Toxicology and Applied Pharmacology 272 (2013) 199–207

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Toxicology and Applied Pharmacology

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Proteomic analysis of rat cerebral cortex following subchronicacrolein toxicity

Marzieh Rashedinia a, Parisa Lari a, Khalil Abnous b,⁎, Hossein Hosseinzadeh c,⁎a Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iranb Pharmaceutical Research Center, Department of Medicinal Chemistry, Mashhad University of Medical Sciences, Mashhad, Iranc Pharmaceutical Research Center, Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

⁎ Corresponding authors at: Pharmaceutical ResePharmacodynamics and Toxicology, School of PharmMedical Sciences, Mashhad, Iran. Fax: +98 511882

E-mail addresses: Abnouskh@mums.ac.r (K. Abnous(H. Hosseinzadeh).

0041-008X/$ – see front matter © 2013 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.taap.2013.05.029

a b s t r a c t

a r t i c l e i n f o

Article history:Received 19 February 2013Revised 14 May 2013Accepted 21 May 2013Available online 3 June 2013

Keywords:AcroleinProteomicsNeurotoxicityOxidative stressMalondialdehydeGlutathione

Acrolein, a member of reactive α,β-unsaturated aldehydes, is a major environmental pollutant. Acrolein isalso produced endogenously as a toxic by-product of lipid peroxidation. Because of high reactivity, acroleinmay mediate oxidative damages to cells and tissues. It has been shown to be involved in a wide variety ofpathological states including pulmonary, atherosclerosis and neurodegenerative diseases.In this study we employed proteomics approach to investigate the effects of subchronic oral exposures to3 mg/kg of acrolein on protein expression profile in the brain of rats. Moreover effects of acrolein onmalondialdehyde (MDA) levels and reduced glutathione (GSH) content were investigated.Our results revealed that treatment with acrolein changed levels of several proteins in diverse physiologicalprocess including energy metabolism, cell communication and transport, response to stimulus and metabolicprocess. Interestingly, several differentially over-expressed proteins, including β-synuclein, enolase andcalcineurin, are known to be associated with human neurodegenerative diseases. Changes in the levels ofsome proteins were confirmed by Western blot. Moreover, acrolein increases the level of MDA, as a lipidperoxidation biomarker and decreased GSH concentrations, as a non-enzyme antioxidant in the brain ofacrolein treated rats.These findings suggested that acrolein induces the oxidative stress and lipid peroxidation in the brain, and sothat may contribute to the pathophysiology of neurological disorders.

© 2013 Elsevier Inc. All rights reserved.

Introduction

Acrolein (CH2_CH\CHO) is a highly reactive molecule amongthe reactive α,β-unsaturated aldehydes that is widely distributed inthe environment as a common contaminant. Acrolein is producedby different sources including incomplete combustion, overheatedcooking oils, cigarette smoke, automobile exhaust and industrialwaste and emission (Aldini et al., 2011). Generally, human exposureto a large amount of acrolein is through cigarette smoke. It is reportedthat 3-hydroxypropyl mercapturic acid, the main urinary metaboliteof acrolein in urine of smokers, is about twice as much as in non-smokers (Stevens and Maier, 2008). Acrolein is also, a high-priorityair and water toxic according to Environmental Protection Agencyclassification (DeWoskin et al., 2003). Toxic effects of acrolein mayoccur following inhalation, oral and dermal exposures (Faroon et al.,

arch Center, Department ofacy, Mashhad University of3251.), Hosseinzadehh@mums.ac.ir

rights reserved.

2008). Metabolism of oxazaphosphorines, as antitumor drugs, is anoth-er source of acrolein. Moreover acrolein is generated endogenously,mainly by fragmentation of polyunsaturated fatty acids during lipidperoxidation reactions and metabolism of amino acids and polyamines(Aldini et al., 2011). Acrolein is one of the most reactive aldehydes andsoft electrophiles that readilymakes covalent adducts with nucleophilicsites of proteins, phospholipids and nucleic acids. Free thiol groups ofcysteine residues, amino groups of histidine and lysine residues of pro-teins are potential sites for acrolein adduct via Michael-type reactions(LoPachin et al., 2008a). Acrolein adduct impairs structure and functionof biomolecules which are considered to be involved in many patho-logical conditions like neurodegenerative and cardiovascular diseases(Aldini et al., 2011).

A growing number of evidences indicate that acrolein is signifi-cantly elevated in the brains or spinal cords of people who have neu-rologic disorders. It may mediate oxidative stress related tissuedamages and play an important role in the pathophysiology of neu-rodegenerative disease (Jomova et al., 2010; Singh et al., 2010) Tak-ing into account that the brain is rich in polyunsaturated fatty acids,interaction of oxygen-derived free radicals with polyunsaturated fattyacids is expected to form a variety of highly reactive aldehydes suchas acrolein. Moreover lipid peroxidation products could induce free

200 M. Rashedinia et al. / Toxicology and Applied Pharmacology 272 (2013) 199–207

radical chain reactions and increase the level of oxidative stress in tissue(Reed, 2011). Consequently acrolein is a product and initiator of lipidperoxidation (Kehrer and Biswal, 2000). Acrolein adducts have beenproposed as a biomarker in pathological conditions related to oxidativestress such as spinal cord injury, cerebral ischemia, Alzheimer's disease(AD) and diabetic nephropathy (Calingasan et al., 1999; Mello et al.,2007). Various studies in the fields of neuroscience and toxicology indi-cate that acrolein can cause nerve terminal damages through formationof adducts with presynaptic proteins related to vital neuronal functionswhich is also an early neuropathogenic event in neurodegenerative dis-ease (LoPachin et al., 2008b).

Owing to its ubiquitous presence in the environment and high re-activity, toxic effects of acrolein on various cells and organs havebeen extensively studied. It has been reported that inhalation ofacrolein following smoke may be a significant contributor to seriouslung injury and death (Ghilarducci and Tjeerdema, 1995). Inhaledacrolein can be absorbed andmay induce systemic effects by increas-ing platelet activation as a contributing factor to the prothromboticrisk in human and also, may induce atherosclerosis and coronary ar-tery disease (Sithu et al., 2010). Moreover, human endothelial cellsare particularly sensitive to acrolein. Acrolein adducts may causesystemic endothelial cell dysfunction and atherosclerosis (Uchida,2000). In addition, other injuries like bladder hemorrhagic cystitisand hepatotoxicity can also occur by acrolein generated from somedrugs such as anti-cancer agents cyclophosphamide and ifosfamide(Batista et al., 2006). Furthermore, acrolein is a pathogenic factor inboth multiple sclerosis and spinal cord injury, causing progressivemyelin damage and functional loss by perpetuating oxidative stress(Leung et al., 2011).

It has been suggested that alkylation reaction at various nucleo-philic sites, oxidative injury and disturbed cell redox balance follow-ing the increased generation of reactive oxygen species (ROS) anddepletion of glutathione (GSH) are implicated in acrolein cytotoxicity(Kehrer and Biswal, 2000; Uchida, 1999). Acrolein dysregulates majorcellular pathways in the process of apoptosis, transcription, cellcycle control, protein biosynthesis and cell signaling (Thompsonand Burcham, 2008). Proteomic analysis of isolated synaptosomesfrom Mongolian gerbils forebrains, showed that acrolein selectivelymodified synaptosomes proteins related to fundamental neuronalfunctions. All of these carbonylated proteins are involved in vitalcellular functions including energy metabolism, protein synthesis,neurotransmission, cytoskeletal integrity and neuronal plasticity (Melloet al., 2007).

