C-Phycocyanin protects SH-SY5Y cells from oxidative

Brain Research Bulletin 89 (2012) 159–167

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C-Phycocyanin protects SH-SY5Y cells from oxidative injury, rat retina from transient ischemia and rat brain mitochondria from Ca2+ /phosphate-induced impairment Javier Marín-Prida a , Giselle Pentón-Rol b , Fernando Postalli Rodrigues c , Luciane Carla Alberici c , Karina Stringhetta d , Andréia Machado Leopoldino d , Zeki Naal c , Ana Cristina Morseli Polizello c , Alexey Llópiz-Arzuaga b , Marcela Nunes Rosa e , José Luiz Liberato e , Wagner Ferreira dos Santos e , Sergio Akira Uyemura d , Eduardo Pentón-Arias b , Carlos Curti c , Gilberto L. Pardo-Andreu a,∗ a

Center for Research and Biological Evaluations, Institute of Pharmacy and Food, University of Havana, Ave. 23 e/214 y 222, PO Box 430, Havana, Cuba Center for Genetic Engineering and Biotechnology, Ave. 31 e/158 y 190, PO Box 6162, Havana, Cuba Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ave. Café s/n, 14040-903 Ribeirão Preto, São Paulo, Brazil d Department of Clinical, Toxicological and Bromatological Analysis, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ave. Café s/n, 14040-903 Ribeirão Preto, São Paulo, Brazil e Department of Biology, Faculty of Philosophy, Sciences, and Literature of Ribeirão Preto, University of São Paulo, Ave. Bandeirantes 3900, PO Box 14040-901, Ribeirão Preto, São Paulo, Brazil b c

a r t i c l e

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Article history: Received 6 July 2012 Received in revised form 9 August 2012 Accepted 30 August 2012 Available online 7 September 2012 Keywords: C-Phycocyanin SH-SY5Y Transient rat retinal ischemia Rat brain mitochondria Antioxidant Neuroprotection

a b s t r a c t Oxidative stress and mitochondrial impairment are essential in the ischemic stroke cascade and eventually lead to tissue injury. C-Phycocyanin (C-PC) has previously been shown to have strong antioxidant and neuroprotective actions. In the present study, we assessed the effects of C-PC on oxidative injury induced by tert-butylhydroperoxide (t-BOOH) in SH-SY5Y neuronal cells, on transient ischemia in rat retinas, and in the calcium/phosphate-induced impairment of isolated rat brain mitochondria (RBM). In SH-SY5Y cells, t-BOOH induced a significant reduction of cell viability as assessed by an MTT assay, and the reduction was effectively prevented by treatment with C-PC in the low micromolar concentration range. Transient ischemia in rat retinas was induced by increasing the intraocular pressure to 120 mmHg for 45 min, which was followed by 15 min of reperfusion. This event resulted in a cell density reduction to lower than 50% in the inner nuclear layer (INL), which was significantly prevented by the intraocular pre-treatment with C-PC for 15 min. In the RBM exposed to 3 mM phosphate and/or 100 ␮M Ca2+ , C-PC prevented in the low micromolar concentration range, the mitochondrial permeability transition as assessed by mitochondrial swelling, the membrane potential dissipation, the increase of reactive oxygen species levels and the release of the pro-apoptotic cytochrome c. In addition, C-PC displayed a strong inhibitory effect against an electrochemically-generated Fenton reaction. Therefore, C-PC is a potential neuroprotective agent against ischemic stroke, resulting in reduced neuronal oxidative injury and the protection of mitochondria from impairment. © 2012 Elsevier Inc. All rights reserved.

1. Introduction

Abbreviations: t-PA, tissue plasminogen activator; C-PC, C-Phycocyanin; tBOOH, tert-butylhydroperoxide; RBM, rat brain mitochondria; DMEM, Dulbecco’s Modified Eagle Medium; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide; PBS, phosphate buffered saline; DMSO, dimethyl sulfoxide; IOP, intraocular pressure; INL, inner nuclear layer; EGTA, ethylene glycol tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; SIM, standard incubation medium; H2 DCFDA, dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; , mitochondrial membrane potential; TPP+ , tetraphenylphosphonium; MPT, mitochondrial permeability transition. ∗ Corresponding author. Tel.: +53 72038472; fax: +53 72038472. E-mail address: [email protected] (G.L. Pardo-Andreu). 0361-9230/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2012.08.011

Ischemic stroke remains a major cause of death and disability worldwide (Mukherjee and Patil, 2011; WHO, 2011). Although significant progress has been made to clarify the mechanisms involved in the ischemic stroke cascade, the development of new and effective treatments has been largely unsuccessful. Thrombolysis with tissue plasminogen activator (t-PA) is currently the only approved human pharmacological treatment, but it is restricted to a minority of patients due to its short therapeutic window (4.5 h) (Fonarow et al., 2011; Maiser et al., 2011). An alternative approach known as neuroprotection has been focused on protecting the neurovascular unit by blocking the main pathogenic processes that causes

