Cerebrospinal fluid of newly diagnosed amyotrophic lateral sclerosis patients exhibits abnormal levels of selenium species including elevated selenite.

(1)

Cerebrospinal

ﬂuid

of

newly

diagnosed

amyotrophic

lateral

sclerosis

patients

exhibits

abnormal

levels

of

selenium

species

including

elevated

selenite

Marco

Vinceti

a,

*

,

Nikolay

Solovyev

b,c

,

Jessica

Mandrioli

d

,

Catherine

M. Crespi

e

,

Francesca

Bonvicini

a

_,

_Elisa

_Arcolin

a

_,

_Eleni

_{Georgoulopoulou}

d

_,

_Bernhard

_Michalke

b

a

CREAGEN–Environmental,GeneticandNutritionalEpidemiologyResearchCenter,DepartmentofDiagnostic,ClinicalandPublicHealthMedicine, UniversityofModenaandReggioEmilia,Modena,Italy

b_Research_Unit_Analytical_{BioGeoChemistry,}_Helmholtz_Zentrum_Mu¨nchen,_German_Research_Center_for_{Environmental}_Health_(GmbH),_Munich,_Germany c_Department_of_Analytical_Chemistry,_St._Petersburg_State_University,_St._Petersburg,_Russia

d

DepartmentofNeuroscienceandSt.Agostino-EstenseHospital,UniversityofModenaandReggioEmilia,Modena,Italy

e

DepartmentofBiostatistics,FieldingSchoolofPublicHealth,UniversityofCaliforniaLosAngeles,LosAngeles,CA,UnitedStates

1. Introduction

Selenium(Se),anessentialtracemineral,hasbothnutritional and toxicological properties, with a broad range of biological activitieswhichstronglydependonitschemicalform.Inorganic speciesof Se aregenerally moretoxic and oflower nutritional valuethantheorganic species(Vincetietal.,2009;Valdiglesias etal.,2010;NogueiraandRocha,2011),insomecasesexhibiting

entirelydifferenteffects(Borellaetal.,1996;Hoefigetal.,2011; NogueiraandRocha,2011;Bodnaretal.,2012;Bitencourtetal.,in press), and moregenerally,each Secompound hasspecific and distinctive biological properties. Unfortunately, most human studieshaveinvestigated theoverallSecontentofbody tissues withoutassessingthedifferentchemicalspeciesofthemetalloid. Moreover, thelevelsofSeand ofspecificSe compoundsinthe differentbodytissuesmayconsiderablyvaryandarefrequently uncorrelated,hamperingorevenprecludingassessmentoftheir concentrationsintargetorgansandcompartmentsofrelevancefor specificdiseasesonthebasisofperipheralindicatorsofexposure (suchasbloodortoenailsSe) ordietaryintakeofthemetalloid (Behneetal.,2010;Dennertetal.,2011;Vincetietal.,2012b).This maybeparticularlytruewhenattemptingtoassessinvolvementof Sespeciesintheetiologyofhumanneurologicaldiseases:arecent Sespeciationstudieshasfoundthatwhileperipheral(blood)levels of some organic Se compounds tend toreflect central nervous

ARTICLE INFO Articlehistory: Received1March2013 Accepted23May2013 Availableonline31May2013 Keywords:

Selenium Selenite Selenoprotein-P

Amyotrophiclateralsclerosis Case–controlstudy Cerebrospinalﬂuid Risk Environment Speciationanalysis ABSTRACT

Exposuretoselenium,andparticularlytoitsinorganicforms,hasbeenhypothesizedasariskfactorfor amyotrophiclateralsclerosis(ALS),afastprogressingmotorneurondiseasewithpoorlyunderstood etiology.However,noinformationisknownaboutlevelsofinorganicandsomeorganicseleniumspecies in the central nervous system of ALS patients, and recent observations suggest that peripheral biomarkersofexposureareunabletopredicttheselevelsforseveralSespeciesincludingtheinorganic forms.Usingahospital-referredcase–controlseriesandadvancedseleniumspeciationmethods,we comparedthechemicalspeciesofseleniumincerebrospinalfluidfrom38ALSpatientstothoseof38 referenceneurologicalpatientsmatchedonageandgender.Wefoundthathigherconcentrationsof inorganic selenium in theform of selenite and of human serum albumin-boundselenium were associatedwithincreasedALSrisk(relativerisks3.9(95%confidenceinterval1.2–11.0)and1.7(1.0–2.9) for 0.1mg/Lincrease). Conversely, lower concentrations of selenoprotein P-bound selenium were associatedwithincreasedrisk(relativerisk0.2for1mg/Lincrease,95%confidenceinterval0.04–0.8). Theassociationswerestrongeramongcasesage50yearsorolder,whoarepostulatedtohavelowerrates ofgeneticdiseaseorigin.Theseresultssuggestthatexcessseleniteandhumanserumalbumin bound-seleniumandlowlevelsofselenoproteinP-boundseleniuminthecentralnervoussystem,whichmaybe related,mayplayaroleinALSetiology.

ß2013TheAuthors.PublishedbyElsevierInc.

*Corresponding author at: CREAGEN – Dipartimento di Medicina Clinica, DiagnosticaediSanita` Pubblica,Universita` diModenaeReggioEmilia,ViaCampi 287,41125Modena,Italy.Tel.:+390592055481;fax:+390592055483.

E-mailaddress:marco.vinceti@unimore.it(M.Vinceti).

ContentslistsavailableatSciVerseScienceDirect

NeuroToxicology

0161-813Xß2013TheAuthors.PublishedbyElsevierInc.

http://dx.doi.org/10.1016/j.neuro.2013.05.016

Open access under CC BY-NC-ND license.

(2)

system(CNS)levels,nosuchcorrelationexistsforotherorganic species such as selenoprotein P, overall Se content and the inorganic forms selenate and selenite (Solovyev et al., 2013), conﬁrmingprevious observationson overallSe(Michalke etal., 2009).Otherobservationsappeartoconﬁrmtheindependenceof brainselenoproteinPcomparedwithitsplasmacirculatinglevels (Scharpfetal.,2007).

Amyotrophic lateral sclerosis (ALS), a severe degenerative motorneurondiseaseoflargelyunknownetiologyapartfromthe fewcasesofgeneticorigin,isamongthediseaseswhoseetiologyis hypothesizedtoberelatedtooverexposuretoselenium(Vinceti et al., 2012a). This hypothesis was ﬁrst proposed based on observationofaclusterofALScasesinaseleniferousareaofSouth Dakota,andanexcessincidenceofALSinanItalian population consuming drinking water with high concentrations of the inorganicSespeciesselenate(KilnessandHochberg,1977;Vinceti et al., 1996, 2010b). Consistent with the hypothesis is the demonstrationofSe-inducedneurotoxicityinlaboratorystudies (Ammar and Couri,1981; Nogueira et al.,2003; Maraldiet al., 2011; Estevez et al., 2012)and its selective toxicityon motor neuronsinspeciessuchasswine(Casteignauetal.,2006;Raber etal.,2010;Vincetietal.,2012a),generallyshowingamuchhigher orevenanexclusivetoxicityofinorganicspeciescomparedwith the organic ones. Moreover, recent epidemiologic observations suggestdeleteriouseffectofSeonneurologicalend-pointssuchas visualevokedpotentials(Saint-Amouretal.,2006)and,evenatlow dosesofexposure,neonatalneurologicalandbehavioral develop-ment(Yangetal.,2013).

Weconductedacase–controlstudytoexaminethehypothesis thatSespeciesandparticularlytheinorganiconesareassociated withALSriskbyusingaCNSbiomarkerofexposure,cerebrospinal ﬂuid(CSF),whichappearstoplayakeyroleinassessingexposure to etiopathogenetic and therapeutic factors in this disease (Tarasiuk et al., 2012; von Neuhoff et al., 2012; Wilson et al., 2013;Wineretal.,2013).

2. Methods

2.1. Studyparticipants

ALSpatientswererecruitedfromacaseseriesofresidentsofthe Emilia-Romagnaregion,northernItaly,whowerediagnosedwith clinicallydeﬁniteorclinicallyprobableALSusingtherevisedEl EscorialCriteria(Georgoulopoulouetal.,2011)attheALSCenterof theModenaUniversityNeurologicalDepartmentfromMay1998 to April 2011, and who underwent lumbar puncture during diagnostic procedures. This group comprised 72 consecutive patients withsporadic ALS,some of whom wereenrolled in a previousstudy(Mandriolietal.,2006).Thirty-eightpatientshadat least1mLCSFavailablewhenthepresentstudywasdesignedand composed the enrolled cases. We recently performed genetic screeningincludingallthemajorgenesthatareimplicatedinALS (SOD1,C9ORF72,FUS,TDP43)in12ofthesepatients,generallythe youngerones,withnonetestingpositive.Wewerenotallowedto performgeneticanalysisintheremainingpatientsbecauseoflack ofinformedconsentand/oravailablebloodsamples.

