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Brain Research Bulletin 89 (2012) 159– 167

Contents lists available at SciVerse ScienceDirect

Brain Research Bulletin

jo u r n al hom epage : www.elsev ier .com/ locate /bra inresbul l

esearch report

-Phycocyanin protects SH-SY5Y cells from oxidative injury, rat retina fromransient ischemia and rat brain mitochondria from Ca2+/phosphate-inducedmpairment

avier Marín-Pridaa, Giselle Pentón-Rolb, Fernando Postalli Rodriguesc, Luciane Carla Alberici c,arina Stringhettad, Andréia Machado Leopoldinod, Zeki Naalc, Ana Cristina Morseli Polizelloc,lexey Llópiz-Arzuagab, Marcela Nunes Rosae, José Luiz Liberatoe, Wagner Ferreira dos Santose,ergio Akira Uyemurad, Eduardo Pentón-Ariasb, Carlos Curti c, Gilberto L. Pardo-Andreua,∗

Center for Research and Biological Evaluations, Institute of Pharmacy and Food, University of Havana, Ave. 23 e/214 y 222, PO Box 430, Havana, CubaCenter for Genetic Engineering and Biotechnology, Ave. 31 e/158 y 190, PO Box 6162, Havana, CubaDepartment 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, BrazilDepartment of Clinical, Toxicological and Bromatological Analysis, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ave. Café s/n,4040-903 Ribeirão Preto, São Paulo, BrazilDepartment of Biology, Faculty of Philosophy, Sciences, and Literature of Ribeirão Preto, University of São Paulo, Ave. Bandeirantes 3900, PO Box 14040-901,ibeirão Preto, São Paulo, Brazil

r t i c l e i n f o

rticle history:eceived 6 July 2012eceived in revised form 9 August 2012ccepted 30 August 2012vailable online 7 September 2012

eywords:-PhycocyaninH-SY5Yransient rat retinal ischemiaat brain mitochondriantioxidant

a b s t r a c t

Oxidative stress and mitochondrial impairment are essential in the ischemic stroke cascade and even-tually lead to tissue injury. C-Phycocyanin (C-PC) has previously been shown to have strong antioxidantand neuroprotective actions. In the present study, we assessed the effects of C-PC on oxidative injuryinduced by tert-butylhydroperoxide (t-BOOH) in SH-SY5Y neuronal cells, on transient ischemia in ratretinas, and in the calcium/phosphate-induced impairment of isolated rat brain mitochondria (RBM). InSH-SY5Y cells, t-BOOH induced a significant reduction of cell viability as assessed by an MTT assay, andthe reduction was effectively prevented by treatment with C-PC in the low micromolar concentrationrange. Transient ischemia in rat retinas was induced by increasing the intraocular pressure to 120 mmHgfor 45 min, which was followed by 15 min of reperfusion. This event resulted in a cell density reductionto lower than 50% in the inner nuclear layer (INL), which was significantly prevented by the intraocu-lar pre-treatment with C-PC for 15 min. In the RBM exposed to 3 mM phosphate and/or 100 �M Ca2+,

europrotection C-PC prevented in the low micromolar concentration range, the mitochondrial permeability transition asassessed by mitochondrial swelling, the membrane potential dissipation, the increase of reactive oxygenspecies levels and the release of the pro-apoptotic cytochrome c. In addition, C-PC displayed a stronginhibitory effect against an electrochemically-generated Fenton reaction. Therefore, C-PC is a potentialneuroprotective agent against ischemic stroke, resulting in reduced neuronal oxidative injury and the

ia fro

protection of mitochondr

Abbreviations: t-PA, tissue plasminogen activator; C-PC, C-Phycocyanin; t-OOH, tert-butylhydroperoxide; RBM, rat brain mitochondria; DMEM, Dulbecco’sodified Eagle Medium; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-

etrazolium bromide; PBS, phosphate buffered saline; DMSO, dimethyl sulfoxide;OP, intraocular pressure; INL, inner nuclear layer; EGTA, ethylene gly-ol tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;IM, standard incubation medium; H2DCFDA, dichlorodihydrofluorescein diac-tate; DCF, dichlorofluorescein; �� , mitochondrial membrane potential; TPP+,etraphenylphosphonium; MPT, mitochondrial permeability transition.∗ Corresponding author. Tel.: +53 72038472; fax: +53 72038472.

E-mail address: gilbertopardo@infomed.sld.cu (G.L. Pardo-Andreu).

