Research Article Differential Effects of Methyl-4 ...

Hindawi Publishing CorporationJournal of ToxicologyVolume 2013 Article ID 347312 10 pageshttpdxdoiorg1011552013347312

Research ArticleDifferential Effects of Methyl-4-Phenylpyridinium IonRotenone and Paraquat on Differentiated SH-SY5Y Cells

Joatildeo Barbosa Martins1 Maria de Lourdes Bastos1

Feacutelix Carvalho1 and Joatildeo Paulo Capela12

1 REQUIMTE (Rede de Quımica e Tecnologia) Laboratorio de Toxicologia Departamento de Ciencias BiologicasFaculdade de Farmacia Universidade do Porto Rua de Jorge Viterbo Ferreira 228 4050-313 Porto Portugal

2 Faculty of Health Sciences University Fernando Pessoa Rua Carlos da Maia 296 4200-150 Porto Portugal

Correspondence should be addressed to Joao Barbosa Martins joao b martinshotmailcom and Joao Paulo Capelajoaocapelaffuppt

Received 15 November 2012 Revised 28 January 2013 Accepted 28 January 2013

Academic Editor Lucio Guido Costa

Copyright copy 2013 Joao Barbosa Martins et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Paraquat (PQ) a cationic nonselective bipyridyl herbicide has been used as neurotoxicant to modulate Parkinsonrsquos disease inlaboratory settings Other compounds like rotenone (ROT) a pesticide and 1-methyl-4-phenylpyridinium ion (MPP+) have beenwidely used as neurotoxicants We compared the toxicity of these three neurotoxicants using differentiated dopaminergic SH-SY5Y human cells aiming to elucidate their differential effects PQ-induced neurotoxicity was shown to be concentration andtime dependent being mitochondrial dysfunction followed by neuronal death On the other hand cells exposure toMPP+ inducedmitochondrial dysfunction but not cellular lyses Meanwhile ROT promoted bothmitochondrial dysfunction and neuronal deathrevealing a biphasic pattern To further elucidate PQ neurotoxic mechanism several protective agents were used SH-SY5Y cellspretreatmentwith tiron (TIR) and 2-hydroxybenzoic acid sodium salt (NaSAL) both antioxidants andN

120596-nitro-L-argininemethyl

ester hydrochloride (L-NAME) a nitric oxide synthase inhibitor partially protected against PQ-induced cell injury Additionally1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenyl-propyl)piperazine (GBR 12909) a dopamine transporter inhibitor andcycloheximide (CHX) a protein synthesis inhibitor also partially protected against PQ-induced cell injury In conclusion wedemonstrated that PQMPP+ and ROT exerted differential toxic effects on dopaminergic cells PQ neurotoxicity occurred throughexacerbated oxidative stress with involvement of uptake through the dopamine transporter and protein synthesis

1 Introduction

Parkinsonrsquos disease (PD) is considered the second mostcommon neurodegenerative disorder worldwide affecting05 to 1 of the population aged between 65 and 69 yearsand 1 to 3 of the population over 80 years [1] PDdevelops from a loss of nigrostriatal neuromelanin-con-taining dopaminergic neurons whose cell bodies lay in thesubstantia nigra pars compacta (SNpc) [2] This nigrostri-atal pathway is essential for a normal motor function andmovement control PD is thought to have a multifactorialetiology frequently including genetic and environmentalfactors [2 3] Several neurotoxic chemicals to dopaminergicneurons leading to PD-like symptoms have been used to

study this disease The synthetic compounds 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP) and 1-methyl-4-phenyl-4-propionoxy-piperidine (MPPP) were the first tobe associated with PD symptoms as described by Langstonand Ballard [4] MPTP enters the blood-brain barrier andis metabolized in glial cells by monoamine oxidases to 1-methyl-4-phenyl-23-dihydropyridium (MPDP+) which issubsequently oxidized to MPP+ [5] Next MPP+ enters thedopaminergic cell via dopamine transporter (DAT) [5] accu-mulates inside themitochondria and interferes with complexI of mitochondrial transport chain inhibiting its activity[6] This reduces ATP cellular stores promoting reactiveoxygen species (ROS) formation and consequentially leadingto neuronal death [5] Another substance known to promote

2 Journal of Toxicology

Parkinsonism is the organic pesticide rotenone (ROT) whichis used worldwide as an insecticide and to eliminate nuisancefish populations in lakes and reservoirs [3] It is used tostudy PD given its ability to cross biological membranesnot depending on transporters inhibiting mitochondrialcomplex I [7] which increases the rate of mitochondrial ROSrelease leading to cell apoptosis [3 8] Despite their use asPD models neither ROT nor MPP+ were correlated with thesporadic occurrence of PD [8] These two neurotoxicants arecurrently used to study PD by means of in vivo and in vitroapproaches however none of these substances reproducescompletely all the clinical features observed in PD [8 9]

The lack of adequate cellular andor animal models ofPD has prompted the screening of many putative neurotoxiccompounds Paraquat (PQ) has received recently a wideattention as a possible inducer of PD In fact occupationalexposure especially in farming has been associated withParkinsonism [10] and also it was demonstrated that patientswho died from PQ poisoning had severe brain damage [11]This was corroborated by studies in animals where besidesthe affection of the lung which is the main target organfor PQ toxicity it was also demonstrated to be toxic todopaminergic neurons [12 13] Several mechanisms havebeen postulated for PQ-induced neurotoxicity includingincrease in ROS formation excitotoxicity andmitochondrialcomplex I inhibition [14ndash16] Nonetheless the mechanismsby which PQ promotes neurotoxicity still remain to be fullyelucidated

The present work aimed to study the differences interms of dopaminergic neurotoxic profile among PQ ROTand MPP+ in a cell culture model with a dopaminergicphenotype Also we intended to provide further insightsinto the mechanism of PQ-induced neurotoxicity For thatpurpose human dopaminergic SH-SY5Y-differentiated cellswere used

2 Materials and Methods

21 Materials The reagents for cell culture were obtainedfrom Gibco (Invitrogen Paisley UK) Dulbeccorsquos ModifiedEagleMedium (D-MEM) nonessential amino acids (NEAA)phosphate-buffered saline (PBS) trypsinEDTA and penicil-linstreptomycin 48 Multiwell plates and 35mm plates wereobtained from Corning Costar (Corning NY USA) 25 cm3flasks were obtained from TPP (Trasadingen Switzerland)Fuchs-Rosenthal Counting Chamber was obtained fromCarl-Rhode (Germany) Other reagents namely trypanblue solution (04) dimethyl sulfoxide (DMSO) retinoicacid (RA) 12-O-tetradecanoyl-phorbol-13-acetate (TPA)3-[45-dimethylthiazol]-25-diphenyltetrazolium (MTT)sodium dodecyl sulfate (SDS) enzyme-standard forkinetic lactate dehydrogenase (LDH)-assay 120573-nicotinamideadenine dinucleotide reduced form (120573-NADH) 111015840-dim-ethyl-441015840-bipyridinium dichloride (Paraquat or PQ) roten-one (ROT) 1-methyl-4-phenyl-pyridinium ion (MPP+)N-acetylcysteine (NAC) tiron (TIR) 1-(2-[bis(4-flu-orophenyl)methoxy]ethyl)-4-(3-phenyl-propyl)piperazine(GBR 12909) cycloheximide (CHX) 2-hydroxybenzoic

acid sodium salt (NaSAL) and 119873120596-nitro-L-arginine methyl

ester hydrochloride (L-NAME) were obtained from Sigma-Aldrich (St Louis MO USA)

22 Cell Line and Culture Conditions Human neuroblas-toma SH-SY5Y cells (ATCC Manassas VA USA) wereusedThrough differentiation (see protocol below) SH-SY5Ycells are able to express dopaminergic markers as well astyrosine hydroxylase DAT and higher ability to accumulatedopamine and exhibit extended neurites [17 18] In this waythe cells are no longer immature mitosis rate is reducedand also differentiation agents can mimic factors secreted byastrocytes in a the natural brain environment In other wordsdifferentiation can provide a cellular model more similar todopaminergic neurons [17 18] The cells were grown in D-MEM containing 45mgL D-glucose 2mM L-glutamine110mgL sodium pyruvate phenol red supplemented with10 fetal bovine serum 100 120583gmL penicillin 100120583gmLstreptomycin and 1 NEAA under an atmosphere of 5CO295 air at 37∘C