The control population consisted of patients residing in the Emilia-Romagnaregionwhowereadmittedtothesame depart-mentbetween1999and2010,inclusive,andunderwentlumbar puncturebecauseofsuspectedbutlaterunconﬁrmedneurological disease,andhadasampleofatleast1mLofCSFstillavailablein September2011.Amongtheseindividuals,werandomlyselected 38subjectsmatched1:1toALScasesonage(10years,inmost cases 5years) and gender. Signs or symptoms that led to neurological examination and lumbar puncture were: headache (n=17),paresthesias(n=5),diplopia(n=6),vertigo(n=3)andother

(n=7). All control patients were subsequently discharged from hospitalwithoutadiagnosisofamajordisease;ﬁnaldiagnosiswas primary headache in 13 individuals, and other diagnoses with negativeinstrumentaltestsintheremaining25individuals.

Informedconsentfordiagnosticlumbarpuncturewasobtained from all patients, and utilization of theCSF specimens for the presentstudywasapprovedbytheModenaEthicalCommittee. 2.2. Samplecollection

Approximately6mLofCSFwerecollectedbylumbarpuncture fromeachpatientandimmediatelystoredat 808Cin polypro-pylenetubes.A1mLaliquotwastransportedbyaircourierdeep frozenindryicetotheMunichlaboratory,andkeptcontinuously frozenuntiluse.Foranalysis,sampleswereslowlythawedina refrigeratorat48C,vortexedandsubsequentlyanalyzed. 2.3. Chemicals

Suprapure grade chemicals were used throughout. Selenite, selenate, selenomethionine (Se-Met), selenocystine (Se-Cys), thioredoxin reductase (EC 1.8.1.9.)-bound selenium (Se-TrxR), glutathioneperoxidase(EC232-749-6)-boundselenium(Se-GPx), humanserumalbumin(HSA)andTrisbufferwereorderedfrom Sigma–Aldrich,Deisenhofen,Germany.CertiﬁedSeandRhstock standards (1000mg/L) were purchased from CPI International, SantaRosa,CA,USA.Ammoniumacetate(NH4Ac)andaceticacid

(HAc)wereobtainedfromMerck,Darmstadt,Germany.Arliqand

methane (99.999% purity) were purchased from Air Liquide, Gro¨benzell,Germany.Seleniteandselenatestocksolutionswere preparedataconcentrationof1000mgSe/Lbydissolvingin Milli-Q water (18.2M

V

cm, Milli-Q system, Millipore, Bedford, MA, USA). HSA was prepared at a concentration of 1000mg/L. Preparation of Se-HSA was performed by mixing 10mg Se/L selenitewiththisstocksolutionandincubatingforatleast14days. WorkingstandardsofSespecieswereprepareddailyfromtheir stockstandardsolutionsbyappropriatedilutionwithMilli-QH2O.

SelenoproteinP-boundSe(SePP)isnotcommerciallyavailableasa standard compound, but it can be prepared fromserum using afﬁnitychromatography(AFC):TheAFC-SePPfractionwaspuriﬁed byamass-calibratedsizeexclusionchromatography(SEC)column, wheretheSePPfractionelutedataRTcalculatedfor61.8kDa. 2.4. Seleniumspeciation

WedeterminedtotalSeandtheSespeciesselenite,selenate, Se-Met,Se-Cys,Se-TrxR,Se-GPx,SePPandSe-HSAintheCSFsamples usinghighpressureliquidchromatography(HPLC)coupledwith inductivelycoupledplasmadynamicreactioncellmass spectrom-etry(ICP-DRC-MS)accordingtomethodologiespreviously estab-lishedfor biologicalmatrices,speciﬁcallyforCSF(Michalkeand Berthele,2011;Solovyevetal.,2013).Sespeciationwasconducted by strong anion exchange (SAX)-ICP-DRC-MS (Michalke and Berthele, 2011). The SAX separation followed Xu et al. (2008)

butwasslightlymodiﬁedforbaselineseparationofcloseeluting peaks.

AKnauer1100SmartlineinertSeriesgradientHPLCsystemwas connected to an anion exchange column ProPac SAX-10 (2502mm I.D.) from Thermo (Dionex Idstein, Germany) for species separation.Thesample volumewas100

m

l.Themobile phaseswere:eluent A:10mM Tris–HAc,pH8.0;andeluent B: A+500mMNH4Ac,pH8.0.Gradientelutionexpressedas%-eluent

A:0–3min 100%;3–10min 100–60%; 10–23min 60–45%; 23– 26min 45–43%; 26–28min 43–0%; 28–52min 0%; 52–60min 100%.Theﬂowratewas0.20mLmin 1_._For_internal

(3)

concentration, total ﬂow rate: 0.25mLmin 1₎ _and _directed _to

ICP–MS.

Total Se was analyzed by ﬂow injection (FI)-ICP-DRC-MS (Michalke et al., 2009). A Knauer 1100 Smartline inert Series HPLCsystemequippedwithvacuumdegasserandanelectronic valvewitha25

m

linjectionloop(PerkinElmer,Rodgau-Ju¨gesheim, Germany)wasdirectlycoupledtoICP-DRC-MS.Theﬂowratewas 1mLmin 1_of_50%_eluents_A_and_B,_each,_from_SAX_separation.

CSF-sampleswerediluted1:1.1byadding1

m

g/LRh(ﬁnalconc.)as internalstandard.

The experimental settings chosen for ICP-DRC-MS (Perkin ElmerNexIon)afteroptimizationwere:radiofrequency power: 1250W,plasmagasflow:15LAr/minauxiliary gasflow:1.05L Ar/min,nebulizergasflow:0.98LAr/min,dailyoptimized,dwell time300ms,ionsmonitored:78Se,80Se,103Rh,DRCreactiongas: CH4reactionat0.58mLmin 1,DRCrejectionparameterq:0.6.

PeakquantificationfromFI-quantificationandfrom chromato-grams was done by comparing peak areas with peak area calibrationcurvesfromFI-ICP-DRC-MS.Standardadditionmethod wasusedforstandard-retention-timematchedidentificationofSe speciesandasQCmeansinquantification.Speciesidentitywas furtherconfirmedusinga2DapproachofSAX-capillary electro-phoresis(CE)-ICP-DRC-MS:SAXfractionswereanalyzedwith CE-ICP-DRC-MS.Inbothseriallyappliedseparationtechniques(SAX, CE)theobservedpeaksmatchedretention-ormigrationtimesof respective standard compounds, in accordance to a parallel investigation(Solovyev et al., 2013). Species identification was regarded as acceptable when species matched the standard compounds with both chromatography/electrophoretic techni-ques (match in first and second technique). Details of these procedureshavebeenpublishedelsewhere(Solovyevetal.,2013). RhandSedatafileswereexportedfromtheNexIonsoftwareand processedwithPeakfitTM_software_for_peak_area_integration._For

eachsample(orstandard)aquotientofSe-peakareatoRh-peak area was calculated and taken as the result corrected for the internalstandard(Rh).

Inthese analyses,in additiontowell-known Seorganic and inorganicspecies,wedetected13peaksattributabletoextremely smallamountsofSeofunknownform;thesewereaddedtoknown inorganicandorganicspeciestocomputetotalSe.Nodetectable traceofSe-Cyswasfoundinanysample.Limitofdetectionforall SespeciesinCSFwas0.01

m

g/L,lowerthanthatweobtainedina recentpilotstudyin neurologicallyhealthy Germanindividuals (Solovyevetal.,2013).

2.5. Qualitycontrol

FortotalSedetermination,qualitycontrolwasperformedby analyzingthecontrolmaterials‘‘humanserum’’and‘‘urine’’from RECIPE,Munich,Germany.Controlmaterialswerereconstituted as indicated on ﬂask labels and the resulting solutions were diluted1/50 (serum)or1/10(urine)withMilli-Q waterbefore measurements.Seconcentrationswerefoundtobe623

m

g/L (serum,n=3) and243

m

g/L(urine,n=3),comparedto manu-facturer’starget meanvalues of62and23 (range: 16–30)

m

g/L, respectively.

Inaprecedinginter-laboratorycomparisonourSePP quanti-ﬁcationwas compared tothe immunoassaymethod for SePP determinationbyHollenbachetal.(2008).Therethereliability of our SePP determination was veriﬁed: The immunologically determinedSePPconcentrationinapooledCSFsampleprepared for the inter-laboratory comparison was 0.54nM/L (9.5%) SePP (expressed as total protein). According to literature data regarding SePP (10 Se atoms per SePP and molecular weight MW(SePP)=61kDa (Ballihaut et al., 2012)) this SePP-protein concentration refers to 0.430.04

m

g Se/L at SePP. This value

correspondedwelltotheSAX-ICP-DRC-MSvaluefromourmethod at0.410.01

m

gSe/LatSePPpeak:Thusourmethodwasveriﬁed byanindependentreferencemethod.