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ischemic injury (Ginsberg, 2008). Reactive oxygen species (ROS) generated soon after ischemia, during reperfusion and thereafter are considered crucial mediators of ischemic injury (Saito et al., 2005). Mitochondria, an important source of ROS, contribute significantly to the elevation of these oxidants after an ischemic event (Allen and Bayraktutan, 2009). Thus, antioxidant-based neurovascular protective strategies may be potential treatments to expand the number of currently approved therapies (Jung et al., 2010). Previous studies have demonstrated that C-Phycocyanin (CPC), the main phycobiliprotein of cyanobacteria Spirulina platensis, exerts potent antioxidant, anti-inflammatory and neuroprotective actions in different experimental conditions (Romay et al., 2003). Phycobiliproteins are light-harvesting complexes composed of apo-proteins covalently attached to open chain tetrapyrrole chromophores, named phycocyanobilins. C-PC has ␣ and ␤ polypeptides subunits (18–20 kDa) (Pentón-Rol et al., 2011a); one phycocyanobilin is attached at cysteine 84 in the ␣-chain and two phycocyanobilins are attached at cysteines 84 and 155 in the ␤-chain (Padyana et al., 2001; Pentón-Rol et al., 2011a). Recent results from our group have shown that the administration of C-PC either prophylactically or therapeutically exhibited beneficial effects against global cerebral ischemia–reperfusion injury in gerbils (Pentón-Rol et al., 2011a). These effects were partially explained by the C-PC ability to counteract oxidative damages. In the present study, we characterized the in vitro protective effects of C-PC against tert-butylhydroperoxide (t-BOOH)-induced oxidative injury in the SH-SY5Y human neuroblastoma cell line and in vivo against transient ischemia in rat retinas. The effects of C-PC were also examined in isolated rat brain mitochondria (RBM) with a Ca2+ /phosphate overload, a condition that mimics the mitotoxic environment that results from an ischemic injury.

(PBS) pH 7.2 (200 ␮L/well), and MTT (freshly prepared at 0.5 mg/mL in serum-free medium) was added at 200 ␮L/well and incubated for 4 h at 37 ◦ C. After MTT withdrawal, 200 ␮L of dimethyl sulfoxide (DMSO) was added to each well to solubilize the formazan crystals, and the absorbance of each well was measured at 570 nm in a microplate reader (Varian Cary 50, Australia). The results were expressed as the percent of absorbance relative to the undamaged control cells. 2.5. Induction of transient retinal ischemia A transient ischemia in the rat retina was induced as previously described, with some modifications (Beleboni et al., 2006). Male Wistar rats weighing 250–300 g at the time of surgery (n = 5 per group) were anesthetized with 450 mg/Kg i.p. urethane (Acrôs Organic, USA) (Meyer, 2008), and the anterior chamber of the left eye was cannulated with a 27-gauge needle attached to a manometer, a pump and an air reservoir. The intraocular pressure (IOP) was raised to 120 mmHg for 45 min. Afterwards the needle was withdrawn, and the IOP was normalized for a 15 min reperfusion period. The drug was intraocularly injected 15 min prior to the ischemic insult. The vehicle group was injected with sterile PBS at pH 7.2. The right-side, untouched retinas served as control (n = 10). 2.6. Histological assessment After the reperfusion period, the animals were sacrificed, and both eyes were rapidly enucleated, placed in the fixation solution (25.5 mL ethanol 80%, 3 mL formaldehyde 37% and 1.5 mL glacial acetic acid) for 24 h and transferred to 80% ethanol for another 4 days (Krinke, 2000). The dissected retina was dehydrated in graded ethanol and xylene and embedded in paraffin. Retinas were sectioned at 5 ␮m, approximately 1 mm from the optic nerve, and stained with hematoxylin and eosin. For each experimental group, five microscopic fields (magnification 400×) of one sagittal section of a retina were captured by a digital camera (DFC300 FX, DM 5000 B microscope, Q-Win software, Leica Microsystems, Germany). The number of viable cells, those that were not eosinophilic, was manually counted in the established areas (200 ␮m × 50 ␮m) of the inner nuclear layer (INL) using ImageJ 1.41 software (National Institutes of Health, USA). Cells showing atrophy, shrinkage, nuclear pyknosis, dark cytoplasmic coloration or ambient empty spaces were considered damaged (Beleboni et al., 2006). The results were expressed as a percentage of the number of viable cells in the control retinas.