Studying the cellular effects of acrolein in murine myocardiumand cardiac mitochondria demonstrated that acrolein could induceleft ventricular dilatation and dysfunction. Proteomic approach re-vealed that differentially over-expressed proteins were involved inmyocardial contraction and energy metabolism (Luo et al., 2007).

In this study we hypothesized that exposure to the environmentalsource of acrolein may contribute to onset or progress of pathologicalconditions in neurological disorders. So that levels of lipid peroxida-tion and GSH content were measured. Moreover differentially over-expressed proteins in brain cortex after chronic exposure to acroleinwere identified using a proteomics approach.

Materials and methods

Animal treatment. Adult male Wistar rats weighing 270–300 g andapproximately 4 months old, were provided by the Animal Centerof School of Pharmacy, Mashhad University of Medical Sciences,Iran. Rats were housed at temperature of 25 ± 2 °C on a 12-hlight/dark cycle with free access to food and water. Experimentswere conducted according to Ethical Committee Acts of MashhadUniversity of Medical Sciences for care and use of laboratory animals(# 89609).

Pilot study. 24 rats were randomly divided in 4 groups (n = 6):Control group, that received distilled water, and acrolein groups, thatreceived 1, 3, and 5 mg/kg/day doses of fresh aqueous solutions of acro-lein (Sigma) by gavage for 30 days. With 5 mg/kg/day three animalsdied within the first 72 h. Only one rat in the group that received3 mg/kg/day acrolein, died up at the end of experiment. Cerebral cortexwas chosen for this study because of higher tissue availability andhigher susceptibility to oxidative stress (Venkateshappa et al., 2012).

MDA levels in cerebral cortex tissues were also measured in acro-lein treated groups and compared with that of in the control group.Our results showed that MDA levels were significantly higher in ratswho received 3 mg/kg/day compared with control, but did not signif-icantly changed in dose of 1 mg/kg/day. So that based on these pilotexperiments, a dose of 3 mg/kg was selected for study.

Levels of lipid peroxidation. To measure the levels of MDA as an im-portant oxidative stress marker, cerebral cortex tissues were homog-enized in 1.15% KCl to make a 10% (w/v) solution at 4 °C. Total proteinconcentrations were determined using Bio-Rad protein assay kit(#500-0002). Then 0.5 mL of supernatant was added to 1 mL 0.6%thiobarbituric acid (TBA) and 3 mL 1% phosphoric acid (Merck) andheated in boiling water bath for 45 min. After cooling to room tem-perature, 4 mL of n-butanol was added and centrifuged at 6000 g.Concentration of thiobarbituric acid reactive substances (TBARS) inn-butanol phase was determined by measuring absorbance at 532 nmusing a microplate reader (Synergy H4 Hybrid Reader-Biotek, USA).Concentrations of MDA in samples were reported as nmol/mg protein(Yavuz et al., 1997).

Levels of reduced glutathione. GSH, as a non-enzymatic antioxidantagent, was measured using the method of Moron et al. (1979). Briefly,tissues were homogenized in phosphate buffer (0.1 M, pH = 7.4) andtotal protein concentrations were determined. 500 μL of tissue homog-enates was mixed with 500 μL of trichloroacetic acid (10%TCA) andvortexed. Trichloroacetic acid was added to homogenate samples toprecipitate protein but not GSH. After centrifugation, 500 μL of superna-tants were mixed with reaction buffer containing 2.5 mL 0.3 M phos-phate buffer pH 8.4 and 500 μL DTNB [5,5′ dithiobis-(2-nitrobenzoicacid)]. Absorbancewasmeasured at 412 nmwithin 5 min using a spec-trophotometer. Levels of GSH were expressed as nmol/mg protein.

Sample preparation and protein extraction for two-dimensional gel elec-trophoresis. 2D-gel electrophoresis procedures were performed asprevious protocols with little modification (Fountoulakis et al., 2005).Briefly, cerebral cortex tissues were homogenized in 2 mL cold lysisbuffer containing 7 M urea, 2 M thiourea, 4% CHAPS (Sigma Aldrich),2% dithiothreitol (DTT; Merck) 0.5% carrier ampholyte (pH 3–10,Bio-Rad) and complete protease inhibit or cocktail (Sigma P8340)using Polytron Homogenizer (IKA®T10, Germany) and sonicated threetimes each time 10 s on ice using a probe sonicator (UP100H, Germany).Crude extractswere shaken for 2 h at 4 °C for protein release and centrifugedat 20,000 ×g for 20 min. Total protein concentrations were measured usingBradford method (protein assay kit; Bio-Rad).

For isoelectric focusing, 150 μg of protein was loaded on to each17 cm 3–10 nonlinear IPGs (Bio-Rad, USA). Strips were activelyrehydrated in the protean IEF cell (Bio-Rad, USA) for 12 h at 50 V.

Isoelectric focusing was performed at 20 °C in 4 steps as follow:200 V for 1 h; 500 V for 1 h; 1000 V for 1 h and 8000 V for 6 huntil 55,000 Vh. For each group three independent experimentswere performed. Before the second dimension, the IPG strips wereequilibrated for 15 min in 37.5 mM Tris–HCl pH 8.8, 6 M urea, 30%(v/v) glycerol, 2% (w/v) SDS, and 1% (w/v) DTT. Strips were thenre-equilibrated for 15 min in the same buffer containing 2.5% (w/v)iodoacetamide instead of DTT. The second-dimension was performedin 12% SDS-poly acrylamide gels. Electrophoresis was carried out for

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30 min at 15 mA then 8 h at 25 mA per gel at 20 °C until the trackingdye reached the end of the gel.

Protein visualization and analysis. Following electrophoresis, gelswere fixed in a methanol/acetic acid solution and protein spotswere visualized by a MS-compatible silver staining (Chevallet et al.,2006). All gels were scanned on Image scanner III (Epson, Japan);Image Master Platinum 6.0 software (GE Healthcare, USA) was usedto analyze intensities of protein spots in control gels to correspondingspots in acrolein gels to find differentially expressed proteins. Proteinintensities were normalized against the total volumes of all thedetected spots. Student's t-test was performed to analyze statisticallydifferent intensities in treatment and control groups. P-values b0.05were considered as statistically different.

In gel digestion, mass analysis and data base searching for protein iden-tification. Protein spots with more than 1.5 fold change were candi-date for identification by MALDI-TOF/TOF. Spots were excised andtransferred to a microtube containing 1% acid acetic and sent to theCenter for Genomic Sciences at the University of Hong Kong for in-geldigestion, identification and characterization by MALDI-TOF/TOF(Matrix Assisted Laser Desorption Ionization-Time of Flight Analyzer).

Briefly, gel plugs were destained, washed and then reduced with10 mM DTT and treated with 55 mM iodoacetamide in dark to alkyl-ate all reduced cysteines. Then the mixture was diluted with 10 mMammonium biocarbonate before digestion. 12.5 ng/μL of sequencinggrade trypsin (Promega) was added to cover gel plugs and incubatedat 4 °C. After 30 min, trypsin was removed and replaced with 20 μL of10 mM ammonium bicarbonate and incubated at 37 °C for overnight.Digestion was stopped by adding 2 fractions of 5% formic acid/50%acetonitrile (Sigma Aldrich, St. Louis, MO, USA) and dehydratedwith 100% acetonitrile. The extracted peptide mixtures were drieddown by SpeedVac and the resuspended in 0.1% formic acid (SigmaAldrich, St. Louis, MO, USA) and followed by μC18 ZipTip (Millipore).