23 Experimental Protocol Stock cultures of SH-SY5Y cells(passages 20 to 45) were maintained in 25 cm3 flasksand grown until confluence (70ndash80 confluence) Cellswere washed with PBS trypsinized (trypsinEDTA 005002wv) counted by trypan blue exclusion using a Fuchs-Rosenthal counting chamber and subcultured at the densityof 25 000 cellscm2 in 48-well culture plates SH-SY5Y cellswere differentiated in order to enhance the dopaminergicphenotype by the addition of 10120583MRA to the medium for 3days afterwhich themediumwas addedwith 70 nMTPAandkept for another 3 days in accordance with previous works[17 18] Finally the medium was removed and replaced with250120583L fresh medium alone or with the drugs PQ (100 500and 1000120583M) MPP+(100 500 and 1000120583M) and ROT (110 and 100 120583M) Concentrations of the neurotoxicants wereselected in accordancewith previous studies [19ndash24]The cellswere exposed to the drugs for different time periods (24 48and 72 h) ROT was initially prepared in DMSO and thendiluted in medium in order to obtain a final concentration of01 of DMSO in the culture medium which was not toxic tocells To avoid variations among cell cultures that could inter-fere in the toxicity outcome results for the concentration-toxicity curves of the 3 toxins were obtained from several cellcultures resulting from different passages and were seededin different days Cell culture procedures and validation ofthe differentiation protocol of SH-SY5Y cells were previouslypublished by our group [18] After selecting the concentra-tion and time of exposure through screening experimentsthe antioxidants NAC (1mM) [25] and TIR (100120583M and1mM) [26] the specific DAT inhibitor GBR 12909 (1120583M)[23] the protein synthesis inhibitor CHX (18 nM) [27] theradical scavenger NaSAL (100120583M and 1mM) [28] and thenonselective nitric oxide synthase (NOS) inhibitor L-NAME(1 120583M) [29] were added 30minutes before exposure of cells toPQThese concentrations of the pharmacological antagonistswere selected according to the drug selectivity to the target

Journal of Toxicology 3

and also taking into account previous cell culture studiesmentioned above

24 Life-Death Assays Cell cultures were assessed mor-phologically by phase contrast microscopy at 3 differenttime points (24 48 and 72 h) Cell damage was assessedquantitatively by the measurement of LDH release into themedium (as a measure of cell membrane integrity) using akinetic measurement assay Also mitochondrial dysfunctionwas assessed by the measurement of MTT salt metabolismassay

241 Lactate Dehydrogenase Kinetic Assay The quantifica-tion of LDH activity was made by a colorimetric methodwhich is based on the reversible reduction of pyruvate tolactate in the presence of 120573-NADH as described by Capela etal 2006 After exposure to drugs two aliquots (50120583L each)of themediumwere removed to which a previously prepared015mgmL 120573-NADH solution was added at room tempera-ture in a 96-well microplate Finally pyruvate 227mM wasadded to start the reaction NADH oxidation to NAD+ wasmeasured at 340 nm using a colorimetric 96-well plate reader(BioTek Instruments VT USA) The delta increase in LDHrelease into the medium was calculated by subtraction ofthe respective controls which were untreated cells culturedalongside the treated cells LDH values are expressed in unitsper liter (UL) calculated based on a LDH standard solutionactivity of 500UL [29]

242 MTT Assay This colorimetric assay relies on theability of viable but not dead cells to convert a solubleyellow tetrazoliumdyeMTT into an insoluble blue formazanproduct that can be measured at 550 nm Given the removalof 100 120583L of culture medium used for the LDH assay eachwell was added 50120583L fresh medium to obtain 200120583L of cellmediumperwell towhich theMTT solutionwas added (finalconcentration of MTT 500120583gmL) Subsequently cells wereincubated at 365∘C for 3 hours The reaction was stopped byadding an equal volume of 10 SDS in 001M hydrochloricacid solution followed by an overnight incubation at 365∘CFinally formazan was detected at 550 nm using a colorimet-ric 96-well plate reader as previously described [29] Theviability of untreated control cells was set to 100 and theeffects resulting from toxicant exposure were expressed as thepercentage of the respective controls

25 Statistical Analysis Results are presented as mean plusmnSEM from at least 3 different independent experimentsThe means for concentrationtime graphics were comparedusing the two-way ANOVA followed by the Bonferronipost hoc test once a significant 119875 had been obtained Themeans for the tested neuroprotectors were compared usingthe one-way ANOVA followed by Student-Newman-Keulspost hoc test once a significant 119875 had been obtained Detailsof the statistical analyses are described in each figure legendSignificance was accepted when 119875 was less than 005

3 Results

31 Paraquat Was Toxic to SH-SY5Y Cells in a Concentration-and Time-Dependent Manner The exposure of differentiatedSH-SY5Y cells to PQ (100 120583M 500120583M and 1000120583M) for24 h did not result in elevation of LDH release at anyconcentration tested (data not shown) However at 48 h therewas a significant LDH release with 500120583M which was evenmore marked with 1000 120583M exposure As expected LDHrelease into themediumwasmore pronounced at 72 h for 500and 1000120583M Thus the loss of cellular viability occurred ina concentration- and time-dependent manner (Figure 1(a))Mitochondrial dysfunction was assayed by the MTT assayIn contrast to cellular death evaluated by the LDH releasemitochondrial dysfunction was already visible at the 24 htime-point for the 500120583M and 1000 120583M concentrations Forthe 100 120583M concentration PQ decreased significantly MTTreduction at 48 h but especially at 72 h Therefore it couldbe clearly observed that mitochondrial dysfunction alsooccurred in a concentration- and time-dependent manner(Figure 1(b))

32 MPP+-Induced Mitochondrial Dysfunction in SH-SY5YCells Following exposure of SH-SY5Y cells to MPP+(100 120583M 500120583M and 1000 120583M) there was no increase in cellinjury at any time-point or concentration tested as assessedby the LDH release into themedium (data not shown)On theother hand MPP+ proved to induce mitochondrial dysfunc-tion (Figure 2) In fact there was significant mitochondrialdysfunction already after 24 h of exposure to 500120583M and1000 120583M concentrations There was no apparent increasein the toxicity by extending the exposure period to 48 hHowever at 72 hMTTmetabolism decreased significantly forall concentrations tested especially for 1000120583M Only at thispoint the toxicity of 1000120583M was significantly higher than500120583M (Figure 2) Thus only longer periods of exposureresulted in a concentration-dependent effect

33 Rotenone Induced a Biphasic Pattern of Neurotoxicity inSH-SY5Y Cells Exposure of SH-SY5Y cells to ROT (1 120583M10 120583M and 100 120583M) did not result in LDH release into themedium at the 24 h time-point (data not shown) Interest-ingly ROT exhibited a biphasic pattern of cellular lyses atthe 48 h time-point (Figure 3(a)) At this time-point 1120583MROT led to a significant reduction of LDH release into themedium to levels below those of controls Meanwhile the10 120583M concentration elicited an increase in LDH releasewhichwas significantly higher than that observed for 100120583MThus cell injury occurred in a concentration-independentmanner After 72 h exposure there was a concentration-dependent toxicity Curiously values for LDH release at48 h and 72 h for the 10 120583M concentration were very similar(Figure 3(a))

Exposure for 24 h to 10 120583M and 100 120583M ROT did notinduce mitochondrial dysfunction (Figure 3(b)) After 48 hexposure there was an increase of mitochondrial dysfunctionfrom 1120583M to 10 120583M in which values were similar to those ofthe 100120583Mconcentration However after 72 h exposure there