2. Materials and methods

2.7. Isolation of rat brain mitochondria

2.1. Reagents and compounds

Mitochondria were isolated as previously described, with minor modifications (Mirandola et al., 2010). Briefly, two male Wistar rats weighing 200–240 g were sacrificed by decapitation, and their brains were rapidly removed and placed in 10 mL of ice-cold isolation buffer (225 mM mannitol, 75 mM sucrose, 1 mM ethylene glycol tetraacetic acid (EGTA), 0.1% de-fatted bovine serum albumin (BSA), and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); pH 7.2). The olfactory bulb, cerebellum and underlying regions were discarded, and the remaining forebrains were split using surgical scissors and washed twice in isolation buffer. The brain tissue was manually homogenized in 20 mL of the isolation buffer using a glass Dounce homogenizer (20–25 strokes). The homogenate was centrifuged at 4 ◦ C (2000 × g, 3 min) in a Hitachi RT15A5 rotor (Japan), and the resulting supernatant was further centrifuged (12,000 × g, 8 min). The pellet was gently resuspended in 10 mL of isolation buffer containing 20 ␮L of 10% digitonin (to induce synaptosomal lysis) using a fine-tipped brush and centrifuged at 12,000 × g for 10 min. The dark pellet was resuspended in 10 mL of isolation medium without EGTA and BSA and centrifuged again (12,000 × g, 10 min), and the resulting pellet was resuspended in 200 ␮L of standard incubation medium (SIM: 225 mM mannitol, 75 mM sucrose, and 10 mM K+ HEPES; pH 7.2). The total protein concentration was determined by the Biuret method with BSA as a standard and was approximately 30–50 mg/mL for each preparation. All procedures were performed at 4 ◦ C.

All chemicals used were of the highest grade available and purchased from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise specified. C-PC was purified by ionic exchange, as previously described (Pentón-Rol et al., 2011a). An A620 /A280 absorbance ratio of 4.58 was obtained, indicative of higher than 90% purity (Patil and Raghavarao, 2007). C-PC was dissolved in sterile phosphate-buffered saline (PBS) pH 7.2, filtered through a 0.2 ␮m syringe filter under sterile conditions (Sartorius Minisart® , Germany) and stored in the dark at 4 ◦ C until use. 2.2. Animals Rats were obtained from the Animal Breeding Center at the Ribeirão Preto Campus, University of São Paulo (USP). The animals were maintained under standard laboratory conditions (60% humidity, 22 ± 1 ◦ C, and 12 h light/darkness cycle) with free access to food and water. All procedures were performed in compliance with the USP guidelines and with the European Communities Council Directive of 24 November 1986 (86/609/EEC) for animal experimentation. 2.3. Cell culture SH-SY5Y human neuroblastoma cells (Rio de Janeiro Cell Bank, Brazil) were expanded in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 100 IU/mL penicillin G, 100 ␮g/mL streptomycin and 0.25 ␮g/mL amphotericin B at 37 ◦ C in 5% CO2 /95% air. The medium was changed every two days, and the cells were subcultured every four days by trypsinization and re-plated in T-25 or T-75 flasks (5 or 15 mL final volume, respectively) (BD Falcon, USA) at 4 × 105 cells/mL. 2.4. Cell treatment and viability assay SH-SY5Y cells between passages three and seven were cultured in quadruplicate in 96 wells plates (BD Falcon, USA) at 4 × 104 cells/200 ␮L/well for 24 h. The cells were incubated with freshly prepared t-BOOH in complete medium at different concentrations for 6 h, or pre-treated with C-PC for 24 h followed by the incubation with fresh medium containing C-PC and 25 ␮M t-BOOH for another 6 h. Cell viability was measured by the quantity of blue formazan products obtained from the colorless 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) by mitochondrial dehydrogenases, which are only active in viable cells. After the incubation time, the cells were gently washed with sterile phosphate buffered saline

2.8. Mitochondrial assays The experiments using isolated RBM were performed at 30 ◦ C with continuous magnetic stirring in the SIM described above. The respiratory control ratio (state 3/state 4 respiratory rates) was over 4; this ratio was determined after monitoring oxygen consumption with an oxygraph equipped with a Clark-type oxygen electrode (Hansatech Instruments Ltd., UK) and with 1 mg/mL of RBM plus 5 mM succinate, 2 ␮M rotenone, 800 ␮M ADP and 2 mM KH2 PO4 . Oligomycin at 1 ␮g/mL was used at state 4. Mitochondrial swelling was estimated from the decrease in the apparent absorbance at 540 nm using a Model U-2910 Hitachi spectrophotometer (Japan). ROS generation in RBM was monitored using 2 ␮M of dichlorodihydrofluorescein diacetate (H2 DCFDA) (Invitrogen, Life Technology Co., USA). The changes in the fluorescence of the DCF product were monitored using an F-4500 fluorescence spectrophotometer (Hitachi, Japan) at 503/529 nm excitation/emission wavelengths, and data were expressed as differences with respect to the initial value (Esposti, 2002; Rodrigues et al., 2011). The mitochondrial membrane potential ( ) was estimated by continuously monitoring the concentration of the lipophilic cation tetraphenylphosphonium chloride (TPP+ ) in the extramitochondrial medium (Ross et al., 2005) with a TPP+ selective electrode (World Precision Instruments Inc., USA)