361-9230 © 2012 Elsevier Inc. ttp://dx.doi.org/10.1016/j.brainresbull.2012.08.011

Open access under the Elsevier OA license.

m impairment.© 2012 Elsevier Inc.

1. Introduction

Ischemic stroke remains a major cause of death and disabilityworldwide (Mukherjee and Patil, 2011; WHO, 2011). Although sig-nificant progress has been made to clarify the mechanisms involvedin the ischemic stroke cascade, the development of new and effec-tive treatments has been largely unsuccessful. Thrombolysis withtissue plasminogen activator (t-PA) is currently the only approvedhuman pharmacological treatment, but it is restricted to a minority

Open access under the Elsevier OA license.

of patients due to its short therapeutic window (4.5 h) (Fonarowet al., 2011; Maiser et al., 2011). An alternative approach knownas neuroprotection has been focused on protecting the neurovas-cular unit by blocking the main pathogenic processes that causes

dx.doi.org/10.1016/j.brainresbull.2012.08.011http://www.sciencedirect.com/science/journal/03619230http://www.elsevier.com/locate/brainresbullmailto:gilbertopardo@infomed.sld.cudx.doi.org/10.1016/j.brainresbull.2012.08.011http://www.elsevier.com/open-access/userlicense/1.0/http://www.elsevier.com/open-access/userlicense/1.0/

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60 J. Marín-Prida et al. / Brain Re

schemic injury (Ginsberg, 2008). Reactive oxygen species (ROS)enerated soon after ischemia, during reperfusion and thereafterre considered crucial mediators of ischemic injury (Saito et al.,005). Mitochondria, an important source of ROS, contribute sig-ificantly to the elevation of these oxidants after an ischemic eventAllen and Bayraktutan, 2009). Thus, antioxidant-based neurovas-ular protective strategies may be potential treatments to expandhe number of currently approved therapies (Jung et al., 2010).

Previous studies have demonstrated that C-Phycocyanin (C-C), the main phycobiliprotein of cyanobacteria Spirulina platensis,xerts potent antioxidant, anti-inflammatory and neuroprotectivections in different experimental conditions (Romay et al., 2003).hycobiliproteins are light-harvesting complexes composed ofpo-proteins covalently attached to open chain tetrapyrrole chro-ophores, named phycocyanobilins. C-PC has � and � polypeptides

ubunits (18–20 kDa) (Pentón-Rol et al., 2011a); one phyco-yanobilin is attached at cysteine 84 in the �-chain and twohycocyanobilins are attached at cysteines 84 and 155 in the-chain (Padyana et al., 2001; Pentón-Rol et al., 2011a). Recent

esults from our group have shown that the administration of-PC either prophylactically or therapeutically exhibited bene-cial effects against global cerebral ischemia–reperfusion injury

n gerbils (Pentón-Rol et al., 2011a). These effects were partiallyxplained by the C-PC ability to counteract oxidative damages.

In the present study, we characterized the in vitro protectiveffects of C-PC against tert-butylhydroperoxide (t-BOOH)-inducedxidative injury in the SH-SY5Y human neuroblastoma cell line and

n vivo against transient ischemia in rat retinas. The effects of C-PCere also examined in isolated rat brain mitochondria (RBM) with

Ca2+/phosphate overload, a condition that mimics the mitotoxicnvironment that results from an ischemic injury.

. Materials and methods

.1. Reagents and compounds

All chemicals used were of the highest grade available and purchased fromigma–Aldrich (St. Louis, MO, USA) unless otherwise specified. C-PC was purifiedy ionic exchange, as previously described (Pentón-Rol et al., 2011a). An A620/A280bsorbance ratio of 4.58 was obtained, indicative of higher than 90% purity (Patil andaghavarao, 2007). C-PC was dissolved in sterile phosphate-buffered saline (PBS)H 7.2, filtered through a 0.2 �m syringe filter under sterile conditions (Sartoriusinisart® , Germany) and stored in the dark at 4 ◦C until use.

.2. Animals

Rats were obtained from the Animal Breeding Center at the Ribeirão Preto Cam-us, University of São Paulo (USP). The animals were maintained under standard

aboratory conditions (60% humidity, 22 ± 1 ◦C, and 12 h light/darkness cycle) withree access to food and water. All procedures were performed in compliance withhe USP guidelines and with the European Communities Council Directive of 24ovember 1986 (86/609/EEC) for animal experimentation.

