Chemico-Biological Interactions 174 (2008) 163–176

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Chemico-Biological Interactions

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Alterations in phospholipid and fatty acid lipid profiles in primaryneocortical cells during oxidant-induced cell injury

Brianna Peterson, Kyndra Stovall, Prashant Monian,James L. Franklin, Brian S. Cummings ∗

Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, Athens, GA 30602-2352, United States

a r t i c l e i n f o

Article history:Received 22 January 2008Received in revised form 19 May 2008Accepted 22 May 2008Available online 29 May 2008

Keywords:PhospholipidsOxidantsFatty acidsNeuronsPhospholipase A2

Cell deathElectrospray ionization-mass spectrometry

a b s t r a c t

Specific phospholipids and fatty acids altered during oxidant-induced neuronal cell injurywere determined using electrospray ionization mass spectrometry (ESI-MS) and ion trap-ping. The oxidants hydrogen peroxide (H2O2, 0–1000 �M) and tert-butylhydroperoxide(TBHP, 0–400 �M) induced time- and concentration-dependent increases in reactiveoxygen species in primary cultures of mouse neocortical cells as determined by 2′,7′-dichlorofluorescein diacetate staining and thiobarbituric acid formation. ESI-MS analysisof 26 m/z values, representing 42 different phospholipids, demonstrated that H2O2 andTBHP increased the abundance of phospholipids containing polyunsaturated fatty acids,but had minimal affect on those containing mono- or di-unsaturated fatty acids. Theseincreases correlated to time-dependent increase in 16:1-20:4, 16:0-20:4, 18:1-20:4 and18:0-20:4 phosphatidylcholine. Oxidant exposure also increased mystric (14:0), palmitic(16:0), and stearic (18:0) acid twofold, oleic acid (18:1) two- to threefold, and arachidonicacid (20:4) fourfold, compared to controls. Increases in arachidonic acid levels occurredprior to increases in the phospholipids, but after increases in ROS, and correlated toincreases in oxidized arachidonic acid species, specifically [20:4-OOH]–H2O–, 20:4-OH–,and Tri–OH-20:4-arachidonic acid. Treatment of cells with methyl arachidonyl flourophos-phonate an inhibitor of Group IV and VI PLA2, decreased oxidant-induced arachidonic acid

release, while bromoenol lactone, an inhibitor of Group VI PLA2, did not. Collectively, thesedata identify phospholipids and fatty acids altered during oxidant treatment of neuronsand suggest differential roles for Group IV and VI PLA2 in oxidant-induced neural cellinjury.

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Abbreviations: PLA2, phospholipase A2; cPLA2, cytosolic phospho-ipase A2; iPLA2, Ca2+-independent phospholipase A2; BEL, bromoenolactone; DCFH-DA, 2′ ,7′-dichlorofluorescein diacetate; ESI-MS, electro-pray ionization-mass spectrometry; MTT, 3-(4,dimethylthiazolyl-2)-2,5-iphenyltetrazolium bromide; PtdCho, phosphatidylcholine; PUFAs, polynsaturated fatty acids; ROS, reactive oxygen species; TBARS, thiobarbi-uric acid substances.∗ Corresponding author at: 371 College of Pharmacy, University of Geor-

ia, Athens, GA 30602, United States. Tel.: +1 706 542 3792;ax: +1 706 542 5358.

E-mail address: bsc@rx.uga.edu (B.S. Cummings).

009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.cbi.2008.05.028

© 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

One of the physiological functions of lipids is to actas a barrier to the outside environment while maintain-ing fluidity and permeability to ions. This function isinherently linked to the type of phospholipids presentin the cellular membrane, and their interactions with

each other. Oxidant-induced alterations in phospho-lipid composition can lead to decreases in membraneintegrity, cell injury and even death. This is especiallytrue of neurons because they have high levels of oxy-gen consumption, high iron content, and low levels of

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antioxidants, such as catalase, glutathione peroxidase,and superoxide dismutase (SOD) [1]. This combinationmakes neurons relatively more vulnerable to oxidativestress and lipid peroxidation, compared to their non-neuronal counterparts (i.e. glial cells, epithelial cells, etc.)[2].

Oxidative stress occurs when the balance between freeradical production (molecules with an unpaired electron)and cellular defense mechanisms are disturbed [3]. Fol-lowing stroke, or other types of traumatic brain injury,there is a rapid increase in the amount of free radicalspresent. This combined with a lack of oxygen, results indecreased levels of ATP. ATP-dependent pumps such asthe Na/K/ATPase pump fail, allowing an influx of sodium,chlorine and calcium into neurons, coupled to an efflux ofpotassium, depolarizing the membrane [4]. The increasein calcium can activate lipid metabolism enzymes suchas phospholipase C (PLC) and phospholipase A2 (PLA2),which may accelerate the production of reactive oxygenspecies.

Increased ROS production may induce neuronal celldeath and injury by inducing lipid peroxidation [5]. Lipidperoxidation occurs as a result of attack of membranelipids [6]. Reportedly, the lipid membranes most suscep-tible to oxidation are those that contain polyunsaturatedfatty acids (PUFAs), which contain multiple carbon–carbondouble bonds [6]. The brain may be more susceptible toROS-induced cell injury because it has a higher degreeof these types of phospholipids compared to other celltypes, suggesting a greater likelihood of lipid peroxi-dation [2]. In support of this hypothesis, studies showthat high amounts of lipid peroxides are released afterbrain injury [7,8], and lipid peroxides are found in theplasma of stroke victims [9]. The exact phospholipidsaltered during ROS injury and lipid peroxidation are notknown.

Another class of lipids proposed to be involved inoxidant-induced neuronal cell injury are those resultingfrom the release of arachidonic acid. Arachidonic acid isa 20 carbon long fatty acid containing four double bonds(20:4). It is present in numerous glycerophospholipidsat the sn-2 position. It is released from glycerophos-pholipids by the actions of PLA2 [10]. Arachidonic acidrelease can stimulate oxidant-induced cell death [11,12]and may mediate oxidant-induced injury in the brain. How-ever, the mechanisms and proteins involved are not fullyunderstood.

