one in which CF is located at one end of the Hb central cavity, between the a chains and external to oxygen-linked chloride sites which are present in the a chains between a1-Val1 and the hydroxyl group of a1-Ser131 and between a1-Val1 and a2-Arg141. For comparison, the corresponding region in the T-state and the R-state are shown in Fig. 2C and D, respectively. On the whole analysis of docking results suggests that CF molecule has higher specificity for R-state Hb than for T-state Hb.

Moreover, we have to outline that in the T-state the CF molecule interacts with the Hb molecule in a region very near to the haeme (Fig. 2B). This could in some way lead to an alteration of the redox properties of the haeme-iron which may become more susceptible towards oxidation. While the docking methodology used to study the binding of CF to Hb allows for flexibility of the ligands, it does not include protein mobility. In order to take into account the protein flexibility and evaluate the dynamic stability of the predicted CF/Hb interactions, the lowest energy conformations obtained from docking were submitted to MD simulations for 5 ns at 300 K. For comparison, MD simulations on the CFefree Hb structures were also performed. MD trajectories suggest that both oxy- and deoxyHb produce stable complexes with CF. The rms deviation (RMSD) of each system through the 5 ns trajectory was computed with respect to its corresponding initial structure. The Ca RMSD values fluctuate for the last 2 ns of the MD run around values of 1.8  0.1 Å (CFebound oxyHb), 2.5  0.2 Å (CFebound deoxyHb) and 2.3  0.1 Å (CFefree oxy- and deoxyHb). To distinguish how protein flexibility is altered by the presence of the bound CF, the rms fluctuations (RMSF) of the Ca residues for the free and bound Hbs were calculated and plotted as a function of the residue number. The profiles of the atomic fluctuations of the

Table 4 Effect of CF concentration on met-Hb and haemolysis levels in RBCs. Met-Hb and haemolysis levels were measured at the end of the incubation time of erythrocytes without (control) and with (CF) 100 mM CF. Each point is the mean value, obtained from four different experimentations (n ¼ 4). Conditions

Incubation time (min)

Met-Hb %

Haemolysis %

Control CF Control CF Control CF

15 15 90 90 Over night Over night

0 1.4  1 0 17.6  2 0 18.2  2

0 0 0 0 0 21.23  2

E. Tellone et al. / Biochimie 94 (2012) 393e402

Fold of control

1.3

1.2

1.1

1.0

0.9 0

90

100

80

90

70

80

40 30

70 50 40 30

10

20 0 10 20 30 40 50 60 70 80 90 100 Concentration (µM)

Fig. 5. Scavenger activity of (-) CF, (:) butylated hydroxy anisole, (C) butylated hydroxy toluene and (A) ascorbic acid against DPPH radical at 517 nm, in the range of concentration 0e100 mM. Each point is the mean value, obtained from three different experimentations (n ¼ 3).

Caffeine Trolox

60

20 0

100

drives RBC towards an increase of the oxidative stress which in turn may cause a significant superoxide release. It may useful to recall that the damaging activity of superoxide derives from its potential role as a generator of hydroxyl radicals via the reactive oxygen species (ROS) chain. Indeed, molecules which are able to destabilize the haemeeprotein interactions and erythrocytic abnormalities (such as those observed in sickle cell anaemia, thalassaemia and glucose-6-phosphate dehydrogenase deficiency) decrease the erythrocyte lifespan due to an increase in the met-Hb content and oxidative stress [21]. This in turn may activate caspase 3 with a consequent membrane derangement and haemolysis [19,41,42]. Paradoxically our experiments on caspase 3 activity on RBC incubated with CF suggested an unexpected antioxidant action of the xanthine. In this light we have tested the antioxidant properties of CF in order to clarify the mechanism of its scavenger action. We have investigated the effect of CF (at a concentration of 100 mM) with respect to t-BHT (at the same concentration 100 mM), a known generator of ROS, which therefore may lead to activation of caspase 3. The results obtained indicate that CF protects the erythrocytes against cytotoxic molecules and from the apoptotic effects of t-BHT, inducing about 10% inhibition of caspase 3 activation (Fig. 4). The caspase 3 activity has been tested also in the presence of a well

110

50

50

Fig. 6. Effect of CF (0e100 mM) on superoxide anion radical generation. Each point is the mean value, obtained from three different experimentations (n ¼ 3).