α-Cyano-4-hydroxycinnamic acid (CHCA), (Sigma Aldrich, St. Louis,MO, USA) was used as the MALDI matrix at a concentration of10 mg/mL in 50% water/acetonitrile and 0.1% formic acid. Sampleswere spotted and dried before applying matrix. All mass spectrawere acquired on 4800MALDI TOF/TOF Analyzer (ABSciex, Framingham,MA) in positive ion Reflector mode. Typical spectra were obtained byaveraging 500 acquisitions in Reflector mode and in MSMS modewith the minimum possible laser energy in order to maintain thebest resolution. Precursor ions with a charge state of 1+ werefragmented via post source decay (PSD). Peak list was generated byData Explorer (ABSciex, Framingham, MA) and searched using Mas-cot (version 2.1.0, Matrix Science, London, UK). Database search set-tings were as follows: +1 monoisotopic peaks were searched with amass tolerance of 75 ppm for precursor masses; ±0.2 Da for MS/MS.Trypsin was selected as the enzyme while allowing one missedcleavage, cysteine carbamidomethylation as fixed modification andmethionine oxidation as variable modification; Rattus taxonomy filterwas applied when searching against SwissProt and NCBInr proteindatabases.

Western blotting. Tissues were lysed in homogenization buffercontaining 50 mM Tris–HCl; pH = 8.8, 2 mM EDTA, 10 mM NaF,2% sodium deoxycholate, 2 mM EGTA, 0.1% SDS, 1% Triton X-100, 10%v/v 2-mercaptoethanol, 1 mM Na3VO4, 10 mM β-glycerophosphate(Sigma), 2 μl complete protease inhibitor cocktail (Sigma P8340)and 1 mM phenylmethylsulfonyl fluoride (PMSF) using Polytronhomogenizer and sonication. After centrifugation at 16,000 g for 10 minat 4 °C, supernatants were transferred to clean microtubes and proteincontents were measured using BioRad protein assay kit. Supernatantswere mixed with equal volumes of sample buffer containing 100 mMTris-base, 4% w/v SDS, 20% v/v glycerol, 10% v/v 2-mercaptoethanoland 0.2% w/v bromophenol blue. The same amounts of total proteins

from each sample were loaded on to SDS-PAGE. After electrophoresis,proteins were transferred to polyvinylidene difluoride (PVDF) mem-branes (Bio-Rad, USA). Membranes were blocked for 3 h in Tris-buffered saline–Tween 20 (TBST) containing 5% skimmedmilk powder.Blots were incubated with four primary monoclonal antibodies:PP2BA: Pan-Calcineurin A antibody (Cell Signaling), RGDIR1: RhoGDIantibody (Cell Signaling), SYUB: α/β-Synuclein (Syn 205) antibody(Cell Signaling) and anti mouse actin antibody (cell signaling) accordingto manufacturer's protocols. Membranes were washed three times andincubated with recommended dilutions of appropriate horseradishperoxidase-conjugated secondary antibodies (cell signaling) for 1 h.Protein bands were visualized using ECL detection reagent kit (Pierce,USA) and Alliance 4.7 gel doc (UK).

The intensities of the chemiluminescence bands were analyzedusing UVband software (UVITEC, UK). All bands were normalizedagainst corresponding beta actin intensities.

Classification of proteins. The PANTHER online database (http://www.pantherdb.org) was used for functional classification of signifi-cantly altered proteins (Mi et al., 2005). PANTHER uses a library of pro-tein families and subfamilies and a set of ontology terms to describeassociations between protein sequences and protein functions, biologi-cal process and biological pathways.

Functional and protein network analysis. Identified proteins weresubmitted to STRING database v. 9 (http://string-db.org/) to figure outfunctional association network of the differentially expressed proteinsin cerebral cortex of acrolein treated rats and control.

Results

Effects of acrolein on GSH and MDA levels

The lipid peroxidation was analyzed by measuring the MDA levelin the cerebral cortex (Table 1). MDA level was significantly higher,73.2%, in acrolein treated rats. Moreover chronic treatment of acroleinresulted in 12.42% decline in cerebral cortex GSH (Table 1).

2-D gel and mass spectrometry analysis of acrolein-effects on the cerebralcortex protein expression profile and identification of differentiallyexpressed proteins

Differentially expressed proteins in the cerebral cortex of acroleintreated and control rats were identified and analyzed using 2-D PAGEand MALDI TOF/TOF. Fig. 1 shows 2-D gel images of proteins isolatedfrom the brain of control (Fig. 1, A) and acrolein (Fig. 1, B) treatedrats. More than 600 protein spots were detected per gel, also 230well matched spots across acrolein and control gels were analyzed forcomparative proteomics study. Protein expression profile of brain cortexin treated rats revealed that expression ofmore than 40 proteinswas re-markable as compared with the control group. Among altered proteins,23 protein spots show significant changes and were quantitatively highenough to be identified by MALDI-TOF-TOF. MS data were analyzedusingMASCOT search engine. All 23 spotswere identifiedwith high con-fidence according to MASCOT search (Table 2). The Confidence Interval(C.I.) of the protein score and total ion score was ≥99 for alanine-tRNAligase, cytoplasmic (SYAC) and 100% for other protein spots. Image anal-ysis of gels from acrolein treated group indicated that among alteredprotein expression of 22 proteins significantly increased, from 1.51 to11.27 fold, and while the level of glutamine synthetase (GLNA) was sig-nificantly lower by 5.88 fold compared to control gels (P b 0.05). Someof differentially expressed proteins are shown in Fig. 1.More detailed in-formation about these identified proteins can be found in Table 2. Theselisted information are: name of proteins, theoretical and experimentalmolecular weights (MWs), isoelectric points (pIs), percent of sequencecoverage, number of matched peptides, the sequence of matched peptide

Table 1Effects of acrolein (4 weeks) on MDA and GSH levels in the cerebral cortex.

Group MDA (nmol/mg) GSH (nmol/mg)

Control 1.16 ± 0.19 38.39 ± 1.75Acrolein 3 mg/kg 2.01 ± 0.32** 34.1 ± 3.32*

Data are shown as mean ± SD, n = 6. The differences between means of acrolein andcontrol rats were analyzed by independent t test. *P b 0.05 and **P b 0.01 wereconsidered statistically significant.

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with the highest ion score and C.I. = 100% and also biological function ofproteins.

Western blotting

Based on the fold change of proteins and biological function fol-lowing proteins were selected for further evaluation with westernblotting, serine/threonine-protein phosphatase 2B (PP2BA), RhoGDP-dissociation inhibitor 1 (RGDIR1) and Beta-synuclein (SYUB).Anti-β actin antibody was used to normalize the optical densityvalues. Densitometric analysis indicated that trends of changeswere approximately the same those proteins detected in the 2D gelanalyses. Expression levels of PP2BA, RGDIR and SYUB significantlyincreased by 27.48%, 23.84% and 32.8% respectively, in the cerebralcortex of acrolein treated rats (Fig. 2). The fold change differencesbetween western blot and silver stained 2D gels (Table 2) are prob-ably due to imperfect correlation between intensities of the spotsand amount of proteins in each spot in the later method.

Pathway analysis

The PANTHER database, (http://panther.appliedbiosystems.com/),was used to investigate the molecular function, biological process andsignaling pathway associated with each identified protein.

