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ADVANCESIN MOLECULAR TOXICOLOGY EDITEDBY
JAMESC.FISHBEIN
DepartmentofChemistryandBiochemistry
UniversityofMaryland,BaltimoreCounty Baltimore,USA
Elsevier
Radarweg29,POBox211,1000AEAmsterdam,TheNetherlands LinacreHouse,JordanHill,OxfordOX28DP,UK 32,JamestownRoad,LondonNW17BY,UK 525BStreet,Suite1900,SanDiego,CA92101-4495,USA 30CorporateDrive,Suite400,Burlington,MA01803,USA
Firstedition2010
Copyright 2010,ElsevierB.V.Allrightsreserved
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Noresponsibilityisassumedbythepublisherforanyinjuryand/ordamage topersonsorpropertyasamatterofproductsliability,negligenceor otherwise,orfromanyuseoroperationofanymethods,products, instructionsorideascontainedinthematerialherein.Becauseofrapid advancesinthemedicalsciences,inparticular,independentverificationof diagnosesanddrugdosagesshouldbemade
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ISBN:978-0-444-53584-9
ISSN:1872-0854
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PrintedandboundinGreatBritain 1011121310987654321
WojciechBal85
AmandaC.Bryant-Friedrich127
PhilipC.Burcham1
RobertCheng157
WilmarieFlores-Santana157 SharonGlynn157
PeterJ.Henry1
RobertJ.Turesky37
DavidA.Wink157
ChristineC.Winterbourn183
PREFACE Thisfourthvolumeintheseriesoffersapotpourriofchemistriesrelevantto thebroadanddiversedisciplineofMolecularToxicology.Fromsingleions tosilicananomaterials,thesereviewsencompassthebestinmechanistic considerationsinthediscipline.TheEditorhopesyouenjoythebreadthof thisspectrum.
Thisvolumeopenswithareviewofthechemistryandtoxicologyofa smallbuthighlyreactivecompoundthatispervasiveinthehumanenvironment—acrolein.Burcham,Thompson,andHenrysurveyanextensive recentliteratureonitsactivity,particularlyasapulmonarytoxicant.There hasbeenarapidlyevolvingliteratureonthechemicalreactivityofacrolein withbiomoleculesanditsactivityinavarietyofmodels.Butastheauthors demonstrate,ourunderstandingofthedetailedmolecularbasisforthe modeofactionofacroleininlungtoxicityisrelativelyfragmentary.
Consumptionofcookedpoultry,meat,andfishleadstowidespread humanexposuretoheterocyclicaromaticamines.Anumberofsuch compoundshavebeendemonstratedtobepowerfulmutagensandcarcinogens.SinceSugimuraandcoworkersdemonstratedtheactivityin charredfishandmeatmorethanthreedecadesago,therehasbeena growingliteratureonthemetabolismandactivationofthesecompounds thathavedemonstratedarisenotonlyfromtheinitialsourcesbutfrom dieselandcigarettecombustionandendogenousreactionsaswell.In Chapter2,Tureskyreviewsthisclassofcompounds,theirmetabolism andactivation,theproductsofreactiveintermediatesinteractingwith biomolecules,andpotentialbiomarkersfordeterminingtheircontribution tothehumancancerburden.
Nickelandcadmiummakeuptinyfractionsoftheearth’scrustbutthey arekeyelementsinmanyimportantindustrialapplicationsleadingto significanthumanexposure.Thisiscompoundedbythepresenceofsignificantquantitiesoftheseelementsinproductsofcombustion—cigarettes smoke,coal,anddieselflyash.InChapter3,KurowskaandBaltakea novelviewofcommonalitiesinboththeexposure,absorption,andmolecularbasisofthetoxicologicalactivitiesofnickelandcadmiuminthis comprehensivereview.
RadicalintermediatesandtheirconsequencesarethefocusofChapter4 whichconcernsoxidativedamagetothesugarmoietyofDNA.Fromthe fivesimplecarbon-centeredradicalsderivearatherlargearraydamage products,someofwhicharesuitablyfunctionalizedtoformfurther adducts.Thecomplexityofthischemistryiswellelaboratedhere.The
needtoconsiderthemetaboliccontextinwhichtheradicalisformedand thedegreetowhichthiscandetermineitssubsequentfateandtheultimate ‘‘fingerprint’’oftheinitialoxidationisemphasized.