Besides,thegoodconcordanceofourSespeciesconcentrations in CSF and serum from this and previous papers with some availableSe-speciesconcentrationsinliteratureunderlinesfurther thatSespeciesdeterminationswithourmethodareatleastoverall OK.Thisisthemoreimportantaswiththesamemethodcasesand controlsweremeasuredandthedifferencesbetweenbotharethe relevantissueinthispaper.

ForrecoverydeterminationduringSAX-ICP-DRC-MSsingleSe species (10

m

gSe/L)wereanalyzedand peak Seconcentrations werequantifiedandrelatedtotheinjectedSeamounts(=100%). Analogously, CSF sampleswere quantified for total Se (=100%) beforeinjectionandcomparedtothesumofelutedandquantified chromatographic peaks.Recoveries were959% forCSF, 106 10%forGPx,978%forselenite,1028%forTrxR,898%for Se-HSA,and8413%forSePP,thelatterlowerrecoverybeingexplained bystabilityproblemsaccompaniedbyanincreasingselenatesignal, asexpected(MichalkeandBerthele,2011).

2.6. Statisticalanalysis

Analyses were conducted for each Se species and for total inorganicSe(sumofseleniteandselenate),totalorganicSe(sumof SePP,Se-GPx,Se-TrxR,Se-MetandSe-HSA),andtotalSe(sumof inorganic,organicandunknownforms).InclusionofSe-HSAinthe organiccategorywasbasedonthepresumptionthatnearlyallof thisSewasorganic,mostlikelyintheformofSe-Met(Xuetal., 2008), thoughtraceamounts ofinorganic Sein this compound cannotbeentirelyexcluded(Haratakeetal.,2008).

WetesteddifferencesindistributionofSespeciesincasesand controlsusingtheWilcoxonsigned-ranktest.We estimatedthe relativerisk(RR)ofALS,asexpressedbytheoddsratio,associated withaone-unitincreaseinsingleSespeciesorcategoriesusing conditionallogisticregressionmodels.SomeSespecies distribu-tionshadoutliersathighvalues,whichwasaconcernastheycould actashighleveragepointsandbeundulyinfluentialinregression models, especially given the small sample size. We used two approaches to address such outliers: (1) diagnostics were conducted to identify highly influential observations, assessed by delta beta statistics (Hosmer and Lemeshow, 2000) and observationswithhighinfluencestatistics(deltabeta>0.1)were omittedandthemodelwasrefitand(2)themodelswerefitusing Sespeciesconcentrationvaluesthatwerewinsorizedbysetting dataexceedingthe95thpercentiletothe95thpercentile.Bothsets ofresultsarepresented.Analyseswererepeatedinthesubgroupof matchedpairsinwhichthecasewasdiagnosedatage50orolder. Sensitivityanalyseswereconductedbyselectivelyomittingfrom analysescontrolpatientswithspecificsymptomsandsignsleading toneurologicalexamination(specifically,the17subjectssuffering fromheadache,the5withparesthesiasandthe6withdiplopia). 3. Results

The38ALScasesincluded16menand22women,withmean ageof55.5years(range30.7–76.4years),whounderwentlumbar punctureduringtheirdiagnosticprocessforneurologicalsignsand symptomsofuncertainorigin.Sevenofthesepatientswerelater diagnosedashavingALSwithbulbaronsetand31ashavingspinal onsetofthedisease,inallcasesataveryearlyclinicalstage.The38 age-andgender-matchedcontrolshadmeanage52.6years(range 30.2–85.5years).

Table 1 summarizes the distribution of the inorganic and organicSespecieswedetermined.Oftheinorganicforms,selenite –butnotselenate–levelswereshiftedtowardhighervaluesin

(4)

cases,asshownbymedianvalues.LevelsoftotalorganicSewere loweramongcasesthanamongcontrols,andthiswasparticularly trueforSePP.Overall,levelsoforganicSecompoundswerehigher thanlevelsofinorganiccompounds;asaresult,totalSewaslower amongcasesthanamongcontrols. Sinceonlyfoursubjectshad detectableSe-Metlevels,thisspecieswasnotanalyzedseparately

insubsequentanalyses.Medianvaluesincontrolswereroughly comparabletoCSFSespecieslevelsrecentlydetectedinaseriesof 24 patients with unspeciﬁc neurological complaints from the Department of Neurology of the Technical University Munich (Solovyev et al., 2013), which reported median levels of total Se=0.861, SePP=0.474, Se-GPx=0.036, Se-TrxR=0.035, Se-HSA=0.068,selenite=0.046,andselenatelowerthanthe detec-tionlimit(allconcentrationsexpressedas

m

g/L).

Inconditionallogisticregressionmodelsomittinggrossoutliers from analysis, the relative risk (RR) of ALS increased with increasing selenite content of CSF but not selenate content (Table2).TheRRincreasedwithincreasingSe-HSAconcentration and decreased with increasing SePP concentration. The RR similarly decreased with increasing total organic Se, both unadjusted and adjusted for inorganic Se, and with increasing totalSe.Inanalysesrestrictedtocase–controlpairsinwhichthe casewasdiagnosedatage50orolder,associationsweregenerally stronger.Allpatientsomittedasoutliershaddeltabetastatistics (indicatingthechangeinthebetacoefﬁcientwhentheobservation isomitted)thatweregrosslylarge comparedtothoseforother observations(forexample,deltabetasof0.13and0.45forselenite, whereas the other 74 observations had values between 0 and 0.036), and were abovethe 95thpercentile of their Se species distributions.Comparingincludedsubjectstopatientsexcludedas outliers,mediantimefromonsettolumbarpuncturewas15vs.13 months,respectively,andmediansurvivalafterdiagnosiswas44 vs.45months,respectively.

Results were similar when conducting the analyses using winsorized distributions of the Se species (Table 3), but with estimatesgenerallyclosertothenullvalue,withtheexceptionof analysesfortotalinorganicSeamongtheolderageatdiagnosis subsample,whichshowedstrongerassociationsinthewinsorized analysis.

Insensitivityanalysesselectivelyomittingsubgroupsofcontrol subjects with speciﬁc neurological symptoms and signs, no substantiallydifferentresultsemerged.

4. Discussion

OverallSecontentinbodytissuessuchasblood,nails,brainand spinal cord of ALS patients has previously been investigated (Mitchelletal., 1991; Moriwakaet al., 1993; Inceet al.,1994; Bergomietal.,2002),butthegrowingawarenessofthedifferent toxicological and physiological roles of the various Se species

Table1

DistributionofSespecies(mg/L)innewlydiagnosedamyotrophiclateralsclerosis cases(n=38)andcontrols(n=38).

Sespecies Percentile 5th 50th 95th Selenite Controls 0 0.026 0.180 Cases 0 0.051 0.169 Selenate Controls 0 0 0.089 Cases 0 0 0.089 Selenomethioninea Controls 0 0 0.305 Cases 0 0 0 Thioredoxinreductase-boundSe Controls 0 0.050 0.389 Cases 0 0.043 0.214 Glutathioneperoxidase-boundSe Controls 0 0.021 0.164 Cases 0 0.026 0.120

Humanserumalbumin-boundSe

Controls 0 0.087 0.258 Cases 0 0.127 0.359 SelenoproteinP-boundSe Controls 0.036 0.856 8.121 Cases 0.093 0.577 2.196 TotalinorganicSe Controls 0 0.034 0.180 Cases 0 0.059 0.277 TotalorganicSe Controls 0.053 1.084 8.284 Cases 0.287 0.765 2.508 TotalSeb Controls 0.140 1.100 8.380 Cases 0.320 0.765 2.660 a

Onlyfournonzerovaluesofthiscompounddetected.

b

SumoftotalinorganicandtotalorganicSeandunknownforms.