100

60

25

Concentration (µM)

Abs decrease at 532 (%)

Abs decrease at 517 nm (%)

CFeoxyHb complex were found to be very similar to those of the unbound protein (Fig. 3A). These calculations also reveal that the binding of CF to the a chains of deoxyHb (binding site 1, Table 2) more significantly affects Hb residues (Fig. 3B). Interestingly, important long-range allosteric effects are observed (Fig. 3B) and residues located in helices B, E and F of b subunits have among the greatest RMSF values; in particular a value of 1.5 Å was obtained for the (F8) proximal histidine b2-His92 (0.8 Å in the CF-free structure), and a value of 1.5 and 1.7 Å for the (E7) distal histidines b2- and b1His63, (0.8 and 0.9 Å in the CF-free structure, respectively). The molecular dynamics simulation studies, therefore, further support the hypothesis that CF may destabilize haemeeprotein interactions. Since the oxygen-linked transition of Hb influences the anion exchanger activity of band 3 protein in RBC [20], the kinetics of sulphate flux across the erythrocyte membrane in the presence of CF was investigated (Table 3). It can be seen that CF does modulate the function of the protein since its rate constant changes from 0.012 min1 (control value) to 0.034 min1 in the presence of 100 mM CF in the oxygenated erythrocytes (HOS, high-oxygenation state, about 90% saturation). The same type of experiments performed on the deoxygenated RBC (LOS, low-oxygenation state, about 15% saturation) show that the rate constant goes from 0.0052 min1 in the absence of CF to 0.0062 min1 in the presence of CF. Statistical analysis further supports this hypothesis with a P < 0.05. Of particular interest is the negligible effect of CF shown under LOS conditions when the deoxyHbeCF interaction is greatly reduced. This, in fact, suggests an indirect effect of the alkaloid, which would influence band 3 function through Hb and not by a direct interaction with the membrane protein. Phosphatase activity in the presence of CF was tested in order to exclude any possible involvement of the phosphorylation state of the erythrocyte membrane. The data obtained did not reveal any alterations of the phosphorylation-dephosphorylation balance (data not shown). However, the remarkable modulation of CF on anion flux measured in HOS and its virtual absence in LOS led us to investigate the possible role of caspase 3 in the functional modulation of the exchanger. The results obtained both in LOS and HOS erythrocytes treated with 100 mM CF or 100 mM t-BHT clearly show that, in contrast to the t-BHT, CF does not induce caspase 3 activation (Fig. 4). However, RBC incubated with CF show a higher than normal met-Hb concentration (Table 4). This change in met-Hb values

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10 0

0 10 20 30 40 50 60 70 80 90 100 Caffeine [µM]

Fig. 7. Effect of CF (0e100 mM) and trolox on deoxyribose degradation assay. Results are means  S.D of three parallel measurements (n ¼ 3).

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Fig. 8. CF influences on metabolism and signal cascade activation on RBCs (the schematic representation take into account only the reaction products involved in the CF effects). aebec: metabolic pathways of peroxynitrite (ONOO); bold arrows indicate the shift of metabolism in the presence of CF.

