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Research Report

Pyruvate protects mitochondria from oxidative stress inhuman neuroblastoma SK-N-SH cells

Xiaofei Wanga, Evelyn Pereza, Ran Liua, Liang-Jun Yana,Robert T. Malletb, Shao-Hua Yanga,⁎aDepartment of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Blvd.,Fort Worth, TX 76107-2699, USAbDepartment of Integrative Physiology, University of North Texas Health Science Center, Fort Worth, TX 76107-2699, USA

A R T I C L E I N F O

⁎ Corresponding author. Fax: +1 817 735 0485.E-mail address: syang@hsc.unt.edu (S.-H.Abbreviations: ROS, reactive oxygen spe

tetrazolium bromide; DCFH-DA, 2,7-dichlortetramethylrhodamine; FRET, fluorescence resaline; HBSS, Hanks-buffered salt solution; H

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.11.032

A B S T R A C T

Article history:Accepted 12 November 2006Available online 15 December 2006

Oxidative stress is implicated in neurodegenerative diseases including stroke, Alzheimer'sdisease and Parkinson's disease, and has been extensively studied as a potential target fortherapeutic intervention. Pyruvate, a natural metabolic intermediate and energy substrate,exerts antioxidant effects in brain and other tissues susceptible to oxidative stress. Wetested the protective effects of pyruvate on hydrogen peroxide (H2O2) toxicity in humanneuroblastoma SK-N-SH cells and the mechanisms underlying its protection. Hydrogenperoxide insult resulted in 85% cell death, but co-treatment with pyruvate dose-dependently attenuated cell death. At concentrations of ≥1 mM, pyruvate totally blockedthe cytotoxic effects of H2O2. Pyruvate exerted its protective effects even when itsadministration was delayed up to 2 h after H2O2 insult. As a scavenger of reactive oxygenspecies (ROS), pyruvate dose-dependently attenuated H2O2-induced ROS formation,assessed from 2,7-dichlorofluorescein diacetate fluorescence. Furthermore, pyruvatesuppressed superoxide production by submitochondrial particles, and attenuatedoxidative stress-induced collapse of the mitochondrial membrane potential. Collectively,these results suggest that pyruvate protects neuronal cells through its antioxidant actionson mitochondria.

© 2006 Elsevier B.V. All rights reserved.

Keywords:PyruvateMitochondriaOxidative stressNeuroprotectionHydrogen peroxideSuperoxide

1. Introduction

The central nervous system is particularly vulnerable tooxidative damage due to its high energy expenditure andoxygen demand. Elevated concentrations of free radicalsand resultant oxidative damage, such as lipid peroxidation

Yang).cies; SMPs, submitochoofluorescein diacetate; Dsonance energy transfer;2O2, hydrogen peroxide; F

er B.V. All rights reserved

and protein carbonylation, have been repeatedly demon-strated in neurodegenerative disorders, such as Alzheimer'sdisease, Parkinson's disease and ischemic stroke (Andersen,2004). Accordingly, antioxidants have been extensivelystudied as potential therapies for various neurodegenerativediseases.

ndrial particles; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylHR, dihydrorhodamine 123; NAO, nonyl acridine orange; TMR,ΔΨm, mitochondrial membrane potential; PBS, phosphate-bufferedBS, fetal bovine serum

.

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Mitochondria, which generate ATP by oxidative phosphor-ylation to meet the brain's energy demands, rank among theprincipal intracellular targets of reactive oxygen species (ROS).These unstable compounds inflict oxidative damage oncellular proteins, lipids, and nucleic acids. Oxidative damageto mitochondrial membranes, enzymes and respiratory chaincomponents culminate in the impairment of mitochondrialATP production. Moreover, ROS are known to interact withphysiologic signaling mechanisms and can initiate apoptoticor necrotic cell death. Indeed, mitochondria dysfunction isassociated with various neurodegenerative diseases and brainischemic injury (Beal, 2005; Dawson and Dawson, 2003;Fiskum, 2000; Fiskum et al., 2004).