.3. Cell culture

SH-SY5Y human neuroblastoma cells (Rio de Janeiro Cell Bank, Brazil) werexpanded in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) containing 10%etal bovine serum (Gibco, USA), 100 IU/mL penicillin G, 100 �g/mL streptomycinnd 0.25 �g/mL amphotericin B at 37 ◦C in 5% CO2/95% air. The medium was changedvery two days, and the cells were subcultured every four days by trypsinization ande-plated in T-25 or T-75 flasks (5 or 15 mL final volume, respectively) (BD Falcon,SA) at 4 × 105 cells/mL.

.4. Cell treatment and viability assay

SH-SY5Y cells between passages three and seven were cultured in quadrupli-ate in 96 wells plates (BD Falcon, USA) at 4 × 104 cells/200 �L/well for 24 h. Theells were incubated with freshly prepared t-BOOH in complete medium at differentoncentrations for 6 h, or pre-treated with C-PC for 24 h followed by the incubation

ith fresh medium containing C-PC and 25 �M t-BOOH for another 6 h. Cell viabilityas measured by the quantity of blue formazan products obtained from the color-

ess 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) byitochondrial dehydrogenases, which are only active in viable cells. After the incu-

ation time, the cells were gently washed with sterile phosphate buffered saline

Bulletin 89 (2012) 159– 167

(PBS) pH 7.2 (200 �L/well), and MTT (freshly prepared at 0.5 mg/mL in serum-freemedium) was added at 200 �L/well and incubated for 4 h at 37 ◦C. After MTT with-drawal, 200 �L of dimethyl sulfoxide (DMSO) was added to each well to solubilizethe formazan crystals, and the absorbance of each well was measured at 570 nm ina microplate reader (Varian Cary 50, Australia). The results were expressed as thepercent 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, withsome modifications (Beleboni et al., 2006). Male Wistar rats weighing 250–300 g atthe time of surgery (n = 5 per group) were anesthetized with 450 mg/Kg i.p. uret-hane (Acrôs Organic, USA) (Meyer, 2008), and the anterior chamber of the left eyewas cannulated with a 27-gauge needle attached to a manometer, a pump and anair 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 minreperfusion period. The drug was intraocularly injected 15 min prior to the ischemicinsult. 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 wererapidly enucleated, placed in the fixation solution (25.5 mL ethanol 80%, 3 mLformaldehyde 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 ingraded ethanol and xylene and embedded in paraffin. Retinas were sectioned at5 �m, approximately 1 mm from the optic nerve, and stained with hematoxylin andeosin. 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 num-ber of viable cells, those that were not eosinophilic, was manually counted in theestablished areas (200 �m × 50 �m) of the inner nuclear layer (INL) using ImageJ1.41 software (National Institutes of Health, USA). Cells showing atrophy, shrinkage,nuclear pyknosis, dark cytoplasmic coloration or ambient empty spaces were con-sidered damaged (Beleboni et al., 2006). The results were expressed as a percentageof the number of viable cells in the control retinas.

2.7. Isolation of rat brain mitochondria

Mitochondria were isolated as previously described, with minor modifications(Mirandola et al., 2010). Briefly, two male Wistar rats weighing 200–240 g were sac-rificed by decapitation, and their brains were rapidly removed and placed in 10 mLof ice-cold isolation buffer (225 mM mannitol, 75 mM sucrose, 1 mM ethylene gly-col tetraacetic acid (EGTA), 0.1% de-fatted bovine serum albumin (BSA), and 10 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); pH 7.2). The olfac-tory bulb, cerebellum and underlying regions were discarded, and the remainingforebrains 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 aglass 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 supernatantwas further centrifuged (12,000 × g, 8 min). The pellet was gently resuspended in10 mL of isolation buffer containing 20 �L of 10% digitonin (to induce synaptosomallysis) using a fine-tipped brush and centrifuged at 12,000 × g for 10 min. The darkpellet was resuspended in 10 mL of isolation medium without EGTA and BSA andcentrifuged again (12,000 × g, 10 min), and the resulting pellet was resuspended in200 �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 bythe Biuret method with BSA as a standard and was approximately 30–50 mg/mL foreach preparation. All procedures were performed at 4 ◦C.