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Figure 1 PQ toxicity occurred in a concentration- and time-dependent manner in differentiated SH-SY5Y cells Cells were incubated with100 500 and 1000120583M of PQ for 24 48 and 72 h (a) Increase in LDH release into the medium in differentiated SH-SY5Y cells The deltaincrease in LDH release in units per liter (UL) into the medium was calculated by subtraction of the respective controls (results were pooledfrom 3 different experiments each experiment having 6 different culture wells per condition) (b)Mitochondrial dysfunction in differentiatedSH-SY5Y cells evaluated by the MTT test The viability of untreated control cells was set to 100 and all treatments were expressed as thepercentage of the respective controls (results were pooled from 3 different experiments each experiment having 6 different culture wells percondition) The means were compared using the two-way ANOVA test followed by the Bonferroni post hoc test (lowast119875 lt 005 lowastlowastlowast119875 lt 0001concentration versus control 119875 lt 0001 concentration versus concentration)

was a concentration-dependent toxicity with the highestlevels of mitochondrial dysfunction attaining values below50 at 100 120583M Comparing the exposure during 24 or 48 hmostly 10 120583M and 100 120583M ROT induced a time-dependentincrease in mitochondrial dysfunction (Figure 3(b))

34 Tiron and Sodium Salicylate Provided Protection againstParaquat Neurotoxicity To carry out neuroprotective exper-iments in human neuroblastoma-differentiated SH-SY5Ycells we selected the 500120583M PQ concentration and the 72 hexposure period since under these conditionsmitochondrialdysfunction reached about 50of controls and cell injurywassignificant The cells were preincubated with NAC (1mM)TIR (100 120583M and 1mM) and NaSAL (100 120583M 1mM and10mM) 30 minutes before exposing cells to PQ Putativeprotective drugs alone were also tested for their respectivetoxicities (Figure 4)

TIR and NaSAL both antioxidants provided partialprotection against 500120583M PQ neurotoxicity as revealedby the LDH assay (Figure 4) The protective action of thereferred compounds was only detected in cell injury revealedby the LDH assay since these compounds did not offer anyprotection against mitochondrial dysfunction as revealed bythe MTT assay (data not shown) For both TIR and NaSALthe protective effect was higher at 100120583M of both protectorscomparatively to the 1mMconcentration (Figure 4) Interest-ingly NaSAL alone at the 1mM concentration showed lowerLDH release than controls seemingly to reduce cell deathoccurring in untreated cells (Figure 4)

NAC an antioxidant and glutathione precursor didnot provide any protection against PQ-induced toxicity asrevealed both by LDH and MTT assays (Figure 4)

35 GBR 12909 Cycloheximide and L-NAME Provide Pro-tection against Paraquat Neurotoxicity GBR 12909 (1120583M)a dopamine transporter blocker CHX (18 nM) a proteinsynthesis inhibitor and L-NAME (1 120583M) a NOS inhibitorprovided significant protection against PQ neurotoxicity asrevealed by the LDH assay (Figure 5) CHX proved to bethe most effective On the other hand none of the testeddrugs proved protection against mitochondrial dysfunctioninduced by PQ (data not shown)

4 Discussion

The key findings of our study conducted in differentiateddopaminergic SH-SY5Y cells were as follows (1) PQ-inducedneurotoxicity was concentration and time dependent pro-moting a delayed type of cell death with an early mito-chondrial dysfunction (2) MPP+ promoted mitochondrialdysfunction especially at higher times of exposure butnot cellular lyses (3) ROT-induced neurotoxicity showed abiphasic pattern promoting a delayed type of cell death atlater time-points of exposure and higher concentrations (4)PQ neurotoxicity could be partially prevented by antioxi-dants DAT inhibitors NOS inhibitors and protein synthesisinhibitors

MPP+ ROT and PQare viewed as experimental toxicantsto study PD mechanisms in vitro [9 30] MPP+ and ROT


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Figure 2 MPP+-induced mitochondrial dysfunction in differenti-ated SH-SY5Y The cells were incubated with 100 500 and 1000 120583Mof MPP+ and the MTT test was performed at 24 48 and 72 htime-points Mitochondrial dysfunction in differentiated SH-SY5Ycells evaluated by the MTT test The viability of untreated controlcells was set to 100 and all treatments were expressed as thepercentage of the respective controls (results were pooled from 3different experiments each experiment having 6 different culturewells per condition) The means were compared using the two-way ANOVA followed by the Bonferroni post hoc test (lowastlowastlowast119875 lt0001 concentration versus control 119875 lt 001 119875 lt 0001concentration versus concentration)

are commonly used instead of PQ because of their well-known mechanisms of toxicity In addition studies usingMPP+ and ROT have already given significant insights intothe molecular mechanisms of dopaminergic neuronal death[24 31] However there is a need for more reliable culturedcell models of PD Several studies using SH-SY5Y cellswith these neurotoxicants have been published without everhaving fully elucidated their differential effects concerning tocell death and optimal concentrations In the present studywe compared the neurotoxicity of PQ with that of ROT andMPP+ using differentiated dopaminergic SH-SY5Y cells Ourdata demonstrated that PQ toxicity involved mitochondrialdysfunction and cell death The increase in LDH release andmitochondrial dysfunction after PQ exposure occurred in aconcentration and time dependent manner and was morepronounced at late times of exposure We verified the exis-tence of two events an earlier damage to the mitochondrialelectronic transport chain that may precede a later mem-brane burst typical of cellular lyses This cellular membranedisruption may be also promoted by external aggressions orother unknown mechanisms promoted by PQ Our resultsare in accordance with our analysis since mitochondrialdysfunction occurred at 24 h and LDH was not measurableat this time point At higher PQ concentrations and times ofexposure once cells are not intact the mitochondrial chain isnot functional thus leading to low MTT metabolism

When compared to MPP+ PQ induced a more pro-nounced mitochondrial dysfunction and cell injury for

the same concentrations and time of exposureMitochondrialdysfunction may corroborate the hypothesis that PQ likeMPP+ inhibits the complex I of the mitochondrial chain[6 23] Our study showed that in the same concentrationrange (100 to 1000 120583M) PQ but not MPP+ induced cell lysesand LDH release This event occurred several hours aftermitochondrial dysfunction since LDH release became onlyevident after 48 h exposure In factMPP+ exposed cellsmighthave lost their metabolic capacity but yet not their membraneintegrity suggesting delayed cell death Not only SH-SY5Y-differentiated cells seem to be more susceptible to PQ butalso PQ is more stable in solution than MPP+ which isphotosensitive and may lose activity when in solution withlonger exposure periodsTherefore PQmay represent severaladvantages over MPP+ to study Parkinsonrsquos mechanisms invitro namely when using cultured cells

Concerning ROT a widely used toxicant to promotedopaminergic toxicity it showed a biphasic pattern of neu-rotoxicity Our results are not in agreement with thosepresented by Molina-Jimenez [19] who observed that ROTcaused a proportional time- and dose-dependent decrease inSH-SY5Y cellular viability This can be explained by a differ-ent cell culture protocol namely their FBS removal and lowercellular density Herein ROT at 1120583M for 48 h reduced celllyses and promoted lower LDH leakage compared to controlcells an effect no longer seen at latter times of exposureOn the other hand the MTT assay proved mitochondrialdysfunction after 48 h exposure to 1120583M This may be con-nected to a late death pattern as ROT is postulated to be ahigh affinity complex I inhibitor [7] and to induce apoptosisvia activation of caspase 3 apoptosis pathway [20] In factROT binds near to the quinone-binding site and blockselectron transport preventing NADH from being convertedto ATP in this way the proton pumping is compromisedwhich culminates in increased superoxide generation andconsequent cell death [32] Cell membrane rupture occursseveral hours after compromising the metabolic activity atlower concentrations though at higher concentrations itmay demonstrate toxicity by other means [8] Other studiesshowed that ROT causes apoptosis at low doses and necrosiswhen applied at high doses thus corroborating our results[33] We demonstrated in SH-SY5Y cells that PQ promoteda more defined pattern of time-concentration-dependentcell death meanwhile ROT induced a biphasic pattern oftoxicity Moreover PQ chloride salt is water soluble whileROT has to be prepared in DMSO which is an hazardoussubstanceTherefore PQmay represent advantages over ROTto study Parkinsonrsquosmechanisms in vitro at least in SH-SY5Y-differentiated cells