An aliphatic monocarboxylate and glycolytic end product,pyruvate enters mitochondria via the inner membrane mono-carboxylate transporter and assumes a central role in cellularenergy production. Moreover, pyruvate embodies antioxidantproperties due to its α-keto-carboxylate structure, enabling it to

Fig. 1 – Cytoprotective effect of pyruvate against H2O2-induced cH2O2 (150 μM) exposure for 18 h induced 85% cell death. Co-treasurvival (a). Delayed pyruvate administration 30 min (b), 1 h (c) aagainst oxidative insult. Values aremeans±SEM from 8 experimewas replicated at least 3 times. ***p<0.001 vs. H2O2+vehicle.

directly neutralize peroxides and peroxynitrite, and itsmitochon-drial metabolism, which generates NADPH to maintain glu-tathione reducing power (Mallet et al., 2005). Pyruvate has beenshown to be neuroprotective in various in vitro and in vivomodelsof oxidative stress. In neuronal cell cultures, pyruvate protectsagainst various insults such as β-amyloid (Alvarez et al., 2003),H2O2 (Desagher et al., 1997; Jagtap et al., 2003; Yoo et al., 2004),mitochondrial toxins (Mazzio and Soliman, 2003b) and zinc (Chenand Liao, 2003; Kawahara et al., 2002). In in vivomodels, pyruvateand its derivatives have been shown to protect against cerebralischemic injury (Leeet al., 2001;Monganet al., 2001;Yuet al., 2005)and zinc toxicity (Lee et al., 2001).

This study tested the effects of pyruvate against oxidativestress in human neuroblastoma SK-N-SH cells, and sought todefine the role ofmitochondria inmediating cytoprotection bypyruvate. We demonstrated that pyruvate remarkably abro-gated H2O2 toxicity in SK-N-SH cells by its direct antioxidantprotection of the mitochondria.

ell death. Cell viability was determined by calcein AM assay.tment of sodium pyruvate dose-dependently increased cellnd 2 h (d) after H2O2 also afforded significant protectionnts per group. In this figure and in Figs. 2–5, each experiment

Fig. 3 – Pyruvate suppression of hydrogen peroxide-inducedintracellular (a) and mitochondrial (b) ROS formation. (a)150 μM H2O2 increased intracellular ROS accumulation,indicated by DCF fluorescence, which plateaued at 45–60minof exposure. Pyruvate co-treatment at 1 and 2 mM, but not atsub-millimolar concentrations, sharply lowered intracellularROS formation. (b) 150 μM H2O2-induced mitochondrial ROSproduction, assessed from DHR fluorescence, plateaued at30 min of exposure. Pyruvate (1–4 mM) concentration-

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2. Results

2.1. Cytoprotection by pyruvate against oxidative stress

Exposure of SK-N-SH cells to 150 μM H2O2 for 18 h resulted in85% cell death as measured by calcein AM assay (Fig. 1b).Sodium pyruvate co-treatment dose-dependently increasedcell survival at concentrations from 100 μM to 4 mM. Atconcentrations ≥1 mM, pyruvate essentially prevented H2O2-induced cell death (Fig. 1a).

Additional experiments tested whether pyruvate couldprotect cells even when its administration was delayed up to2 h after H2O2 exposure. Administration of sodium pyruvate at30 min (Fig. 1b) or 1 h (Fig. 1c) after H2O2 provided significantprotection against the oxidative insult. When given 1 h afterH2O2, 100 μM and 1 mM sodium pyruvate increased cellsurvival from 16±2% to 55±3% and 79±3%, respectively. Evenwhen administered 2 h after H2O2, pyruvate provided sig-nificant protection against H2O2-induced cytotoxicity, albeitwith decreased efficacy (Fig. 1d).

The cytoprotective effects of pyruvate were also examinedby MTT assay (Fig. 2). Exposure of cells to 150 μM H2O2 for 18 hdecreased MTT reduction to 37±1% of baseline. Pyruvate co-treatment dose-dependently mitigated the loss of MTTreduction caused by H2O2: 0.1 mM pyruvate increased MTTreduction to 49±3% of baseline, and 1–4 mM pyruvate nearlyrestored the activity to c. 90% of baseline (Fig. 2).