2.8. Mitochondrial assays

The experiments using isolated RBM were performed at 30 ◦C with continuousmagnetic stirring in the SIM described above. The respiratory control ratio (state3/state 4 respiratory rates) was over 4; this ratio was determined after monitoringoxygen 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 KH2PO4. Oligomycin at 1 �g/mL was usedat state 4. Mitochondrial swelling was estimated from the decrease in the apparentabsorbance at 540 nm using a Model U-2910 Hitachi spectrophotometer (Japan).ROS generation in RBM was monitored using 2 �M of dichlorodihydrofluoresceindiacetate (H2DCFDA) (Invitrogen, Life Technology Co., USA). The changes in thefluorescence of the DCF product were monitored using an F-4500 fluorescence spec-trophotometer (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 (�� ) wasestimated by continuously monitoring the concentration of the lipophilic cationtetraphenylphosphonium chloride (TPP+) in the extramitochondrial medium (Rosset al., 2005) with a TPP+ selective electrode (World Precision Instruments Inc., USA)

search Bulletin 89 (2012) 159– 167 161

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Fig. 1. Protective effects of C-PC against oxidative injury in SH-SY5Y humanneuroblastoma cells. (A) Viability of cells exposed to different concentrations oftert-butylhydroperoxide (t-BOOH) for 6 h. (B) Effects of a pre-treatment with C-PCfor 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

J. Marín-Prida et al. / Brain Re

onnected to the oxygraph. For the calculation of �� , the matrix volume of theBM was assumed to be 1.5 �L/mg protein. The membrane potential was calcu-

ated assuming that the TPP+ distribution between the mitochondria and mediumollowed the Nernst equation. Corrections were made as previously described forhe binding of TPP+ to the mitochondrial membrane (Jensen et al., 1986). Data werexpressed as a percentage of �� in relation to the level reached before Ca2+addition.

.9. Assessment of the release of cytochrome c from mitochondria

The release of cytochrome c was assessed in the same conditions as used inhe swelling experiments. The mitochondrial suspension was centrifuged (9000 × g,

min) at 4 ◦C, and the supernatant (100 �L) was supplemented with 2 �L of proteasenhibitor cocktail (Sigma–Aldrich, USA), incubated with 25 �L of loading buffer (50%lycerol, 10% SDS, 0.05% bromophenol blue, 25% mercaptoethanol) at 95 ◦C for 5 min,ubjected to 12% SDS-PAGE and transferred to a nitrocellulose membrane. A mouseonoclonal antibody against cytochrome c (1:1000, BD Biosciences Pharmingen,SA), a mouse-specific secondary antibody conjugated with horseradish peroxidase

1:1000, Santa Cruz Biotechnology, USA) and the ECLTM Systems (GE HealthCare, UK)ere used for detection. The films were placed in a digital flatbed scanner (HP Scanjet

770, USA) for image acquisition. Densitometry was performed using the ImageJ.41 software (National Institutes of Health, USA). The results were presented as aercent of the Ca2+/phosphate damaged controls.

.10. Electrochemical assays

Electrochemical assays were conducted with a BAS CV-27 potentiostat (Bio-nalytical System, Inc., USA) using conventional electrochemical cells with threelectrodes and an Omnigraphic 100 recorder (Texas Instruments, USA). A glassyarbon electrode with a 0.0314 cm2 area, polished with a 1 �m alumina–wateruspension and rinsed thoroughly with water and acetone was used as workinglectrode. A platinum wire was used as the counter electrode, and all potentialsere referenced to a [Ag/AgCl/KCl (sat)] electrode without regard for the liquid junc-

ion potential. The electrolyte supporting solution was obtained by mixing 100 mMCl and 100 mM chloroacetic acid pH 3.3, and Fe3+-EDTA (0.1 mM) was formed fromquimolar additions of Fe(NO3)3 and Na2H2EDTA. The 100 mM hydroperoxide stockolution was prepared by dissolving 30% H2O2 in the electrolyte supporting solution.ach sample was deoxygenated with argon for 20 min before performing electro-hemical measurements. Cyclic voltammetry was conducted at a final volume of.0 mL for all experiments with a potential sweep rate of 30 mV/s and a potentialindow between −0.4 V and 0.4 V. Control experiments were measured in ESS in

he presence of Fe3+-EDTA or H2O2, in the presence of both and without C-PC or oxy-en (Argon purge). The voltammetric backgrounds were measured in the presencef Fe3+-EDTA but in the absence of any peroxides or oxygen (N2 purge).