Differentially expressed proteins were classified in 11 pathwaysincluding; cell communication, cellular process, transport, cellular com-ponent organization, systemprocess, response to stimulus, developmen-tal process, generation of precursor metabolites and energy, metabolicprocess, cell cycle, and immune system process. All proteins were cate-gorized in four main clusters as listed: 1) Generation of precursor

Fig. 1. Two-dimensional gel images of cerebral cortex proteins in rat. Control (A) and ac

metabolites and energy, 2) response to stimulus and cellular immunesystem process, 3) cell communication and transport and 4) metabolicprocess (Fig. 3).

Interaction network

To get a deeper insight of potential relationship and specific cellularpathways that proteins are involved, STRING database and a functionalannotation space, Kyoto Encyclopedia of Genes and Genome (KEGG)pathway enrichment were used. STRING identified the main interac-tion between identified proteins whichwere differentially expressed(Fig. 4). The KEGG analysis shows altered proteins enriched in glycolysis/gluconeogenesis, citrate cycle (TCA cycle), glutathione metabolism, me-tabolism of xenobiotics by cytochrome P450, amyotrophic lateral sclero-sis (ALS), Alzheimer's disease, long-termpotentiation, axon guidance andneurotrophin signaling pathways.

Discussion

Although neurotoxicity of acrolein is very well documented invitro (Kehrer and Biswal, 2000; Singh et al., 2010), little is known aboutits effects on the central nervous system and in the neural pathologyafter oral exposure.

In the current study proteomics approach was employed to iden-tify if protein expressions were changed in response to chronic oralexposure to acrolein. Our results showed that expressions of someproteins involved in diverse physiological processes in the cortex ofacrolein treated rats changed as compared with control. Moreover,increased MDA and decreased GSH concentration were observed inbrain of acrolein treated rats. Previous studies also indicated that sys-tematic exposure to acrolein resulted in significant decrease in gluta-thione levels and elevation of lipid peroxidation (Arumugam et al.,1999). It has been reported that exposure to acrolein lead to time-and dose-dependent ROS production and lipid peroxidation in spinalcord tissue (Luo and Shi, 2004).

Acrolein, as a byproduct of brain lipid peroxidation, is significantlyelevated in the brains or spinal cords of people who have AD, Parkinson'sdisease (PD), amyotrophic lateral sclerosis, and other neurologic disor-ders. It was indicated that oxidative stress has been implicated in acroleinrelated cytotoxicity and neural cell damages (Luo and Shi, 2005). Epide-miological studies have shown that exposures to many environmental

rolein (B). Labeled spots are significantly altered after treatment (P b 0.05, n = 3).

Table 2Differentially expressed proteins in the cerebral cortex of rats as identified by MALDI-TOF/TOF and MASCOT software.

Protein name(entry name)/gene name

Swiss Protaccessionnumber

TheoreticalMW(Da)/pI

ExperimentalMW(kDa)/pIa

Fold-change(acrolein/control)

PROTEINSCOREb %sequenecoverage

Matchedpeptides

Expectation-valuec Matched peptide sequenced Biological function ormolecular pathways(PANTHER classification)

Alanine-tRNA ligase,cytoplasmic (SYAC)/Aars

P50475 107,521/5.4 128/5.4 2.03 76 1 1 (1) 0.00071 R.AVFDETYPDPVR.V Metabolic process

78 kDa glucose-regulatedprotein (GRP78)/Hspa5

P06761 72,473/5.07 84/4.6 1.52 250 4 2 (2) 1.1E−10 R.IINEPTAAAIAYGR.E Protein folding, immunesystem and response tostimulus, metabolic process

Dihydrolipoyllysine-residueacetyltransferase (ODP2)/Dlat

P08461 67,636/8.76 72/5.7 2.01 193 5 3 (2) 9.9E−06 R.VAPTPAGVFIDIPISNIR.R Energy metabolism,metabolic process

Serine/threonine-proteinphosphatase 2B catalytic subunitalpha isoform (PP2BA)/Ppp3ca

P63329 59,290/5.58 65/5.6 3.79 197 5 3 (2) 2.9E−05 K.LFEVGGSPANTR.Y Immune system, cellcommunication, metabolicprocess

V-type proton ATPase subunit B,brain (VATB2)/Atp6v1b2

P62815 56,857/5.57 58/5.7 3.57 266 5 2 (2) 1.1E−06 R.IPQSTLSEFYPR.D Cell transport, Energymetabolism, Metabolic process

Dihydropyrimidinase-relatedprotein 2 (DPYL2)/Dpysl2

P47942 62,637/5.95 71/6.5 2.03 637 12 5 (5) 3.5E−13 R.NLHQSGFSLSGAQIDDNIPR.R Metabolic process

Dynamin-1 (DYN1)/Dnm1 P21575 97,576/6.44 119/7.5 2.19 112 2 2 (1) 0.034 K.HIFALFNTEQR.N Cell communication andtransport

Actin-related protein 3 (ARP3)/Actr3 Q4V7C7 47,783/5.61 50/5.7 4.38 387 18 4 (4) 1.7E−11 R.AEPEDHYFLLTEPPLNTPENR.E Cell communication andtransport

Alpha-enolase (ENOA)/Eno1 P04764 47,440/6.16 52/6.4 1.86 362 13 4 (4) 2.1E−11 R.AAVPSGASTGIYEALELR.D Metabolic process,Energy metabolism

Guanine nucleotide-binding proteinG(o) subunit alpha (GNAO)/Gnao1

P59215 40,613/5.34 42/4.8 3.87 415 18 5 (4) 4E−09 R.IGAADYQPTEQDILR.T Cell communication andtransport, signal transduction

Isocitrate dehydrogenase [NAD]subunit alpha, mitochondrial(IDH3A)/Idh3a

Q99NA5 40,044/6.47 41/5.7 1.82 332 12 4 (3) 3.8E−07 R.IAEFAFEYAR.N Metabolic process, Energymetabolism

Prohibitin (PHB)/Phb P67779 29,858/5.57 30/5.5 1.51 460 20 5 (4) 6.4E−09 R.KLEAAEDIAYQLSR.S Metabolic process, Cell cycleRho GDP-dissociation inhibitor 1(RGDIR1)/Arhgdi

Q5XI73 23,449/5.12 25/4.7 4.84 248 18 4 (3) 1.9E−06 R.VAVSADPNVPNVIVTR.L Cell communication, signaltransduction

Thioredoxin-dependent peroxidereductase, mitochondrial(PRDX)/Prdx3

Q9Z0V6 28,562/7.14 23/6.4 3.5 190 8 2 (2) 6.1E−09 R.GLFIIDPNGVIKHLSVNDLPVGR.S Immune system andresponse to stimulus, metabolicprocess

Glutathione S-transferase(GSTM4)/Gstm3

P08009 25,835/6.84 24/8.1 9.2 293 24 5 (4) 6.3E−05 K.ITQSNAILR.Y Immune system andresponse to stimulus

Glutathione S-transferaseP(GSTP1)/Gstp1

P04906 23,652/6.89 22/8.1 3.72 431 30 4 (3) 3.1E−12 K.FEDGDLTLYQSNAILR.H Immune system andresponse to stimulus,metabolic process

Beta-synuclein (SYUB)/Sncb Q63754 14,495/4.48 16/4.1 7.96 577 30 4 (4) 1.4E-20 K.TKEQASHLGGAVFSGAGNIAAATGLVK.K Cell communicationand transport

Superoxide dismutase [Cu-Zn](SOD)/Sod1

P07632 16,072/5.88 13/6.2 2.83 472 32 4 (4) 3.3E−09 R.VISLSGEHSIIGR.T Immune system andresponse to stimulus

Calmodulin (CALM)/Calm1 P62161 16,826/4.09 11/3.7 11.27 207 10 2 (2) 4.1E−10 R.VFDKDGNGYISAAELR.H Cell communication andsignal transduction