Nitricoxide,redoxinflammation,andcancerarethetopicsofChapter 5.ChengandcoauthorselaboratethefundamentalchemistryofNOandits Janus-likecharacterinbothnormalandaberrantbiochemicalcontexts. Thismodestdiatomicradicalmanifestsaprofusionofhomeostaticand toxicroles.Itsroleincarcinogenesisandmetastaticprocesses,aformof molecularhijacking,isdiscussed.Akeyinsighttothecontrastingbehaviors isthatNOlevels—‘‘dose’’—canbeakeydeterminantto‘‘outcome.’’
BiologicalthiolsandtheirredoxchemistryarethefocusofNagyand WinterbourninChapter6.Theapproachisfundamental,asiswarranted. Thebalanceofnumerousredoxcouplesisintegraltothedetectionof oxidativestressandnumerousmodificationsofsmallmoleculeandprotein thiolsarebothkeysignalingandenzymeactivity-modifyingevents.Activityofthiolsastargetsisoftenmodulatedbymicroenvironmentthatcontrols nucleophilicityandselectivity.Theextentofpreviouslylittle-recognized arrayofposttranslationalmodificationsofproteincysteineresiduesisdocumented.Finally,thenewlyrecognizedactivityofH2Sasakeysignaling agentactingatsulfhydrylsisdiscussed.
Nanomaterialsareacurrentfocusofmaterialsdevelopmentastheyoffer uniquebio-andphysicochemicalpropertiesthatcansignificantlyimpacton thepropertiesofthematerialsthemselvesandtheagentswithwhichthey maybecoupledorattached.Withthesenewpropertiesaccruenew manifestationsoftoxicity.AnaspectofthisisthefocusofPetushkovand coauthorsinChapter7.Thisincludesasurveytoxicityofzeolitesand nanoscalesilicalitesandmesoporoussilicatesaswellastheimpactofsurface functionalization.Theinvolvementofreactiveoxygenspeciesintoxication isemphasizedandthenumerousvariablesthatimpactonthisactivityare emphasized.Thisunderscorestheneedinthisarea,respectingmaterialsfor whichapplicationisconceived,forcarefulcharacterizationofthephysicochemicalcharacteristicsoftheconstructs.
TheeditorwishestothankalltheauthorsfordistinguishedcontributionsandacknowledgesthetalentedElsevierstaffwhohaveputthis volumetogether.
InvitedReview AcroleinandtheLung:Chemical,Molecular,and PathologicalAspects
PhilipC.Burcham, ColinAThompson, and PeterJ.Henry
Contents
1.Introduction2
1.1.Scopeofreview2
1.2.Sourcesofairborneacrolein2
1.3.Involvementinrespiratorydiseases4
2.ChemicalToxicologyofAcrolein6
2.1.Chemicalproperties6
2.2.Chemistryofproteindamage7
2.3.ChemistryofDNAdamagebyacrolein10
2.4.Macromolecularcross-links12
3.CellularToxicologyofAcrolein13
3.1.Proteintargets13
3.2.Transcriptionalresponsestoacrolein16
3.3.Moleculareffectsofacrolein19
3.4.Mutagenicityofacrolein22
3.5.Celldeathpathways24
4.RespiratoryEffectsofAcuteInhaledAcrolein24
4.1.Bronchoconstriction25
4.2.Airwayshyperresponsiveness25
4.3.ProtectiveCOX-2-mediatedcounterresponses26
5.Conclusions27 Acknowledgments28 References28
PharmacologyandAnaesthesiologyUnit,SchoolofMedicineandPharmacology,theUniversityofWestern Australia,Crawley,WA,Australia
Correspondingauthor.Tel.:61893462986
Emailaddress:Philip.burcham@uwa.edu.au
AdvancesinMolecularToxicology,Volume4
2010ElsevierB.V. ISSN1872-0854,DOI10.1016/S1872-0854(10)04001-4Allrightsreserved.