Table2

Relativerisks(RR)ofamyotrophiclateralsclerosisassociatedwith1mg/La_increase_in_{cerebrospinal}_ﬂuid_{concentration}_of_Se_species:_analysis_removing_observations_with

extremelyhighinﬂuencestatistics.b

Sespecies Allsubjects(38cases,38controls) Casesdiagnosed50years(28cases,28controls) RR(95%CI) P-value #Cases/#controls RR(95%CI) P-value #Cases/#controls Selenite 3.9(1.2–11.0) .02 36/36 8.2(1.5–46.2) .02 26/26

Selenate 0.9(0.2–4.2) .93 38/38 3.8(0.4–39.6) .26 27/27 Thioredoxinreductase-boundSe 1.0(0.9–1.1) .13 37/37 1.1(1.0–1.2) .12 26/26 Glutathioneperoxidase-boundSe 1.0(0.9–1.1) .47 38/38 1.0(0.9–1.2) .91 28/28 Humanserumalbumin-boundSe 1.7(1.0–2.9) .05 37/37 2.2(1.0–4.7) .04 27/27 SelenoproteinP-boundSe 0.2(0.04–0.8) .03 37/37 0.1(0.01–0.8) .03 27/27 TotalinorganicSe

Crude 1.7(0.8–3.5) .18 36/36 2.9(1.0–8.4) .06 26/26 AdjustedfororganicSe 1.9(0.8–4.8) .17 36/36 4.1(0.8–21.3) .09 26/26 TotalorganicSe

Crude 0.3(0.1–1.0) .04 36/36 0.2(0.03–0.95) .04 26/26 AdjustedforinorganicSe 0.3(0.1–0.9) .04 36/36 0.1(0.02–0.96) .05 26/26 TotalSe 0.3(0.1–0.9) .03 37/37 0.3(0.1–1.1) .08 27/27

a

Unitincreaseis0.1mg/Lforselenate,selenite,totalinorganicSeandhumanserumalbuminbound-Seand1mg/Lforallothercompounds.

b

Resultsobtainedfromconditionallogisticregressionmodels.ToreducetheinﬂuenceofextremeoutliersinthedistributionsofsomeSespecies,observationswith unusuallyhighinﬂuencestatistics(deltabeta>0.1)wereomitted,resultinginsmallersamplesizesforsomeanalyses.

(5)

makessuchan approachinadequate.Unlikeprevious investiga-tions,weassessedwhetherspecificSecompoundsandparticularly theinorganicspeciesrepresentedariskfactorforALS,promptedby suggestivefindingsof epidemiologicstudies(Kilnessand Hoch-berg,1977;Vincetietal.,1996,2010b)andanimalandlaboratory investigations(Vincetietal.,2009;Maraldietal.,2011;Estevez etal.,2012).Tothebestofourknowledge,thepresentstudyisthe firstonSespeciationinALSandmoregenerallyinhumandiseases, amongthelargenumberofepidemiologicstudiesonhealtheffects ofSe(Vincetietal.,2009;Dennertetal.,2011).Ourresultsindicate theimportanceofSespeciation,despitetheanalyticalcomplexity andcost(Borellaetal.,1996;Vincetietal.,2009;Valdiglesiasetal., 2010;NogueiraandRocha,2011);RRestimatesfortotalSewere opposite tothose for seleniteor totalinorganic Se, confirming recentresultsontotalCSFSefromNorwegianALSpatients(Roos etal.,2013),andthusananalysisbasedontotalSewouldhaveled to opposite conclusions. This indicates that findings may be seriouslymisleadingifassessmentisbasedonoverallSecontentof bodytissuesandnotspecificSespecies,highlightingtheneedto focus on Se homeostasis, speciation and metabolism when investigationtherelationbetweenthismetalloidandCNSdiseases (Vincetietal.,2012a;SteinbrennerandSies,inpress).

UseofCSF,anervoussystemindicatorofgrowinginterestin ALS(Sussmuthetal.,2008;Gladmanetal.,2012;Mendoncaetal., 2012;Tarasiuketal.,2012;vonNeuhoffetal.,2012;Wilsonetal., 2013;Wineretal.,2013),asabiomarkerofexposureratherthan peripheral indicatorssuch asblood or toenails is anadditional strengthofthepresentstudy.Inourrecentstudyontherelation amongCSFandotherindicatorsinneurologicallyhealthysubjects (Solovyev et al., 2013), positive associations between CSF and blood levels werefound only for Se-GPx, Se-TrxRand Se-HSA, whiletherewaslittle orno correlationbetween CSFand blood levels for other organic forms such as SePP, for selenite and selenate (correlation coefficients 0.03 and 0.23, respectively, unpublisheddatabyN.S.andB.M.),andtotalSe.Similarly,SePPin thebrainhasbeenfoundtobeindependentofplasmacirculating levels(Scharpfetal.,2007).Thesefindingsindicatetheinadequacy ofstudiesofinorganicSenot basedon biomarkersofexposure directlyassociatedwithtargettissues.However,CSFisveryrarely used as a biomarker in epidemiologic studies, since it can be collected only following specificmedical indications, making it infeasibletousenotonlyin population-basedbutalsoin most hospital-basedinvestigations.

PreviousstudiesonCNSSecontentinALSpatients,whichhave generallyshownhigheroverallSelevelsinbrainandspinalcord

compared withcontrols (Mitchellet al.,1991; Moriwakaet al., 1993;Inceetal.,1994;Markesberyetal.,1995)andhighlyvariable GPxactivity(Przedborskietal.,1996),wereconductedindeceased subjects, and may therefore have suffered from the potential inadequacy of post-mortem tissues in reﬂecting antecedent Se contentandenzymeactivities.Thesestudieswerealsolimitedby smallsamplesizeandpotentialselectionbias.

OurresultsindicatemajorabnormalitiesofSecompoundsin ALS,andsupportthehypothesisthatinorganicSeandspecifically selenite may play a key role in theneurodegenerative process characterizingALS,inlinewithepidemiologicevidence implicat-ing overexposure to environmental Se (Kilness and Hochberg, 1977)andtodrinkingwaterwithhighcontentofinorganicSeas selenate (Vinceti et al., 1996, 2010b). Evidence supporting biological plausibility of a relation between Se species, and seleniteinparticular,andALShasbeenprovidedfromobservations inswineindicatingthatafeworganicandparticularlyinorganicSe speciesselectivelykillmotorneurons(Raberetal.,2010;Vinceti et al., 2012a), a nematode model in which selenitespecifically affectedmotorfunction(Estevezetal.,2012)andrecentinvitro observations(Maraldietal.,2011).InorganicSeformsandselenite in particular are well recognized in laboratory studies to be considerablymoretoxicthanorganicspecies(Benkoetal.,2012; Peyroche et al., 2012; Vinceti et al., 2013), particularly for neurotoxic effects (Ammar and Couri, 1981; Tsunoda et al., 2000), even though they are retained in humans (Thomson etal.,1993)andanimals(Vinceti etal.,2009)ataconsiderably lowerratethanorganiccompounds.Selenite,andotherSespecies (Zhengetal.,2012),caninduceoxidativestressandmitochondrial damage(BelyaevaandSaris,2011;Maetal.,2011),andbothfree radical damage and mitochondrial abnormalities have been implicated inALS etiopathogenesis(Rossi etal., 2012). Selenite in vitro has also been shown to induce other biochemical alterations suggested to occur in ALS, such as copper/zinc superoxide-dismutase (SOD1) translocation into mitochondria andincreasedlevelsofreactiveoxygenspeciesandofinducible nitricoxidesynthase,withenhancedsusceptibilitytoSetoxicity bynervouscells comparedwithothercelltypes(Maraldietal., 2011). Low-dose Seexposure in humans mayalso induceDNA damage(Karunasingheetal.,2012)andP38-P53activation(Chen et al., 2010) as confirmed by laboratory studies using selenite (Rudolf et al., 2008), and these mechanisms are among those suggested as involved in ALS etiopathogenesis (Kim and Choi, 2010;Coppede,2011;Hanadaetal.,2013).Finally,severalstudies have suggestedaninhibitoryeffectofseleniteoninflammatory

Table3

Relativerisks(RR)ofamyotrophiclateralsclerosisassociatedwith1mg/La

increaseincerebrospinalﬂuidconcentrationofSespecies:analysisusingwinsorizedvariables.b

Sespecies Allsubjects(38cases,38controls) Casesdiagnosed50years(28cases,28controls) RR(95%CI) P-value #Cases/#controls RR(95%CI) P-value #Cases/#controls Selenite 1.9(0.8–4.6) .13 38/38 3.9(1.1–13.0) .03 28/28

Selenate 0.9(0.2–4.4) .88 38/38 1.9(0.3–13.6) .54 28/28 Thioredoxinreductase-boundSe 1.0(0.9–1.0) .37 38/38 1.0(0.9–1.1) .65 28/28 Glutathioneperoxidase-boundSe 1.0(0.9–1.1) .73 38/38 1.0(0.9–1.2) .58 28/28 Humanserumalbumin-boundSe 1.5(0.9–2.4) .11 38/38 1.9(1.0–3.6) .05 28/28 SelenoproteinP-boundSe 0.3(0.08–0.8) .02 38/38 0.2(0.06–0.9) .03 28/28 TotalinorganicSe