2.2. Effect of pyruvate on hydrogen peroxide-induced freeradical generation

The antioxidant effects of pyruvate on intracellular andmitochondrial reactive oxygen species (ROS) were respectivelyexamined by monitoring DCFH-DA and DHR fluorescence.

Fig. 2 – Cytoprotective effect of pyruvate against H2O2 insult.Cell viability was assessed by MTT assay. Exposure to150 μM H2O2 for 18 h markedly decreased MTT reduction to37.0±0.8% of baseline. Pyruvate co-treatment at ≥0.1 mMameliorated H2O2-induced loss of MTT reduction. Values aremeans±SEM from 8 experiments per group. **p<0.01 vs.H2O2+vehicle; ***p<0.001 vs. H2O2+vehicle.

dependently suppressed mitochondrial ROS formationinduced by H2O2. Values are means±SEM from 8experiments per group. *p<0.05 vs. H2O2+vehicle, ***p<0.001vs. H2O2+vehicle.

DCFH-DA fluorescence revealed a progressive increase inintracellular ROS to 609±12% of H2O2-free control following1 h of exposure to 150 μM H2O2 (Fig. 3a). Co-treatment with 1and 2 mM sodium pyruvate sharply attenuated intracellularROS accumulation to 220±5% and 126±5% of control, respec-tively (Fig. 3a). DHR fluorescence, a measure of mitochondrialROS, revealed a similar pattern of responses to H2O2 andsodium pyruvate (Fig. 3b). H2O2 alone increased DHR fluores-cence up to 10-fold within 30 min. Pyruvate dose-dependentlyattenuatedmitochondrial ROS generation (Fig. 3b), although toa somewhat lesser extent than its suppression of intracellularROS (Fig. 3a).

2.3. Effect of pyruvate on superoxide production bysubmitochondrial particles

Inhibition of mitochondrial respiratory complexes canincrease UO2

− production. We assessed effects of pyruvate onUO2

− production by SMP under three conditions: no mitochon-drial complex inhibitor; mitochondrial complex I inhibitor,

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rotenone; mitochondrial complex III inhibitor, antimycin A. Inthe absence of respiratory complex inhibitors, UO2

− generationrate was 0.79±0.09 pmol mg protein−1 min−1; treatment with0.1 and 1mMpyruvate lowered the rate to 0.38±0.10 and 0.50±0.07 pmol mg protein−1 min−1, respectively (Fig. 4a). In thepresence of respiratory complex inhibitors, rotenone (Fig. 4b)and antimycin A (Fig. 4c), UO2

− production increased to 1.16±0.12 and 1.31±0.06 pmol mg protein−1 min−1, respectively.Pyruvate at 0.1 and 1 mM markedly reduced UO2

− generation inthe presence of the respiratory inhibitors (Figs. 4b, c).

2.4. Effect of pyruvate on mitochondrial membranepotential

In the FRET assay, 30 min of exposure to 3.0 mM H2O2 causedΔΨm collapse in SK-N-SH cells. As expected, the concentrationof H2O2 required to cause acute collapse of ΔΨm (i.e., 3 mM)wassubstantially more than that required for long-term cytotoxi-city (i.e., 0.15 mM). H2O2 rapidly induced ΔΨm collapse,evidenced by the increase of NAO fluorescence due to efflux

Fig. 4 – Pyruvate attenuates UO2− formation by submitochondrial

complex inhibitors. Rotenone, a respiratory complex I inhibitorformation over the basal, inhibitor-free rate (a). Basal and respi0.1 and 1 mM pyruvate. Values are means±SEM from 4 to 5 excontrol.

of TMR from the mitochondrial matrix. Pyruvate concentra-tion-dependently reduced the magnitude of mitochondrialdepolarization induced by H2O2 (Fig. 5a).