As mentioned above, the exact phospholipids targetedduring oxidant-induced neuronal cell injury and death, andthe mechanisms by which they are altered are not wellunderstood [13]. This question is of clinical importance asstudies suggest that both phospholipids and fatty acids arealtered in patients suffering from a variety of neurodegen-erative pathologies such as stroke and Alzheimer’s disease[11,14,15]. Recent advances in electrospray ionization-massspectrometry (ESI-MS) now allow for the rapid and accurate

analysis of phospholipids and fatty acids in low amountsof material [16]. Therefore, the goal of this study was touse ESI-MS to identify specific phospholipids and fattyacids altered in a model of oxidant-induced neuronal cellinjury.

teractions 174 (2008) 163–176

2. Materials and methods

2.1. Materials

Horse serum and fetal bovine serum were purchasedfrom American Type Culture Collection (Manassas, VA).Neurobasal medium, B-27 supplement, and dichloroflu-orescein were obtained from Invitrogen (Carlsbad, CA).cPLA2 assay kit and bromoenol lactone (BEL) were obtainedfrom Cayman Chemical Co. (Ann Arbor, MI). Biolumines-cent somatic cell ATP assay kit and all other chemicals wereobtained from Sigma Chemical (St. Louis, MO).

2.2. Isolation and culture of mouse neocortical primarycells

Primary cultures were prepared from embryonic day 17Swiss Webster mice as described previously [17]. Pregnantmice were sacrificed by CO2 asphyxiation, and embryosremoved by laparotomy under sterile conditions. Corticeswere collected in an isolation buffer consisting of 15 mLKrebs buffer (1 M NaCl; 0.045 M KCl, 8.5 mM NaH2PO4·H2O,0.12 M d-glucose, 0.2 mM phenol red, and 0.2 M HEPES,pH 7.4), 135 mL distilled H2O, 0.45 g bovine serum albu-min and 1.2 mL of 3.82% MgSO4. The cortex was strippedof meninges, and the neocortex collected from each hemi-sphere. The neocortices were minced by trituration witha Pasteur pipette and centrifuged at 1334 × g for 5 min.The supernatant was aspirated and the pellet was incu-bated for 25 min at 37 ◦C in 30 mL of isolation buffercontaining 7.5 mg trypsin. A solution of 15 mL of isolationbuffer containing 1.2 mg DNase (150 KU) and 9.0 mg soy-bean trypsin inhibitor (>7700 BAEE units/mg) was addedand the cells were centrifuged at 1334 × g for 5 min. Thesupernatant was aspirated and cells were dissociated bytwo successive trituration and sedimentation steps in iso-lation buffer containing soybean trypsin inhibitor andDNase. The cells were centrifuged at 1334 × g for 7 min andresuspended in Eagles’ minimal essential medium supple-mented with 2 mM l-glutamine, 10% fetal bovine serum,10% horse serum, 100 IU/mL penicillin and 0.10 mg/mLstreptomycin, pH 7.4. Cells were plated onto poly-l-lysinecoated plates at a density of 1.45 × 106 cells/mL andincubated at 37 ◦C in a 5% CO2 and 95% O2 humidityatmosphere. Cytosine arabinoside (10 �M) was added toculture medium on day 2 to prevent proliferation of non-neuronal cells. Media was changed on day 7 and 10 byhalf addition with Neurobasal media supplemented with2% B-27 supplement, 1% penicillin/streptomycin and 0.1%glutamine.

2.3. Phase contrast images

After treatment, cells were washed twice with PBS andfixed for 10 min using 10% (v/v) buffered formalin. After fix-ing, cells were washed three times with PBS and cover slips

applied. Images were taken using a Nikon TE300 invertedmicroscope (Nikon, Melville, NY) equipped with a CCDcamera (MicroMAX, Princeton Instruments, Trenton, NJ)using Metamorph software (Universal Imaging Corpora-tion, Westchester, PA).

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.4. Measurement of MTT staining

All experiments measuring 3-(4, dimethylthiazolyl-2)-, 5-diphenyltetrazolium bromide (MTT) staining werearried out in the linear working range of MTT. Cellsere seeded in 12-well plates at a concentration of

.45 × 106 cells/well and allowed to grow until day in vitroDIV) 12, prior to treatment for 24 h. At the desired timeoints, 30 �L of 5 mg/mL MTT was added to each well, theells incubated for 2 h, after which the media was removed,.2 mL of DMSO added to dissolve the resulting purple for-azan, and absorbance was read at 544 nm with FLUOstarPTIMA plate reader (BMG Labtechnologies, Inc., Durham,C).

.5. Measurement of reactive oxygen species

The generation of reactive oxygen species (ROS)as measured using the fluorescent dye 2′,7′-ichlorofluorescein diacetate (DCFH-DA). Cultures in2-well plates were preloaded with 10 �M DCFH-DA for0 min at 37 ◦C. After incubation, the media was aspiratednd replaced with fresh media. Cells were pretreated withEL for 30 min and then dosed with H2O2 (0–1000 �M) andBHP (0–400 �M). After treatment, media was aspiratednd cells detached using 0.01 M Tris–HCl with 0.5% Triton-100 (pH 7.4). A 100 �L aliquot was used to measureuorescence at excitation 485 nm and emission 520 nm.easurements were normalized with the protein content

f each sample.

.6. Measurement of ATP levels

ATP levels were measured using the adenosine 5′-riphosphate (ATP) Bioluminescence Assay Kit fromigma–Aldrich (St. Louis, MO) according to manufacturer’snstructions. Briefly, after oxidant exposure, cells wereysed using 100 �L somatic cell ATP releasing reagent pro-ided by the kit and 50 �L of the cell lysate was added to0 �L deionized water and 100 �L releasing agent. A 100 �Lliquot of this mixture was added to 100 �L of luciferaseubstrate and bioluminescence was measured. The cellularTP level was reported as percentage of control cells.

.7. Measurement of TBARS in neurons

The amount of lipid peroxidation was assayed usingthiobarbituric acid assay. Cultures were incubated with

xidants, the medium was aspirated and 2 mL 2.5% (w/v)richloroacetic acid was added and cells were removed bycraping. Cells were centrifuged at 2641 × g for 5 min. A00 �L aliquot of the cell supernatant was added to 500 �Lf a 0.67% (w/v) 2-thiobarbituric acid solution and vortexed.he suspension was incubated at 95 ◦C for 10 min. After theuspension cooled, a 200 �L aliquot was used to measurehe absorbance at 532 nm.