known irreversible inhibitor, Z-DEVD-FMK and in the presence of Trolox, a water-soluble derivative of vitamin E. As shown in Fig. 4 both compounds completely avoided caspase 3 activation. In particular the experiment performed with specific caspase 3 inhibitor (Z-DEVD-FMK) shows the absence of non-specific hydrolysis of the substrate, further supporting our experimental evidences. DPPH radical scavenging activity of CF is shown in Fig. 5. The addition of increasing concentrations (up to 100 mM) of CF to DPPH solution did not induce a significant decrease of absorbance at 517 nm. Hence the activity is clearly lower with respect to that of ascorbic acid and synthetic antioxidant (butylated hydroxy toluene and butylated hydroxy anisole) tested under the same experimental conditions. The effects of increasing CF concentrations (0e100 mM) on superoxide anion were also analyzed and the results are presented in Fig. 6. In the PMS/NADH-NBT system, NBT is reduced by the superoxide anion produced via the dissolved oxygen and the PMS/ NADH coupling reaction. Interestingly, CF induced an increase of the absorbance value at 560 nm at all concentrations tested. In particular, the observed increase was w1.053, 1.138 and 1.186 fold in the presence of 25, 50, and 100 mM of CF respectively. In order to better characterize the CF scavenging activity of hydroxyl radicals we performed the deoxyribose assay. This assay mimics the Fenton type reaction in vitro, generating the hydroxyl radical that is able to degrade the sugar deoxyribose into fragments. As shown in Fig. 7, CF exhibited a powerful and concentration dependent scavenging activity towards the hydroxyl radical. Although the hydroxyl radical scavenging ability of the different CF concentrations were lower than that of trolox, a commercial antioxidant, however the alkaloid was able to eliminate about 85% of the hydroxyl radical just at a concentration of 44 mM. In fact, CF IC50 of 16.2 mM was only 1.6 fold higher than that of trolox. 4. Conclusion As already observed with some drugs such as Bezafibrate, Gemfibrozil and Resveratrol and in line with the global allostery

theory by Tsuneshige et al. [18], CF interacts both with the T and R states of the Hb molecule [17,18,39,43e45]. In this respect, the different values of the rate constant (Table 3) measured for the two oxygenation states (HOS and LOS) of the erythrocyte, may well be due to the different free binding energies between oxyHbeCF and deoxyHbeCF as confirmed by the docking procedure and the resulting complexes with CDB3 [46]. Thus, the presence of CF would promote a rapid oxygenation in the lung by maintaining a gradual release of oxygen to tissue level. It should be noted that the CF destabilization of the haemeeprotein interactions, the consequent formation of superoxide and the observed pro-oxidant effect induced by CF (Figs. 2B and 6, Table 4), are almost counterbalanced by the highly efficient antioxidant activity with respect of the hydroxyl radical (one of the most harmful oxygen species converging in the ROS chain from several pathways) (Fig. 8). Hence we have to consider that destabilizing events, if not promptly neutralized, could lead the RBC towards the molecular derangement of the membrane and a substantial haemolysis due to caspase 3 activation (performed by ROS) which causes CDB3 cleavage. All this does indeed occur in a sort of harmful and vicious cycle fuelled by the preferential channelling of G6P in the EmbdeneMeyerhof pathway (EMP) at the expense of the pentose phosphate pathway (PPP) with a consequent further increase of the oxidative stress [19,39,41]. The scavenger activity of CF with respect to the hydroxyl radical, appears even at low concentration of CF, about 20 mM, an amount comparable to that contained in plasma of an ordinary coffee consumer [2]. These findings were further supported by the absence of any caspase 3 activation. Our data show that moderate CF amounts may prevent some membrane derangements. At this regard, it is important to remember that the positive effects of CF (at low doses) reduce risk of Alzheimer’s disease and type 2 diabetes insurgence and contribute to weight control [47e49]. The above considerations are consistent with the stability observed in the phosphorylation-dephosphorylation balance of RBC, since any alteration of this status would increase the oxidative stress and would contribute to caspase 3 activation [41], phosphatidylserine exposure at the cell surface, cell shrinkage and