Crude 1.4(0.7–2.9) .30 38/38 3.2(1.1–9.4) .04 28/28 AdjustedfororganicSe 1.7(0.8–3.9) .18 38/38 4.6(1.1–20.2) .04 28/28 TotalorganicSe

Crude 0.4(0.2–0.9) .03 38/38 0.4(0.2–1.1) .07 28/28 AdjustedforinorganicSe 0.4(0.2–0.9) .03 38/38 0.3(0.1–1.0) .05 28/28 TotalSe 0.5(0.2–1.0) .05 38/38 0.5(0.2–1.1) .09 28/28

a_Unit_increase_is_0.1_m_g/L_for_selenate,_selenite,_total_inorganic_Se_and_human_serum_albumin_bound-Se_and₁_m_g/L_for_all_other_compounds.

b _Results_obtained_from_conditional_logistic_regression_models._To_reduce_the_inﬂuence_of_extreme_outliers_in_the_{distributions}_of_some_Se_species,_Se_variables_were

(6)

process(Rusoloetal.,2013;Shanuetal.,2013),whichinturnmay influencetherelativedistributionofseleniumamongbodytissues (Maehira et al., 2002), and inflammation has been recently suggestedtoplayapotentialroleinALSetiologyandpathogenesis (Evans etal.,2013).Therefore,thepossibilityofa linkbetween alteredSecompoundscontentinCNSandneuroinflammationin ALSetiologymeritsfurtherconsideration.

Inthepreviousepidemiologicstudyonconsumersofdrinking waterwithhighSecontentintheReggioEmiliaarea(Vincetietal., 1996), excess ALS risk was associated to high levels of Se as selenate,theusualformofthemetalloidinundergroundwaters (Vinceti etal.,2010b),while inthepresentstudytheincreased concentrationswerefoundonlyforselenite.However,toxicityof thesetwoinorganicSespecies,whosedietaryintakeisusuallyvery low,iscomparableinsomestudiesthoughnotinall,withgeneral highertoxicityforselenite(Spyrouetal.,1996;Weilleretal.,2004; Lunoeetal.,2011;Maraldietal.,2011;Alturkmanietal.,2012; Benko et al., 2012; Tsai et al., 2013). Moreover, a conversion betweenthesetwo inorganicSeforms,i.e. fromthehexavalent species(selenate)tothetetravalentone(selenite),hasbeenshown tooccur asa chemical or a biologicalprocess (Hagemanet al., 2013)anditmightalsooccurinthehuman(Gammelgaardetal., 2012),thoughlimitedinformationonthisissueisavailable.

The levels of selenite found in our study of CSF from ALS patientswereclosetothose(0.316

m

g/L)foundinaninvitrostudy toinduceapoptosisinculturedmousecorticalneurons(Xiaoetal., 2006).Toxiceffectsofseleniteappeartobehighlydependenton celltype(Maraldietal.,2011)andpossibly,innervouscells,onthe brain region involved (Zia and Islam, 2000). An additional mechanismsuggestedtolinkexcessSetoALSriskisnon-speciﬁc incorporationofSeintoproteins(Bell,2009).

ThecauseoftheelevatedCSFselenitecontentdetectedinour ALSpatientsmaybeattributabletoseveralcauses,suchasexcess exposuretothecompoundfromenvironmentalsources,abnormal and enhanced localization of this compound in the CNS, and variationsin responseand repair pathwaysto selenite-induced DNA damage (Manikova et al., 2012) or alteration of its accumulation,detoxification ortoxicity (Sun et al., 2013), with thepossibleinvolvementofgeneticfactors.Aboutthelatter,an interplaybetween excessselenite exposureand genetics might occurinsporadicALSaccordingtothe‘low-penetrance suscepti-bilitymodel,’whichhypothesizessomeinvolvementofgeneticas wellasenvironmentalfactorseveninthenon-familialformofthe disease(Sabatelli et al.,2013). Unfortunately, we didnot have availableinourpatientsperipheralindicatorsofSeexposuresuch aslevelsofbloodortoenailsSespecies,whichmighthavehelped, despite the limited ability of these biomarkers in reflecting exposuretoinorganicSecompared withorganic(Vinceti etal., 2001,2009), toassess thepossibleenvironmental originof the excess CNS selenite content we detected. Ascertainment of historicalresidenceofstudypatientsthroughtheRevenueAgency oftheMinistryofFinance,whichmaintainsrecordsofhistorical residencenationwideandcouldbeaccessedforresearchpurposes byoneofus(Vincetietal.,2012c),allowedustoruleoutthatanyof the study subjects had resided in Reggio Emilia area where drinkingwaterwithhighselenitecontentwasdistributedinthe 1970sand1980s(Vincetietal.,2010a).However,wedidnothave available any specific information about personal sources of drinkingwaterconsumptionandotherpotentialenvironmentalor occupationalexposuresofstudysubjectstoSespecies.

OurobservationofaninverseassociationbetweenALSriskand levelsoforganic Secompoundsand particularlySePP,withthe exception of Se-HSA, may imply a deﬁciency in antioxidant responsetofreeradicaldamage(Mandriolietal.,2006)ormore generallyahigherriskofneurodegenerationduetoadeﬁciencyof proteinssuchastheantioxidantenzymeGPx(Wirthetal.,2010)

andofSePP(Caitoetal.,2011),whichisconsideredamajorsource ofSeforSe-dependentenzymesynthesisandalsohasantioxidant propertiesitself(Scharpfetal.,2007;Meplanetal.,2009).Reasons for decreased organic Se levels in CSF of our ALS patients are unclear,sincetheycannotbeascribedtoalowSesupply,duetothe higheramountofinorganicSeavailable.Twopossiblyinterrelated hypothesesmaybeadvanced:theroleofunknowngeneticor non-geneticfactorsinreducingtheantioxidantresponse,ortheability ofpro-oxidantSespecies,includingselenite,toreduceexpression ofSe containingenzymes(Tallandiniet al.,1996; Brancoet al., 2012; Medeiroset al.,2012), possibly throughtheinduction of oxidativestress(SuryoRahmantoetal.,2012).Ontheotherhand, inotherinvestigationsselenitehasbeenshowntoincreasebrain GPxactivityaswellaslipidperoxidation(Glaseretal.,2010),the lattereffectbeingpossiblythecauseoftheformer,andinhuman andinvitrostudiesitappearstoincreaseSePPlevels(Meplanetal., 2009;Hoeﬁgetal.,2011).

Recentanimalandhumanstudiesinvestigatingtherelationof SePP with neurological or other diseases have yielded sharply differentresults(Bellingeret al.,2008,2012;Yang etal.,2011; Raman et al., 2012; Takata et al., 2012), not unexpectedly in selenium research, making it difficult to assess the biological significanceofourfindings.ThehypotheticalroleofSePPinCNS diseasesisintriguingandcontroversial,rangingfrombeneficialas suggestedbyanimal studies(Ramanetal.,2012)topotentially deleteriousinhumanneurodegenerativediseases(Bellingeretal., 2008,2012).Scharpfetal.investigatedthedistributionofSePPin thehumanCNS:theyfoundveryunevenlocalization,withhigh SePPimmunoreactivityinspinal,cranialandparticularly pyrami-dalmotorneurons,anddetectedevidencesupportingacrucialrole of SePPexpression andactivity in humanbrain (Scharpf et al., 2007).

Weobservedanunexpectedpositiveassociationbetween Se-HSA and ALS risk. The exact nature of Se-HSA is unclear, but availableevidencesuggeststhatitismostlycomposedoforganic Se (Xuet al., 2008), though the occurrence of inorganicforms cannotberuled out (Haratakeet al.,2008). Despitethelack of informationaboutthebiologicalpropertiesofthisspeciesofthe metalloid,weconsiderthisﬁndingworthfurtherinvestigationin futurehumanstudiesontheroleofSeinneurodegeneration. Se-HSAmightactaslong-termstorageofthemetalloidforfurther selenoproteinsynthesis,thussuggesting thepossibility thatthe decreasedSePPlevelsfoundincasescouldbeaconsequence,ona hypothetical basis, of a decreased Se re-mobilization and availabilityfromSe-HSA.ExcessSeexposureinChineseindividuals hasbeenassociatedwithpreferentiallocalizationofSeinthe Se-HSA fraction, while in Se-deﬁcient or adequate subjects the greatestSeamountwasintheSePPfraction(Guetal.,1998).

WefoundastrongerpositiveassociationbetweenCSFselenite andALSriskamongoldersubjects.Apossiblereasonforenhanced riskwitholderagemaybethedecreasedroleofgeneticfactors supposedtobemoreimportantintriggeringearlieronsetdisease (Chio et al., 2012), indicating a greater role of environmental variablesinoldersubjects.