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connected to the oxygraph. For the calculation of , the matrix volume of the RBM was assumed to be 1.5 ␮L/mg protein. The membrane potential was calculated assuming that the TPP+ distribution between the mitochondria and medium followed the Nernst equation. Corrections were made as previously described for the binding of TPP+ to the mitochondrial membrane (Jensen et al., 1986). Data were expressed as a percentage of in relation to the level reached before Ca2+ addition. 2.9. Assessment of the release of cytochrome c from mitochondria The release of cytochrome c was assessed in the same conditions as used in the swelling experiments. The mitochondrial suspension was centrifuged (9000 × g, 6 min) at 4 ◦ C, and the supernatant (100 ␮L) was supplemented with 2 ␮L of protease inhibitor cocktail (Sigma–Aldrich, USA), incubated with 25 ␮L of loading buffer (50% glycerol, 10% SDS, 0.05% bromophenol blue, 25% mercaptoethanol) at 95 ◦ C for 5 min, subjected to 12% SDS-PAGE and transferred to a nitrocellulose membrane. A mouse monoclonal antibody against cytochrome c (1:1000, BD Biosciences Pharmingen, USA), a mouse-specific secondary antibody conjugated with horseradish peroxidase (1:1000, Santa Cruz Biotechnology, USA) and the ECLTM Systems (GE HealthCare, UK) were used for detection. The films were placed in a digital flatbed scanner (HP Scanjet 3770, USA) for image acquisition. Densitometry was performed using the ImageJ 1.41 software (National Institutes of Health, USA). The results were presented as a percent of the Ca2+ /phosphate damaged controls. 2.10. Electrochemical assays Electrochemical assays were conducted with a BAS CV-27 potentiostat (Bioanalytical System, Inc., USA) using conventional electrochemical cells with three electrodes and an Omnigraphic 100 recorder (Texas Instruments, USA). A glassy carbon electrode with a 0.0314 cm2 area, polished with a 1 ␮m alumina–water suspension and rinsed thoroughly with water and acetone was used as working electrode. A platinum wire was used as the counter electrode, and all potentials were referenced to a [Ag/AgCl/KCl (sat)] electrode without regard for the liquid junction potential. The electrolyte supporting solution was obtained by mixing 100 mM KCl and 100 mM chloroacetic acid pH 3.3, and Fe3+ -EDTA (0.1 mM) was formed from equimolar additions of Fe(NO3 )3 and Na2 H2 EDTA. The 100 mM hydroperoxide stock solution was prepared by dissolving 30% H2 O2 in the electrolyte supporting solution. Each sample was deoxygenated with argon for 20 min before performing electrochemical measurements. Cyclic voltammetry was conducted at a final volume of 5.0 mL for all experiments with a potential sweep rate of 30 mV/s and a potential window between −0.4 V and 0.4 V. Control experiments were measured in ESS in the presence of Fe3+ -EDTA or H2 O2 , in the presence of both and without C-PC or oxygen (Argon purge). The voltammetric backgrounds were measured in the presence of Fe3+ -EDTA but in the absence of any peroxides or oxygen (N2 purge). 2.11. Statistical analysis The software program GraphPad Prism 5.0 (GraphPad Software Inc., USA) was used for statistical analysis. The data were expressed as the mean ± SEM. Comparisons between the different groups were performed by the one-way analysis of variance (ANOVA) followed by the Newman–Keuls multiple comparison test. Differences were considered statistically significant at p < 0.05.

3. Results 3.1. C-PC protected SH-SY5Y cells from t-BOOH-induced death Fig. 1A shows that the viability of SH-SY5Y cells was significantly reduced after exposing them to t-BOOH, and pre-treatment with CPC prevented this effect (Fig. 1B). C-PC at 50 ␮M almost completely prevented the cell viability reduction and did not present cytotoxic effects, demonstrating the safety of the compound. This result demonstrates that C-PC has a protective effect against the oxidative neurotoxic injury induced by t-BOOH. We have used Trolox, a water-soluble analogue of vitamin E, as a control antioxidant to show that ROS play a major role in t-BOOH cytotoxicity in SH-SY5Y cells (Lee et al., 2000). 3.2. C-PC protected rat retinas from transient ischemia To assess whether C-PC protects neuronal viability against a focal stroke in vivo, we performed a proof-of concept experiment for transient ischemia in rat retinas. Fig. 2 shows that the rat retina was highly susceptible to an ischemia–reperfusion injury primarily at the INL in our experimental conditions, which exhibited less than a 50% reduction in viable cells; neuronal death was also accompanied

Fig. 1. Protective effects of C-PC against oxidative injury in SH-SY5Y human neuroblastoma cells. (A) Viability of cells exposed to different concentrations of tert-butylhydroperoxide (t-BOOH) for 6 h. (B) Effects of a pre-treatment with C-PC for 24 h followed by a co-incubation with 25 ␮M of t-BOOH for 6 h. Trolox (500 ␮M) was used as a control antioxidant. Cell viability was assessed by an MTT assay and expressed as a percent of the control. Data are presented as the mean ± SEM of three independent experiments. Different letters: p < 0.05 according to the ANOVA and post hoc Newman–Keuls tests.