.8. Bligh–Dyer lipid extraction

Cellular phospholipids were extracted using chloroformnd methanol according to the method of Bligh and Dyer

teractions 174 (2008) 163–176 165

[18]. Cells were grown for 12 days in T25 flasks. After treat-ment, media was removed and cells were washed with PBS.3 mL of methanol:water (2.0:0.8, v/v) was added to the flaskand cells were removed with a cell scraper. The liquid wastransferred to a glass test tube and 1.25 mL chloroform wasadded. Tubes were vortexed for 30 s and allowed to sit for10 min on ice. Tubes were centrifuged at 213 × g for 3 minand the bottom chloroform layer was transferred to a newtest tube. The extraction steps were repeated a second timeand the chloroform layers combined. The collected chlo-roform layers were dried under argon, reconstituted with100 �L of methanol: chloroform (2:1, v/v), and stored at−20 ◦C.

2.9. Lipid phosphorus assay

Lipid phosphorus was quantified using malachite green[19]. Lipid extract (10 �L) was dried down under argon ina test tube. Perchloric acid (200 �L) was added to the tube,and heated at 140 ◦C for 2–3 h. After this time, 1 mL of dH2Owas added to the tube while vortexing. Reagent C (1.5 mLof 4.2 g ammonium molybdate tetrahydrate in 100 mL 5NHCl and 0.15 g malachite green oxalate in 300 mL ddH2O)was added and vortexed. Tween 20 (200 �L of 1.5% v/v) wasadded and vortexed. After 25 min of sitting at room temper-ature, a 200 �L aliquot was used to measure the absorbanceat 590 nm.

2.10. Characterization and quantitation of cellularphospholipids using electrospray ionization-massspectrometry (ESI-MS)

Lipid extract samples (500 pmol/�L) were prepared byreconstituting in chloroform: methanol (2:1, v/v). Massspectrometry was performed as described previously [20].Samples were analyzed using a LCT Premier time of flightmass spectrometer (Waters, Milford, MA) equipped withan electrospray ion source. Samples (5 �L) were intro-duced by means of a flow injector into the ESI sourceat a rate of 0.2 mL/min. The elution solvent was acetoni-trile:methanol:water (2:3:1, v/v/v) containing 0.1% (w/v)ammonium formate (pH 6.4). The mass spectrometer wasoperated in the positive ionization mode. The flow rate ofnitrogen drying gas was 10 L/min at 80 ◦C. The capillaryand cone voltages were set at 2.5 kV and 30 V, respectively.As previously described [20], qualitative identification ofindividual phospholipid molecular species was based ontheir calculated theoretical monoisotopic mass values andquantification was done by comparison to the most abun-dant phospholipid in each sample, which correspondedto m/z 760 or [(34:1 (16:0-18:1)] phosphatidylcholine(PtdCho). MassLynx 4.0 software was used for dataanalysis.

MSnth fragmentation was performed on a Trap XCTion-trap mass spectrometer (Agilent Technologies, SantaClara, CA) equipped with an ESI source. The analyte was

introduced by direct injection from the HPLC system. Thenitrogen drying gas flow rate was 8.0 L/min at 350 ◦C, neb-ulizer pressure was 30 psi, and capillary voltage was 3 kV.The ion source and ion optic parameters were optimizedwith respect to the positive molecular ion of interest.

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2.11. Characterization and quantification of fatty acidsusing ESI-MS

Lipid extracts (500 pmol/�l) were prepared by recon-stituting in chloroform:methanol (2:1, v/v). A deuteratedpalmitic acid solution (5 �L of 0.12 mM) was added asan external standard to each sample. Samples were ana-lyzed using a LCT Premier time of flight mass spectrometer(Waters, Milford, MA) equipped with an electrospray ionsource. Samples (5 �L) were introduced by means of a flowinjector into the ESI source at a rate of 0.2 mL/min. The elu-tion solvent was acetonitrile:methanol:water (2:3:1, v/v/v)containing 0.1% (w/v) ammonium formate (pH 6.4). Themass spectrometer was operated in the negative ioniza-tion mode. The ESI conditions were as follows: nitrogenflow rate, 10 L/min at 80 ◦C; capillary voltage, 2.5 kV;source block temperature, 120 ◦C; desolvation temperature,350 ◦C; cone voltage, 40 V.

2.12. Statistical analysis

Cortical cells isolated from embryos of one pregnantmouse were pooled and represent one experiment (n = 1).Data are represented as the average ± the S.E.M. of at leastthree separate experiments (n = 3). The appropriate analy-sis of variance (ANOVA) was performed for each data setusing GraphPad Prism software. Individual means werecompared using Fisher protected least significant differ-ence test with P < 0.05 being considered indicative of astatistically significant difference between mean values.

3. Results

3.1. Cell injury and ROS formation in primary cultures ofmouse neocortical cells

To establish a model to assess the effect of oxidants onlipids, primary cultures of mouse neocortical cells wereexposed to H2O2 and TBHP. These toxicants were chosenbecause our previous studies demonstrate that they inducelipid peroxidation, arachidonic acid release, ROS formation,and cell death [21–24]. Further, these oxidants are knownto alter PLA2 activity in several models [22,25,26].

Treatment of cells with 500 �M H2O2 for 24 h alteredcellular morphology, causing loss of neurite attachmentand granulation, compared to control cells (Fig. 1A and B).Treatment of cells with 200 �M TBHP also induced neuritedetachment and granulation after 24 h (Fig. 1C). Analysisof MTT absorbance demonstrated both H2O2 and TBHPinduced concentration- and time-dependent decrease inMTT staining after 24 h (Fig. 1D and E). Significant decreasesin MTT staining were observed for concentrations of H2O2above 500 �M and TBHP concentrations above 200 �Mafter 24 h, as compared to control. Decreases in MTT werenot detected after either 2 or 4 h of exposure.