Wemustrecognizelimitationsofthepresentstudy,including thepossibilitythatthediseaseprocessinfluencedthecontentofSe species in CSF, and possibly in different ways for different compounds. The possibility of reverse causality is inherent in studies conducted with biomarkers in patients suffering from disease; however, in the present study CSF sampling was conducted at a very early stage of the disease, i.e. during the clinicalprocessleadingtotheitsfirstdiagnosis,thusdecreasing theriskof disease-inducedalterationsin metabolismorintake. Furthermore,theextenttowhichinorganicSespecies,whichare notsynthesizedbytheorganismbutratherabsorbed,mayreflect long-termenvironmentaloverexposureisunknown,duetoalack

(7)

ofstudiesontheirpersistenceordistributioninCSForotherCNS areas.Weselectedascontrolsubjectsneurologicalpatientswhose diagnosis was benign or null after their diagnostic process; however,wecannotentirelyruleoutselectionbiasinthereferent population,possiblymediatedbysomeriskfactorsaffectingtheir conditions that are correlated with differential exposure to Se species. However, there is no a priori plausibility to such a hypothesis,andoursensitivityanalyseswereunabletoidentify trendssupportingthispossibility.Selectionbiasisunlikelytohave occurredintheALSpatientgroup,astheywereselectedamonga consecutivepatientseriesadmittedtothesamehospitalascontrol patients and from thesame underlying population, which was furthermorequitehomogeneousastogeneticandenvironmental lifestylefactors.ALSpatientsundergoinglumbarpunctureforCSF samplingduringthe diagnosticprocess weremorelikely to be youngerandcharacterizedbypyramidalsignsthanALScasesnot undergoingthisprocedurewhowerethereforenotincludedinthe present study; however, there is no reason to suspect an association between these clinical features and alterations in singleSespecies,andexcessCSFselenitecontentwasconfirmed (andevenenhanced)inourolderstudygroup.Finally,studysize wasnotlargeenoughtoyieldstatisticallypreciseriskestimates,as reflectedbywideconfidenceintervalssurroundingtheRRs,and thedataincludedoutliers,whichweattemptedtodealwithby usingmultipleapproachestotheanalysis.

Inconclusion,ourresultsindicateadirectrelationbetweenALS riskandtheconcentrationofseleniteinCSFofnewlydiagnosed ALSpatients,aswellaninverseassociationwiththeorganicSe formSePPwhichmightbearelatedphenomenon,supportingthe hypothesisthatoverexposuretoselenitemaybeanetiologicalrisk factor in the disease. Selenite may trigger neurodegenerative effectsthroughitspowerfultoxicity,whichappearstobeunique among toxic chemicals, being highly speciﬁc toward motor neuronsinsomeanimalstudies.

Conﬂictofinterest Nonedeclared. Acknowledgements

Financial supportto this study was provided by the Pietro ManodoriFoundationofReggioEmilia,theNational,Modenaand ReggioEmiliasectionsoftheItalianAmyotrophicLateralSclerosis Association(AISLA)andtheLocalHealthUnitofReggioEmiliato Dr.Vinceti,andbytheUSNationalInstituteofHealthtoDr.Crespi (grantNIHUL1TR000124).

References

AlturkmaniHJ,ZgheibC,ZoueinFA,AlshaaerNE,KurdiM,BoozGW.Selenateenhances STAT3transcriptionalactivityinendothelialcells:differentialactionsofselenate andseleniteonLIFcytokinesignalingandcellviability.JInorgBiochem2012; 109:9–15.

AmmarEM,CouriD.Acutetoxicityofsodiumseleniteandselenomethionineinmice afterICVorIVadministration.Neurotoxicology1981;2:383–6.

BallihautG,KilpatrickLE,KilpatrickEL,DavisWC.MultipleformsofselenoproteinPina candidatehumanplasmastandardreferencematerial.Metallomics2012;4:533–8.

BehneD,AlberD,KyriakopoulosA.Long-termseleniumsupplementationofhumans: seleniumstatusandrelationshipsbetweenseleniumconcentrationsinskeletal muscleandindicatormaterials.JTraceElemMedBiol2010;24:99–105.

BellEA.The discoveryofBMAA, and examplesofbiomagniﬁcationand protein incorporationinvolvingothernon-proteinaminoacids.AmyotrophLateralScler 2009;10(Suppl.2):21–5.

BellingerFP,HeQP,BellingerMT,LinY,RamanAV,WhiteLR, etal.Associationof selenoproteinpwithAlzheimer’spathologyinhumancortex.JAlzheimersDis 2008;15:465–72.

BellingerFP,RamanAV,RueliRH,BellingerMT,DewingAS,SealeLA,etal.Changesin selenoproteinPinsubstantianigraandputameninParkinson’sdisease.J Parkin-sonsDis2012;2:115–26.

BelyaevaEA,SarisNE.Mechanism(s)oftoxicactionofZnandselenite:astudyonAS-30D hepatomacellsandisolatedmitochondria.BiochemResInt2011;2011:387297.

BenkoI,NagyG,TanczosB,UngvariE,SztrikA,EszenyiP, etal.Subacutetoxicityof nano-seleniumcomparedtootherseleniumspeciesinmice.EnvironToxicolChem 2012;31:2812–20.

BergomiM,VincetiM,NacciG,PietriniV,BratterP,AlberD, etal.Environmental exposuretotraceelementsandriskofamyotrophiclateralsclerosis:a population-basedcase–controlstudy.EnvironRes2002;89:116–23.

BitencourtP,BelleL,BonfantiG,CargneluttiL,deBonaK,SilvaP, etal.Differential effectsoforganicandinorganicseleniumcompoundsonadenosinedeaminase activityandscavengercapacityincerebralcortexslicesofyoungrats.HumExp Toxicol2013(inpress).

BodnarM,KonieczkaP,NamiesnikJ.Theproperties,functions,anduseofselenium compoundsinlivingorganisms.JEnvironSciHealthCEnvironCarcinogEcotoxicol Rev2012;30:225–52.

BorellaP,BargelliniA,MediciCI.Chemicalformofseleniumgreatlyaffectsmetal uptake andresponses bycultured humanlymphocytes.BiolTraceElem Res 1996;51:43–54.

BrancoV,CanarioJ,LuJ,HolmgrenA,CarvalhoC.Mercuryandseleniuminteractionin vivo:effectsonthioredoxinreductaseandglutathioneperoxidase.FreeRadicBiol Med2012;52:781–93.

CaitoSW,MilatovicD,HillKE,AschnerM,BurkRF,ValentineWM.Progressionof neurodegenerationandmorphologicchangesinthebrainsofjuvenilemicewith selenoproteinPdeleted.BrainRes2011;1398:1–12.

CasteignauA,FontanA,MorilloA,OliverosJA,SegalesJ.Clinical,pathologicaland toxicologicalﬁndingsofaiatrogenicseleniumtoxicosiscaseinfeederpigs.JVet MedAPhysiolPatholClinMed2006;53:323–6.

ChenQ,WangZ,XiongY,ZouX,LiuZ.Comparativestudyofp38MAPKsignal transductionpathwayofperipheralbloodmononuclearcellsfrompatientswith coal-combustion-typeﬂuorosiswithandwithouthighhairseleniumlevels.IntJ HygEnvironHealth2010;213:381–6.

ChioA,CalvoA,MazziniL,CantelloR,MoraG,MogliaC,etal.ExtensivegeneticsofALS: apopulation-basedstudyinItaly.Neurology2012;79:1983–9.

CoppedeF.AnoverviewofDNArepairinamyotrophiclateralsclerosis.SciWorldJ 2011;11:1679–91.

DennertG,ZwahlenM,BrinkmanM,VincetiM,ZeegersMP,HorneberM.Seleniumfor preventingcancer.CochraneDatabaseSystRev2011;5:CD005195.

EstevezAO,MuellerCL,MorganKL,SzewczykNJ,TeeceL,Miranda-VizueteA, etal.

SeleniuminducescholinergicmotorneurondegenerationinCaenorhabditis ele-gans.Neurotoxicology2012;33:1021–32.

EvansMC,CouchY,SibsonN,TurnerMR.Inﬂammationandneurovascularchangesin amyotrophiclateralsclerosis.MolCellNeurosci2013;53:34–41.

GammelgaardB,SturupS,ChristensenMV.Humanurinaryexcretionandmetabolism of(82)Se-enrichedseleniteandselenatedeterminedbyLC-ICP-MS.Metallomics 2012;4:149–55.

GeorgoulopoulouE,VincetiM,BonviciniF,SolaP,GoldoniCA,DeGirolamoG, etal.

ChangingincidenceandsubtypesofALSinModena,Italy:a10-yearsprospective study.AmyotrophLateralScler2011;12:451–7.