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eryptosis, giving rise to a process very similar to erythrocyte senescence [5,50,51]. Furthermore, it isn’t speculative to assert the existence for CF of wide unknown human population “niches” who unconsciously make therapeutic use of the drug (like resveratrol in the French paradox). On the whole the effects of CF on RBCs can be summarized as follows: 1. CF modulates the anion flux strongly in HOS, responding adequately to the exo- and endogenous CO2 production; 2. CF binds Hb with a higher specificity for R-state than T-state Hb and destabilizes the Hb molecule leading to the appearance of met-Hb and superoxide; 3. CF shows a pro-oxidant activity with respect of superoxide, which is compensated by a strong antioxidant capacity towards the most harmful of oxygen radicals, the hydroxyl one. In this way, it contributes to prevent caspase 3 activation (Fig. 8). In this respect, “caspase silence” is certainly promoted by the overall efficiency of the antioxidant activity performed by CF and it also ensures the structural integrity of CDB3, which is essential not only for physiological anion exchange through B3, but also for the time-dependent “control” of the G6P flux towards EMP or PPP. This time-dependent metabolic modulation is necessary for an adequate production of ATP, useful as a signalling molecule that is “free” to leave the erythrocyte [52]. In turn ATP once released into the lumen can regulate vascular resistance via stimulation of purinergic receptors located on vascular endothelial cells, leading to the synthesis of NO [28]. Thus, CF indirectly affects NO production in the capillary endothelium by NO synthase, also allowing the microcirculation of senescent RBC. Indeed, Zeling et al. [53] have recently demonstrated that the release of ATP from RBC requires an increased binding of nitritemodified Hb to the RBC membrane. This would displace glycolytic enzymes from CDB3 resulting in the formation of an ATP “pool” which can be released into the extracellular medium. Thus the integrity of CDB3 is also at the base of the ATP/NO bidirectional flow between RBCs and the vascular epithelium, through a mutual metabolic modulation of their synthesis and relative paracrine signal. In addition, we have to outline some positive effects specifically related to the direct and indirect antioxidant action of CF. Hence, in HOS CF increases anion kinetics by a factor of about 3, performing a further and indirect antioxidant action through the rapid removal of CO2 by preventing the subsequent formation of secondary radicals such as carbonate and nitrogen dioxide (Fig. 8). Alternatively, peroxynitrite or peroxynitrous acid may also undergo homolytic fission to generate one-electron oxidants, hydroxyl and nitrogen dioxide radicals (with a 30% yield) [54]. However, this event does not perturb too much the erythrocyte, because of the high degree of scavenger efficiency against the hydroxyl radical exerted by CF. In addition, we cannot overlook the third path that peroxynitrite can follow. In fact, the fast isomerization to nitrate by reaction with oxyHb and the concomitant formation of met-Hb and superoxide [55], would also explain the higher met-Hb values observed in the presence of CF. Last but not least it would give further support to the observed stability of pro-caspase. Indeed, the superoxide produced can be converted to peroxynitrite or it could take the Fenton or HabereWeiss reactions path inducing the production of the hydroxyl radical which however is readily neutralized by CF. On the whole, further studies are needed to describe and clarify the different functions and the molecular targets of this interesting molecule in order to gain insight into its possible therapeutic use.