Treatment of cells with H2O2 resulted in concentration-

and time-dependent increases in DCFH-DA staining, with500 �M H2O2 causing a 75% increase in staining after 2 h,as compared to controls cells (Fig. 2A). Treatment of cellswith TBHP also increased DCFH-DA staining; with 100 �MTBHP causing 200% increases after 2 h and almost a 300%

teractions 174 (2008) 163–176

increase after 4 h (Fig. 2B). Increasing the time and concen-tration of TBHP exposure significantly increased DCFH-DAstaining. H2O2 did not significantly increase TBARS forma-tion after 2 or 4 h even at high concentrations (1000 �M,Fig. 2C). However, TBHP significantly increased TBARS for-mation above controls at concentrations as low as 100 �Mafter just 2 h (Fig. 2D).

Treatment of cells with 500 �M H2O2 decreased ATP lev-els 60% after 2 h of exposure. In contrast, treatment of cellswith 100 �M TBHP treatment decreased ATP levels 60%,but only after 4 h of exposure. Decreases in ATP were time-dependent. These data demonstrate that oxidant-inducedincreases in DCFH-DA staining and TBARS formation corre-late to altered cell morphology, and decreases in ATP levelsand MTT staining (Fig. 3). Based on these data we chose500 �M H2O2 and 100 �M TBHP to test the effect of oxi-dants on phospholipids and fatty acids.

3.2. Lipidomic analyses of primary neocortical cells

To identify phospholipids and fatty acids altered by oxi-dants during neuronal cell death we performed ESI-MS.This technique allows for rapid identification of multiplephospholipid species in small amounts of material [16].To our knowledge, ESI-MS has never been used to assessalterations in phospholipid profiles in neurons after oxidantexposures.

Fig. 4A represents typical ESI-MS spectra for controlcells while Fig. 4B represents the ESI-MS spectra for cellsexposed to 500 �M H2O2. Inorganic phosphorus assayswere performed to ensure that equal amounts of lipids wereanalyzed. Spectra for TBHP exposed cells were similar toH2O2 (data not shown). We initially analyzed 26 m/z valuescorresponding to approximately 42 possible phospholipids(Tables 1 and 2). Preliminary identification was based ontheoretical m/z values reported in Taguchi et al. [20] andLipids Maps (www.lipidmaps.org/). Peaks of interests werefurther identified using ESI-MS–MS and ESI-MS–MS–MS inboth the positive and negative modes (see below).

The average signal intensity (i.e. the average total ioncount or total abundance) can be used to estimate globalchanges in cellular phospholipid levels. Control cells hadan average signal intensity of 7.48 ± 0.23. In contrast,cells treated with 500 �M H2O2 for 24 h had an aver-age signal intensity of 9.81 ± 0.48, while those exposed to100 �M TBHP for 24 h had a value of 8.44 ± 0.36 (Table 1).The average signal intensity for m/z values correspond-ing to phospholipids that contain PUFAs (those containingthree or more double bonds) increased from 2.38 ± 0.09in control cells to 4.82 ± 0.52 in H2O2 treated cells, and3.34 ± 0.05 in TBHP-treated cells. In contrast, the averagesignal intensity for m/z values representing mono- or di-saturated species (i.e. those containing 0–2 double bonds)was unchanged among groups.

Data in Table 1 suggests that treatment of neuronswith oxidants preferentially increases the abundance of

PUFA-containing phospholipids. To study this hypothesis,changes in the levels of individual phospholipids wereassessed based on their theoretical m/z values (Table 2). Asin Table 1, these phospholipids were grouped into mono/di-unsaturated species or into those that contain PUFAs. H2O2

B. Peterson et al. / Chemico-Biological Interactions 174 (2008) 163–176 167

Fig. 1. Morphological and biochemical changes in primary neocortical cultures exposed to oxidants. Cultures were treated with either solvent control (A),500 �M H2O2 (B) or 200 �M TBHP (C) for 24 h. After treatment, cells were washed, fixed, and phase contrast images taken using a Nikon TE300 invertedm g of celo st threed

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icroscope at 20× magnification. Panels D and E represents MTT staininr 24 h. Data in D and E are represented as the average ± S.E.M. of at leaifferent from each other (P < 0.05).

reatment increased the level of only two di-unsaturatedontaining species; 34:1 and 36:2 PtdCho. These phos-holipid represent either 18:0-18:1 or 16:0-20:1 PtdCho34:1) and 18:1-18:1 or 18:0-18:2 PtdCho (36:2). Treatmentf cells with TBHP for 24 h only slightly altered mono/di-nsaturated phospholipids.

In contrast to mono/di-unsaturated phospholipidpecies, H2O2 and TBHP significantly increased the levelsf almost all phospholipid that contained PUFAs two- toightfold compared to controls (Table 2). H2O2 appearedo induce greater increases in these phospholipids than

BHP. Highest among these phospholipids were those with/z values of 754, 780, 782, 804, 808 and 810. Four of these

UFAs-species corresponded to phospholipids containingrachidonic acid at the sn-2 ester bond (780, 782, 808 and10). This suggests that treatment of neurons with oxi-

ls exposed to increasing concentrations of H2O2 (D) or TBHP (E) for 2, 4separate experiments. Means with different subscripts are significantly

dants increases the levels of arachidonic acid-containingphospholipids after 24 h.

ESI-MS–MS–MS was used to verify that the phos-pholipids present at m/z 780, 782, 808 and 810 containphosphatidylcholine and arachidonic acid (Fig. 5). ESI-MS–MS of peaks at the m/z of 808 resulted in thefragmentation products of 749.7 and 625.7 (Fig. 5A). Thesevalues correspond to the loss of a choline group (−59) and aphosphocholine head group (−183). A secondary round ofMS on the resulting 625.7 peak resulted in the appearanceof a major peak at m/z 303, which corresponds to arachi-

donic acid (Fig. 5B). Analysis of peaks at m/z 780, 782, and810 yielded similar results and demonstrated that all thesephospholipids contained phosphocholine head groups andarachidonic acid at the sn-2 ester bond. Based on these data,and analysis of the resulting lysophospholipid groups (data

168 B. Peterson et al. / Chemico-Biological Interactions 174 (2008) 163–176

ROS forr 0–400perimen

Fig. 2. Oxidant-induced ROS formation in primary neocortical cultures.TBARS (C and D) formation after exposure to 0–1000 �M H2O2 (A and C) oData are represented as the average ± S.E.M. of at least three separate exother (P < 0.05).

not shown), these PUFAs species were identified as 16:1-

20:4, 16:0-20:4, 18:1-20:4 and 18:0-20:4 PtdCho. Thesepeaks are labeled with an asterisk in Fig. 4.