Phosphatidylethanolamine-bindingprotein 1 (PEBP1)/Pebp1

P31044 20,902/5.48 21/5.1 1.87 445 25 4 (4) 3.9E−13 K.GNDISSGTVLSEYVGSGPPKDTGLHR.Y Cell communication andsignal transduction

Glutamine synthetase (GLNA)/Glul P09606 42,981/6.64 45/7.6 0.17 546 14 5 (5) 9.4E−16 R.LTGFHETSNINDFSAGVANR.S Metabolic processPhosphoglycerate kinase 1(PGK1)/Pgk1

P16617 44,909/8.02 45/8.4 1.51 556 22 5 (5) 4E−15 K.DCVGSEVENACANPAAGTVILLENLR.F Energy metabolism,Metabolic process

ATP synthase subunit alpha,mitochondrial (ATPA)/Atp5a1

P15999 59,830/9.22 54/8.3 1.87 516 10 5 (5) 2.1E−10 R.EAYPGDVFYLHSR.L Cell transport, Energymetabolism, Metabolic process

a Experimental MW/pI was estimated in gel.b Protein score [PMF + MS/MS] = protein score [PMF] + total ion score [MS / MS]. It is the Mascot probability based scoring that reports scores as −10 ∗ log10 (P), where P is the probability that the observed match is a random event.

The total ion score is calculated by weighting ion scores for all individual peptides matched to the protein that is associated with this peptide and MS/MS spectrum. Ion scores for duplicated matches are excluded in the calculation.c The probability of an incorrect identification associated with matched peptide with the highest ion score and C.I. % = 100.d The sequence of matched peptide with the highest ion score and C.I. % = 100.

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pollutant, such as air pollution, diet, pesticide and metals are potentialrisk factors for various neurodegenerative disorders (Migliore andCoppedè, 2009). Exposure to acrolein, like most of the noted pollutants,induces oxidative stress and may lead to neurological disorders associat-ed with oxidative stress and enhanced lipid peroxidation.

Brain tissue is highly susceptible to oxidative damage because ofthe lack of antioxidant enzymes, high rate of oxygen consumption,abundance of catalytic transition metals and high content of oxidiz-able substrates like polyunsaturated fatty acids. Numerous studieshave confirmed that oxidative stress is associated with brain aging,leading to morphological changes and metabolic deficits (Chakrabartiet al., 2011).

Acrolein, as strong electrophile aldehydes, reacts with cysteine, his-tidine, and lysine residues, producing protein-carbonyl derivatives.Thus it has been reported that acrolein can potentially interact andalter structure of some proteins including albumin α-1-proteinase,human serum albumin and axonal cytoskeletal proteins (Pocernichet al., 2001). So that acrolein may potentially hurt neurons by bindingto a range of proteins and indirectly lead to oxidative stress, whichcan contribute to neuronal dysfunction and cell death (Pocernichet al., 2001). Beside administration of acrolein, it can be endogenouslygenerated under oxidative stress condition as a product of lipid peroxi-dation. Recently, an in vivo study showed that chronic oral exposure toacrolein decreases the activity of superoxide dismutase and increasesthe level of MDA (Huang et al., 2013). So that simple measurement ofacrolein–protein adduct in-vivo may not show direct interaction ofexact administrated acrolein with proteins. Instead, radio labeled acro-lein can be used to show the ability of acrolein to pass through theblood–brain barrier, and so it interacts with the cortex proteins.

Proteomic analysis in our study revealed that there is a good agree-ment between well-known biomarkers of human neurodegenerativediseases and cellular responses to acrolein. Although chronic intoxica-tion with acrolein did not display symptoms of human neurodegenera-tive diseases, some molecular mechanisms were shown to be commonamong neurological disorders and environmental pollutant toxicities.We showed that 23 proteins are up-regulated in acrolein treated rats.These proteins were classified to four main clusters according toPANTHER database: 1) Generation of precursormetabolites and energy,2) cell response to stimuli, 3) cell communication and transport and4) metabolic process (Fig. 3).

Fig. 2. Western blot analysis of PP2BA: Pan-Calcineurin A, RGDIR1: RhoGDI, and SYUB:α/β-Synuclein in the cerebral cortex tissues of acrolein treated and control rats (A).Plot expresses protein levels relative to control (B). Differences between means ofacrolein and control were analyzed by independent t test. * P b 0.05, ** P b 0.01 wereconsidered statistically significant.

Acrolein effects on generation of precursor metabolites and energy

Glucose metabolism is a vital process in the brain that influencesmany normal cellular processes from synthesis of neurotransmitterto ATP production (Hoyer, 2012). Impairment of mitochondrial bio-energetic capacity has a major role in the pathogenesis of neurode-generative disorders (Butterfield and Lange, 2009).

Our results showed that several enzymes are important in the glyco-lytic process, tricarboxylic acid (TCA) cycles and ATP production wereup-regulated in acrolein treated rats. These proteins are: isocitratedehydrogenase 3 alpha (IDH3A), ATP synthase subunit alpha (ATPA),dihydrolipoyllysineal-residue acetyl transferase (ODP2, is a compo-nent of pyruvate dehydrogenase complex), phosphoglycerate kinase1(PGK1), V-type proton ATPase subunit B (VATB2) and α-enolase(ENOLA).

Acrolein damages structure and function of mitochondrial mem-brane by stimulating ROS generation leading to impaired oxidativephosphorylation and energy production (Arumugam et al., 1999; Luoet al., 2005). Furthermore, some proteins which were modified byacrolein-adducts, are involved in energy metabolism (e.g. mitochondrialcreatine kinase-2, ATP synthase) in myocardial cells and tissue exposedto acrolein (Luo et al., 2007). Up-regulation of glycolytic enzymes inthe cerebral cortex of the brain, as a major consumer organ of glucose,probably is an effort to compensate the mounting energy deficit.

Effect of acrolein on cellular response to stimuli

Response to stress is an evolutionary conservedmechanism that letscells to protect themselves against unfavorable effects of environmentalagents or metabolic conditions. Our results showed that glutathionelevels were significantly diminished in the brain of acrolein treatedrats. Acrolein, as a strong electrophile, rapidly reacts with nucleophiles,mainly cellular thiols, such as GSH. Oxidative stress induces depletion ofglutathione, making neural cells even more susceptible to oxidants(Kehrer and Biswal, 2000; Starke et al., 1997). Moreover, conjugationof acrolein with GSH produces GS-propionaldehyde metabolite whichis a substrate for aldehyde dehydrogenase and probably for aldehydeoxidase. The oxidation of GS-propionaldehyde by these enzymes resultsin O2• and probably HO• formation, that may induce lipid peroxidation(Adams and Klaidman, 1993).

Acrolein has been reported to induce the expression of antioxidant re-sponse element dependent genes and transcription of stress-responseproteins, resulting in protection of cells against electrophile stress byupregulation of phase II biotransformation enzymes such as superoxidedismutase, GSH peroxidase, GSH reductase, thioredoxin, and thioredoxinreductase (Stevens and Maier, 2008).

In this study, we have reported a significant increase in the expres-sion of glutathione S-transferases (GST), superoxide dismutase (SOD),78 kDa glucose-regulated protein (GRP78), and thioredoxin-dependentperoxide reductase (PRDX) in the cortex of acrolein treated rats.

GSTs catalyze reduction of free radicals by conjugation of reducedglutathione to electrophilic groups on substrate molecules, includingbyproducts of oxidative stress to make them more soluble and to fa-cilitate their elimination (Hayes et al., 2005).