Comparing the toxicity of ROT and MPP+ in SH-SY5Y-differentiated cells one can conclude that ROT promoted ahigher grade of mitochondrial dysfunction and cell injurythan MPP+ In fact 10 times lower concentrations of ROTand shorter exposure times were required for achieving thedegree of toxicity seen with MPP+ One possible explanationmay be that ROT does not need a transporter to enterthe cells as it crosses membranes by diffusion [7] Ourresults are in accordance with Richardson and collabora-tors who demonstrated that higher MPP+concentrations


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Figure 3 ROT-induced neurotoxicity showed a biphasic pattern with mitochondrial dysfunction and cell death in differentiated SH-SY5YcellsThe cells were incubated with 1 10 and 100120583Mof ROT and thenMTT and LDH tests were performed at 24 48 and 72 h time points (a)Increase in LDH release in units per liter (UL) into the medium in differentiated SH-SY5Y cells The delta increase in LDH release into themediumwas calculated by subtraction of the respective controls (results were pooled from 3 different experiments each experiment having 6different culture wells per condition) (b) Mitochondrial dysfunction in differentiated SH-SY5Y cells evaluated by the MTT test The viabilityof untreated control cells was set to 100 and all treatments were expressed as the percentage of the respective controls (results were pooledfrom 3 different experiments each experiment having 6 different culture wells per condition) The means were compared using the two-wayANOVA followed by the Bonferroni post hoc test (lowastlowastlowast119875 lt 0001 concentration versus control 119875 lt 001 119875 lt 0001 concentration versusconcentration)

were required to achieve the same degree of complex Iinhibition showing that ROT has a higher complex I affinity[8] In contrast to ROT the concentrations of MPP+ usedin our SH-SY5Y-differentiated cells did not result in celllyses during the 72 h of exposure Other studies using SH-SY5Y have reported cell death using higher concentrations ofMPP+ up to 10mM [22 34 35] Moreover differentiated SH-SY5Y cells have an enhanced resistance towards neurotoxinsOur group recently showed that differentiation of SH-SY5Ycells with RA and TPA led to SH-SY5Y neurons withhigher ability to accumulate dopamine and higher resistancetowards dopamine neurotoxicity [18] Importantly a study byCheung and coauthors demonstrated that differentiation ofSH-SY5Y cells with RA conferred higher resistance towardsMPP+ toxicity [36] Thus using higher concentrations ofMPP+and longer periods of exposure should be necessary toobtain significant cell lyses

Our study demonstrated that several compounds couldpartially prevent or slow PQ-induced toxicity NaSAL isknown to prevent PQ-induced apoptosis in the rat lung [37]In fact one of the proposed mechanisms of PQ neurotoxicityis oxidative stress by producingROS such as superoxide anion(O2

∙minus) hydroxyl radicals (OH∙) and hydrogen peroxide(H2O2) which increase lipid peroxidation increase oxidation

of proteins DNA and iron content and promote a markeddecrease in reduced glutathione (GSH) and GSHglutathionedisulfide ratio [23 38] Oxidative stress is also a typical featureof PD brains [39] Salicylates are important HO∙ scavengersand therefore reduce PQ toxicity [37] On the other hand

salicylates may also form stable complexes with PQ [40]These two proposed mechanisms may be the explanation ofthe neuroprotection obtained by us with NaSAL against PQ-induced injury Interestingly NaSAL did not prevent mito-chondrial dysfunction On the other hand LDH values werereduced possibly because direct cellular membrane damagewas prevented either by complex forming or oxidative radicalreduction Still PQ may promote toxicity by other meansonce neurotoxicity was not fully prevented

Another antioxidant used TIR partially prevented PQ-induced neurotoxicity TIR is a known scavenger of O

2

∙minusacting both at extracellular and intracellular levels O

2

∙minus thatoriginated from PQ redox cycle can be scavenged by TIRtherefore affording protection Also O

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∙minus can be convertedfurther to H

2O2and to HO∙ through Fenton-type reactions

using Fe2+ promoting more oxidative stress [41] HoweverTIR only partially prevented cell lyses and not mitochondrialdysfunction promoted by PQ which may indicate that itsneurotoxicity occurred by additional mechanisms

PQ-induced cell death might occur through the forma-tion of reactive nitrogen species (RNS) which also promoteoxidative stress The NOS inhibitor L-NAME also atten-uated PQ toxicity Nitric oxide produced by NOS formsperoxynitrite anion by reacting with O

2

∙minus produced in theredox-cycle of PQ crosses membranes and can easily oxidizeproteins lipids RNA and DNA [42] Overall the protectiveeffects of the two antioxidants and the NO synthesis inhibitorcorroborate the hypothesis that oxidative stress is a majormechanism of PQ neurotoxicity [23 42 43]


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Figure 4 TIR and NaSAL decreased PQ-induced LDH release intothe medium while NAC afforded no protection Differentiated SH-SY5Y cells were pretreated with TIR (100120583M and 1mM) NaSAL(100120583M 1mM) and NAC (1mM) 30 minutes prior to culturesexposure to PQ for 72 h The delta increase in LDH release inunits per liter (UL) into the medium was calculated by subtractionof the respective controls (results were pooled from 3 differentexperiments each experiment having 6 different culture wells percondition) The means were compared using one-way ANOVAfollowed by Student-Newman-Keuls post hoc test (lowastlowastlowast119875 lt 0001 PQversus control +++119875 lt 0001PQversus PQplus protector $119875 lt 005$$119875 lt 001 PQ plus protector low concentration versus PQ plusprotector high concentration)

Our study revealed that CHX a protein synthesisinhibitor protected against PQ-induced neurotoxicityThere-fore PQ-induced cell death might involve proteins whosesynthesis is prevented by CHX Protein synthesis is involvedin many processes of programmed and nonprogrammed celldeath and therefore PQ-induced dopaminergic death mightinvolve those processes [44]

GBR 12909 conferred partial protection against PQ-induced neurotoxicity revealing that PQ entrance into thedopaminergic neuron might occur through the DAT trans-porter which is corroborated by Yang andTiffany-Castiglioni[23] Since PQ is structurally very similar to MPP+ it may aswell use dopamine transporters [45] though this still remainsa controversial issue as there are contrary opinions like thatof Richardson and collaborators [8] who claim that PQtoxicity is independent of DAT expression or Ramachandiranand coauthors [46] who claim that PQ does not require afunctional dopamine transporter for dopaminergic toxicityIn fact we found that despite DAT blocking PQ still exertsneurotoxicity so it is suggested that it may nevertheless enterthe cell or exert toxic effects on the outside leading to cellularlyses

Other studies have evaluated the effects of the phar-macological agents that we used against PQ neurotoxicityto circumvent MPP+ or ROT neurotoxicity L-NAME didnot present any significant protection to MPP+ toxicity in

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minus20

Co

PQ 5

00 120583

M

GBR

1 120583M

GBR

1 120583M

+PQ

CHX

18 n

M

CHX

18 n

M+

PQ

L-N

AM

E 1 120583

M

L-N

AM

E 1 120583

M+

PQ

lowastlowastlowast

+++

++++

Figure 5 CHX GBR 12909 and L-NAME decreased PQ-inducedLDH release into the medium Differentiated SH-SY5Y cells werepretreated with GBR 12909 (1120583M) CHX (18 nM) and L-NAME(1 120583M) 30 minutes prior to cultures exposure to PQ for 72 h Thedelta increase in LDH release in units per liter (UL) into themedium was calculated by subtraction of the respective controls(results were pooled from 3 different experiments each experimenthaving 6 different culture wells per condition) The means werecompared using one-way ANOVA followed by Student-Newman-Keuls post hoc test (lowastlowastlowast119875 lt 0001 PQ versus control ++119875 lt 001+++119875 lt 0001 PQ versus PQ plus protector)