Time-dependent studies were conducted to assess thekinetics involved in the increases of arachidonic acid-

Fig. 3. Effect of oxidants on intracellular ATP levels in primary neocorticalcultures. Cultures were treated with 500 �M H2O2 or 100 �M TBHP for0–4 h, prior to analysis of intracellular ATP levels. Data are represented asthe average ± S.E.M. of at least three separate experiments. Means withdifferent subscripts are significantly different from each other (P < 0.05).

mation was measured by either DCFH-DA fluorescence (A and B) or by�M TBHP (B and D). Data in C and D are derived from 2 h exposures only.ts. Means with different subscripts are significantly different from each

containing phospholipids after oxidant exposure (Fig. 6).Treatment of cells with 500 �M H2O2 for 2–8 h did not sig-nificantly increase the level of any phospholipid studied(Fig. 6A). However, 16:0-20:4 and 18:1-20:4 PtdCho levelsincreased after 16 h and continued to rise at 24 h. Similarpatterns were seen for 16:1-20:4 and 18:0-20:4 PtdCho.Treatment of cells with 100 �M TBHP for 2, 4 and 8 h alsodid not increase any phospholipid studied (Fig. 6B). How-ever, 16:1-20:4 PtdCho levels were increased after 16 h ofexposure, while 16:0-20:4 and 18:1-20:4 were increasedafter 24 h.

The above data demonstrate that treatment of pri-mary cultures of mouse neocortical cells with oxidantsincreases the levels of arachidonic acid-containing phos-pholipids in a time-dependent manner. To investigate themechanisms involved we tested the effect of inhibitors ofCa2+-independent and cytosolic phospholipase A2 (PLA2)on phospholipid profiles in both untreated and treatedcells. Treatment of cells with 2.5 �M bromoenol lac-tone (BEL), a selective inhibitor of Ca2+-independentPLA2, decreased the ability of H2O2 to increase all phos-pholipids analyzed (Fig. 6C). BEL also did not prevent

TBHP-induced increases in these phospholipids. Treat-ment of cells with methylarachidonyl flourophosphonate,an inhibitor of cytosolic PLA2 (cPLA2), prior to oxidantexposure did not alter oxidant-induced alterations in anyarachidonic acid-containing phospholipid studied (Fig. 6D).

B. Peterson et al. / Chemico-Biological Interactions 174 (2008) 163–176 169

F l culturea indicate

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ig. 4. Positive-ion ESI-MS spectra of oxidant treated primary neocorticand cells treated with 500 �M H2O2 for 24 h (B). Peaks marked with an *

either BEL, nor MAFP altered lipid profiles in the absencef oxidants (data not shown).

.3. Changes in fatty acids in treated primary neocorticalells

Because the majority of phospholipids altered by oxi-ants contained arachidonic acid, and because arachidoniccid is released by actions of PLA2, the effect of oxidantsnd PLA2 inhibitors on fatty acid release was determinedsing ESI-MS. Fig. 7 represents ESI-MS spectra of lipidxtracts isolated from oxidant-treated cells as assessed inhe negative ion mode. Fig. 7A represents control spec-ra while Fig. 7B represents spectra from cells exposed to00 �M H2O2 for 24 h. Spectra for cells treated with TBHPere similar to cells treated with H2O2 (data not shown).

able 3 lists the fatty acids analyzed and the effect of both2O2 and TBHP on their average signal intensity (in per-ent of control) compared to control cells after 24 h. Theatty acid most altered upon exposure to oxidants wasrachidonic acid (m/z 303). Time-dependent studies were

able 1hanges in average signal intensity of phospholipids in neocortical cells after 24 h

Control

ll phospholipidsb 7.48 ± 0.23c

UFA-containing species 2.38 ± 0.09ono/di-unsaturated-containing species 5.10 ± 0.14

a Signal intensity refers to the average total ion count for all phospholipids idenb Lipids were identified based on theoretical m/z values of all phospholipid studc Values represent the average signal intensity ± the SEM of at least three (n = 3* Indicates significant (P < 0.05) difference from control.

s. Comparison of the mass spectra of phospholipids for control cells (A)those that increased after treatment with oxidant.

conducted by exposing cells to 500 �M H2O2 and 100 �MTBHP for 2, 4, 8, 16 and 24 h (Fig. 8). H2O2 treatment sig-nificantly increased arachidonic acid levels threefold after4 h of exposure. Increasing the time-period of exposure didnot further increase arachidonic acid levels. Exposure ofcells to TBHP also significantly increased arachidonic acidlevels above controls starting at 4 h, after which arachi-donic acid levels remained steady up to 24 h. Treatmentof cells with BEL prior to oxidant exposure did not inhibitoxidant-induced increases in arachidonic acid after 24 h(Fig. 8A). In contrast, pretreatment with MAFP reducedoxidant-induced increases in arachidonic acid at this sametime point (Fig. 8B).

3.4. Oxidized arachidonic acid products

In addition to increases in arachidonic acid levels, oxi-dant exposure increased the abundance of species presentat m/z values that correspond to oxidized arachidonic acidproducts. For example, there was an increase in [20:4-OOH]–H2O, 20:4-OH, and tri-OH-20:4 (m/z 317, 319, and

exposure to oxidantsa

H2O2 (500 �M) TBHP (100 �M)

9.81 ± 0.48* 8.44 ± 0.36*

4.83 ± 0.52* 3.34 ± 0.55*

4.98 ± 0.13 5.10 ± 0.26

tified in each class.ied and by comparison to internal standards.

) measurements for 26 m/z values.