Superoxide dismutase SOD catalyzes the reduction of free radicalsand is responsible for the removal of lipid oxidation products (Hayeset al., 2005). GST and SOD over-expression in acrolein treated ratsmay protect neural cells against oxidation by free radicals.

GRP78/Bip, a member of endoplasmic reticulum (ER) chaperones,was also over-expressed in the brain of acrolein treated rats. GRP78that facilitates protein folding is reported to be higher under stress

Fig. 3. Functional classification of identified proteins in the cerebral cortex of rat. Pie chart shows PANTHER classifications according to the biological process related to each protein.Numbers indicate the percentage of protein against the total number of proteins.

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conditions such as oxidative stress, calcium homeostasis perturbationand glucose starvation, protecting neurons against these conditions(Ito et al., 2001; Yu et al., 1999).

PRDXalso known as thioredoxin peroxidase, a member of thethioredoxin system (TXR), was also up-regulated in acrolein treatedrats. TXR is known to play an important role in the enzymatic removalof ROS and contribute to defend the cells of an organism against

Fig. 4. Functional association network between the differentially expressed proteins in the STbetween nodes show types of evidence for the association. Protein–protein interaction modes btially expressed proteins and more associated protein in cell (B).

oxidative stress by their capability to repair the catalytic activity ofglutathione peroxidases. Furthermore, it is indicated that expressionof PRDX could promote neuronal cell survival and protect neuralcells against oxidative stress (Ichimiya et al., 1997).

Increased expression of antioxidant proteins provides a safeguardmechanism in the brain against acrolein oxidation. We concludedthat higher levels of MDA and low GSH content after exposure to

RING database. Proteins are shown by gene names. Nodes are proteins and colored linesetween the identified differentially expressed proteins (A). Interaction between differen-

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acrolein may stimulate expression of oxido-redox active enzymes tocompensate oxidative stress.

Effect of acrolein on cell communication and transport proteins

All differentially expressed proteins in control and acrolein treatedrats with a role in synaptic function and/or synaptic formation wereclassified as cell communication protein. These up-regulated proteinswere identified as: Beta-synuclein (SYUB), calmodulin (CALM),guanine nucleotide-binding protein G subunit alpha (GNAO),phosphatidylethanolamine-binding protein 1 (PEBP1), Rho GDP-dissociation inhibitor 1(RGDIR1), serine/threonine-protein phosphatase2B catalytic subunit alpha (PP2BA), dynamin-1 (DYN1), actin-relatedprotein 3 (ARP3), VATB2 and ATPA.

α-Synuclein (α-SYN) inclusions, found in a significant percentageof neurodegenerative disorders, could impair cellular functions byobstructing normal cellular trafficking, axonal transport and alsomay damage proteasome function (Waxman and Giasson, 2009).

Study on α-SYN in PD suggested that acrolein could be conjugatedtoα-SYN proteins and change their conformation resulting in the accu-mulation in dopamine neurons of aged and PDpatients.Moreover, acro-lein adducts with 20S proteasome proteins could reduce proteasomeactivity and increases α-SYN (Shamoto-Nagai et al., 2007). Our resultsupports an increase in the relative levels ofα/β-SYN in acrolein treatedrats compared to the controls. The effect of acrolein on the protein levelsof α/β-SYN was also confirmed by Western blotting.

Calcium dyshomeostasis and disruption of Ca2+ signaling havebeen linked to neuronal dysfunction, brain aging and many neurode-generative diseases (Berridge, 2010; Berrocal et al., 2012). In ourstudy, over-expression of two important calcium dependent proteins,calcineurin and calmodulin were observed after exposure to acroleinin rats.

Calcineurin or PP2BA as a Ca2+/calmodulin-dependent protein isthe most abundant and the only calcium-activated phosphatase inthe brain. The over-expression of PP2BA has been linked tomitochondri-al dysfunction, increased superoxide levels and promoted production ofreactive oxygen species. Inhibition of PP2BA activity is neuroprotective inischemia models (Bales et al., 2010; Robinson et al., 2011).

Western blot analysis also confirmed an increase of PP2BA in acro-lein treated rats.

Calmodulin is a small calcium binding and multifunctional proteinthat transduces calcium signals by binding to calcium ions, altering itsaffinity to a variety of target proteins; including large number of enzymeion channels, protein kinases and phosphatases (Chin andMeans, 2000).

Expression of RGDIR1, a member of Rho GTPase family, increased inacrolein treated group. This protein regulatesmembrane trafficking, cyto-skeletal organization, cell adhesive interactions, cellmigration andneuro-transmitter release. RGDIR1is a negative regulator of Rho GTPase activityand promote rearrangement of cytoskeleton and neural developmentand also, has antiapoptotic activity. Also, dihydropyrimidinase-relatedprotein 2 (DPYL2) plays a similar role in neuronal connection, cell com-munication and migration (Ciavardelli et al., 2010). Thus increaseRGDIR1 and DPYL2 levels may have a neuroprotective effect on cerebralcortex in acrolein treated rat (Kaibuchi et al., 1999).Western blot analysisconfirmed an increase of RGDIR1 in acrolein treated rats. The changes inabundance of proteins associated with endocytosis and vesicle traffick-ing, can affect normal synaptic function of brain in response to acroleintoxicity.

ARP 3 stimulates actin polymerization and elongation process ofaxons and dendrites in neurons (Weitzdoerfer et al., 2002). Ourstudy showed that ARP 3 is up-regulated in acrolein treated brains.Induction of ARP 3 subunit may result in abnormality in actin poly-merization, impaired neurite extension and morphological changes.Because of a significantly high amount of accessible nucleophilic tar-get sites, such as Cys and His residues, actin is a promising target for

α,β-unsaturated aldehyde, and scavenging reactive electrophilic al-dehydes, such as acrolein (Dalle-Donne et al., 2007).

Effect of acrolein on metabolic enzymes

Fourteen of identified proteins were involved in cell metabolism.Some of these proteins have already been discussed.

Prohibitin (PHB) acts as a membrane-bound chaperone for thestabilization of mitochondrial proteins that induces correct foldingof newly synthesized subunits of mitochondrial enzymes. Over-expression of PHB protects the mitochondria against oxidative stressdamages in cardiomyocyte and suppresses the mitochondria-mediatedapoptosis pathway (Liu et al., 2009).

Increase in levels of PHB, ATP synthase and SOD2 has also beenreported in the frontal cortices of PD and AD patients (Ferrer et al.,2007). Up-regulation of PHB in this study may be considered as partof cellular defense against oxidative stress following acrolein toxicity.

Glutamine synthase (GLNA) catalyzes the rapid amidation of glu-tamate to glutamine and restores the optimal level of ammonia andglutamate in neurons and modulates excitotoxicity that may resultfrom the impairment of this cycle (Smith et al., 1991). We showedthat GLNA was down-regulated in acrolein treated versus controlrats. Due to the central role of GLNA in brain function, numerousstudies have shown that GLNA is particularly sensitive to inactivationby oxidant agents and ROS generating systems (Prince et al., 1995)and GLNA activity is a good indicator for evaluation of brain damagesfollowing oxidative stress (Castegna et al., 2002).

Conclusion

Exogenous acrolein may additionally act with endogenous unsat-urated aldehydes to increase cellular damage and thereby acceleratepathogenic processes in oxidative stress.