Pyruvate protection of ΔΨm during H2O2 exposure wasconfirmed by confocal microscopy, using JC1 as ΔΨm

indicator (Fig. 5b). H2O2 insult induced ΔΨm collapse,indicated by the progressive loss of red J-aggregate fluores-cence and increase of green monomer fluorescence, produ-cing a shift from red to yellow in the confocal images (Fig.5b). The ΔΨm collapse induced by H2O2 was suppressed by2 mM pyruvate.

3. Discussion

Two important findings resulted from this study. First,pyruvate exerts significant protection of neuronally derivedSK-N-SH cells against H2O2-induced oxidative stress in bothco-treatment as well as delayed treatment protocols. Con-cordant with reports of other investigators, the cytoprotective

particles in the absence (a) and presence (b, c) of respiratory(b) and antimycin D, a complex III inhibitor (c) increased UO2

−

ratory inhibitor-enhanced UO2− formation was suppressed by

periments per group. *p<0.05 vs. control, ***p<0.001 vs.

Fig. 5 – Pyruvate mitigates H2O2-induced mitochondrial depolarization. (a) Mitochondrial membrane potential collapseinduced by 3 mM H2O2 and the effect of pyruvate. H2O2 caused ΔΨm collapse, evidenced by the increase of NAO fluorescence asthe consequence of efflux of TMR. Pyruvate dose-dependently attenuated H2O2-induced mitochondrial depolarization. Valuesare means±SEM from 6 to 8 experiments. **p<0.01 vs. vehicle, ***p<0.001 vs. vehicle. (b) Confocal microscopic images ofmitochondrial membrane depolarization induced by hydrogen peroxide and the effect of pyruvate. Confocal microscopicimages show the same field of cells viewed before and at 10, 20, and 30 min after H2O2 (2 mM) exposure with vehicle (upper) or2mM sodium pyruvate (lower) co-treatment. After exposure to H2O2, aggregated JC1 (in red) becamemonomeric and fluorescedgreen upon mitochondrial depolarization, as evidenced by the increase of yellow (merging of red and green) in the time seriesimages. Pyruvate co-treatment minimized mitochondrial depolarization induced by H2O2.

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effects of pyruvate are concentration-dependent, and areafforded by physiological (high μM) to pharmacological (lowmM) concentrations (Desagher et al., 1997; Jagtap et al., 2003).The therapeutic window of pyruvate administration extendsup to 2 h after H2O2 insult, suggesting pyruvate protects cellsby mechanisms other than its direct nonenzymatic reactionwith H2O2. Second, pyruvate cytoprotection against oxidativeinsult ismediated through its direct action inmitochondria, asevidenced by its dampening of mitochondrial ROS generationand its stabilization of mitochondrial membrane potentialmaintenance under oxidative stress.

The SK-N-SH neuroblastoma cell line was developed byBiedler et al. (1973), and is used extensively as a target cell linein cytotoxicity assays. SK-N-SH cells exhibit a neuronalphenotype with expression of multiple neurochemical mar-kers (Biedler et al., 1978). SK-N-SH cells respond to numerousinsults including β-amyloid, mitochondrial permeability tran-sition, and serum deprivation, indicating that this cell linecould be very useful in the assessment of neurotoxicity andneuroprotection (Ba et al., 2003). SK-N-SH cells have been used

by this and other laboratories (Ba et al., 2004; Green et al., 2001;Gridley et al., 1998; Wallace et al., 2006; Wang et al., 2006;Watters and Dorsa, 1998; Wen et al., 2004; Xia and Krukoff,2004; Zaulyanov et al., 1999) as an in vitro model for studyingpotential neuroprotection mechanisms.