SH-SY5Y cells [47] whereas GBR 12909 blocked its neu-rotoxic effects [17] Meanwhile CHX reduced proapoptoticproteins production in response to MPP+-induced toxicity[48] and also CHX blocked MPP+-induced cell death inthe dopaminergic cell line MN9D [49] In rats NaSALsignificantly attenuated striatal dopamine depletion causedby intrastriatal MPP+ infusion [50] In what concerns ROTCHX was demonstrated to reduce cell death in culturedcortical neurons [51] In another study GBR 12909 signifi-cantly prevented the ROT-induced decrease of cell viability inPC12 cells [52] In rats NaSAL demonstrated neuroprotectiveefficacy against ROT toxicity [53] Also in oral cancer celllines ROT-induced apoptosis was inhibited by TIR [54]

5 Conclusions

The results obtained in the present study showed that PQpromoted a diverse profile of toxicity when compared toMPP+ and ROT and could present advantages over those twotoxicants for modelling PD in vitro In fact PQ in differenti-ated dopaminergic SH-SY5Y cells promoted a concentration-and time-dependent toxicity promoting a delayed type ofcell death with mitochondrial dysfunction leading to cellularlyses We further studied the mechanisms of PQ-inducedtoxicity and demonstrated that antioxidants NOS inhibitorsDAT inhibitors and protein synthesis inhibitors could beuseful tools in preventing dopaminergic toxicity at least invitro


Abbreviations

CHX CycloheximideDAT Dopamine transporterD-MEM Dulbeccorsquos Modified Eagle MediaDMSO Dimethyl sulfoxideGBR 12909 1-(2-[Bis(4-fluorophenyl)methoxy]ethyl)-4-

(3-phenyl-propyl)piperazineGSH Reduced glutathioneH2O2 Hydrogen peroxide

LDH Lactate dehydrogenaseL-NAME 119873

120596-Nitro-L-arginine methyl ester

hydrochlorideMPP+ 1-Methyl-4-phenyl-pyridinium ionMPPP 1-Methyl-4-phenyl-4-propionoxy-piperidineMPTP 1-Methyl-4-phenyl-1236-

tetrahydropyridineMTT 3-[45-Dimethylthiazol]-25-diphenyltetra-

zoliumNAC 119873-AcetylcysteineNADH Nicotinamide adenine dinucleotide reduced

formNaSAL 2-Hydroxybenzoic acid sodium saltNEAA Nonessential amino acidsNOS Nitric oxide synthaseO2

∙minus Superoxide anionOH∙ Hydroxyl radicalPBS Phosphate-buffered salinePD Parkinsonrsquos diseasePQ ParaquatRA Retinoic acidRNS Reactive nitrogen speciesROS Reactive oxygen speciesROT RotenoneSDS Sodium dodecyl sulfateSNpc Substantia nigra pars compactaTIR TironTPA 12-O-Tetradecanoyl-phorbol-13-acetate

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work received financial support from ldquoFundacao para aCiencia e Tecnologiardquo Portugal (Project PTDCSAU-FCF1029582008) under the frame of ldquoPrograma OperacionalTematico Factores de Competitividade (COMPTE) doQuadro Comunitario de Apoio IIIrdquo and ldquoFundoComunitarioEuropeu (FEDER) (FCOMP-01-0124-FEDER-011079)rdquo J PCapela was the recipient of a Postdoc grant from ldquoFunda-cao para a Ciencia e a Tecnologiardquo Portugal (SFRHBPD307762006)

References

[1] L M de Lau and M M Breteler ldquoEpidemiology of ParkinsonrsquosdiseaserdquoThe Lancet Neurology vol 5 no 6 pp 525ndash535 2006

[2] P Piccini D J Burn R Ceravolo D Maraganore and D JBrooks ldquoThe role of inheritance in sporadic Parkinsonrsquos diseaseevidence from a longitudinal study of dopaminergic function intwinsrdquo Annals of Neurology vol 45 no 5 pp 577ndash582 1999

[3] R Betarbet T B Sherer G MacKenzie M Garcia-Osuna AV Panov and J T Greenamyre ldquoChronic systemic pesticideexposure reproduces features of Parkinsonrsquos diseaserdquo NatureNeuroscience vol 3 no 12 pp 1301ndash1306 2000

[4] J W Langston and P A Ballard Jr ldquoParkinsonrsquos dis-ease in a chemist working with 1-methyl-4-phenyl-1256-tetrahydropyridinerdquoThe New England Journal of Medicine vol309 no 5 article 310 1983

[5] R J Smeyne and V Jackson-Lewis ldquoThe MPTP model ofParkinsonrsquos diseaserdquo Molecular Brain Research vol 134 no 1pp 57ndash66 2005

[6] K Suzuki Y Mizuno and M Yoshida ldquoEffects of 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP)-like compounds onmitochondrial respirationrdquo Advances in Neurology vol 53 pp215ndash218 1990

[7] J T Greenamyre R Betarbet and T B Sherer ldquoThe rotenonemodel of Parkinsonrsquos disease genes environment and mito-chondriardquo Parkinsonism and Related Disorders vol 9 supple-ment 2 pp S59ndashS64 2003

[8] J R Richardson Y Quan T B Sherer J T Greenamyre and GWMiller ldquoParaquat neurotoxicity is distinct from that ofMPTPand rotenonerdquo Toxicological Sciences vol 88 no 1 pp 193ndash2012005

[9] J Bove D Prou C Perier and S Przedborski ldquoToxin-inducedmodels of Parkinsonrsquos diseaserdquo NeuroRx vol 2 no 3 pp 484ndash494 2005

[10] J M Gorell C C Johnson B A Rybicki E L Peterson and RJ Richardson ldquoThe risk of Parkinsonrsquos disease with exposure topesticides farming well water and rural livingrdquoNeurology vol50 no 5 pp 1346ndash1350 1998

[11] J T Hughes ldquoBrain damage due to Paraquat poisoning afatal case with neuropathological examination of the brainrdquoNeuroToxicology vol 9 no 2 pp 243ndash248 1988

[12] M J Kang S J Gil and H C Koh ldquoParaquat inducesalternation of the dopamine catabolic pathways and glutathionelevels in the substantia nigra of micerdquo Toxicology Letters vol188 no 2 pp 148ndash152 2009

[13] A L McCormack MThiruchelvam A B Manning-Bog et alldquoEnvironmental risk factors and Parkinsonrsquos disease selectivedegeneration of nigral dopaminergic neurons caused by theherbicide paraquatrdquo Neurobiology of Disease vol 10 no 2 pp119ndash127 2002

[14] K Shimizu K Matsubara K Ohtaki S Fujimaru O Saito andH Shiono ldquoParaquat induces long-lasting dopamine overflowthrough the excitotoxic pathway in the striatum of freelymoving ratsrdquo Brain Research vol 976 no 2 pp 243ndash252 2003

[15] T Tawara T Fukushima N Hojo et al ldquoEffects of paraquaton mitochondrial electron transport system and catecholaminecontents in rat brainrdquo Archives of Toxicology vol 70 no 9 pp585ndash589 1996

[16] D Bagchi M Bagchi E A Hassoun and S J Stohs ldquoIn vitroand in vivo generation of reactive oxygen species DNA dam-age and lactate dehydrogenase leakage by selected pesticidesrdquoToxicology vol 104 no 1ndash3 pp 129ndash140 1995

[17] S P Presgraves T Ahmed S Borwege and J N Joyce ldquoTer-minally differentiated SH-SY5Y cells provide a model systemfor studying neuroprotective effects of dopamine agonistsrdquoNeurotoxicity Research vol 5 no 8 pp 579ndash598 2003


[18] P S Ferreira T B Nogueira V M Costa et al ldquoNeurotoxicityof ldquoecstasyrdquo and its metabolites in human dopaminergic differ-entiated SH-SY5Y cellsrdquo Toxicology Letters vol 216 no 2-3 pp159ndash170 2013

[19] M FMolina-JimenezM I Sanchez-Reus and J Benedi ldquoEffectof fraxetin and myricetin on rotenone-induced cytotoxicity inSH-SY5Y cells comparison with N-acetylcysteinerdquo EuropeanJournal of Pharmacology vol 472 no 1-2 pp 81ndash87 2003