.11. Statistical analysis

The software program GraphPad Prism 5.0 (GraphPad Software Inc., USA) wassed for statistical analysis. The data were expressed as the mean ± SEM. Com-arisons between the different groups were performed by the one-way analysisf variance (ANOVA) followed by the Newman–Keuls multiple comparison test.ifferences were considered statistically significant at p < 0.05.

. Results

.1. C-PC protected SH-SY5Y cells from t-BOOH-induced death

Fig. 1A shows that the viability of SH-SY5Y cells was significantlyeduced after exposing them to t-BOOH, and pre-treatment with C-C prevented this effect (Fig. 1B). C-PC at 50 �M almost completelyrevented the cell viability reduction and did not present cyto-oxic effects, demonstrating the safety of the compound. This resultemonstrates that C-PC has a protective effect against the oxida-ive neurotoxic injury induced by t-BOOH. We have used Trolox,

water-soluble analogue of vitamin E, as a control antioxidant tohow that ROS play a major role in t-BOOH cytotoxicity in SH-SY5Yells (Lee et al., 2000).

.2. C-PC protected rat retinas from transient ischemia

To assess whether C-PC protects neuronal viability against aocal stroke in vivo, we performed a proof-of concept experiment for

ransient ischemia in rat retinas. Fig. 2 shows that the rat retina wasighly susceptible to an ischemia–reperfusion injury primarily athe INL in our experimental conditions, which exhibited less than a0% reduction in viable cells; neuronal death was also accompanied

expressed as a percent of the control. Data are presented as the mean ± SEM ofthree independent experiments. Different letters: p < 0.05 according to the ANOVAand post hoc Newman–Keuls tests.

by edema and layer disorganization. Pre-treatment with C-PC pre-vented INL cell loss, indicating that C-PC not only protects neuronsin vitro, but also in in vivo ischemic conditions.

3.3. C-PC prevented the mitochondrial permeability transition inRBM

A key pathogenic event in a stroke is the opening of pores in theinner mitochondrial membrane; the mitochondrial permeabilitytransition (MPT) (Hirsch et al., 1998). In the next set of experi-ments, we assessed the effect of C-PC on the MPT as estimated bythe swelling of the RBM induced by Ca2+ and inorganic phosphate(Pi), as previously described (Berman et al., 2000) (Fig. 3A). Compar-isons among the initial decreases in the slopes of the absorbancesat 540 nm revealed that C-PC prevented MPT in the low micromo-lar concentration range (Fig. 3B). This suggests that the inhibitionof MPT is involved in the mechanisms that are responsible for theC-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 inbrain mitochondria (Maciel et al., 2001). Our results showed thatCa2+ overload induced a significant increase in ROS levels in RBM(Fig. 4A), and pre-treatment with C-PC at low micromolar concen-tration 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 approximately10 min, and then 100 �M of Ca2+ was added (Fig. 5). Ca2+ overloadproduced a �� dissipation of approximately 30%; this dissipation

162 J. Marín-Prida et al. / Brain Research Bulletin 89 (2012) 159– 167

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 ofthe 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: outernuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer and GCL: ganglion cell layer. Scale bar: 50 �m.

J. Marín-Prida et al. / Brain Research Bulletin 89 (2012) 159– 167 163

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 succi-nate and 2 �M rotenone followed by the addition of 100 �M CaCl2 (Ca2+) for another1 min. Then, 3 mM KH2PO4 (Pi) was added to the mitochondrial suspension, andabsorbance was monitored at 540 nm for 10 min (A). The rates of swelling (slope oftDD

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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 suc-cinate, 2 �M rotenone, 3 mM KH2PO4 (Pi) and 2 �M H2DCFDA, and fluorescencemonitoring was then initiated. After 5 min, 100 �M CaCl2 (Ca2+) was added to thesuspension, which was monitored for another 10 min (A). Comparisons in fluores-cence intensity were made at this point (B). Data are expressed as the mean ± SEM forthree independent experiments. Different letters: p < 0.05 according to the ANOVAand post hoc Newman–Keuls tests.

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), andthe % of �� dissipation after Ca2+ addition (insert). The �� values before and afterCa2+ 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, respec-tively. The RBM (2 mg/mL) were incubated in 1 mL of SIM with 5 mM succinate and

he decrease in absorbance in the first 5 min after Pi addition) were compared (B).ata are expressed as the means ± SEM of at least three independent experiments.ifferent letters: p < 0.05 according to the ANOVA and post hoc Newman–Keuls tests.

as effectively prevented by treatment with either C-PC or thelassical MPT inhibitor cyclosporin A (Fig. 5).