by edema and layer disorganization. Pre-treatment with C-PC prevented INL cell loss, indicating that C-PC not only protects neurons in vitro, but also in in vivo ischemic conditions. 3.3. C-PC prevented the mitochondrial permeability transition in RBM A key pathogenic event in a stroke is the opening of pores in the inner mitochondrial membrane; the mitochondrial permeability transition (MPT) (Hirsch et al., 1998). In the next set of experiments, we assessed the effect of C-PC on the MPT as estimated by the swelling of the RBM induced by Ca2+ and inorganic phosphate (Pi), as previously described (Berman et al., 2000) (Fig. 3A). Comparisons among the initial decreases in the slopes of the absorbances at 540 nm revealed that C-PC prevented MPT in the low micromolar concentration range (Fig. 3B). This suggests that the inhibition of MPT is involved in the mechanisms that are responsible for the C-PC neuroprotective effects. 3.4. C-PC prevented an increase in ROS levels in RBM MPT has been associated with an increase in ROS levels in brain mitochondria (Maciel et al., 2001). Our results showed that Ca2+ overload induced a significant increase in ROS levels in RBM (Fig. 4A), and pre-treatment with C-PC at low micromolar concentration range prevented this effect (Fig. 4B). 3.5. C-PC prevented dissipation in RBM To assess the effect of C-PC on Ca2+ -promoted dissipation, RBM were pre-incubated with 30 ␮M of C-PC for approximately 10 min, and then 100 ␮M of Ca2+ was added (Fig. 5). Ca2+ overload produced a dissipation of approximately 30%; this dissipation

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Fig. 2. Histological evaluation (hematoxylin and eosin staining) of the effects of C-PC on transient ischemia in rat retinas. The panels show representative photographs of the sagittal sections of retina from undamaged controls (A), and retinas that underwent ischemia–reperfusion (I-R) treated with vehicle (B), 10 ␮M C-PC (C), 50 ␮M C-PC (D) or 100 ␮M C-PC (E). Morphometric analysis of INL regions is shown in (F). Different letters: p < 0.05 according to the ANOVA and post hoc Newman–Keuls tests. ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer and GCL: ganglion cell layer. Scale bar: 50 ␮m.


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Fig. 4. Effects of C-PC on the increase in mitochondrial ROS levels induced by Ca2+ . The RBM (1 mg/mL) were incubated for 5 min at 30 ◦ C in SIM plus C-PC, 5 mM succinate, 2 ␮M rotenone, 3 mM KH2 PO4 (Pi) and 2 ␮M H2 DCFDA, and fluorescence monitoring was then initiated. After 5 min, 100 ␮M CaCl2 (Ca2+ ) was added to the suspension, which was monitored for another 10 min (A). Comparisons in fluorescence intensity were made at this point (B). Data are expressed as the mean ± SEM for three independent experiments. Different letters: p < 0.05 according to the ANOVA and post hoc Newman–Keuls tests.

Fig. 3. Effects of C-PC on mitochondrial swelling induced by Ca2+ /phosphate. RBM (1 mg/mL) were pre-incubated for 5 min at 30 ◦ C with C-PC in SIM with 5 mM succinate and 2 ␮M rotenone followed by the addition of 100 ␮M CaCl2 (Ca2+ ) for another 1 min. Then, 3 mM KH2 PO4 (Pi) was added to the mitochondrial suspension, and absorbance was monitored at 540 nm for 10 min (A). The rates of swelling (slope of the decrease in absorbance in the first 5 min after Pi addition) were compared (B). Data are expressed as the means ± SEM of at least three independent experiments. Different letters: p < 0.05 according to the ANOVA and post hoc Newman–Keuls tests.

was effectively prevented by treatment with either C-PC or the classical MPT inhibitor cyclosporin A (Fig. 5). 3.6. C-PC prevented mitochondrial cytochrome c release in RBM Cytochrome c release from mitochondria plays an important role in cell death (Li et al., 1997). Our results showed that CPC prevented the oxidative stress-induced cell viability decrease in SH-SY5Y cells (Fig. 1). To assess the potential involvement of cytochrome c release inhibition in this effect, we used Western blot to detect the free cytochrome c in the supernatant fraction obtained after the mitochondrial swelling experiments (Fig. 6). The Ca2+ /Pioverload induced a substantial release of cytochrome c from RBM, but this release was prevented by C-PC at low micromolar concentrations. The effect of C-PC was significantly higher than the effect of the antioxidant Trolox. 3.7. C-PC inhibited the Fenton reaction H2 O2 is regarded as a key mediator in oxidative stress because it is highly stable under physiological conditions (Bergendi et al., 1999). When reacting with metals such as iron, H2 O2 yields a hydroxyl through the Fenton reaction (Winterbourn, 1995). Hydroxyls are highly reactive radicals that can initiate lipid

Fig. 5. Effects of C-PC on dissipation induced by Ca2+ . Effects in the absence (A) or presence of 30 ␮M C-PC (B), in the presence of 1 ␮M of cyclosporine A (CsA, C), and the % of dissipation after Ca2+ addition (insert). The values before and after Ca2+ addition for (A), (B) and (C), in mV, were: −154.06 ± 4.43 and −108.90 ± 3.14; −153.49 ± 2.81 and −143.96 ± 2.17; and −159.52 ± 4.12 and −153.83 ± 5.26, respectively. The RBM (2 mg/mL) were incubated in 1 mL of SIM with 5 mM succinate and 2 ␮M rotenone in the presence or the absence of C-PC; this was performed after the addition (indicated by the arrows) of both 0.5 ␮M TPP+ at a 1.5 ␮M final concentration and 100 ␮M CaCl2 (Ca2+ ). Data are expressed as the mean ± SEM of three independent experiments. Different letters: p < 0.05 according to the ANOVA and post hoc Newman–Keuls tests.