[20] T Pan P Rawal Y Wu W Xie J Jankovic and WLe ldquoRapamycin protects against rotenone-induced apoptosisthrough autophagy inductionrdquoNeuroscience vol 164 no 2 pp541ndash551 2009

[21] K Y Chau L V P Korlipara J M Cooper and A HV Schapira ldquoProtection against paraquat and A53T alpha-synuclein toxicity by cabergoline is partially mediated bydopamine receptorsrdquo Journal of the Neurological Sciences vol278 no 1-2 pp 44ndash53 2009

[22] J R Mathiasen B A W McKenna M S Saporito et alldquoInhibition of mixed lineage kinase 3 attenuates MPP+-inducedneurotoxicity in SH-SY5Y cellsrdquo Brain Research vol 1003 no1-2 pp 86ndash97 2004

[23] W Yang and E Tiffany-Castiglioni ldquoThe bipyridyl herbicideparaquat produces oxidative stress-mediated toxicity in humanneuroblastoma SH-SY5Y cells relevance to the dopaminer-gic pathogenesisrdquo Journal of Toxicology and EnvironmentalHealthmdashPart A vol 68 no 22 pp 1939ndash1961 2005

[24] H An I S Kim S Koppula et al ldquoProtective effects ofGastrodia elataBlume onMPP+-induced cytotoxicity in humandopaminergic SH-SY5Y cellsrdquo Journal of Ethnopharmacologyvol 130 no 2 pp 290ndash298 2010

[25] J P Capela C Macedo P S Branco et al ldquoNeurotoxicitymechanisms of thioether ecstasy metabolitesrdquo Neurosciencevol 146 no 4 pp 1743ndash1757 2007

[26] J Yamada S Yoshimura H Yamakawa et al ldquoCell permeableROS scavengers Tiron and Tempol rescue PC12 cell deathcaused by pyrogallol or hypoxiareoxygenationrdquo NeuroscienceResearch vol 45 no 1 pp 1ndash8 2003

[27] M A Lopes A Meisel U Dirnagl F D Carvalho and M DL Bastos ldquoDoxorubicin induces biphasic neurotoxicity to ratcortical neuronsrdquo NeuroToxicology vol 29 no 2 pp 286ndash2932008

[28] J T Bau and E U Kurz ldquoSodium salicylate is a novel catalyticinhibitor of human DNA topoisomerase II alphardquo BiochemicalPharmacology vol 81 no 3 pp 345ndash354 2011

[29] J P Capela K Ruscher M Lautenschlager et al ldquoEcstasy-induced cell death in cortical neuronal cultures is serotonin2A-receptor-dependent and potentiated under hyperthermiardquoNeuroscience vol 139 no 3 pp 1069ndash1081 2006

[30] K Nakamura D Tsuchiya M Inden and T Taniguchi ldquoInvitro neurodegeneration model dopaminergic toxin-inducedapoptosis in human SH-SY5Y cellsrdquo International CongressSeries vol 1260 pp 287ndash290 2004

[31] D V G DeAndrade DMadureira deOliveria G Barreto et alldquoEffects of the extract of Anemopaegma mirandum (Catuaba)on Rotenone-induced apoptosis in human neuroblastomas SH-SY5Y cellsrdquo Brain Research vol 1198 pp 188ndash196 2008

[32] A J Lambert and M D Brand ldquoInhibitors of the quinone-binding site allow rapid superoxide production from mito-chondrial NADHubiquinone oxidoreductase (complex I)rdquoTheJournal of Biological Chemistry vol 279 no 38 pp 39414ndash39420 2004

[33] A Hartley J M Stone C Heron J M Cooper and A H VSchapira ldquoComplex I inhibitors induce dose-dependent apop-tosis in PC12 cells relevance to Parkinsonrsquos diseaserdquo Journal ofNeurochemistry vol 63 no 5 pp 1987ndash1990 1994

[34] K E Beck L A De Girolamo M Griffin and E E Billett ldquoTherole of tissue transglutaminase in 1-methyl-4-phenylpyridinium(MPP+)-induced toxicity in differentiated human SH-SY5Yneuroblastoma cellsrdquo Neuroscience Letters vol 405 no 1-2 pp46ndash51 2006

[35] C P Fall and J P Bennett ldquoCharacterization and time courseofMPP+-induced apoptosis in human SH-SY5Yneuroblastomacellsrdquo Journal of Neuroscience Research vol 55 no 5 pp 620ndash628 1999

[36] Y T Cheung W K W Lau M S Yu et al ldquoEffects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitromodel in neurotoxicity researchrdquo NeuroToxicology vol 30 no1 pp 127ndash135 2009

[37] R J Dinis-Oliveira C Sousa F Remiao et al ldquoSodiumsalicylate prevents paraquat-induced apoptosis in the rat lungrdquoFree Radical Biology andMedicine vol 43 no 1 pp 48ndash61 2007

[38] M P Mattson ldquoNeuronal life-and-death signaling apoptosisand neurodegenerative disordersrdquo Antioxidants and Redox Sig-naling vol 8 no 11-12 pp 1997ndash2006 2006

[39] P Jenner ldquoOxidative stress as a cause of Parkinsonrsquos diseaserdquoActa Neurologica Scandinavica Supplement vol 84 no 136 pp6ndash15 1991

[40] R J Dinis-Oliveira P G de Pinho A C S Ferreira et alldquoReactivity of paraquat with sodium salicylate formation ofstable complexesrdquo Toxicology vol 249 no 2-3 pp 130ndash1392008

[41] R Franco S Li H Rodriguez-Rocha M Burns and MI Panayiotidis ldquoMolecular mechanisms of pesticide-inducedneurotoxicity relevance to Parkinsonrsquos diseaserdquo Chemico-Biological Interactions vol 188 no 2 pp 289ndash300 2010

[42] M J La Voie and T G Hastings ldquoPeroxynitrite- and nitrite-induced oxidation of dopamine implications for nitric oxide indopaminergic cell lossrdquo Journal of Neurochemistry vol 73 no6 pp 2546ndash2554 1999

[43] W L Yang and A Y Sun ldquoParaquat-induced free radicalreaction in mouse brain microsomesrdquo Neurochemical Researchvol 23 no 1 pp 47ndash53 1998

[44] C Harms M Lautenschlager A Bergk et al ldquoDifferentialmechanisms of neuroprotection by 17 120573-estradiol in apoptoticversus necrotic neurodegenerationrdquo Journal of Neurosciencevol 21 no 8 pp 2600ndash2609 2001

[45] B K Barlow M J Thiruchelvam L Bennice D A Cory-Slechta N Ballatori and E K Richfield ldquoIncreased synapto-somal dopamine content and brain concentration of paraquatproduced by selective dithiocarbamatesrdquo Journal of Neurochem-istry vol 85 no 4 pp 1075ndash1086 2003

[46] S Ramachandiran J M Hansen D P Jones J R Richardsonand G W Miller ldquoDivergent mechanisms of paraquat MPP+and rotenone toxicity oxidation of thioredoxin and caspase-3 activationrdquo Toxicological Sciences vol 95 no 1 pp 163ndash1712007

[47] H S Lee C W Park and Y S Kim ldquoMPP+ increases thevulnerability to oxidative stress rather than directly mediatingoxidative damage in human neuroblastoma cellsrdquo ExperimentalNeurology vol 165 no 1 pp 164ndash171 2000

[48] J Dennis and J P Bennett ldquoInteractions among nitric oxide andBcl-family proteins after MPP+ exposure of SH-SY5Y neural


cells II exogenous NO replicates MPP+ actionsrdquo Journal ofNeuroscience Research vol 72 no 1 pp 89ndash97 2003

[49] W S Choi L M T Canzoniero S L Sensi et al ldquoCharacteriza-tion of MPP+-induced cell death in a dopaminergic neuronalcell line role of macromolecule synthesis cytosolic calciumcaspase and Bcl-2-related proteinsrdquo Experimental Neurologyvol 159 no 1 pp 274ndash282 1999