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Acknowledgements Computational resources for molecular dynamics simulations were provided by CASPUR (Consorzio interuniversitario per le Applicazioni di Supercalcolo Per Università e Ricerca). References [1] J.W. Daly, Caffeine analogs: biomedical impact, Cell. Mol. Life Sci. 64 (2007) 2153e2169. [2] L.A. Horrigan, J.P. Kelly, T.J. Connor, Immunomodulatory effects of caffeine: friend or foe? Pharmacol. Ther. 111 (2006) 877e892. [3] Y.R. Lou, Y.P. Lu, J.G. Xie, M.T. Huang, A.H. Conney, Effects of oral administration of tea, decaffeinated tea, and caffeine on the formation and growth of tumors in high-risk SKH-1 mice previously treated with ultraviolet B light, Nutr. Cancer 33 (1999) 146e153. [4] N. Teramoto, T. Yunoki, K. Tanaka, M. Takano, I. Masaki, Y. Yonemitsu, K. Sueishi, Y. Ito, The effects of caffeine on ATP-sensitive K(þ) channels in smooth muscle cells from pig urethra, Br. J. Pharmacol. 131 (3) (2000) 505e513. [5] E. Floride, M. Föller, M. Ritter, F. Lang, Caffeine inhibits suicidal erythrocyte death, Cell. Physiol. Biochem. 22 (2008) 253e260. [6] M.R. Clark, R.C. Unger, S.B. Shohet, Monovalent cation composition and ATP and lipid content of irreversibly sickled cells, Blood 51 (6) (1978) 1169e1178. [7] M.R. Clark, N. Mohandas, C. Feo, M.S. Jacobs, S.B. Shohet, Separate mechanisms of deformability loss in ATP-depleted and Ca-loaded erythrocytes, J. Clin. Invest. 67 (1981) 531e539. [8] F. Greer, R. Hudson, R. Ross, T. Graham, Caffeine ingestion decreases glucose disposal during a hyperinsulinemic-euglycemic clamp in sedentary humans, Diabetes 50 (2001) 2349e2354. [9] A.I. Spiff, A.A. Uwakwe, Activation of human erythrocyte glutathione, J. Appl. Sci. Environ. Manag. 7 (2) (2003) 45e48. [10] T. Efferth, U. Fabry, P. Glatte, R. Osieka, Expression of apoptosis-related oncoproteins and modulation of apoptosis by caffeine in human leukemic cells, J. Cancer Res. Clin. Oncol. 121 (1995) 648e656. [11] N. Shinomiya, T. Takemura, K. Iwamoto, M. Rokutanda, Caffeine induces S-Phase apoptosis in cis-diamminedichloroplatinum-treated cells, whereas cis-diamminedichloroplatinum induces a block in G2-M, Cytometry 27 (1997) 365e373. [12] W.S. El-Deiry, T. Tokino, V.E. Velculescu, D.B. Levy, R. Parsons, J.M. Trent, D. Lin, W.E. Mercer, K.W. Kinzler, B. Vogelstein, WAF1, a potential mediator p53 tumor suppression, Cell 75 (1993) 817e825. [13] L.H. Hartwell, M.B. Kastan, Cell cycle control and cancer, Science 266 (5192) (1994) 1821e1828. [14] Z. He, W.Y. Ma, T. Hashimoto, A.M. Bode, C.S. Yang, Z. Dong, Induction of apoptosis by caffeine is mediated by the p53, Bax, and caspase 3 pathways, Cancer Res. 63 (2003) 4396e4401. [15] J.J. Stephanos, Drugeprotein interactions: two-site binding of heterocyclic ligands to a monomeric haemoglobin, J. Inorg. Biochem. 62 (1996) 155e169. [16] Y.Q. Wang, H.M. Zhang, Q.H. Zhou, Studies on the interaction of caffeine with bovine haemoglobin, Eur. J. Med. Chem. 44 (2009) 2100e2105. [17] M. Laberge, I. Kovesi, T. Yonetani, J. Fidy, R-state hemoglobin bound to heterotropic effectors: model of the DPG, IHP, and RSR 13 binding sites, FEBS Lett. 579 (2005) 627e635. [18] A. Tsuneshige, S. Park, T. Yonetani, Heterotropic effectors control the hemoglobin function by interacting with its T and R states, a new view on the principle of allostery, Biophys. Chem. 98 (2002) 49e63. [19] S. Ficarra, E. Tellone, B. Giardina, R. Scatena, A. Russo, F. Misiti, M.E. Clementi, D. Colucci, E. Bellocco, G. Laganà, D. Barreca, A. Galtieri, Derangement of erythrocytic AE1 in beta-thalassemia by caspase 3: pathogenic mechanisms and implications in red blood cell senescence, J. Membr. Biol. 228 (1) (2009) 43e49. [20] A. Galtieri, E. Tellone, L. Romano, F. Misiti, E. Bellocco, S. Ficarra, A. Russo, D. Di Rosa, M. Castagnola, B. Giardina, I. Messana, Band-3 protein function in human erythrocytes: effect of oxygenationedeoxygenation, Biochim. Biophys. Acta 1564 (1) (2002) 214e218. [21] B. Giardina, I. Messana, R. Scatena, M. Castagnola, The multiple functions of haemoglobin, Crit. Rev. Biochem. Mol. Biol. 30 (1995) 165e196. [22] A. Russo, E. Tellone, S. Ficarra, B. Giardina, E. Bellocco, G. Lagana’, U. Leuzzi, A. Kotyk, A. Galtieri, Band 3 protein function in teleost fish erythrocytes: effect of oxygenationedeoxygenation, Physiol. Res. 57 (1) (2008) 49e54. [23] E. Clementi, B. Giardina, D. Colucci, A. Galtieri, F. Misiti, Amyloid-beta peptide affects the oxygen dependence of erythrocyte metabolism: a role for caspase 3, Int. J. Biochem. Cell. Biol. 39 (2007) 727e735. [24] D. Mandal, P.K. Moitra, S. Saha, J. Basu, Caspase 3 regulates phosphatidylserine externalization and phagocytosis of oxidatively stressed erythrocytes, FEBS 513 (2002) 184e188. [25] D. Mandal, V. Baudin-Creuza, A. Bhattacharyya, S. Pathak, J. Delaunay, M. Kundu, J. Basu, Caspase 3-mediated proteolysis of the N-terminal cytoplasmic domain of the human erythroid anion exchanger 1 (band 3), J. Biol. Chem. 278 (2003) 52551e52558. [26] P. Matarrese, E. Straface, D. Pietraforte, L. Gambardella, R. Vona, A. Maccaglia, M. Minetti, W. Malorni, Peroxinitritre induces senescence and apoptosis of red blood cells through the activation of aspartyl and cysteinyl protease, FASEB J. 19 (2005) 416e418.