1.Introduction 1.1.Scopeofreview Thethree-carbon a,b-unsaturatedaldehydeacroleinwasfirstidentified 170yearsagoasathermaldecompositionproductofglycerine,thename assignedinrecognitionofitspungentodor(‘‘acrid’’)andoil-likeconstituency(‘‘oleum’’) [1].Amongthemostnoxioussubstancesknowntotoxicology,recentyearshavewitnessedaresurgenceofinterestinacrolein, partlydrivenbydebateconcerningitscontributiontosmoking-relatedlung cancer [2].Therenewedinterestalsoreflectsthegrowingawarenessthat acroleinformsendogenouslyduringoxidativestress,suggestingarolein variousdegenerativedisorders [3].Therisingprofileisincreasingthe applicationofmodernmoleculartoolstothestudyofthecellulareffects ofacrolein,anoutcomethatshouldhelpclarifyitsroleinspecificdiseases. Inthefaceofarapidlyexpandingliterature,thisreviewfocusesonthe chemicalandcellulareffectsofacroleinwithinthecontextofitsemerging roleasamediatorofhumanlungdisease.Asavolatile,airbornetoxicant, thelungistheprimaryportofentryforenvironmentalacrolein,ensuring respiratoryepitheliumbearsthebruntofitscell-damagingproperties.An overarchinggoalistoreviewexistingknowledgewithaviewtosynthesizingunderstandingsofthechemicalreactivityofacroleinanditsmolecular effectsatthecellularlevelwithitstoxicologicalandpathologicaleffectson theintactlung.Whenviewedfromthisstandpoint,thestateofknowledge concerningacroleinandthelungappearssurprisinglyrudimentary.
1.2.Sourcesofairborneacrolein Althoughitformsubiquitouslyduringarangeofnaturalandhuman activities,mostatmosphericacroleinlikelyderivesfromthecombustion oforganicmatter.Basedonairmeasurementswithintwooutdoorurban settingsover1961–1980,theUSEnvironmentalProtectionAgency reportedaverageambientconcentrationsof14.3 mgm –3 (6.2ppb) [4]. Correspondingindoorconcentrationscanbe2–20-foldhigher,withthe higherlevelstypicallyrecordedinsmokyindoorenvironments,poorly ventilatedkitchens,anddwellingsheatedwithwoodstoves[4,5].Formationduringcombustionoforganicmatterensuresparticularlifestyleor occupationalpracticescanheightenhumanexposuretoacrolein.For example,asaby-productofthecombustionofsugarsandtoalesserextent glycerolwithintobacco,inexcessof100–200 mgacroleincanbereleased fromaburningcigarette[1,4].Cigarettesmokerslikelyinhaleupto 100nmolacrolein/puff,withevenhigherlevelspresentinsidestream smoke[1,5,6].Acroleinlevelsinsmokeformeduponburningothertypes
oforganicmattercanexceedthoseintobaccosmoke [5].Wood,vegetation,fossilfuels,andconstructionmaterialsareallsourcesforsmoke-borne acrolein,withespeciallyhighyieldsreleasedfromsmoulderingsynthetic polymerssuchaspolyethyleneandpolypropylene [7].
Whilemeasuringairborneacroleinisstraightforward,clarifyingthe concentrationsachievedinlungtissueduringeitherchronicoracute exposuretoacrolein-containingsmokeismoreproblematic.Thispartly reflectsthetechnicalchallengesaccompanyingmeasurementofinhaled toxicantsinthefluidsthatlinetherespiratorytract,theinitialpointof contactforinhaledacrolein.Conventionallavagetechniquesusedtocollect airwayandbronchoalveolarliquidsamplescauseunpredictabledilutionof lungfluids,renderingtoxicantconcentrationsunreliable [8].Furthermore, thereactivityofacroleinwithfluidproteinsandepithelialcellconstituents ensuresanysuchmeasurementof‘‘free’’acroleininrespiratoryfluidsisof questionabletoxicologicalsignificance.Notwithstandingthesechallenges, someworkershaveestimatedthatsmokingasinglecigaretteachieves ‘‘micromolar’’acroleinconcentrationsinrespiratoryfluids,althoughthe quantitativebasisfortheseestimatesisuncertain [9].Concentrationsof acroleinwithinrespiratoryfluidsofheavy,long-termsmokers,orvictimsof high-dose,acutesmokeintoxication[e.g.,smokeinhalationvictims(SII)], areentirelyunknown.Futureprogressawaitstranslationofknowledgeof thechemistryofacroleinadductionofbiologicalmacromolecules(e.g., DNA,protein—seeSection 2 below)intorobustanalyticalmethodsthat allowthe‘‘biologicallyeffectivedose’’tobeestimatedforacroleinunder differentexposureconditions(Figure1).