In this study proteomics approach was implied to identify proteinsinvolved in acrolein toxicity. Our data showed that, acrolein inducedglutathione depletion resulting in enhanced lipid peroxidation inthe cerebral cortex. Proteomic analysis revealed that the levels of anumber of proteins were increased following exposure to acrolein.These proteins are involved in energy metabolism, antioxidant de-fense, cell communication and transport and also, cell metabolism.This studymay improve our understanding of mechanisms of acroleintoxicity and reveal other probable mechanism or cellular pathwaysinvolve in toxicity of the reactive aldehyde acrolein particularly inCNS.

Conflict of interest statement

All authors have no financial or personal relationships with otherpeople or organizations that could improperly influence our work.

Acknowledgments

Authors appreciate the Vice Chancellor of Research, MashhadUniversity of Medical Sciences for the financial support and Centerfor Genomic Sciences at University of Hong Kong for performingMALDI-TOF/TOF. The results of this research are part of a Ph.D. thesis.

References

Adams, J.D., Klaidman, L.K., 1993. Acrolein-induced oxygen radical formation. FreeRadic. Biol. Med. 15, 187–193.

Aldini, G., Orioli, M., Carini, M., 2011. Protein modification by acrolein: relevance topathological conditions and inhibition by aldehyde sequestering agents. Mol.Nutr. Food Res. 55, 1301–1319.

Arumugam, N., Thanislass, J., Ragunath, K., Niranjali Devaraj, S., Devaraj, H., 1999. Acro-lein-induced toxicity—defective mitochondrial function as a possible mechanism.Arch. Environ. Contam. Toxicol. 36, 373–376.

207M. Rashedinia et al. / Toxicology and Applied Pharmacology 272 (2013) 199–207

Bales, J.W., Ma, X., Yan, H.Q., Jenkins, L.W., Dixon, C.E., 2010. Expression of proteinphosphatase 2B (calcineurin) subunit A isoforms in rat hippocampus after trau-matic brain injury. J. Neurotrauma 27, 109–120.

Batista, C., Brito, G., Souza, M., Leitão, B., Cunha, F., Ribeiro, R., 2006. A model ofhemorrhagic cystitis induced with acrolein in mice. Braz. J. Med. Biol. Res. 39,1475–1481.

Berridge, M.J., 2010. Calcium hypothesis of Alzheimer's disease. Pflugers Arch. Eur. J.Physiol. 459, 441–449.

Berrocal, M., Sepulveda, M.R., Vazquez-Hernandez, M., Mata, A.M., 2012. Calmodulinantagonizes amyloid-β peptides-mediated inhibition of brain plasma membraneCa2+-ATPase. Biochim. Biophys. Acta Mol. Basis Dis. 1822, 961–969.

Butterfield, D.A., Lange, M.L.B., 2009. Multifunctional roles of enolase in Alzheimer'sdisease brain: beyond altered glucose metabolism. J. Neurochem. 111, 915–933.

Calingasan, N.Y., Uchida, K., Gibson, G.E., 1999. Protein-bound acrolein: a novel markerof oxidative stress in Alzheimer's disease. J. Neurochem. 72, 751–756.

Castegna, A., Aksenov, M., Aksenova, M., Thongboonkerd, V., Klein, J.B., Pierce, W.M.,Booze, R., Markesbery, W.R., Butterfield, D.A., 2002. Proteomic identification of ox-idatively modified proteins in Alzheimer's disease brain. Part I: creatine kinase BB,glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. Free Radic.Biol. Med. 33, 562–571.

Chakrabarti, S., Munshi, S., Banerjee, K., Thakurta, I.G., Sinha, M., Bagh, M.B., 2011.Mitochondrial dysfunction during brain aging: role of oxidative stress and modu-lation by antioxidant supplementation. Aging Dis. 2, 242.

Chevallet, M., Luche, S., Rabilloud, T., 2006. Silver staining of proteins in polyacrylamidegels. Nat. Protoc. 1, 1852–1858.

Chin, D., Means, A.R., 2000. Calmodulin: a prototypical calcium sensor. Trends Cell Biol.10, 323.

Ciavardelli, D., Silvestri, E., Del Viscovo, A., Bomba, M., De Gregorio, D., Moreno, M., DiIlio, C., Goglia, F., Canzoniero, L., Sensi, S., 2010. Alterations of brain and cerebellarproteomes linked to Aβ and tau pathology in a female triple-transgenic murinemodel of Alzheimer's disease. Cell Death Dis. 1, e90.

Dalle-Donne, I., Carini, M., Vistoli, G., Gamberoni, L., Giustarini, D., Colombo, R., MaffeiFacino, R., Rossi, R., Milzani, A., Aldini, G., 2007. Actin Cys374 as a nucleophilictarget of α, β-unsaturated aldehydes. Free Radic. Biol. Med. 42, 583–598.

DeWoskin, R.S., Greenberg, M., Pepelko, W., Strickland, J., 2003. Toxicological review ofacrolein. Support of Summary Information on the Integrated Risk Information System(IRIS) (CAS No. 107-02-8). US Environmental Protection Agency, Washington, DC.

Faroon, O., Roney, N., Taylor, J., Ashizawa, A., Lumpkin, M., Plewak, D., 2008. Acroleinhealth effects. Toxicol. Ind. Health 24, 447–490.

Ferrer, I., Perez, E., Dalfó, E., Barrachina, M., 2007. Abnormal levels of prohibitin and ATPsynthase in the substantia nigra and frontal cortex in Parkinson's disease. Neurosci.Lett. 415, 205–209.

Fountoulakis, M., Tsangaris, G.T., Maris, A., Lubec, G., 2005. The rat brain hippocampusproteome. J. Chromatogr. B 819, 115–129.

Ghilarducci, D.P., Tjeerdema, R.S., 1995. Fate and effects of acrolein. Rev. Environ.Contam. Toxicol. 144, 95–146.

Hayes, J.D., Flanagan, J.U., Jowsey, I.R., 2005. Glutathione transferases. Annu. Rev.Pharmacol. Toxicol. 45, 51–88.

Hoyer, S., 2012. Oxidative metabolism deficiencies in brains of patients withAlzheimer's disease. Acta Neurol. Scand. 94, 18–24.

Huang, Y.-J., Jin, M.-H., Pi, R.-B., Zhang, J.-J., Ouyang, Y., Chao, X.-J., Chen, M.-H., Liu, P.-Q.,Yu, J.-C., Ramassamy, C., 2013. Acrolein induces Alzheimer's disease-like pathologiesin vitro and in vivo. Toxicol. Lett. 217, 184–191.

Ichimiya, S., Davis, J.G., O'rourke, D.M., Katsumata, M., Greene, M.I., 1997. Murinethioredoxin peroxidase delays neuronal apoptosis and is expressed in areas ofthe brain most susceptible to hypoxic and ischemic injury. DNA Cell Biol. 16,311–321.

Ito, D., Tanaka, K., Suzuki, S., Dembo, T., Kosakai, A., Fukuuchi, Y., 2001. Up-regulation ofthe Ire1-mediated signaling molecule, Bip, in ischemic rat brain. Neuroreport 12,4023–4028.

Jomova, K., Vondrakova, D., Lawson, M., Valko, M., 2010. Metals, oxidative stress andneurodegenerative disorders. Mol. Cell Biochem. 345, 91–104.

Kaibuchi, K., Kuroda, S., Amano,M., 1999. Regulation of the cytoskeleton and cell adhesionby the Rho family GTPases in mammalian cells. Annu. Rev. Biochem. 68, 459–486.