170 B. Peterson et al. / Chemico-Biological Interactions 174 (2008) 163–176

ortical cm/z 808.7 resul20:4 Ptd

Fig. 5. ESI-MS–MS (A) and ESI-MS–MS–MS (B) of m/z 808 in primary neoccells were subjected to ESI-MS analysis in tandem with ion selection formass units, respectively (A). Further fragmentation of the peak at m/z 625acid (B). The putative structure of the phospholipid is shown in (A) (18:1-

351, respectively), compared to control cells (Fig. 9). Theearliest that these species were detected was 16 h, andthat was only in TBHP-treated cells (data not shown). Nei-ther BEL (Fig. 9A) nor MAFP (Fig. 9B) pretreatment reducedoxidant-induced increases in oxidized arachidonic acidproducts.

4. Discussion

Several studies demonstrate that neuronal phospho-lipids and fatty acids are altered after oxidant exposure[7,8]. It is hypothesized that oxidant-induced alterations inlipids arise from ROS-mediated increases in lipid peroxida-tion [6]. The lipid membranes most susceptible to oxidationare reportedly those that contain PUFAs [6]. However, fewstudies have identified the exact PUFA-containing phos-pholipids altered. The present study used ESI-MS to identify

these phospholipids, as well as fatty acids, altered duringoxidant-induced injury in primary cultures of mouse neo-cortical cells.

Prior to measuring oxidant-induced alterations in lipidprofiles we first established a neuronal cell model of

ells exposed to 500 �M H2O2 for 24 h. Phospholipids isolated from treated(MS/MS) resulting in peaks at m/z 749.7 and 625.7, a loss of 59 and 183

ted in the appearance of a peak at m/z 303, corresponding to arachidonicCho).

oxidant-injury using H2O2 and TBHP. Data derived usingthese oxidants demonstrated that both oxidants increasedROS formation within 2 h of treatment, which was followedby decreases in ATP between 2 and 4 h. This was followedby decreases in MTT staining and eventual loss of cell mor-phology. It should be noted that these data are not novel. Infact, they agree with previous studies demonstrating sim-ilar findings [27–29]. However, these data establish a timeframe to address the effect of oxidants on the phospholipidprofile of the cells using ESI-MS.

We used ESI-MS to assess changes in multiple phospho-lipids at 2, 4, 8, 16 and 24 h. These time points correspondto initial injury (2 and 4 h) and subsequent loss of cell func-tion and morphology (16 and 24 h). Using ESI-MS allowedfor rapid and quantitative screening of almost 50 differ-ent phospholipid species, as well as fatty acids, in the lowamounts of material typically present in primary cultures.

The use of primary cultures allowed for a more relevantmodel than cell lines, which are typically immortalized,cancerous in origin or otherwise transformed.

The level of at least six phospholipids changedtwofold or more after oxidant exposure. ESI-MS–MS–MS

B. Peterson et al. / Chemico-Biological Interactions 174 (2008) 163–176 171

Fig. 6. Effect of oxidants on phosphatidylcholine species in primary neocortical cultures. Confluent cells were treated with 500 �M H2O2 (A) or 100 �MTBHP (B) in the presence and absence of 2.5 �M BEL (C) or MAFP (D) prior to ESI-MS in tandem with ion selection (MS/MS). Cells in A and B were exposed tooxidants for 2, 4, 8, 16 and 24 h, while samples in C and D were exposed to oxidants for 24 h only. Phospholipids analyzed corresponded to those containing2 as the avs ipts in Cg

da2dr

1i1otwoibdfopo

0:4 at the sn-2 position (m/z 780, 782, 808 and 810). Data are representedignificant difference (P < 0.05) from control. Means with different subscrroup.

emonstrated that four of these phospholipids containedrachidonic acid and were 16:0-, 16:1-, 18:0-, and 18:1-0:4 PtdCho. To our knowledge, this is the first study toemonstrate that these specific phospholipids increase inesponse to oxidant treatment.

We believe that increases in 16:0-, 16:1-, 18:0-, and8:1-20:4 PtdCho are not a global response to oxidant-njury. This is because increases in the abundance of 16:0-,6:1, 18:0-, and 18:1-20:4 PtdCho occurred in the absencef alterations in at least 10 other phospholipids. Further,hese phospholipids increased, as opposed to decreasing,hich is what one would expect if changes were due to

verall loss of membrane integrity. Additionally, oxidant-nduced alterations in these phospholipids were reversedy the iPLA2 inhibitor BEL. In contrast, BEL and MAFP

id not change the levels of any mono/di-unsaturated

atty acid-containing phospholipid, and had little effectn the remaining poly-unsaturated fatty acid-containinghospholipids (data not shown). These data suggest thatxidant-induced neuronal cell injury leads to the remodel-

erage ± S.E.M. of at least three separate experiments. * in A and B indicatesand D are significantly different from each other (P < 0.05) within each

ing of phospholipids and that this remodeling is mediatedby iPLA2.

Arachidonic acid-containing phospholipids were notthe only species altered after oxidant exposure. Indeed,Table 2 demonstrates that almost all PUFA-containingspecies increased after oxidant treatment. Thus, arachi-donic acid-containing phospholipids are not the only targetof oxidants. We focused on 16:0-, 16:1-, 18:0-, and 18:1-20:4 PtdCho as these species demonstrated the largestincrease compared to other phospholipids.

Time-dependent studies demonstrate that arachidonicacid levels increase prior to increases in 16:0-, 16:1, 18:0-,and 18:1-20:4 PtdCho. It is possible that the cell may re-incorporate some of this arachidonic acid back into themembrane, and that this increases the levels of 16:0-, 16:1-

, 18:0-, and 18:1-20:4 PtdCho. This may be an adaptiveresponse to counter the high arachidonic acid levels, or mayrepresent a repair process.