[50] K Sairam K S Saravanan R Banerjee and K P Mohanaku-mar ldquoNon-steroidal anti-inflammatory drug sodium salicylatebut not diclofenac or celecoxib protects against 1-methyl-4-phenyl pyridinium-induced dopaminergic neurotoxicity inratsrdquo Brain Research vol 966 no 2 pp 245ndash252 2003

[51] W Pei A K Liou and J Chen ldquoTwo caspase-mediatedapoptotic pathways induced by rotenone toxicity in corticalneuronal cellsrdquo The FASEB Journal vol 17 no 3 pp 520ndash5222003

[52] Y Sai Q Wu W Le F Ye Y Li and Z Dong ldquoRotenone-induced PC12 cell toxicity is caused by oxidative stress resultingfrom altered dopaminemetabolismrdquoToxicology inVitro vol 22no 6 pp 1461ndash1468 2008

[53] P Thakur and B Nehru ldquoAnti-inflammatory properties ratherthan anti-oxidant capability is the major mechanism of neuro-protection by sodium salicylate in a chronic rotenone model ofParkinsonrsquos diseaserdquo Neuroscience vol 231 pp 420ndash431 2013

[54] J Lee M S Huang I C Yang et al ldquoEssential roles of caspasesand their upstream regulators in rotenone-induced apoptosisrdquoBiochemical and Biophysical Research Communications vol 371no 1 pp 33ndash38 2008

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


Parkinsonism is the organic pesticide rotenone (ROT) whichis used worldwide as an insecticide and to eliminate nuisancefish populations in lakes and reservoirs [3] It is used tostudy PD given its ability to cross biological membranesnot depending on transporters inhibiting mitochondrialcomplex I [7] which increases the rate of mitochondrial ROSrelease leading to cell apoptosis [3 8] Despite their use asPD models neither ROT nor MPP+ were correlated with thesporadic occurrence of PD [8] These two neurotoxicants arecurrently used to study PD by means of in vivo and in vitroapproaches however none of these substances reproducescompletely all the clinical features observed in PD [8 9]

The lack of adequate cellular andor animal models ofPD has prompted the screening of many putative neurotoxiccompounds Paraquat (PQ) has received recently a wideattention as a possible inducer of PD In fact occupationalexposure especially in farming has been associated withParkinsonism [10] and also it was demonstrated that patientswho died from PQ poisoning had severe brain damage [11]This was corroborated by studies in animals where besidesthe affection of the lung which is the main target organfor PQ toxicity it was also demonstrated to be toxic todopaminergic neurons [12 13] Several mechanisms havebeen postulated for PQ-induced neurotoxicity includingincrease in ROS formation excitotoxicity andmitochondrialcomplex I inhibition [14ndash16] Nonetheless the mechanismsby which PQ promotes neurotoxicity still remain to be fullyelucidated

The present work aimed to study the differences interms of dopaminergic neurotoxic profile among PQ ROTand MPP+ in a cell culture model with a dopaminergicphenotype Also we intended to provide further insightsinto the mechanism of PQ-induced neurotoxicity For thatpurpose human dopaminergic SH-SY5Y-differentiated cellswere used

2 Materials and Methods

21 Materials The reagents for cell culture were obtainedfrom Gibco (Invitrogen Paisley UK) Dulbeccorsquos ModifiedEagleMedium (D-MEM) nonessential amino acids (NEAA)phosphate-buffered saline (PBS) trypsinEDTA and penicil-linstreptomycin 48 Multiwell plates and 35mm plates wereobtained from Corning Costar (Corning NY USA) 25 cm3flasks were obtained from TPP (Trasadingen Switzerland)Fuchs-Rosenthal Counting Chamber was obtained fromCarl-Rhode (Germany) Other reagents namely trypanblue solution (04) dimethyl sulfoxide (DMSO) retinoicacid (RA) 12-O-tetradecanoyl-phorbol-13-acetate (TPA)3-[45-dimethylthiazol]-25-diphenyltetrazolium (MTT)sodium dodecyl sulfate (SDS) enzyme-standard forkinetic lactate dehydrogenase (LDH)-assay 120573-nicotinamideadenine dinucleotide reduced form (120573-NADH) 111015840-dim-ethyl-441015840-bipyridinium dichloride (Paraquat or PQ) roten-one (ROT) 1-methyl-4-phenyl-pyridinium ion (MPP+)N-acetylcysteine (NAC) tiron (TIR) 1-(2-[bis(4-flu-orophenyl)methoxy]ethyl)-4-(3-phenyl-propyl)piperazine(GBR 12909) cycloheximide (CHX) 2-hydroxybenzoic

acid sodium salt (NaSAL) and 119873120596-nitro-L-arginine methyl

ester hydrochloride (L-NAME) were obtained from Sigma-Aldrich (St Louis MO USA)

22 Cell Line and Culture Conditions Human neuroblas-toma SH-SY5Y cells (ATCC Manassas VA USA) wereusedThrough differentiation (see protocol below) SH-SY5Ycells are able to express dopaminergic markers as well astyrosine hydroxylase DAT and higher ability to accumulatedopamine and exhibit extended neurites [17 18] In this waythe cells are no longer immature mitosis rate is reducedand also differentiation agents can mimic factors secreted byastrocytes in a the natural brain environment In other wordsdifferentiation can provide a cellular model more similar todopaminergic neurons [17 18] The cells were grown in D-MEM containing 45mgL D-glucose 2mM L-glutamine110mgL sodium pyruvate phenol red supplemented with10 fetal bovine serum 100 120583gmL penicillin 100120583gmLstreptomycin and 1 NEAA under an atmosphere of 5CO295 air at 37∘C

23 Experimental Protocol Stock cultures of SH-SY5Y cells(passages 20 to 45) were maintained in 25 cm3 flasksand grown until confluence (70ndash80 confluence) Cellswere washed with PBS trypsinized (trypsinEDTA 005002wv) counted by trypan blue exclusion using a Fuchs-Rosenthal counting chamber and subcultured at the densityof 25 000 cellscm2 in 48-well culture plates SH-SY5Y cellswere differentiated in order to enhance the dopaminergicphenotype by the addition of 10120583MRA to the medium for 3days afterwhich themediumwas addedwith 70 nMTPAandkept for another 3 days in accordance with previous works[17 18] Finally the medium was removed and replaced with250120583L fresh medium alone or with the drugs PQ (100 500and 1000120583M) MPP+(100 500 and 1000120583M) and ROT (110 and 100 120583M) Concentrations of the neurotoxicants wereselected in accordancewith previous studies [19ndash24]The cellswere exposed to the drugs for different time periods (24 48and 72 h) ROT was initially prepared in DMSO and thendiluted in medium in order to obtain a final concentration of01 of DMSO in the culture medium which was not toxic tocells To avoid variations among cell cultures that could inter-fere in the toxicity outcome results for the concentration-toxicity curves of the 3 toxins were obtained from several cellcultures resulting from different passages and were seededin different days Cell culture procedures and validation ofthe differentiation protocol of SH-SY5Y cells were previouslypublished by our group [18] After selecting the concentra-tion and time of exposure through screening experimentsthe antioxidants NAC (1mM) [25] and TIR (100120583M and1mM) [26] the specific DAT inhibitor GBR 12909 (1120583M)[23] the protein synthesis inhibitor CHX (18 nM) [27] theradical scavenger NaSAL (100120583M and 1mM) [28] and thenonselective nitric oxide synthase (NOS) inhibitor L-NAME(1 120583M) [29] were added 30minutes before exposure of cells toPQThese concentrations of the pharmacological antagonistswere selected according to the drug selectivity to the target







3 Results






Paraquat

500 10000

50

100

Delt

a LD

H (U

L)

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


48 h72 h

(a)

0 500 100020

40

60

80

100

120


48 h24 h 72 h


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Paraquat

Perc

enta

ge o

f con

trols

(MTT

test)

(b)







4 Discussion




0 500 100020

40

60

80

100

120

o

f con

trols

(MTT

test)


48 h24 h 72 h

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

MPP+








Rotenone

50 1000

50

100

Delt

a LD

H (U

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

lowastlowastlowast


48 h72 h

(a)