Kehrer, J.P., Biswal, S.S., 2000. The molecular effects of acrolein. Toxicol. Sci. 57, 6–15.Leung, G., Sun, W., Zheng, L., Brookes, S., Tully, M., Shi, R., 2011. Anti-acrolein treatment

improves behavioral outcome and alleviates myelin damage in experimental auto-immune enchephalomyelitis mouse. Neuroscience 173, 150–155.

Liu, X.H., Ren, Z., Zhan, R., Wang, X.X., Wang, X.M., Zhang, Z.Q., Leng, X., Yang, Z.H., Qian,L.J., 2009. Prohibitin protects against oxidative stress-induced cell injury in cul-tured neonatal cardiomyocyte. Cell Stress Chaperones 14, 311–319.

LoPachin, R.M., Barber, D.S., Gavin, T., 2008a. Molecular mechanisms of the conjugatedalpha, beta-unsaturated carbonyl derivatives: relevance to neurotoxicity and neu-rodegenerative diseases. Toxicol. Sci. 104, 235–249.

LoPachin, R.M., Gavin, T., Barber, D.S., 2008b. Type-2 alkenes mediate synaptotoxicityin neurodegenerative diseases. Neurotoxicology 29, 871–882.

Luo, J., Shi, R., 2004. Acrolein induces axolemmal disruption, oxidative stress, and mito-chondrial impairment in spinal cord tissue. Neurochem. Int. 44, 475–486.

Luo, J., Shi, R., 2005. Acrolein induces oxidative stress in brain mitochondria. Neurochem.Int. 46, 243–252.

Luo, J., Robinson, J.P., Shi, R., 2005. Acrolein-induced cell death in PC12 cells: role ofmitochondria-mediated oxidative stress. Neurochem. Int. 47, 449–457.

Luo, J., Hill, B.G., Gu, Y., Cai, J., Srivastava, S., Bhatnagar, A., Prabhu, S.D., 2007. Mechanismsof acrolein-induced myocardial dysfunction: implications for environmental and en-dogenous aldehyde exposure. Am. J. Physiol. Heart Circ. Physiol. 293, H3673–H3684.

Mello, C.F., Sultana, R., Piroddi, M., Cai, J., Pierce, W.M., Klein, J.B., Butterfield, D.A., 2007.Acrolein induces selective protein carbonylation in synaptosomes. Neuroscience147, 674–679.

Mi, H., Lazareva-Ulitsky, B., Loo, R., Kejariwal, A., Vandergriff, J., Rabkin, S., Guo, N.,Muruganujan, A., Doremieux, O., Campbell, M.J., 2005. The PANTHER database ofprotein families, subfamilies, functions and pathways. Nucleic Acids Res. 33,D284–D288.

Migliore, L., Coppedè, F., 2009. Environmental-induced oxidative stress in neurodegener-ative disorders and aging. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 674, 73–84.

Moron, M.S., Depierre, J.W., Mannervik, B., 1979. Levels of glutathione, glutathione re-ductase and glutathione-S-transferase activities in rat lung and liver. Biochim.Biophys. Acta, Gen. Subj. 582, 67–78.

Pocernich, C.B., Cardin, A.L., Racine, C.L., Lauderback, C.M., Allan Butterfield, D., 2001.Glutathione elevation and its protective role in acrolein-induced protein damagein synaptosomal membranes: relevance to brain lipid peroxidation in neurodegen-erative disease. Neurochem. Int. 39, 141–149.

Prince, G., Delaere, P., Fages, C., Lefrancois, T., Touret, M., Salanon,M., Tardy, M., 1995. Glu-tamine synthetase (GS) expression is reduced in senile dementia of the Alzheimertype. Neurochem. Int. 20, 859–862.

Reed, T.T., 2011. Lipid peroxidation and neurodegenerative disease. Free Radic. Biol. Med.51, 1302–1319.

Robinson, R.A.S., Lange, M.B., Sultana, R., Galvan, V., Fombonne, J., Gorostiza, O., Zhang,J., Warrier, G., Cai, J., Pierce, W.M., 2011. Differential expression and redox proteo-mics analyses of an Alzheimer disease transgenic mouse model: effects of theamyloid-β peptide of amyloid precursor protein. Neuroscience 177, 207–222.

Shamoto-Nagai, M., Maruyama, W., Hashizume, Y., Yoshida, M., Osawa, T., Riederer, P.,Naoi, M., 2007. In Parkinsonian substantia nigra, α-synuclein is modified by acrolein,a lipid-peroxidation product, and accumulates in the dopamine neurons with inhibi-tion of proteasome activity. J. Neural Transm. 114, 1559–1567.

Singh, M., Nam, D.T., Arseneault, M., Ramassamy, C., 2010. Role of by-products of lipidoxidation in Alzheimer's disease brain: a focus on acrolein. J. Alzheimers Dis. 21,741–756.

Sithu, S.D., Srivastava, S., Siddiqui, M.A., Vladykovskaya, E., Riggs, D.W., Conklin, D.J.,Haberzettl, P., O'Toole, T.E., Bhatnagar, A., D'Souza, S.E., 2010. Exposure to acroleinby inhalation causes platelet activation. Toxicol. Appl. Pharmacol. 248, 100–110.

Smith, C., Carney, J., Starke-Reed, P., Oliver, C., Stadtman, E., Floyd, R., Markesbery, W.,1991. Excess brain protein oxidation and enzyme dysfunction in normal aging andin Alzheimer disease. Proc. Est. Acad. Sci. 88, 10540–10543.

Starke, D.W., Chen, Y., Bapna, C.P., Lesnefsky, E.J., Mieyal, J.J., 1997. Sensitivity of proteinsulfhydryl repair enzymes to oxidative stress. Free Radic. Biol. Med. 23, 373.

Stevens, J.F., Maier, C.S., 2008. Acrolein: sources, metabolism, and biomolecular in-teractions relevant to human health and disease. Mol. Nutr. Food Res. 52, 7–25.

Thompson, C.A., Burcham, P.C., 2008. Genome-wide transcriptional responses to acrolein.Chem. Res. Toxicol. 21, 2245–2256.

Uchida, K., 1999. Current status of acrolein as a lipid peroxidation product. TrendsCardiovasc. Med. 9, 109–113.

Uchida, K., 2000. Role of reactive aldehyde in cardiovascular diseases. Free Radic. Biol.Med. 28, 1685–1696.

Venkateshappa, C., Harish, G., Mahadevan, A., Bharath, M.S., Shankar, S., 2012. Elevatedoxidative stress and decreased antioxidant function in the human hippocampusand frontal cortex with increasing age: implications for neurodegeneration inAlzheimer's disease. Neurochem. Res. 37, 1601–1614.

Waxman, E.A., Giasson, B.I., 2009. Molecularmechanisms ofα-synuclein neurodegeneration.Biochim. Biophys. Acta Mol Basis Dis. 1792, 616–624.

Weitzdoerfer, R., Fountoulakis, M., Lubec, G., 2002. Reduction of actin-related proteincomplex 2/3 in fetal Down syndrome brain. Biochem. Biophys. Res. Commun.293, 836–841.

Yavuz, Ö., Türközkan, N., Dogulu, F., Aykol, S., 1997. The effect of 2-chloroadenosine onlipid peroxide level during experimental cerebral ischemia–reperfusion in gerbils.Free Radic. Biol. Med. 22, 337–341.

Yu, Z.F., Luo, H., Fu,W., Mattson, M.P., 1999. The endoplasmic reticulum stress-responsiveprotein GRP78 protects neurons against excitotoxicity and apoptosis: suppression ofoxidative stress and stabilization of calcium homeostasis. Exp. Neurol. 155, 302–314.