Peroxides are generated continuously by cells that con-sume oxygen. Among the different peroxides, H2O2 is the oneformed in the highest quantities (Dringen et al., 2005). H2O2 is amajor reactive oxygen intermediate and a by-product ofnormal cellular metabolism produced by superoxide dismu-tase (SOD) and monoamine oxidase (MAO). H2O2 can easilypenetrate cellular membranes and exert its toxic effectsthrough either the ferrous iron-dependent or UO2

−-drivenformation of the highly reactive hydroxyl radical (UOH),which attacks and modifies lipids, proteins and DNA, or byaltering cytosolic redox status (Halliwell, 1992). Normally,cellular concentrations of H2O2 and redox-active iron are keptlow by efficient disposal of H2O2 and by storage of iron inferritin, respectively. However, in pathological situationsincluding various neurodegenerative diseases, the detoxifi-

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cation of H2O2 is compromised in neurons and other cell types,and H2O2 accumulates and triggers cell death cascades(Dringen et al., 2005; Hampton and Orrenius, 1997). Highconcentrations of H2O2 have been detected in the brain afterischemia–reperfusion injury (Patt et al., 1988; Hyslop et al.,1995). In striatum, H2O2 concentrations could exceed 100 μMafter ischemia–reperfusion insult (Hyslop et al., 1995). Suchconcentrations of H2O2 have been shown to be toxic to PC12cells and rat primary hippocampal neurons (Hyslop et al.,1995). In this study, 18 h of exposure to 150 μM H2O2 caused85% death of SK-N-SH cells. Consistent with reports fromother laboratories (Desagher et al., 1997; Jagtap et al., 2003; Yooet al., 2004), pyruvate dose-dependently prevented H2O2-induced cell death. Moreover, this study demonstrated thatpyruvate afforded significant neuroprotection even when itsadministration was delayed up to 2 h after oxidative insult.

Multiple mechanisms have been suggested to contribute tothe cytoprotective effect of pyruvate. Pyruvate enters cells by aspecific H+-monocarboxylate cotransporter (Garcia et al., 1994;Poole and Halestrap, 1993). A readily oxidized metabolic fuel,pyruvate bolsters cytosolic energy state, thereby providingenergy to maintain cellular functions in the face of metabolicchallenges (Bunger et al., 1989; Mallet and Bunger, 1993). Inaddition, antioxidant actions of pyruvate could be pivotal to itscytoprotection. Pyruvate can directly degrade H2O2 andperoxynitrite in nonenzymatic reactions (Desagher et al.,1997; Mallet and Sun, 2003). Consequently, H2O2 has a shorthalf-life of only a few min when exposed to excess pyruvate(Dringen and Hamprecht, 1997; Mallet et al., 2005). In thisstudy, pyruvate exerted significant cytoprotection even whenits administration was delayed until after the oxidative insult.Pyruvate provided essentially complete cytoprotection whenadministered concomitantly or 30 min after H2O2, and stillafforded significant, albeit incomplete, protection when given1 or 2 h after the oxidant. This delayed pyruvate cytoprotec-tion suggested that mechanisms in addition to direct H2O2

degradation may have contributed to pyruvate's salutaryeffects. Indeed, a recently study also suggested that cytopro-tection could be afforded evenwhen pyruvate was provided 1–3 h after H2O2 insult in primary cortical neurons (Nakamichi etal., 2005). Moreover, pyruvate protection is not limited to H2O2-induced cytotoxicity, and has been demonstrated in responseto various cytochemical insults, including zinc (Kawahara etal., 2002), menadion (Desagher et al., 1997), NMDA (Maus et al.,1999), glutamate (Ruiz et al., 1998), β-amyloid (Alvarez et al.,2003), and 1-methy-4-phenylpyridinium ion (MPP+) (Mazzioand Soliman, 2003a).

In this study, pyruvate's cytoprotective effects wereexamined by calcein AM and MTT assays. Reduction of thetetrazolium salt MTT to a blue formazan product is widelyused for assessing cell survival. The reduction is mainlycatalyzed by dehydrogenases localized in the mitochondria ofviable cells (York et al., 1998). Thus, the cytoprotectionrevealed by MTT assay suggested a direct action of pyruvateon mitochondria. Mitochondria are complex organellesinvolved in oxygen consumption, production of ATP, ROSgeneration and mobilization of calcium (Green and Kroemer,2004; Gunter and Pfeiffer, 1990; Melov, 2000). Pyruvate hasbeen reported to increase contribution of mitochondria toneuronal calcium homeostasis, and thereby mitigate gluta-

mate-mediated neurotoxicity (Ruiz et al., 1998; Villalba et al.,1994). Accordingly, we tested the effects of pyruvate oncellular and mitochondrial ROS generation induced by oxida-tive stress.