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intensity of the cathode current (arrowhead) was substantially increased (from −4 ␮A to approximately −18 ␮A), but that of the anode was not. This indicates that a catalytic mechanism is involved and could be ascribed to the Fenton reaction, represented by Eq. (2). The C-PC in the reaction medium reduced the cathode current to approximately −8 ␮A (trace c), which represents approximately a 55% inhibition of the Fenton reaction.

4. Discussion

Fig. 6. Effects of C-PC on the release of cytochrome c from mitochondria as induced by Ca2+ and phosphate. Representative Western blots and densitometry of free cytochrome c following the incubation of RBM under swelling conditions. Lane 1: control; lanes 2–5: Ca2+ /Pi plus 5, 10, 15 ␮M C-PC and 1 mM Trolox, respectively; lane 6: Ca2+ /Pi. Data are expressed as the mean ± SEM of three independent experiments. Different letters: p < 0.05 according to the ANOVA and post hoc Newman–Keuls tests.

peroxidation followed by membrane disorganization and rupture (Adibhatla and Hatcher, 2010). In this study, we assessed the effect of C-PC on the Fenton reaction using an electrochemical approach (Laine and Cheng, 2009). Fig. 7 (trace a) shows a cyclic voltammogram of the Fe3+ -EDTA/Fe2+ -EDTA oxidation–reduction process that can be represented by Eq. (1). Fig. 7 (trace b) shows the voltammetric behavior of Fe3+ -EDTA in the presence of 4 mM H2 O2 . The

Fig. 7. Effects of C-PC on the Fenton reaction. Cyclic voltammograms of the reversible oxidation–reduction of Fe3+ -EDTA/Fe2+ -EDTA (a, Eq. (1)) and the electrocatalytic reaction between Fe2+ -EDTA and H2 O2 in the absence (b, Eq. (2)) and in the presence of 0.38 mM C-PC (c). Solutions contain 100 mM KCl, 0.1 mM Fe3+ -EDTA and 100 mM chloroacetic acid (pH 3.3). Cyclic voltammograms were performed in the absence of O2 by a prior nitrogen purge, at a scan rate of 100 mV/s, and with a glassy carbon electrode (area 0.0314 cm2 ) as the working electrode. The typical tracings of three independent experiments are shown.

Oxidative stress is a crucial factor in the pathophysiology of ischemic stroke and acts by either damaging cellular structures or participating in signaling pathways (Saito et al., 2005). Compounds inhibiting this neurotoxic process have been considered as promising therapeutic candidates against ischemic stroke (Broussalis et al., 2012). C-PC was shown to have a strong antioxidant activity in several in vivo experiments, including global ischemia–reperfusion damage in the brain (Pentón-Rol et al., 2011a) and the heart (Khan et al., 2006a), doxorubicin-induced injury in cardiomyocytes (Khan et al., 2006b), experimental autoimmune encephalomyelitis (Pentón-Rol et al., 2011b), atherosclerosis (Riss et al., 2007), paraquat-induced lung damage (Sun et al., 2011), activated platelets (Hsiao et al., 2005) and thymic atrophy (Gupta et al., 2011). In vitro, C-PC prevents neurotoxic injury in cerebellar granule cells during conditions of low potassium and serum deprivation (Rimbau et al., 2001) and in SH-SY5Y human neuroblastoma cells subjected to an environment of exogenous iron overload (Bermejo-Bescós et al., 2008). In this study, we assessed the potential of C-PC to protect the SH-SY5Y cell line from the oxidative damage induced by the organic hydroperoxide t-BOOH (Chance et al., 1979). C-PC protected the viability of the SH-SY5Y cells exposed to t-BOOH. Iron can catalyze the conversion of t-BOOH to tert-butylperoxy and tert-butoxy radicals, which initiate lipid peroxidation (Cadenas and Sies, 1982). In primary rat cortical neurons, t-BOOH damage was blocked by a pre-treatment with deferoxamine, an iron specific chelator, indicating the requirement of the intracellular pool of iron for t-BOOH toxicity (Abe and Saito, 1998). Moreover, an increased t-BOOH detoxification activity by glutathione or thioredoxine peroxidases impaired the reductive effect of NAD(P)H on glutathione and thioredoxine and decreased their capacities to remove H2 O2 , particularly in mitochondria (Kowaltowski et al., 2001). Indeed, it has been reported that t-BOOH induces a free radical overproduction in SH-SY5Y cells (Amoroso et al., 1999). Therefore, the potent action of C-PC in scavenging free radicals (Bhat and Madyastha, 2000; Romay et al., 2000) and preserving the endogenous antioxidant systems (Bermejo-Bescós et al., 2008), together with a potential iron chelating capability, may be related to the neuroprotective effects of C-PC reported in this study. Retinal ischemia induced by an IOP increase can produce devastating visual impairment disorders such as glaucoma, which is the second leading cause of blindness worldwide (Resnikoff et al., 2004). The retina is highly sensitive to a transient blockade in blood supply due to its active metabolism and high content of polyunsaturated fatty acids, and therefore, this tissue is very susceptible to oxidative damage (Kuriyama et al., 2001). At the cellular level, accumulating evidence indicates that both glutamate excitotoxicity and oxidative stress are key mediators in ischemia–reperfusion injury to retina (Bonne et al., 1998; Louzada-Junior et al., 1992; Osborne et al., 2004), similar to the damage in an ischemic brain (Allen and Bayraktutan, 2009; Lau and Tymianski, 2010). Therefore, retinal ischemic damage is a suitable in vivo model to assess the protective effects of compounds against focal ischemic stroke. To our knowledge, the present study is the first to reveal that C-PC prevents neuron loss in rat retinas subjected to a transient ischemia. Because oxidative stress may be strongly implicated in the pathogenesis