.6. C-PC prevented mitochondrial cytochrome c release in RBM

Cytochrome c release from mitochondria plays an importantole in cell death (Li et al., 1997). Our results showed that C-C prevented the oxidative stress-induced cell viability decreasen SH-SY5Y cells (Fig. 1). To assess the potential involvement ofytochrome c release inhibition in this effect, we used Western bloto detect the free cytochrome c in the supernatant fraction obtainedfter the mitochondrial swelling experiments (Fig. 6). The Ca2+/Pi-verload induced a substantial release of cytochrome c from RBM,ut this release was prevented by C-PC at low micromolar concen-rations. The effect of C-PC was significantly higher than the effectf the antioxidant Trolox.

.7. C-PC inhibited the Fenton reaction

H2O2 is regarded as a key mediator in oxidative stress because

t is highly stable under physiological conditions (Bergendi et al.,999). When reacting with metals such as iron, H2O2 yields

hydroxyl through the Fenton reaction (Winterbourn, 1995).ydroxyls are highly reactive radicals that can initiate lipid

2 �M rotenone in the presence or the absence of C-PC; this was performed afterthe addition (indicated by the arrows) of both 0.5 �M TPP+ at a 1.5 �M final con-centration and 100 �M CaCl2 (Ca2+). Data are expressed as the mean ± SEM of threeindependent experiments. Different letters: p < 0.05 according to the ANOVA andpost hoc Newman–Keuls tests.

164 J. Marín-Prida et al. / Brain Research

Fig. 6. Effects of C-PC on the release of cytochrome c from mitochondria asinduced by Ca2+ and phosphate. Representative Western blots and densitometryof 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,rph

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espectively; lane 6: Ca2+/Pi. Data are expressed as the mean ± SEM of three inde-endent experiments. Different letters: p < 0.05 according to the ANOVA and postoc Newman–Keuls tests.

eroxidation followed by membrane disorganization and ruptureAdibhatla and Hatcher, 2010). In this study, we assessed the effectf C-PC on the Fenton reaction using an electrochemical approachLaine and Cheng, 2009). Fig. 7 (trace a) shows a cyclic voltam-

ogram of the Fe3+-EDTA/Fe2+-EDTA oxidation–reduction processhat can be represented by Eq. (1). Fig. 7 (trace b) shows the voltam-

etric behavior of Fe3+-EDTA in the presence of 4 mM H2O2. The

ig. 7. Effects of C-PC on the Fenton reaction. Cyclic voltammograms of theeversible oxidation–reduction of Fe3+-EDTA/Fe2+-EDTA (a, Eq. (1)) and the elec-rocatalytic reaction between Fe2+-EDTA and H2O2 in the absence (b, Eq. (2)) and inhe presence of 0.38 mM C-PC (c). Solutions contain 100 mM KCl, 0.1 mM Fe3+-EDTAnd 100 mM chloroacetic acid (pH 3.3). Cyclic voltammograms were performed inhe absence of O2 by a prior nitrogen purge, at a scan rate of 100 mV/s, and with

glassy carbon electrode (area 0.0314 cm2) as the working electrode. The typicalracings of three independent experiments are shown.

Bulletin 89 (2012) 159– 167

intensity of the cathode current (arrowhead) was substantiallyincreased (from −4 �A to approximately −18 �A), but that of theanode was not. This indicates that a catalytic mechanism is involvedand could be ascribed to the Fenton reaction, represented by Eq. (2).The C-PC in the reaction medium reduced the cathode current toapproximately −8 �A (trace c), which represents approximately a55% inhibition of the Fenton reaction.