Formation of ROS is an early cellular response to oxidativestress. Exposure to H2O2 initiated a rapid burst in ROSformation as demonstrated by the DCFH-DA assay. Thefluorogenic compound DCFH-DA has been utilized extensivelyas a marker for overall intracellular oxidative stress, and isthought to reflect the overall oxidative status of the cell (Wangand Joseph, 1999). DCFH is sensitive towards oxidation by H2O2

in combination with cellular peroxidases, peroxidases alone,peroxynitrite or UOH, but is not suitable for measurement ofnitric oxide, hypochlorous acid, or UO2

− in biological systems(Myhre et al., 2003). In this study, pyruvate at low millimolarconcentrations suppressed H2O2-induced ROS formation.However, 100 μM pyruvate, although partially cytoprotective,did not affect intracellular ROS production in response toH2O2. This discrepancy suggested other mechanisms mayhave mediated the cytoprotection by sub-millimolar concen-trations of pyruvate.

Mitochondria are themajor intracellular sources of ROSandalso the major targets of oxidative stress (Fiskum et al., 2004).We used DHR to examine the effect of pyruvate on mitochon-drial ROS generation. DHR is the uncharged and nonfluores-cent reduction product of the mitochondrial-selective dyerhodamine 123 and has been used to detect mitochondrialreactive oxygen and nitrogen species, including UO2

− andperoxynitrite. Similar to its effects on the DCFH-DA assay,pyruvate also inhibitedmitochondrial ROS generation inducedby H2O2. We further tested the effect of pyruvate on UO2

−

generation by submitochondrial particles. Inhibition ofrespiratory complexes I or III inhibition causes electrons toaccumulate within respiratory chain components; theseelectrons canbe addeddirectly to oxygenmolecules to produceUO2

− (St-Pierre et al., 2002; Turrens et al., 1985). Here, inhibitionof complexes I and III induced robust UO2

− generation. Pyruvatenot only inhibited UO2

− production under basal conditions butalso suppressed UO2

− generation induced by either complex I orcomplex III inhibition.

The proposed mitochondria-protective effect of pyruvatewas corroborated by its preservation of ΔΨm, the driving forcefor mitochondrial ATP production. Depolarization and col-lapse of ΔΨm is one of the early events in the apoptotic cascade.Depolarization of ΔΨm can be initiated by opening of mito-chondrial permeability transition pores, followed by release ofpro-apoptotic factors such as cytochrome c from the mito-chondrial inter-membrane space. Pyruvate, a readily oxidizedmetabolic fuel, could bolster cytosolic energy state (Mallet andBunger, 1993; Mallet et al., 2005), thereby providing energy tomaintain cellular functions in the face of oxidative stress(Nicholls et al., 2003). Indeed, we found that pyruvate couldmaintain ATP production compromised by oxidative stress(data not shown). Pyruvate maintained ΔΨm, demonstrated byFRET assay and confocal microscopy of JC1, providing directevidence to support the proposal that pyruvate protectsneurons by stabilizing mitochondrial function.

In summary, the present study demonstrated that pyr-uvate exerts significant protection against oxidative stress inneuroblastoma SK-N-SH cells. Pyruvate suppressesmitochon-

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drial ROS generation and maintains ΔΨm under oxidativestress, indicating that mitochondria are the principal targetsof pyruvate neuroprotection.