of ischemic stroke (Allen and Bayraktutan, 2009), the scavenger activities of C-PC against hydroxyl radicals (Romay et al., 1998), peroxyl radicals (Lissi et al., 2000) and peroxynitrite anions (Bhat and Madyastha, 2001) may be involved in C-PC preventing the neurodegeneration of retinas after ischemia–reperfusion. Accordingly, our results showed an inhibitory effect of C-PC on the Fenton reaction as measured by an electrochemical approach, and this inhibition may be involved in the protection of retina tissue from ischemic damage. Mitochondria are both initiators and targets of oxidative damage, which impairs ATP production, induces MPT and releases pro-apoptotic factors (Sims and Anderson, 2002; Valko et al., 2007). Therefore, in this work, we also studied brain mitochondria as a potential pharmacological target to explain the neuroprotective effects elicited by C-PC. Under various conditions, such as in the presence of Ca2+ and inorganic phosphate, isolated mitochondria undergo MPT. This process is characterized by a Ca2+ -dependent increase in the permeability of the inner mitochondrial membrane, resulting in a dissipation, mitochondrial swelling, and an eventual rupture of the outer mitochondrial membrane (Zoratti and Szabo, 1995). MPT is one of the proposed mechanisms for the release of mitochondrial pro-apoptotic mediators, and its inhibition is a plausible pharmacological target for therapeutic intervention in stroke (Kristal et al., 2004). In our study, MPT was induced by an overload of Ca2+ in the presence of inorganic phosphate (Pi) and estimated by the occurrence of RBM swelling. These conditions mimic those of ischemic neurons, in which a rise in cytosolic Ca2+ and Pi concentrations due to excitotoxic glutamate and increased ATP hydrolysis, respectively, both occur (Nicholls and Budd, 1998; Oliveira and Kowaltowski, 2004). As a result of C-PC treatment, mitochondrial swelling, and therefore MPT, was significantly prevented, and this may constitute an important event contributing to the neuroprotective capability of C-PC. Ischemia induces an abnormal increase in intracellular Ca2+ levels. This cation can cause mitotoxic effects through the activation of degradative and ROS-generating enzymes, which may damage mitochondrial structures (Nakahara et al., 1992; Wingrave et al., 2003). Accordingly, our results showed a significant rise in ROS levels in isolated RBM as a consequence of Ca2+ /Pi overload. The probe that we used for ROS detection, H2 DCFDA, is a membrane-permeable molecule that can be converted by mitochondrial matrix esterases to a non-fluorescent H2 DCF product (LeBel and Ischiropoulos, 1992). Although it is not actually a reliable probe for superoxide and hydrogen peroxide, the most common ROS in biology, it is still widely used (Wardman, 2007). In the presence of different radicals, such as hydroxyl, carbonate, nitrogen dioxide (Wardman, 2007), and peroxynitrite (Jakubowski and Bartosz, 2000), H2 DCF is oxidized to the highly fluorescent DCF product. We assumed that increased H2 DCF oxidation reflects the overall ROS status rather than any reactive species in particular. Therefore, the inhibitory effect of C-PC on mitochondrial ROS levels may reflect its radical scavenging activity as a central mechanism to protect mitochondria against oxidative injury. In this regard, it is worth mentioning that in isolated guinea-pig brain mitochondria, in the absence of nucleotides and respiratory chain inhibitors, H2 O2 generation was decreased by Ca2+ (Komary et al., 2008). However, differences in the experimental conditions, tissues or species used (i.e. guinea-pig brain cortices against rat forebrains) may influence the characteristics of ROS production in brain mitochondria (AdamVizi, 2005). Accordingly, mitochondrial complex I inhibition by rotenone stimulates ROS generation (Barrientos and Moraes, 1999; Liu et al., 2002; Votyakova and Reynolds, 2001) and Ca2+ strongly increases ROS levels as detected with H2 DCFDA in rotenone-treated rat forebrain mitochondria. Apparently, this increase is not associated with mitochondrial Ca2+ accumulation, but with Ca2+ acting