4. Discussion

Oxidative stress is a crucial factor in the pathophysiology ofischemic stroke and acts by either damaging cellular structures orparticipating in signaling pathways (Saito et al., 2005). Compoundsinhibiting this neurotoxic process have been considered as promis-ing therapeutic candidates against ischemic stroke (Broussalis et al.,2012). C-PC was shown to have a strong antioxidant activity inseveral in vivo experiments, including global ischemia–reperfusiondamage 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 encephalomyeli-tis (Pentón-Rol et al., 2011b), atherosclerosis (Riss et al., 2007),paraquat-induced lung damage (Sun et al., 2011), activatedplatelets (Hsiao et al., 2005) and thymic atrophy (Gupta et al., 2011).In vitro, C-PC prevents neurotoxic injury in cerebellar granule cellsduring conditions of low potassium and serum deprivation (Rimbauet al., 2001) and in SH-SY5Y human neuroblastoma cells subjectedto an environment of exogenous iron overload (Bermejo-Bescóset al., 2008). In this study, we assessed the potential of C-PC toprotect the SH-SY5Y cell line from the oxidative damage inducedby the organic hydroperoxide t-BOOH (Chance et al., 1979). C-PCprotected the viability of the SH-SY5Y cells exposed to t-BOOH.Iron can catalyze the conversion of t-BOOH to tert-butylperoxyand tert-butoxy radicals, which initiate lipid peroxidation (Cadenasand Sies, 1982). In primary rat cortical neurons, t-BOOH damagewas blocked by a pre-treatment with deferoxamine, an iron spe-cific chelator, indicating the requirement of the intracellular poolof iron for t-BOOH toxicity (Abe and Saito, 1998). Moreover, anincreased t-BOOH detoxification activity by glutathione or thiore-doxine peroxidases impaired the reductive effect of NAD(P)H onglutathione and thioredoxine and decreased their capacities toremove H2O2, particularly in mitochondria (Kowaltowski et al.,2001). Indeed, it has been reported that t-BOOH induces a freeradical 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 preservingthe endogenous antioxidant systems (Bermejo-Bescós et al., 2008),together with a potential iron chelating capability, may be relatedto the neuroprotective effects of C-PC reported in this study.

Retinal ischemia induced by an IOP increase can produce dev-astating visual impairment disorders such as glaucoma, which isthe second leading cause of blindness worldwide (Resnikoff et al.,2004). The retina is highly sensitive to a transient blockade in bloodsupply due to its active metabolism and high content of polyunsat-urated fatty acids, and therefore, this tissue is very susceptible tooxidative damage (Kuriyama et al., 2001). At the cellular level, accu-mulating evidence indicates that both glutamate excitotoxicity andoxidative stress are key mediators in ischemia–reperfusion injuryto retina (Bonne et al., 1998; Louzada-Junior et al., 1992; Osborneet al., 2004), similar to the damage in an ischemic brain (Allen andBayraktutan, 2009; Lau and Tymianski, 2010). Therefore, retinalischemic damage is a suitable in vivo model to assess the protective

effects of compounds against focal ischemic stroke. To our knowl-edge, the present study is the first to reveal that C-PC preventsneuron loss in rat retinas subjected to a transient ischemia. Becauseoxidative stress may be strongly implicated in the pathogenesis

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f ischemic stroke (Allen and Bayraktutan, 2009), the scavengerctivities of C-PC against hydroxyl radicals (Romay et al., 1998), per-xyl radicals (Lissi et al., 2000) and peroxynitrite anions (Bhat andadyastha, 2001) may be involved in C-PC preventing the neurode-

eneration of retinas after ischemia–reperfusion. Accordingly, ouresults showed an inhibitory effect of C-PC on the Fenton reactions measured by an electrochemical approach, and this inhibitionay be involved in the protection of retina tissue from ischemic

amage.Mitochondria are both initiators and targets of oxidative dam-

ge, which impairs ATP production, induces MPT and releasesro-apoptotic factors (Sims and Anderson, 2002; Valko et al., 2007).herefore, in this work, we also studied brain mitochondria as aotential pharmacological target to explain the neuroprotectiveffects elicited by C-PC.

Under various conditions, such as in the presence of Ca2+ andnorganic phosphate, isolated mitochondria undergo MPT. Thisrocess is characterized by a Ca2+-dependent increase in the per-eability of the inner mitochondrial membrane, resulting in a ��

issipation, mitochondrial swelling, and an eventual rupture of theuter mitochondrial membrane (Zoratti and Szabo, 1995). MPT isne of the proposed mechanisms for the release of mitochondrialro-apoptotic mediators, and its inhibition is a plausible phar-acological target for therapeutic intervention in stroke (Kristal

t al., 2004). In our study, MPT was induced by an overload ofa2+ in the presence of inorganic phosphate (Pi) and estimated byhe occurrence of RBM swelling. These conditions mimic those ofschemic neurons, in which a rise in cytosolic Ca2+ and Pi concen-rations due to excitotoxic glutamate and increased ATP hydrolysis,espectively, both occur (Nicholls and Budd, 1998; Oliveira andowaltowski, 2004). As a result of C-PC treatment, mitochondrialwelling, and therefore MPT, was significantly prevented, and thisay constitute an important event contributing to the neuropro-