402

E. Tellone et al. / Biochimie 94 (2012) 393e402

[27] R. Radi, J.S. Beckman, K.M. Bush, B.A. Freeman, Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide, J. Biol. Chem. 266 (1991) 4244e4250. [28] J.E. Jagger, R.M. Bateman, M.L. Ellsworth, C.G. Ellis, Role of erythrocyte in regulating local O2 delivery mediated by hemoglobin oxygenation, Am. J. Physiol. Heart Circ. Physiol. 280 (2001) H2833eH2839. [29] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson, Using a Lamarckian genetic algorithm and empirical binding free energy function, J. Comput. Chem. 19 (1998) 1639e1662. [30] W.G. Zijlstra, A. Buursma, W.P. Meeuwsen-Van Der Roest, Absorption spectra of human fetal and adult oxyhemoglobin, de-oxyhemoglobin, carboxyhemoglobin and methemoglobin, Clin. Chem. 37 (1991) 1633e1638. [31] R.J. Labotka, Measurement of intracellular pH and deoxyhemoglobin concentration in deoxygenated erythrocytes by phosphorus-31 nuclear magnetic resonance, Biochemistry 23 (1984) 5549e5555. [32] L. Romano, D. Peritore, E. Simone, A. Sidoti, F. Trischitta, P. Romano, Chlorideesulphate exchange chemically measured in human erythrocyte ghosts, Cell. Mol. Biol. 44 (1998) 351e355. [33] B. Giardina, G. Amiconi, Measurement of binding of gaseous and nongaseous ligands to hemoglobin by conventional spectrophotometric procedures, Methods Enzymol. 76 (1981) 417e427. [34] A. Maccaglia, C. Mallozzi, M. Minetti, Differential effects of quercetin and resveratrol on band 3 tyrosine phosphorylation signalling of red blood cells, Biochem. Biophys. Res. Commun. 305 (3) (2003) 541e547. [35] M.S. Blois, Antioxidant determinations by the use of a stable free radical, Nature 181 (1958) 1199e1200. [36] M. Nishimiki, N. Appaji, K. Yagi, The occurrence of superoxide anion in the reaction of reduced phenazine methosulphate and molecular oxygen, Biochem. Biophys. Res. Commun. 46 (1972) 849e854. [37] B. Halliwell, J.M.C. Gutteridge, O.L. Aruoma, The deoxyribose method: a simple test-tube assay for determination of rate constants for reactions of hydroxyl radicals, Anal. Biochem. 165 (1987) 215e219. [38] S.Y. Park, T. Yokoyama, N. Shibayama, Y. Shiro, J.R. Tame, 1.25 Å resolution crystal structures of human haemoglobin in the oxy, deoxy and carbonmonoxy forms, J. Mol. Biol. 360 (2006) 690e701. [39] B. Hess, C. Kutzner, D. van der Spoel, E. Lindahl, GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation, J. Chem. Theory Comput. 4 (2008) 435e447. [40] L.D. Schuler, X. Daura, W.F.V. Gunsteren, An improved GROMOS96 force field for aliphatic hydrocarbons in the condensed phase, J. Comput. Chem. 22 (2001) 1205e1218. [41] A. Galtieri, E. Tellone, S. Ficarra, A. Russo, E. Bellocco, D. Barreca, R. Scatena, G. Laganà, U. Leuzzi, B. Giardina, Resveratrol treatment induces redox stress in