Figure1 Applicationofthe‘‘toxicologicalparadigm’’totheroleofacroleininvarious humanrespiratorydisorders.Acroleinmaycontributetolunginjuryfollowingeither acute,high-doseexposuretosmokeinfirevictims(e.g.,smokeinhalationinjury)or afterchronicexposuretotobaccosmoke(e.g.,COPDorlungcancer).Seetextfor details.
1.3.Involvementinrespiratorydiseases WhiletrialasachemicalwarfareagentinWorldWarIraisedtheprospect ofacute,single-agentexposuretoacrolein,thepotentialforsimilar exposuresinthemoderneraseemlimitedtospecificworkplacesettings (e.g.,factoriesinvolvedinthesynthesisofacrylics,etc).Ongoingconcern overthepulmonaryeffectsofinhaledacroleinthusmostlycentersuponits presenceinsmokeformedfromvario uscombustiblematerials.Acomplex mixtureofparticulates,gases,an dorganics,thecompositionofsmoke variesaccordingtotheflammablematerial,ignitiontemperature,oxygen availability,rateofincineration,etc [10] .Whilethesimultaneousexposuretomultiplecombustionprodu ctscomplicatesevaluationofthe contributionofacroleintosmoketoxi city,possibletoxicologicalinteractionsinvolvingacroleinduringsmokeinhalationarebeyondthescope ofthisreview.
Respiratorydiseasesinwhicharoleforacroleinissuspectedinclude chronicdisordersplaguingindividualsexposedtofirst-orsecond-hand tobaccosmokeoveranextendedperiodaswellasacute,life-threatening disordersseeninfireandSIIvictims.
1.3.1.Chronicobstructivepulmonarydisorder Chronicobstructivepulmonarydisorder(COPD)isadevastatingcondition thatdespitethesuccessofsmokingcessationinitiativesinmanycountriesis ofincreasingglobalsignificance [11].WhileCOPDoccursmostfrequently amongtobaccosmokers,chronicexposuretootherformsofairpollution caninduceasimilarsyndrome.ThelungpathologyunderlyingCOPDisan exaggeratedmanifestationofthelow-gradeinfiltrationofinflammatory cellstothebronchiandperipherallungof‘‘normal’’smokers.Inthe minorityofsmokerswhodevelopCOPDthisprocessisamplifiedand accompaniedbyatissue-remodelingprocessthatproduceslunginjury resemblingchronicbronchitis.OtherfeaturesofCOPDincludemucus hypersecretion,smallairwaysobstruction,emphysematousalveolar damage,andpulmonaryhypertension [12].Theairflowlimitationand ‘‘breathlessness’’thatischaracteristicofCOPDismainlyduetopermanent enlargementofdistalrespiratoryairspacesfollowingdestructionofthe alveolarwalls [12].WhilethepathobiologicalbasisforCOPDiscomplex, itappearsthetransitionfroma‘‘normal’’inflammatoryresponsetoan abnormalinnateandadaptiveimmuneresponseisdrivenbydisparitiesin theprotease–antiproteaseandoxidant–antioxidantbalanceattendingthe influxofneutrophils,macrophages,andlymphocytes [12].Togetherwith proinflammatorycytokineproductionandothertissueresponses,these processestriggerapoptosisandfailureofrepairmechanisms,elicitingalveolardestructionandremodelingofthesmallairways.
IndicationsthatacroleinparticipatesinCOPDincludethefindingthat itisthetobaccosmokeconstituentwhichmoststronglystimulatesmucus hypersecretion.ThemolecularbasisforthisresponseisreviewedinSection 3.2.Acroleinisalsoimplicatedinthemacrophageadhesionandactivation thataccompaniesthealveolardestructionphaseofCOPD [13].Microarray analysisofCOPDlungsamplesalsoidentifiedEgr1,atranscriptionfactor thatisstronglyupregulatedbyacroleininlungcelllines(Section 3.2 below),asahighlypredictivemarkerofCOPD [14].AsEgr1maypromote theexpressionofproinflammatorycytokinesaswellasmetalloproteinase activation[15,16],acroleinmayfacilitateCOPDpathogenesisbydriving theexpressionofthisproinflammatorytranscriptionfactor.