4. Experimental procedures

4.1. Chemicals

Sodium pyruvate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), gentamycin, and H2O2 werepurchased from Sigma (St. Louis, MO, USA). Calcein AM, 2,7-dichlorofluorescein diacetate (DCFH-DA), dihydrorhodamine123 (DHR), nonyl acridine orange (NAO), and tetramethylrho-damine (TMR) were obtained from Molecular Probes (Eugene,OR, USA). Fetal bovine serum (FBS) was purchased fromHyclone (Logan, UT, USA).

4.2. Cell culturing

SK-N-SH human neuroblastoma cells were obtained fromAmerican Type Tissue Collection (Rockville, MD) at passage 38.Cells were grown to monolayer confluency in RPMI-1640media supplementedwith 10% FBS and 20 μgml−1 gentamycinin plastic Nunc 75 cm2 flasks (Fisher Scientific, Orlando, FL) at37 °C under 5% CO2/95% air. Medium was changed 3 times/week. Cells were observed with a phase-contrast microscope(Nikon Diaphot-300). SK-N-SH cells were back-cultured every5–7 days using standard trypsinization procedures tomaintainthe cell line. SK-N-SH cells were used in passages 39–48. Foroxidative insult, H2O2 was diluted with culture media to thedesired concentration before use. Equimolar NaCl served as‘vehicle’ control for sodium pyruvate.

4.3. Cell viability: calcein AM and MTT assays

For viability assay, SK-N-SH cells were plated at a density of20,000 cells/well in 96-well plates 72 h before initiation ofexperiments. Cells were exposed to treatments (H2O2, pyr-uvate) for 18 h. For calcein AM assay, cells were rinsed with 1×phosphate-buffered saline (PBS; pH 7.4) immediately aftertreatment, and calcein AM (25 μM) was added. After incuba-tion for 15 min at 37 °C, calcein AM fluorescence wasdetermined at 485 nm excitation wavelength and 538 nmemission wavelength, using a Biotek FL600 microplate-reader(Highland Park, VT). Percentage viability was calculated bynormalizing values to those of the H2O2-free control group,taken as 100% viable. For MTT assay, MTT (5 mg ml−1) wasadded to cultures, incubated at 37 °C for 3–4 h, and thenculture media was removed. Following overnight solubiliza-tion of the formazan product in 50%N,N-dimethyl formamide,20% sodium dodecyl sulfate, pH 4.8, absorbance values weredetermined at 590 nm.

4.4. Measurement of ROS formation

The extent of cellular oxidative stress was assessed bymonitoring the formation of free radical species using DCFH-DA or DHR. Cells were plated 72 h before initiation of theexperiment at a density of 20,000 cells/well in 96-well plates.

For DCFH-DA assays, cells were incubated with 50 μM DCFH-DA for 45 min. After removal of DCFH-DA medium, cells werewashed twice with 1× PBS (pH 7.4) and incubated with RPMIcontaining 10% FBS and 150 μM H2O2 for 60 min. 2,7-dichlorofluorescein fluorescence was measured at 485 nmexcitation wavelength and 538 nm emission wavelength. ForDHR assay, cells were incubated with 10 μM DHR at 37 °C for30min, thenwashedwith PBS and incubatedwith 150 μMH2O2

as described above. DHR fluorescence was measured at485 nm excitation wavelength and 538 nm emission wave-length. Fluorescence values were expressed as percentages ofuntreated control values.

4.5. Preparation of mitochondria and submitochondrialparticles

SK-N-SH cells were harvested, resuspended in ice-coldmitochondria isolation buffer (0.3 M sucrose, 0.03 M nicotina-mide, 0.02 M EDTA, pH 7.4) and homogenized with a Teflonpiston. Cell homogenates were centrifuged twice at 700×g for10 min. The supernatant was collected and centrifuged at10,000×g for 10 min. The pellet, i.e. the mitochondrial fraction,was resuspended in 30 mM potassium phosphate buffer(pH 7.4) and sonicated 4 times for 1 min with at least 1-minintervals between each sonication. Unbroken mitochondriawere sedimented by centrifuging in mitochondrial isolationsolution at 8000×g for 10 min. The supernatant containing thesubmitochondrial fraction was collected and centrifuged at18,000×g for 60 min. The pellet, enriched in submitochondrialparticles (SMP) was resuspended in 100 mM potassiumphosphate buffer (pH 7.4). Protein concentrations were color-imetrically assayed as described by Bradford (1976), usingbovine serum albumin at concentrations ranging from 0.063 to1 mg ml−1 as standard.