GladmanM,CudkowiczM,ZinmanL.Enhancingclinicaltrialsinneurodegenerative disorders:lessonsfromamyotrophiclateralsclerosis.CurrOpinNeurol2012; 25:735–42.

GlaserV,NazariEM,MullerYM,FeksaL,WannmacherCM,RochaJB, etal.Effectsof inorganicseleniumadministrationinmethylmercury-inducedneurotoxicityin mousecerebralcortex.IntJDevNeurosci2010;28:631–7.

GuQP,XiaYM,HaPC,ButlerJA,WhangerPD.Distributionofseleniumbetweenplasma fractionsinguineapigsandhumanswithvariousintakesofdietaryselenium.J TraceElemMedBiol1998;12:8–15.

HagemanSP,vanderWeijdenRD,WeijmaJ,BuismanCJ.Microbiologicalselenateto seleniteconversionforseleniumremoval.WaterRes2013;47:2118–28.

HanadaT,WeitzerS,MairB,BernreutherC,WaingerBJ,IchidaJ,etal.CLP1linkstRNA metabolismtoprogressivemotor-neuronloss.Nature2013;495:474–80.

HaratakeM,HongohM,MiyauchiM,HirakawaR,OnoM,NakayamaM. Albumin-mediatedseleniumtransferbyaselenotrisulﬁderelaymechanism.InorgChem 2008;47:6273–80.

HoeﬁgCS,RenkoK,KohrleJ,BirringerM,SchomburgL.Comparisonofdifferent selenocompoundswithrespecttonutritionalvaluevs.toxicityusinglivercells inculture.JNutrBiochem2011;22:945–55.

HollenbachB,MorgenthalerNG,StruckJ,AlonsoC,BergmannA,KohrleJ, etal.New assayforthemeasurementofselenoproteinPasasepsisbiomarkerfromserum.J TraceElemMedBiol2008;22:24–32.

HosmerDW,LemeshowSL.Appliedlogisticregression.Hoboken(NJ):JohnWiley& Sons;2000.

IncePG,ShawPJ,CandyJM,MantleD,TandonL,EhmannWD,etal.Iron,seleniumand glutathioneperoxidaseactivityareelevatedinsporadicmotorneurondisease. NeurosciLett1994;182:87–90.

KarunasingheN,HanDY,ZhuS,YuJ,LangeK,DuanH,etal.Serumseleniumand single-nucleotidepolymorphismsingenesforselenoproteins:relationshiptomarkersof oxidativestressinmenfromAuckland,NewZealand.GenesNutr2012;7:179–90.

KilnessAW,HochbergFH.Amyotrophiclateralsclerosisinahighselenium environ-ment.JAMA1977;237:2843–4.

KimEK,ChoiEJ.PathologicalrolesofMAPKsignalingpathwaysinhumandiseases. BiochimBiophysActa2010;1802:396–405.

LunoeK,Gabel-JensenC,SturupS,AndresenL,SkovS,GammelgaardB.Investigationof theseleniummetabolismincancercelllines.Metallomics2011;3:162–8.

MaQ,FangH,ShangW,LiuL,XuZ,YeT,etal.Superoxideﬂashes:earlymitochondrial signalsforoxidativestress-inducedapoptosis.JBiolChem2011;286:27573–81.

(8)

MaehiraF,LuyoGA,MiyagiI,OshiroM,YamaneN,KubaM,etal.Alterationsofserum seleniumconcentrationsintheacutephaseofpathologicalconditions.ClinChim Acta2002;316:137–46.

MandrioliJ,DelRioD,ZiniA,NichelliP,MerelliE,BeltramiD,etal.Totalantioxidant capacityofcerebrospinalﬂuidisdecreasedinpatientswithmotorneurondisease. NeurosciLett2006;401:203–8.

ManikovaD,Vlasakova D, Letavayova L,Klobucnikova V,Griac P,Chovanec M.

Seleniumtoxicitytowardyeastasassessedbymicroarrayanalysisanddeletion mutantlibraryscreen:aroleforDNArepair.ChemResToxicol2012;25:1598–608.

MaraldiT,RiccioM,ZamboninL,VincetiM,DePolA,HakimG.Lowlevelsofselenium compoundsareselectivelytoxicforahumanneuroncelllinethroughROS/RNS increaseandapoptoticprocessactivation.Neurotoxicology2011;32:180–7.

MarkesberyWR,EhmannWD,CandyJM,IncePG,ShawPJ,TandonL, etal.Neutron activationanalysisoftraceelementsinmotorneurondiseasespinalcord. Neu-rodegeneration1995;4:383–90.

MedeirosMC,MelloA,GemelliT,TeixeiraC,deAlmeidaM,deAndradeRB,etal.Effect ofchronicadministrationofthevinylchalcogenide 3-methyl-1-phenyl-2-(phe-nylseleno)oct-2-en-1-oneonoxidativestress indifferentbrain areasofrats. NeurochemRes2012;37:928–34.

MendoncaDM, PizzatiL, Mostacada K,deSMSC, HigashiR, AyresSaL, etal.

Neuroproteomics:aninsightintoALS.NeurolRes2012;34:937–43.

MeplanC,NicolF,BurtleBT,CrosleyLK,ArthurJR,MathersJC,etal.Relativeabundance ofselenoproteinPisoformsinhumanplasmadependsongenotype,Seintake,and cancerstatus.AntioxidRedoxSignal2009;11:2631–40.

MichalkeB,GrillP,BertheleA.AmethodforlowvolumeandlowSeconcentration samplesandapplicationtopairedcerebrospinalﬂuidandserumsamples.JTrace ElemMedBiol2009;23:243–50.

MichalkeB,BertheleA.Contributiontoseleniumspeciationincerebrospinalﬂuid samples.JAnalAtomSpectrom2011;26:165–70.

MitchellJD,EastBW,HarrisIA,PentlandB.Manganese,seleniumandothertrace elementsinspinalcord,liverandboneinmotorneuronedisease.EurNeurol 1991;31:7–11.

MoriwakaF,SatohH,EjimaA,WatanabeC,TashiroK,HamadaT, etal.Mercuryand seleniumcontentsinamyotrophiclateralsclerosisinHokkaido,thenorthernmost IslandofJapan.JNeurolSci1993;118:38–42.

NogueiraCW,MeottiFC,CurteE,PilissaoC,ZeniG,RochaJB.Investigationsintothe potentialneurotoxicity induced bydiselenides in mice and rats.Toxicology 2003;183:29–37.

NogueiraCW,RochaJB.Toxicologyandpharmacologyofselenium:emphasison syntheticorganoseleniumcompounds.ArchToxicol2011;85:1313–59.

PeyrocheG,SaveanuC,DauplaisM,LazardM,BeuneuF,DecourtyL, etal.Sodium selenide toxicity is mediated by O2-dependent DNA breaks. PLoS ONE 2012;7:e36343.

PrzedborskiS,DonaldsonD,JakowecM,KishSJ,GuttmanM,RosoklijaG,etal.Brain superoxidedismutase,catalase,andglutathioneperoxidaseactivitiesin amyo-trophiclateralsclerosis.AnnNeurol1996;39:158–65.

RaberM,SydlerT,WolﬁsbergU,GeyerH,BurgiE.Feed-relatedseleniumpoisoningin swine.SchweizArchTierheilkd2010;152:245–52.

RamanAV,PittsMW,SeyedaliA,HashimotoAC,SealeLA,BellingerFP,etal.Absenceof selenoproteinPbutnotselenocysteinelyaseresultsinsevereneurological dys-function.GenesBrainBehav2012;11:601–13.

RoosPM,VesterbergO,SyversenT,FlatenTP,NordbergM.Metalconcentrationsin cerebrospinalﬂuidand bloodplasmafrompatientswithamyotrophiclateral sclerosis.BiolTraceElemRes2013;151:159–70.

RossiL,ValleC,CarriMT.Alteredgeneexpression,mitochondrialdamageandoxidative stress: converging routes in motor neuron degeneration. Int J Cell Biol 2012;2012:908724.

RudolfE,RudolfK,CervinkaM.Seleniumactivatesp53andp38pathwaysandinduces caspase-independent cell death in cervical cancer cells. Cell Biol Toxicol 2008;24:123–41.

RusoloF,PucciB,ColonnaG,CaponeF,GuerrieroE,MiloneMR, etal.Evaluationof seleniteeffectsonselenoproteinsandcytokinomeinhumanhepatomacelllines. Molecules2013;18:2549–62.

SabatelliM,ConteA,ZollinoM.Clinicalandgeneticheterogeneityofamyotrophic lateralsclerosis.ClinGenet2013;83:408–16.

Saint-AmourD,RoyMS,BastienC,AyotteP,DewaillyE,DespresC,etal.Alterations ofvisualevokedpotentialsinpreschoolInuitchildrenexposedto methylmer-cury and polychlorinated biphenyls from a marine diet. Neurotoxicology 2006;27:567–78.