ective capability of C-PC.Ischemia induces an abnormal increase in intracellular Ca2+

evels. This cation can cause mitotoxic effects through the acti-ation of degradative and ROS-generating enzymes, which mayamage mitochondrial structures (Nakahara et al., 1992; Wingravet al., 2003). Accordingly, our results showed a significant risen ROS levels in isolated RBM as a consequence of Ca2+/Pi over-oad. The probe that we used for ROS detection, H2DCFDA, is a

embrane-permeable molecule that can be converted by mito-hondrial matrix esterases to a non-fluorescent H2DCF productLeBel and Ischiropoulos, 1992). Although it is not actually a reliablerobe for superoxide and hydrogen peroxide, the most commonOS in biology, it is still widely used (Wardman, 2007). In theresence of different radicals, such as hydroxyl, carbonate, nitro-en dioxide (Wardman, 2007), and peroxynitrite (Jakubowski andartosz, 2000), H2DCF is oxidized to the highly fluorescent DCFroduct. We assumed that increased H2DCF oxidation reflects theverall ROS status rather than any reactive species in particular.herefore, the inhibitory effect of C-PC on mitochondrial ROS levelsay reflect its radical scavenging activity as a central mechanism

o protect mitochondria against oxidative injury. In this regard, it isorth mentioning that in isolated guinea-pig brain mitochondria,

n the absence of nucleotides and respiratory chain inhibitors, H2O2eneration was decreased by Ca2+ (Komary et al., 2008). However,ifferences in the experimental conditions, tissues or species usedi.e. guinea-pig brain cortices against rat forebrains) may influencehe characteristics of ROS production in brain mitochondria (Adam-izi, 2005). Accordingly, mitochondrial complex I inhibition byotenone stimulates ROS generation (Barrientos and Moraes, 1999;

iu et al., 2002; Votyakova and Reynolds, 2001) and Ca2+ stronglyncreases ROS levels as detected with H2DCFDA in rotenone-treatedat forebrain mitochondria. Apparently, this increase is not associ-ted with mitochondrial Ca2+ accumulation, but with Ca2+ acting

Bulletin 89 (2012) 159– 167 165

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 associatedwith oxidative damage to mitochondrial membranes (Brustovetskyet al., 2002; Peng and Jou, 2010). These processes were inhibited byCsA and Trolox, an MPT blocker and a water-soluble antioxidant,respectively, indicating that MPT and the oxidative imbalance areimportant in mitochondrial impairment. The protective effect of C-PC 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 neuroprotectiveboth in vitro against t-BOOH-induced oxidative damage in the SH-SY5Y human neuroblastoma cell line and in vivo in rat retina againstischemia–reperfusion injury. In the isolated RBM, C-PC preventedMPT, restored normal ROS levels, and prevented �� dissipationand cytochrome c release. These results indicate that mitochon-drial protection may be a mechanism involved in the effects of C-PCagainst oxidative injury in neuronal cells.

Conflicts of interest

The authors have no potential conflicts of interest related to thesubject of this report.

Acknowledgments

This work was partially supported by a CAPES-Brazil/MES-Cuba(064/09) project.

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C-Phycocyanin protects SH-SY5Y cells from oxidative injury, rat retina from transient ischemia and rat brain mitochondria ...1 Introduction2 Materials and methods2.1 Reagents and compounds2.2 Animals2.3 Cell culture2.4 Cell treatment and viability assay2.5 Induction of transient retinal ischemia2.6 Histological assessment2.7 Isolation of rat brain mitochondria2.8 Mitochondrial assays2.9 Assessment of the release of cytochrome c from mitochondria2.10 Electrochemical assays2.11 Statistical analysis

3 Results3.1 C-PC protected SH-SY5Y cells from t-BOOH-induced death3.2 C-PC protected rat retinas from transient ischemia3.3 C-PC prevented the mitochondrial permeability transition in RBM3.4 C-PC prevented an increase in ROS levels in RBM3.5 C-PC prevented ΔΨ dissipation in RBM3.6 C-PC prevented mitochondrial cytochrome c release in RBM3.7 C-PC inhibited the Fenton reaction

4 DiscussionConflicts of interestAcknowledgmentsReferences