0 50 10020

40

60

80

100

120

o

f con

trols

(MTT

test)


lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

Rotenone

48 h24 h 72 h

lowastlowastlowast

(b)








2


2


2





2



10

30

50

70

minus10

Delt

a LD

H (U

L)

lowastlowastlowast

+++

+++

+++

+++

$$$

Co

PQ 5

00 120583

M

TIR

100 120583

M

TIR

100 120583

M+

PQ

TIR

1 mM

TIR

1 mM+

PQ

NaS

AL

100 120583

M+

PQ

NaS

AL

100 120583

M

NaS

AL

1 mM

NaS

AL

1 mM+

PQ

NAC

1 m

M

NAC

+PQ





0

20

40

60

80

Delt

a LD

H (U

L)

minus20

Co

PQ 5

00 120583

M

GBR

1 120583M

GBR

1 120583M

+PQ

CHX

18 n

M

CHX

18 n

M+

PQ

L-N

AM

E 1 120583

M

L-N

AM

E 1 120583

M+

PQ

lowastlowastlowast

+++

++++



5 Conclusions



Abbreviations












Acknowledgments


References






























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







3 Results






Paraquat

500 10000

50

100

Delt

a LD

H (U

L)

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


48 h72 h

(a)

0 500 100020

40

60

80

100

120


48 h24 h 72 h


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Paraquat

Perc

enta

ge o

f con

trols

(MTT

test)

(b)







4 Discussion




0 500 100020

40

60

80

100

120

o

f con

trols

(MTT

test)


48 h24 h 72 h

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

MPP+








Rotenone

50 1000

50

100

Delt

a LD

H (U

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

lowastlowastlowast


48 h72 h

(a)

0 50 10020

40

60

80

100

120

o

f con

trols

(MTT

test)


lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

Rotenone

48 h24 h 72 h

lowastlowastlowast

(b)








2


2


2





2



10

30

50

70

minus10

Delt

a LD

H (U

L)

lowastlowastlowast

+++

+++

+++

+++

$$$

Co

PQ 5

00 120583

M

TIR

100 120583

M

TIR

100 120583

M+

PQ

TIR

1 mM

TIR

1 mM+

PQ

NaS

AL

100 120583

M+

PQ

NaS

AL

100 120583

M

NaS

AL

1 mM

NaS

AL

1 mM+

PQ

NAC

1 m

M

NAC

+PQ





0

20

40

60

80

Delt

a LD

H (U

L)

minus20

Co

PQ 5

00 120583

M

GBR

1 120583M

GBR

1 120583M

+PQ

CHX

18 n

M

CHX

18 n

M+

PQ

L-N

AM

E 1 120583

M

L-N

AM

E 1 120583

M+

PQ

lowastlowastlowast

+++

++++



5 Conclusions



Abbreviations












Acknowledgments


References






























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Paraquat

500 10000

50

100

Delt

a LD

H (U

L)

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


48 h72 h

(a)

0 500 100020

40

60

80

100

120


48 h24 h 72 h


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Paraquat

Perc

enta

ge o

f con

trols

(MTT

test)

(b)







4 Discussion




0 500 100020

40

60

80

100

120

o

f con

trols

(MTT

test)


48 h24 h 72 h

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

MPP+








Rotenone

50 1000

50

100

Delt

a LD

H (U

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

lowastlowastlowast


48 h72 h

(a)

0 50 10020

40

60

80

100

120

o

f con

trols

(MTT

test)


lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

Rotenone

48 h24 h 72 h

lowastlowastlowast

(b)








2


2


2





2



10

30

50

70

minus10

Delt

a LD

H (U

L)

lowastlowastlowast

+++

+++

+++

+++

$$$

Co

PQ 5

00 120583

M

TIR

100 120583

M

TIR

100 120583

M+

PQ

TIR

1 mM

TIR

1 mM+

PQ

NaS

AL

100 120583

M+

PQ

NaS

AL

100 120583

M

NaS

AL

1 mM

NaS

AL

1 mM+

PQ

NAC

1 m

M

NAC

+PQ





0

20

40

60

80

Delt

a LD

H (U

L)

minus20

Co

PQ 5

00 120583

M

GBR

1 120583M

GBR

1 120583M

+PQ

CHX

18 n

M

CHX

18 n

M+

PQ

L-N

AM

E 1 120583

M

L-N

AM

E 1 120583

M+

PQ

lowastlowastlowast

+++

++++



5 Conclusions



Abbreviations












Acknowledgments


References






























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0 500 100020

40

60

80

100

120

o

f con

trols

(MTT

test)


48 h24 h 72 h

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

MPP+








Rotenone

50 1000

50

100

Delt

a LD

H (U

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

lowastlowastlowast


48 h72 h

(a)

0 50 10020

40

60

80

100

120

o

f con

trols

(MTT

test)


lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

Rotenone

48 h24 h 72 h

lowastlowastlowast

(b)








2


2


2





2



10

30

50

70

minus10

Delt

a LD

H (U

L)

lowastlowastlowast

+++

+++

+++

+++

$$$

Co

PQ 5

00 120583

M

TIR

100 120583

M

TIR

100 120583

M+

PQ

TIR

1 mM

TIR

1 mM+

PQ

NaS

AL

100 120583

M+

PQ

NaS

AL

100 120583

M

NaS

AL

1 mM

NaS

AL

1 mM+

PQ

NAC

1 m

M

NAC

+PQ





0

20

40

60

80

Delt

a LD

H (U

L)

minus20

Co

PQ 5

00 120583

M

GBR

1 120583M

GBR

1 120583M

+PQ

CHX

18 n

M

CHX

18 n

M+

PQ

L-N

AM

E 1 120583

M

L-N

AM

E 1 120583

M+

PQ

lowastlowastlowast

+++

++++



5 Conclusions



Abbreviations












Acknowledgments


References






























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Rotenone

50 1000

50

100

Delt

a LD

H (U

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

lowastlowastlowast


48 h72 h

(a)

0 50 10020

40

60

80

100

120

o

f con

trols

(MTT

test)


lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

Rotenone

48 h24 h 72 h

lowastlowastlowast

(b)








2


2


2





2



10

30

50

70

minus10

Delt

a LD

H (U

L)

lowastlowastlowast

+++

+++

+++

+++

$$$

Co

PQ 5

00 120583

M

TIR

100 120583

M

TIR

100 120583

M+

PQ

TIR

1 mM

TIR

1 mM+

PQ

NaS

AL

100 120583

M+

PQ

NaS

AL

100 120583

M

NaS

AL

1 mM

NaS

AL

1 mM+

PQ

NAC

1 m

M

NAC

+PQ





0

20

40

60

80

Delt

a LD

H (U

L)

minus20

Co

PQ 5

00 120583

M

GBR

1 120583M

GBR

1 120583M

+PQ

CHX

18 n

M

CHX

18 n

M+

PQ

L-N

AM

E 1 120583

M

L-N

AM

E 1 120583

M+

PQ

lowastlowastlowast

+++

++++



5 Conclusions



Abbreviations












Acknowledgments


References






























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


10

30

50

70

minus10

Delt

a LD

H (U

L)

lowastlowastlowast

+++

+++

+++

+++

$$$

Co

PQ 5

00 120583

M

TIR

100 120583

M

TIR

100 120583

M+

PQ

TIR

1 mM

TIR

1 mM+

PQ

NaS

AL

100 120583

M+

PQ

NaS

AL

100 120583

M

NaS

AL

1 mM

NaS

AL

1 mM+

PQ

NAC

1 m

M

NAC

+PQ





0

20

40

60

80

Delt

a LD

H (U

L)

minus20

Co

PQ 5

00 120583

M

GBR

1 120583M

GBR

1 120583M

+PQ

CHX

18 n

M

CHX

18 n

M+

PQ

L-N

AM

E 1 120583

M

L-N

AM

E 1 120583

M+

PQ

lowastlowastlowast

+++

++++



5 Conclusions



Abbreviations












Acknowledgments


References






























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Abbreviations












Acknowledgments


References






























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Differential Effects of Methyl-4 ...

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