1.3.2.Tobacco-relatedlungcancer Lungcancerinsmokersisinsidioussinceittypicallydevelopsoveraperiodof years,partlydrivenbytheaccrualofmutationswithincriticalgrowth regulatorygenes [17].Countlesscareerswithinthetoxicologyandcancer researchcommunitieshavebeendevotedtoclarifyingthepathwaysof metabolicactivation,detoxication,andDNAadductionbyclassictobaccobornecarcinogenssuchaspolycyclicaromatichydrocarbons,nitrosamines, andaromaticamines.Whilethecombustionofacigarettetypicallyreleases justnanogramquantitiesofthesecarcinogens,thecumulativedosesreceived overa40-yearperiodbyapack-a-daysmoker(i.e.,~290,000cigarettesin total)resemblethoseinducingtumorsinrodents[12,18].Howeverlongstandingassumptionsconcerningthetobaccosmokeconstituentsofgreatest toxicologicalsignificancearechallengedbyrecentsuggestionsthatacrolein stronglycontributestolungtumorigenesisinsmokers [19].Comparedto ‘‘classic’’tobaccocarcinogensthatrequireCYP-catalyzedbioactivationto formDNAadducts,acroleinisadirect-actinggenotoxicantthatispresentin smokeat1000–10,000-foldgreaterconcentrations.Itspotentialrolein tobacco-relatedlungcancerisexploredfurtherinSection 3.4.
1.3.3.Smokeinhalationinjury Despitesignificantadvancesinburnsmanagement,thepathologicaleffects ofinhaledsmokeremainsignificantcontributorstomorbidityandmortality amongfirevictims [20].Astheportofentry,thelungisparticularly vulnerabletolife-threateningpulmonaryedemauponinhalationoflarge dosesofsmoke[20,21].TheclinicalprognosisafterSIIisoftenpoor,with mortalityratesof45–78%reportedinsomestudies[20–22].Respiratory failureinsuchpatientscaninvolvealatencyof24–72hfollowedbysevere bronchorrhea,bronchospasm,breathingabnormalities,andretrograde alveolarflooding[20–22].Thepathobiologyofpulmonaryedemain acutelunginjuryinvolveslossofvascularandepithelialpermeabilityas wellasdisruptionofionandfluidtransportersthatnormallymaintain‘‘dry’’
alveolarairspaces[23,24].Productionofproinflammatorycytokinesalso accompaniestheacutephaseofSII [25].
ThedataimplicatingacroleinasamediatorofpulmonaryedemainSII islong-standing,includingearlycanineworkbyZikria etal. whichcomparedtheedematogenicpotentialofsmokeproducedfromdifferentcombustiblematerials [26].Acroleinlevelsinsmokefromburningcottonwaste andwoodwereover50-foldhigherthaninsmokeformedduringkerosene combustion [26].Inclearcontrasttoanimalsexposedtothetwohighyieldingsmoketypes,nofatalitiesoredemaoccurredindogsexposedto kerosene-derivedsmoke [26].Later,usinganesthetizedsheep,Hales etal performedsomecompellingstudieswithsyntheticsmokecontaining differentreactivesmokeconstituentsthatincriminatedacroleinasthe primaryedematogenicconstituent[27–29].Despitetheselong-standing observations,existingSIItherapiesremainpoorlydirectedagainsttherole oftoxicsmokeconstituentssuchasacrolein.