The concentration of oxidants used to induce changesin cellular lipids did not induce 100% cell death. At least

172 B. Peterson et al. / Chemico-Biological Interactions 174 (2008) 163–176

Table 2Effect of oxidants on phospholipid levels in primary cultures of mouse neocortical cells after 24 h exposure

Probable PtdCho speciesa m/z Ctrlb H2O2 (500 �M) TBHP (100 �M)

Mono/di-unsaturated species14:0-16:0 706 100 78 ± 8 86 ± 516:1-16:1/14:1-18:1 730 100 91 ± 18 82 ± 416:0-16:1/14:0-18:1 732 100 80 ± 6 92 ± 416:0-16:0/14:0-18:0 734 100 90 ± 5 91 ± 316:0-18:1/16:1-18:0 746 100 126 ± 19 120 ± 716:1-18:1/16:0-18:2 758 100 132 ± 25 123 ± 1616:0-18:1/16:1-18:0c 760 100 100 10016:0-18:0 762 100 101 ± 6 98 ± 318:0-18:1/16:0-20:1 774 100 164 ± 24* 136 ± 2218:1-18:1/18:0-18:2 786 100 167 ± 41* 152 ± 2418:0-18:1/16:0-20:1 788 100 133 ± 22 133 ± 1118:1-20:2 814 100 112 ± 4 111 ± 218:0-20:1 816 100 164 ± 26 146 ± 25

Polyunsaturated species16:1-18:3 754 100 465 ± 142* 201 ± 81*

16:1-18:2/16:0-18:3 756 100 238 ± 61* 155 ± 5516:0-20:4/16:1-20:3 768 100 236 ± 81* 94 ± 3216:1-20:5 778 100 168 ± 14* 125 ± 1516:0-20:4 780 100 882 ± 379* 247 ± 19916:0-20:4/16:1-20:3 782 100 494 ± 187* 180 ± 11216:0-20:3/18:1-18:2 784 100 269 ± 13* 144 ± 1116:1-22:7 802 100 229 ± 29* 165 ± 3016:1-22:6 804 100 435 ± 126* 254 ± 110*

16:0-22:6/16:1-22:5 806 100 301 ± 45* 185 ± 4418:1-20:4/16:0-22:5 808 100 885 ± 451* 294 ± 188*

18:0-20:4/16:0-22:4 810 100 406 ± 146* 255 ± 66*

18:0-20:3 812 100 181 ± 48 128 ± 15

www.lipthe S.E.M

a Based on theoretical m/z reported in Taguchi et al. [20], Lipids Maps (b Values represent the percent control of the average signal intensity ±c Most abundant phospholipid in control cells.* Indicates significant (P < 0.05) difference from control.

30–40% viability is maintained after 24 h, based on MTTstaining, cell morphology and ATP levels. This supports thehypothesis that increases in 16:0-, 16:1, 18:0-, and 18:1-20:4 PtdCho are an adaptive response to oxidant-injury.Further supporting this hypothesis is the fact that the levelsof these phospholipids increased only after 16 h, which isover 12 h past initial increases in ROS formation and arachi-donic acid release.

The mechanisms by which oxidants induce arachidonic

acid release appear to involve cPLA2. This is supported bythe ability of MAFP, but not BEL, to decrease arachidonicacid levels after oxidant exposure. However, inhibition ofcPLA2 using MAFP did not decrease arachidonic acid release

Table 3Effect of oxidants on fatty acid levels in primary cultures of mouse neocortical ce

Fatty acid species m/z Controlb

14:0-Myristic 227 10016:0-Palmitic 255 10018:1-Oleic 281 10018:0-Stearic 283 10019:0-Nonadecanoic 297 10020:4-Arachidonic 303 10020:3-Homo-�-linolenic 305 10024:1-Nervonic 365 100

a Based on theoretical m/z values and comparison to internal standards.b Values represent percent of control of the average signal intensity ± the S.E

external standard (deuterated palmitic acid).* Indicates significant (P < 0.05) difference from control.

idmaps.org/), and Murphy, 2002.. of at least 3 (n = 3) measurements for 26 m/z values.

to control levels, suggesting the presence of additionalmechanisms. Oxidants alone are capable of oxidizing dou-ble and ester bonds in fatty acids, effecting non-enzymaticcleavage and release of fatty acids [22]. Thus arachidonicacid release may also occur non-enzymatically, via a directattack by ROS.

While MAFP decreased oxidant-induced arachidonicacid release, it did not alter oxidant-induced increasesin 16:0-, 16:1-, 18:0-, and 18:1-20:4 PtdCho. In contrast,

BEL had no effect on arachidonic acid levels, but did alteroxidant-induced increases in the above phospholipids. Thissuggests that the mechanisms that mediate increases in16:0-, 16:1-, 18:0-, and 18:1-20:4 PtdCho involve iPLA2. This

lls after 24 ha

H2O2 (500 �M) TBHP (100 �M)

285 ± 54* 200 ± 42254 ± 55* 216 ± 53436 ± 62* 333 ± 82*

246 ± 31 194 ± 41190 ± 35 121 ± 20886 ± 126* 438 ± 88*

881 ± 335* 293 ± 101*

149 ± 40 111 ± 31

.M. of at least three (n = 3) measurements and normalization against an

B. Peterson et al. / Chemico-Biological Interactions 174 (2008) 163–176 173

F l cultura to the m

mrbftr

wtidpTL[ivt[[li

iapcai

ig. 7. Negative-ion ESI-MS spectra of oxidant treated primary neocorticand cells treated with 500 �M H2O2 for 24 h (B). The peak corresponding

ay be due to the fact that iPLA2 is preferentially utilized toelease lysophospholipid acceptors to which fatty acids cane attached to. In fact, it is believed that this is the primaryunction of iPLA2 [30,31]. Collectively, these data suggesthat cPLA2 and iPLA2 play differential roles in phospholipidemodeling after oxidant exposure.

Increases in 16:0-, 16:1-, 18:0- and 18:1-20:4 PtdChoere not specific to H2O2, as TBHP treatment also increased

hese phospholipids. However, unlike H2O2, BEL did notnhibit phospholipid increases after TBHP exposure. Theseifferences may be explained by the fact that TBHP is a moreotent and lipophilic oxidant, compared to H2O2. Further,BHP induced higher levels of lipid peroxidation than H2O2.ipid peroxides themselves are suggested to inhibit iPLA222,25,26]. Finally, TBHP, but not H2O2, inactivates iPLA2n several types of tissues [22]. The mechanism of inacti-ation involves the formation of a carbon-centered radicalhat may lead to a thiol-alkylation of iPLA2 on cysteines26], and correlates to the genesis of lipid peroxidation22,26]. Thus, the inability of BEL to modulate phospho-ipids in the presence of TBHP may be because it is alreadynactivated.