[42]

[43] [44]

[45] [46]

[47]

[48] [49]

[50]

[51] [52]

[53]

[54]

[55]

red blood cell: a possible role of caspase 3 in metabolism and anion transport, Biol. Chem. 391 (2010) 1057e1065. E. Tellone, S. Ficarra, B. Giardina, R. Scatena, A. Russo, M.E. Clementi, F. Misiti, E. Bellocco, A. Galtieri, Oxidative effect of gemfibrozil on anion influx and metabolism in normal and beta-thalassaemic erythrocytes, physiological implications, J. Membr. Biol. 224 (2008) 1e8. M.F. Perutz, C. Poyart, Bezafibrate lowers oxygen affinity of hemoglobin, Lancet 2 (8355) (1983) 881e882. N. Shibayama, S. Miura, J.R.H. Tame, T. Yonetani, S.Y. Park, Crystal structure of horse carbonmonoxyhemoglobin-bezafibrate complex at 1.55-A resolution, J. Biol. Chem. 277 (41) (2002) 38791e38796. T. Yonetani, M. Laberge, Protein dynamics explain the allosteric behavior of hemoglobin, Biochim. Biophys. Acta 1784 (9) (2008) 1146e1158. E. Tellone, S. Ficarra, R. Scatena, B. Giardina, A. Kotyk, A. Russo, D. Colucci, E. Bellocco, G. Laganà, A. Galtieri, Influence of gemfibrozil on sulphate transport in human erythrocytes during the oxygenationedeoxygenation cycle, Physiol. Res. 57 (2008) 621e629. G.W. Arendash, W. Schleif, K. Rezai-Zadeh, E.K. Jackson, L.C. Zacharia, J.R. Cracchiolo, D. Shippy, J. Tan, Caffeine protects Alzheimer’s mice against cognitive impairment and reduces brain b-amyloid production, Neuroscience 142 (2006) 941e952. R.M. van Dam, F.B. Hu, Coffee consumption and risk of type 2 diabetes, JAMA 294 (1) (2005) 97e104. E. Lopez-Garcia, R.M. van Dam, S. Rajpathak, W.C. Willett, J.E. Manson, F.B. Hu, Changes in caffeine intake and long-term weight change in men and women, Am. J. Clin. Nutr. 83 (2006) 674e680. P. Arese, F. Turrini, E. Schwarzr, Band 3/complement-mediated recognition and removal of normally senescent and pathological human erythrocytes, Cell. Physiol. Biochem. 16 (2005) 133e146. G.J. Bosman, F.L. Willekens, J.M. Were, Erythrocyte aging: a more than superficial resemblance to apoptosis? Cell. Physiol. Biochem. 16 (2005) 1e8. F. Misiti, F. Orsini, M.E. Clementi, D. Masala, E. Tellone, A. Galtieri, B. Giardina, Amyloid peptide inhibits ATP release from human erythrocytes, Biochem. Cell. Biol. 86 (2008) 501e508. C. Zeling, J.B. Bell, J.G. Mohanty, E. Nagababu, J.M. Rifkind, Nitrite enhances RBC hypoxic ATP synthesis and the release of ATP into the vasculature: a new mechanism for nitrite-induced vasodilation, Am. J. Physiol. Heart Circ. Physiol. 297 (2009) H1494eH1503. C. Szabò, H. Ischiropoulos, R. Radi, Peroxynitrite: biochemistry, pathophysiology and development of therapeutics, Nat. Rev. Drug Discov. 6 (2007) 662e680. N. Romero, A. Denicola, R. Radi, Red blood cells in the metabolism of nitric oxide-derived peroxynitrite, IUBMB Life 58 (10) (2006) 572e580.