1.3.4.Cyclophosphamide-inducedlunginjury
Inaminorityofpatientstheanticancerdrugcyclophosphamidecaninduce pulmonarytoxicitythatinvolvesendothelialswelling,intra-alveolarexudation,interstitialinflammation,pulmonaryfibrosis,andfibroblastproliferation [30].AsamajorCYP-derivedmetaboliteofcyclophosphamide,arole foracroleininthepulmonarypathologyissuggestedbylong-standing animalstudies [31]
2.1.Chemicalproperties
Thetoxicityofacroleintowardlungtissuesreflectsitsstrongreactivitywith electron-densecentersintargetmacromolecules.Inchemicalterms,acrolein isatype-2alkene,possessingapolarizablealkene(–C=C–)bondinclose proximitytoanelectron-withdrawingcarbonylgroup. -Electronmobility withinthisconjugatedsystemcreatesastrongelectrondeficiencyatthe terminal b-carbon [32].AccordingtotheHard–Softtheoryusedtoexplain theinteractionofreactiveintermediateswiththeircellulartargets [33], acroleinisa‘‘soft’’electrophilethatreactspreferentiallywith‘‘soft’’nucleophiliccenters.Withlargeatomicradiiandpolarizablevalenceelectrons, sulfur(S)atomsarethesoftestnucleophileswithincellsandtissues [32].In recentstudiesofthereactivityofvarious a,b–unsaturatedcarbonylcompoundswithmodelS-containingnucleophiles,astrongcorrelationwas observedbetweentheelectrophilicindex(o)andfunctionaldeficitselicited bythecarbonylcompoundsinasynaptosomalbioassaysystem [34].Acrolein
2.ChemicalToxicologyofAcrolein
wasmorereactivewithsoftnucleophilesthanothertype-2alkenessuchas 4-hydroxynonenal,methylvinylketone,oracrylamide [34].Asthemajor low-massS-containingcellularnucleophile,glutathioneisakeybiological targetforacrolein,generating S-glutathionylconjugatesthatundergosubsequentrenalproteolysisandN-acetylationtourinarymercapturates [1] Sincetheseconjugatesundergospontaneous b-eliminationreactionsto releaseacroleintheyareproposedtofunctionas‘‘toxictransporters,’’circulatingfromtheportofentrytoinducedamageatdistalsites [35]
2.2.Chemistryofproteindamage Acroleindisplaysstrikingchemicaldiversityduringreactionswithproteins (Figure2).Thefollowingdiscussionwillhighlightthatmoreworkis neededtodeterminewhichofthemultipleproteinadductsformedby acrolein invitro contributetoitspathologicaleffectswithintheintactlung.
Figure2 Proteinmodificationbyacroleinpotentiallygeneratesadiversityofadducts atlysineresiduesaswellasspeciesatcysteine,histidine,andarginineresidues.The invivo significanceofindividualadductsawaitsconfirmationviadefinitiveassay methods.Seetextfordetails.
2.2.1.Cysteine DuetoitsfavoredreactivitywithS-containingnucleophiles,acroleinreadilyformscysteineadductsduringMichaeladditionreactionswithproteins [36–38].Adductionofaccessiblecysteineresiduesonsuchdiversecellular targetsastranscriptionfactors(e.g.,NF-k B),ionchannels(e.g.,TRPA1), andcaspaseslikelyunderliemanyofthecellulareffectsofacrolein[39–41]. Todate,quantitativeinformationconcerningthelevelsofcysteineadducts inlungproteinsfromacrolein-exposedanimalsorhumansislacking. Nonetheless,sinceMichaeladductsatcysteinearereadilyderivatized withthecarbonyl-modifyingreagent2,4-dinitrophenylhydrazine,itis likelythatcysteine–acroleinadductsaremainspeciesdetectedduring Westernblottingwithanti-DNPseruminacrolein-exposedlungcells [42].However,thelackofspecificityofthisapproachensuresMichael adductsathistidineandlysinegroupsarealsodetectedduringtheseassays.
Onefactorcomplicatingdetectionofcysteine–acroleinadductswithin tissuesistheirlikelyinstability.InA549lungcellsallowedto‘‘recover’’ fromabrief(30min)exposuretoacrolein,carbonylatedadductsdecline quicklyover2–3h,withneitherproteasomalnorlysosomalinhibitors slowingtheloss [42].Severalmechanismsmightunderliesuchprotein decarbonylation,includingretrogradeMichaeladditionreactionsaswell asadductconsumptionduringcross-linkingreactions.Recentstudiesofthe fateofacroleinadductsinamodelpeptide(insulin)revealedthatMichael adductsatcysteineunderwentrapidsecondaryreactionstoformSchiff adducts,withmassspectrometry(MS)/MSanalysislocalizingthese speciestotheN-terminusofmodifiedpeptides [37].Priorblockingof theN-terminusincreasedthestabilityofcysteineadductsbyimpeding Schiffadductionoftheterminalnitrogen [37].MinimalSchiffadduction occurredonpeptidesthatdidnotcontaincysteineresidues [37].The authorsconcludedthatproteinscontainingcysteineneartheN-terminal domainmightbeespeciallypronetoadductmigration,apossibilitythatwas confirmedusingtwomodelproteins [37].Whilesuchreactionshaveyetto beconfirmedwithinbiologicalsettings,thepossibilitythatcysteineadducts formedbyacroleinundergosecondaryreactionscomplicatesthestudyof proteinmodificationbythissubstance,andfurtherexplainsthepaucityof quantitativedataconcerningthelevelsofacrolein–cysteineadductswithin animalorhumanlung.