ScharpfM,SchweizerU,ArzbergerT,RoggendorfW,SchomburgL,KohrleJ.Neuronal andependymalexpressionofselenoproteinPinthehumanbrain.JNeuralTransm 2007;114:877–84.

ShanuA,GroeblerL,KimHB,WoodS,WeekleyCM,AitkenJB,etal.Seleniuminhibits renaloxidationandinﬂammationbutnotacutekidneyinjuryinananimalmodel ofrhabdomyolysis.AntioxidRedoxSignal2013;18:756–69.

SolovyevN,BertheleA,MichalkeB.Seleniumspeciationinpairedserumand cerebro-spinalﬂuidsamples.AnalBioanalChem2013;405:1875–84.

SpyrouG,BjornstedtM,SkogS,HolmgrenA.Seleniteandselenateinhibithuman lymphocytegrowthviadifferentmechanisms.CancerRes1996;56:4407–12.

SteinbrennerH,SiesH.Seleniumhomeostasisandantioxidantselenoproteinsinbrain: implicationsfordisordersinthecentralnervoussystem.ArchBiochemBiophys 2013(inpress).

SunK,WuS,WangY,WanX,ThompsonHJ,ZhangJ.High-dosesodiumselenitetoxicity cannotbepreventedbytheco-administrationofpharmacologicallevelsof epi-gallocatechin-3-gallatewhichinturnaggravatesthetoxicity.FoodChemToxicol 2013;52:36–41.

SuryoRahmantoA,PattisonDI,DaviesMJ.Photo-oxidation-inducedinactivationofthe selenium-containingprotectiveenzymesthioredoxinreductaseandglutathione peroxidase.FreeRadicBiolMed2012;53:1308–16.

SussmuthSD,BrettschneiderJ,LudolphAC,TumaniH.BiochemicalmarkersinCSFof ALSpatients.CurrMedChem2008;15:1788–801.

TakataY,KristalAR,SantellaRM,KingIB,DugganDJ,LampeJW, etal.Selenium, selenoenzymes,oxidativestressandriskofneoplasticprogressionfromBarrett’s esophagus: results from biomarkers and genetic variants. PLoSONE 2012; 7:e38612.

TallandiniL,CecchiR,DeBoniS,GalassiniS,GhermandiG,GialanellaG, etal.Toxic levelsofseleniuminenzymesandseleniumuptakeintissuesofamarineﬁsh.Biol TraceElemRes1996;51:97–106.

TarasiukJ,KulakowskaA,DrozdowskiW,KornhuberJ,LewczukP.CSFmarkersin amyotrophiclateralsclerosis.JNeuralTransm2012;119:747–57.

ThomsonCD,RobinsonMF,ButlerJA,WhangerPD.Long-termsupplementationwith selenateandselenomethionine:seleniumandglutathioneperoxidase(EC1.11.1.9) inbloodcomponentsofNewZealandwomen.BrJNutr1993;69:577–88.

TsaiCF,OuBR,LiangYC,YehJY.Growthinhibitionandantioxidativestatusinducedby selenium-enrichedbroccoliextractandselenocompoundsinDNAmismatch re-pair-deﬁcienthumancoloncancercells.FoodChem2013;139:267–73.

TsunodaM,JohnsonVJ,Sharma RP.Increaseindopaminemetabolitesinmurine striatumafteroralexposuretoinorganicbutnotorganicformofselenium.Arch EnvironContamToxicol2000;39:32–7.

ValdiglesiasV,PasaroE,MendezJ,LaffonB.Invitroevaluationofseleniumgenotoxic, cytotoxic,andprotectiveeffects:areview.ArchToxicol2010;84:337–51.

VincetiM,GuidettiD,PinottiM,RovestiS,MerlinM,VescoviL, etal.Amyotrophic lateralsclerosisafterlong-termexposuretodrinkingwaterwithhighselenium content.Epidemiology1996;7:529–32.

VincetiM,WeiET,MalagoliC,BergomiM,VivoliG.Adversehealtheffectsofselenium inhumans.RevEnvironHealth2001;16:233–51.

VincetiM,MaraldiT,BergomiM,MalagoliC.Riskofchroniclow-doseselenium overexposureinhumans:insightsfrom epidemiologyand biochemistry. Rev EnvironHealth2009;24:231–48.

VincetiM,BonviciniF,BergomiM,MalagoliC.Possibleinvolvementofoverexposureto environmentalseleniumintheetiologyofamyotrophiclateralsclerosis:ashort review.AnnIstSuperSanita2010a;46:279–83.

VincetiM,BonviciniF,RothmanKJ,VescoviL,WangF.Therelationbetween amyo-trophiclateralsclerosisandinorganicseleniumindrinkingwater:a population-basedcase–controlstudy.EnvironHealth2010b;9:77.

VincetiM,BottecchiI,FanA,FinkelsteinY,MandrioliJ.Areenvironmentalexposuresto selenium,heavymetalsandpesticidesriskfactorsforamyotrophiclateral sclero-sis.RevEnvironHealth2012a;27:19–41.

VincetiM,CrespiCM,MalagoliC,BottecchiE,FerrariA,SieriS, etal.Acase–control studyoftheriskofcutaneousmelanomaassociatedwiththreeseleniumexposure indicators.Tumori2012b;98:287–95.

VincetiM,RothmanKJ,CrespiCM,SterniA,CherubiniA,GuerraL,etal.Leukemiarisk inchildrenexposedtobenzeneandPM10fromvehiculartrafﬁc:acase–control studyinanItalianpopulation.EurJEpidemiol2012c;27:781–90.

VincetiM,CrespiCM,BonviciniF,MalagoliC,FerranteM,MarmiroliS,etal.Theneed forareassessmentofthesafeupperlimitofseleniumindrinkingwater.SciTotal Environ2013;443:633–42.

vonNeuhoffN,OumeraciT,WolfT,KolleweK,BewerungeP,NeumannB, etal.

MonitoringCSFproteomealterationsinamyotrophiclateralsclerosis:obstacles andperspectivesintranslatinganovelmarkerpaneltotheclinic.PLoSONE 2012;7:e44401.

WeillerM,LattaM,KresseM,LucasR,WendelA.Toxicityofnutritionallyavailable selenium compounds in primary and transformed hepatocytes. Toxicology 2004;201:21–30.

WilsonME,BoumazaI,BowserR.MeasurementofcystatinCfunctionalactivityinthe cerebrospinalﬂuidofamyotrophiclateralsclerosisandcontrolsubjects.Fluids BarriersCNS2013;10:15.

WinerL,SrinivasanD,ChunS,LacomisD,JaffaM,FaganA,etal.SOD1incerebralspinal ﬂuidasapharmacodynamicmarkerforantisenseoligonucleotidetherapy.JAMA Neurol2013;70:201–7.

WirthEK,ConradM, WintererJ,WoznyC,CarlsonBA,RothS, etal.Neuronal selenoproteinexpressionisrequiredforinterneurondevelopmentandprevents seizuresandneurodegeneration.FASEBJ2010;24:844–52.

XiaoR,QiaoJT,ZhaoHF,LiangJ,YuHL,LiuJ,etal.Sodiumseleniteinducesapoptosisin culturedcorticalneuronswithspecialconcomitantchangesinexpressionofthe apoptosis-relatedgenes.Neurotoxicology2006;27:478–84.

XuM,YangLN,WangQQ.Quantiﬁcationofselenium-taggedproteinsinhumanplasma usingspecies-unspeciﬁcisotopedilutionICP-DRC-qMScoupledon-linewithanion exchangechromatography.JAnalAtomSpectrom2008;23:1545–9.

YangSJ,HwangSY,ChoiHY,YooHJ,SeoJA,KimSG,etal.SerumselenoproteinPlevels inpatientswithtype2diabetesandprediabetes:implicationsforinsulin resis-tance,inﬂammation,andatherosclerosis.JClinEndocrinolMetab2011;96:E1325– 9.

YangX,YuX,FuH,LiL,RenT.Differentlevelsofprenatalzincandseleniumhad different effects on neonatal neurobehavioral development. Neurotoxicology 2013;37:35–9.

ZhengS,LiX,ZhangY,XieQ,WongYS,ZhengW, etal.PEG-nanolizedultrasmall seleniumnanoparticlesovercomedrugresistanceinhepatocellularcarcinoma HepG2cellsthroughinductionofmitochondriadysfunction.IntJNanomedicine 2012;7:3939–49.

ZiaS,IslamF.Seleniumalteredthelevelsoflipids,lipidperoxidation,andsulfhydryl groupsinstraitumandthalamusofrat.BiolTraceElemRes2000;77:251–9.