4.6. Superoxide generation by submitochondrial particles

The rate of superoxide (UO2−) generation was measured as

previously described (Yan et al., 2002). SMP were resuspendedin 100 mM phosphate buffer (pH 7.4). The rate of SMPproduction of UO2

− was taken as the rate of SOD-inhibitablereduction of acetylated ferricytochrome c (Azzi et al., 1975).The reaction mixture contained 10 μM acetylated cytochromec, 100 U SOD ml− 1, mitochondrial respiratory complexinhibitors (12 μM rotenone or 1.2 μM antimycin A) and 100 μgSMP protein in 100 mM potassium phosphate buffer (pH 7.4).The reaction was initiated by the addition of 1 mMNADH. Therate of UO2

− formation was measured by monitoring acetylatedcytochrome c reduction at 550 nM (ε=27.7 mM−1 cm−1).

4.7. Mitochondrial membrane potential

Mitochondrial membrane potential (ΔΨm) was monitored inintact cells by two methods: fluorescence resonance energytransfer assay (FRET) and confocal microscopy using JC1, amitochondrial membrane potential-dependent dye. The FRETassay is based on fluorescence resonance energy transferbetween two dyes: nonyl acridine orange (NAO), which stainscardiolipin, a lipid found exclusively in the mitochondrialinner membrane, and tetramethylrhodamine (TMR), a poten-

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tiometric dye taken up by mitochondria in accord withNernstian principles of potential and concentration. Thepresence of TMR quenches NAO emission in proportion toΔΨm, while loss of ΔΨm, with consequent efflux of TMR,dequenches NAO. The high specificity of NAO staining,selective monitoring of the fluorescence emitted by NAO, notby TMR, and the stringent requirement for co-localization ofboth dyes within the mitochondrion, collectively enable theFRET assay to report ΔΨm without the confounding influenceof background signal arising from potentiometric dyeresponding to plasma membrane potential. For FRET assay,cells were trypsinized and plated in clear-bottom, black-walled, 96-well plates at a density of 60,000 cells/well (Costar3606, Corning International, Corning, NY). Cells were loadedwith 86 nM NAO for 5 min and washed 3 times with Hanks-buffered salt solution (HBSS), and then 80 μl of HBSS wasadded into each well followed by 20 μl TMR to a finalconcentration of 150 nM. After 5 min incubation, cells wereexposed to 3 mM H2O2±1 μM–1 mM sodium pyruvate. Incontrol experiments, cells were exposed to neither H2O2 norpyruvate. Mitochondrial membrane potential collapse wasmeasured by monitoring NAO fluorescence at 488 nm excita-tion/525 nm emission wavelengths.

For confocalmicroscopy, cells were seeded on 25mm coverslips at a density of 600,000 cells/well, grown for 24 h, and thenincubated with JC1 (10 μg ml−1) for 2 h. The cover slip waswashed twice and mounted in a cell chamber (ALA ScientificInstruments, Westbury, NY). Serial confocal images weretaken every 10minwith a confocalmicroscopewith excitationat 490 nm and emission at 510 and 590 nm. Mitochondrialmembrane depolarization was induced by adding H2O2 to afinal concentration of 3 mM, with co-treatment with pyruvate(2 mM) or control.

4.8. Data analysis

Data in the figures are presented as mean values±SEM.Sodium pyruvate-treated and control groups were comparedusing one-way ANOVA with Tukey's multiple-comparisonstest. For all tests, P values<0.05 were considered statisticallysignificant.

Acknowledgments

This work was supported by grants from the American HeartAssociation Texas Affiliate and the National Heart, Lung andBlood Institute (HL-071684).

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