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on external sites of the inner mitochondrial membrane (Sousa et al., 2003). Our results also showed that major consequences of MPT are dissipation and release of cytochrome c, which are associated with oxidative damage to mitochondrial membranes (Brustovetsky et al., 2002; Peng and Jou, 2010). These processes were inhibited by CsA and Trolox, an MPT blocker and a water-soluble antioxidant, respectively, indicating that MPT and the oxidative imbalance are important in mitochondrial impairment. The protective effect of CPC on and its inhibitory action on cytochrome c release may, therefore, be indirectly caused by its strong antioxidant properties. In conclusion, we demonstrated that C-PC is neuroprotective both in vitro against t-BOOH-induced oxidative damage in the SHSY5Y human neuroblastoma cell line and in vivo in rat retina against ischemia–reperfusion injury. In the isolated RBM, C-PC prevented MPT, restored normal ROS levels, and prevented dissipation and cytochrome c release. These results indicate that mitochondrial protection may be a mechanism involved in the effects of C-PC against oxidative injury in neuronal cells. Conflicts of interest The authors have no potential conflicts of interest related to the subject of this report. Acknowledgments This work was partially supported by a CAPES-Brazil/MES-Cuba (064/09) project. References Abe, K., Saito, H., 1998. Characterization of t-butyl hydroperoxide toxicity in cultured rat cortical neurones and astrocytes. Pharmacology and Toxicology 83, 40–46. Adam-Vizi, V., 2005. Production of reactive oxygen species in brain mitochondria: contribution by electron transport chain and non-electron transport chain sources. Antioxidants and Redox Signaling 7, 1140–1149. Adibhatla, R.M., Hatcher, J.F., 2010. Lipid oxidation and peroxidation in CNS health and disease: from molecular mechanisms to therapeutic opportunities. Antioxidants and Redox Signaling 12, 125–169. Allen, C.L., Bayraktutan, U., 2009. Oxidative stress and its role in the pathogenesis of ischaemic stroke. International Journal of Stroke 4, 461–470. Amoroso, S., Gioielli, A., Cataldi, M., Renzo, G.D., Annunziato, L., 1999. In the neuronal cell line SH-SY5Y, oxidative stress-induced free radical overproduction causes cell death without any participation of intracellular Ca2+ increase. Biochimica et Biophysica Acta 1452, 151–160. Barrientos, A., Moraes, C.T., 1999. Titrating the effects of mitochondrial complex I impairment in the cell physiology. Journal of Biological Chemistry 274, 16188–16197. Beleboni, R.O., Guizzo, R., Fontana, A.C.K., Pizzo, A.B., Carolino, R.O.G., Gobbo-Neto, L., Lopes, N.P., Coutinho-Netto, J., Santos, W.F., 2006. Neurochemical characterization of a neuroprotective compound from Parawixia bistriata spider venom that inhibits synaptosomal uptake of GABA and glycine. Molecular Pharmacology 69, 1998–2006. Bergendi, L., Benes, L., Duracková, Z., Ferencik, M., 1999. Chemistry, physiology and pathology of free radicals. Life Sciences 65, 1865–1874. Berman, S.B., Watkins, S.C., Hastings, T.G., 2000. Quantitative biochemical and ultrastructural comparison of mitochondrial permeability transition in isolated brain and liver mitochondria: evidence for reduced sensitivity of brain mitochondria. Experimental Neurology 164, 415–425. ˜ E., Fresno, A.M.V., 2008. Neuroprotection by SpirBermejo-Bescós, P., Pinero-Estrada, ulina platensis protean extract and phycocyanin against iron-induced toxicity in SH-SY5Y neuroblastoma cells. Toxicology In Vitro 22, 1496–1502. Bhat, V.B., Madyastha, K.M., 2000. C-Phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro. Biochemical and Biophysical Research Communications 275, 20–25. Bhat, V.B., Madyastha, K.M., 2001. Scavenging of peroxynitrite by phycocyanin and phycocyanobilin from Spirulina platensis: protection against oxidative damage to DNA. Biochemical and Biophysical Research Communications 285, 262–266. Bonne, C., Muller, A., Villain, M., 1998. Free radicals in retinal ischemia. General Pharmacology 30, 275–280. Broussalis, E., Trinka, E., Killer, M., Harrer, A., McCoy, M., Kraus, J., 2012. Current therapies in ischemic stroke. Part B. Future candidates in stroke therapy and experimental studies. Drug Discovery Today, http://dx.doi.org/10.1016/j.drudis.2012.02.011.

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