Collectively, these data suggest that oxidative stressnduces the release of arachidonic acid, which is medi-

ted, in part, by cPLA2. Increases in arachidonic acid occurrior to increases in the level of specific arachidonic acid-ontaining phospholipids. Increases in these phospholipidsppear to be mediated, in part, by iPLA2. This mechanisms illustrated in Fig. 10.

es. Comparison of the mass spectra of phospholipids for control cells (A)/z values for arachidonic acid (303) is marked with an *.

Data in this study do not identify the phospholipidsfrom which arachidonic acid is released or the individ-ual organelle membranes altered (phospholipid pool 1 inFig. 10). Further, we were unable to identify lipids whoselevels decreased in correlation with increases in 16:0-,16:1-, 18:0- and 18:1-20:4 PtdCho (phospholipid pool 3).The ESI-MS method used to assess phospholipids focuseson choline-containing phospholipids. Phospholipids thatcontain ethanolamine and serine are not as well tracked.Thus, it is possible that oxidative stress induces the releaseof arachidonic acid either from phosphatidylethanolamine,serine, or other phospholipids. Future studies usingLC–MS, or HP-TLC, on membranes isolated from dif-ferent cellular organelles, are needed to address thesequestions.

In order for arachidonic acid to be re-incorporated intomembranes it must be esterified to a lysophospholipid. Asmentioned above, one of the primary functions of iPLA2is to generate lysophospholipid acceptors [32]. Inhibitionof iPLA2 alters arachidonic acid-containing phospholipidsin several cell types [32–35]. The ability of BEL to inhibitoxidant-induced increases in 16:0-, 16:1-, 18:0- and 18:1-20:4 PtdCho suggest that iPLA2 plays a similar role inneuronal cells. We do not believe that arachidonic acid is

re-esterified to the same lysophospholipid from which itwas released from as BEL did not alter arachidonic levelsthemselves. Thus, iPLA2 appears to be generating lysophos-pholipids from a second phospholipid pool (phospholipidpool 2 in Fig. 10).

174 B. Peterson et al. / Chemico-Biological Interactions 174 (2008) 163–176

Fig. 8. Effect of oxidants on arachidonic acid levels in primary neocortical cultures. Cultures were treated with 500 �M H2O2 or 100 �M TBHP for 0, 2, 4,8, 16 and 24 h (A), or pretreated with 2.5 �M BEL (B) or MAFP (C) for 30 min prior to 24 h exposure to either oxidant. Following exposure samples wereanalyzed for arachidonic acid (m/z 303) using ESI-MS. Data are represented as the average ± S.E.M. of at least three separate experiments. * in A and Bindicate significant difference (P < 0.05) from control. Means with different subscripts are significantly different from each other (P < 0.05).

Fig. 9. Effect of oxidants on the formation of oxidized arachidonic acid products in primary neocortical cultures. Cultures were treated with 500 �M H2O2

or 100 �M TBHP in the presence and absence of 2.5 �M BEL (A) or MAFP (B) for 24 h prior to ESI-MS analysis of attached cells for [20:4-OOH]–H2O (m/z 317),20:4-OH (m/z 319), and Tri–OH-20:4 (m/z 351). Data are represented as the average ± S.E.M. of at least three separate experiments. Means with differentsubscripts are significantly different from each other (P < 0.05) within each group.

B. Peterson et al. / Chemico-Biological Interactions 174 (2008) 163–176 175

Fig. 10. Proposed mechanism for oxidant-induced increases in 16:0-, 16:1-, 18:0- and 18:1-20:4 PtdCho. (1) Oxidant exposure results in the cPLA2-mediatedrelease of arachidonic acid (phospholipid pool 1). (2) This arachidonic acid may be further oxidized to reactive species which appear after 16 h. (3) Bothu es in ari donic acp ile incre1

mattosdrs

wiltedtii

tdiltpBltwpTscb

n-oxidized and oxidized arachidonic acid induce cell injury. (4) IncreasPLA2 from different phospholipids. (5) The lysophospholipids and arachiool 3). The release of arachidonic acid appears to occur as early as 4 h, wh6:1-, 18:0- and 18:1-20:4 PtdCho.

Oxidized arachidonic acid species increased after treat-ent with both H2O2 and TBHP. These increases do not

ppear to be mediated by either cPLA2 or iPLA2 as nei-her MAFP, nor BEL, altered oxidant-induced increases inhese species. Further, increases in these oxidized speciesccurred after increases in arachidonic acid (data nothown). These data suggests that arachidonic is not oxi-ized while still on the phospholipid or that they areeleased by a different lipase. The origins of these oxidizedpecies are still under study.

Increases in arachidonic-containing phospholipids, asell as un-oxidized and oxidized arachidonic acid, occurred

n the absence of increases in several other phospho-ipids. This specificity may be used in clinical studieso identify biomarkers of oxidant-induced injury. Forxample, the formation of triple hydroxylated arachi-onic acid in detached cells may be used to designests for cells collected in the cerebral spinal fluid ofschemic-stroke patients to assess the type and extent ofnjury.

In conclusion, we used ESI-MS in tandem with ion selec-ion to identify phospholipids whose levels were altereduring oxidant-induced neuronal cell death. Significant

ncreases in select arachidonic acid-containing phospho-ipids were detected. These increases were inhibited byreatment with the iPLA2 selective inhibitor BEL and werereceded by the release of arachidonic acid. However,EL only inhibited H2O2-induced increases in phospho-

ipids, and had no effect on TBHP-induced increases inhese same phospholipids. Increases in arachidonic acidere inhibited by the cPLA2 inhibitor MAFP, and occurred

rior to increases in oxidized arachidonic acid species.hese data suggest that neuronal cells respond to oxidativetress by increasing the production of arachidonic acid-ontaining phospholipids. The reason for this is unclear,ut may be an attempt by neurons to limit the further

[

achidonic acid are balanced by the release of acyl-lysophospholipids byid are re-acylated and esterified into a new phospholipid (phospholipid

ases in oxidized arachidonic acid occur after 16 h, as do increases in 16:0-,

metabolism of arachidonic acid and to preserve membraneintegrity.

Acknowledgements

This work was supported by a Georgia Cancer Coali-tion Distinguished Scholar and University of Georgia JuniorFaculty grant to BSC, an American Foundation for Pharma-ceutical Education and University of Georgia Fellowships toBP, and by NIHRO1NS37110 to JLF.

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