2.2.2.Lysine Thechemistryoflysinemodification,whilewellstudied invitro (Figure2),is alsosubjecttouncertaintyconcerningwhichoftheknownadductsareof in vivo relevance.MSofproductsformedduringreactionsbetweenacroleinand Na-blockedlysineormodelpeptideshaveconfirmedexpectedMichael adductiononthe e-NH2 group(propanal)aswellasabis-Michaeladducted
speciesinvolvingthesamenucleophile[43,44].MinorSchiffbaseformation (aldimine)alsooccurredonlysinesidechainsandtheterminal-NH2 of modelpeptides [44].Whilemono-andbis-Michael-adductedspeciespredominateintheearlystagesofpeptidemodificationbyacrolein,anadditional heterocyclicspeciesalsoforms,Ne-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine, Figure1),withananalogousadductalsodetectedonthe N-terminus [44].Thesecyclicadductsformviacondensationofbis-Michael adductedspecies [43].FDP-lysinereactsrapidlywithglutathione [45],raising thepossibilityitreactswithproteinnucleophilestoformcross-links, althoughacyclicMichaeladductsarealsolikelymediatorsofproteincrosslinking[42,46].Anothercyclicspecies,Ne-(3-methylpyridinium)lysine (MP-lysine, Figure2)formsviasequentialMichaelandSchiffadductionof asinglepeptidyllysinebytwoacroleinmolecules[47,48].
Whetheracrolein–lysineadductsform invivo hasbeenexploredusing antibodiesthatrecognizecycliclysineadducts,withseveralstudiesreportingtheformationofmodifiedproteinsintheaffectedtissuesofseveral degenerativediseases [49].Recently,arabbitpolyclonalantibodyagainst acrolein–lysineadductswasusedtodetectdamagedproteinsinthelungsof miceafter5hexposuretotobaccosmokeoracrolein [50].Acrolein increasedtheadductionintensityofseveralproteins(~75,130,150,and 250kDa)inthelungsofexposedanimals,withmodifiedproteinsalso detectedinplasmaandperipheraltissues [50].Futureidentificationofthe pulmonarytargetsmayprovideusefulinsightsintothepathogenetic mechanismsunderlyingacroleinpneumotoxicity.
Despitethesepromisingobservations,quantitativedataobtainedusing structurallydefinitiveanalyticalmethodsislackinginrelationtotissuelevels ofanyknownacrolein–lysineadduct[43,45,47].Theabsenceofanalytical confirmationofthepresenceofcyclicacrolein–lysineadductswithin biologicalsamplesisproblematic,giventhatantibodiesraisedagainstacrolein-adductedlysinegroupsmayalsodetectacyclicspecies [51].Theadvent ofspecificanalyticalmethodspermittingquantificationofthemultiple acrolein-derivedlysineadductswithinbiologicalsampleswouldprovide importantvalidationofadductionchemistrythattodatehasbeendescribed undertesttubeconditionsorsurmisedonthebasisofimmunochemical data.
2.2.3.Otheraminoacidtargets Recentworkusingpurifiedactinasamodelproteintargetaswellashigh molarratiosofacroleinestablishedthatmodificationofseveralhistidine residues(His40,His87,andHis173)onlyoccurredaftersaturationofadductionofthemostsusceptibleresidue,Cys374 [52].AroleforeitherMichaelorSchiff-typehistidinyladductsintheformationofcross-linkedoligomers wasalsodemonstratedusingthe b–chainofinsulinasamodeltarget [46].
Inthemainhowever,thechemistryofhistidinemodificationbyacroleinis poorlycharacterized.
Themostbasicofallaminoacidsidechains(pKa = 12.48),theguanidinogroupofarginineisfullyprotonatedatphysiologicalpH,afactor thatisthoughttoprecludemodificationbysoftelectrophilessuchas acrolein.DuringourrecentMSstudiesofpeptidemodificationbyacrolein,noadductsweredetectedonarginineresiduesunderexposure conditionsthatcausedextensiveadductionoflysineandN-terminal amines [44].Surprisinglyhowever,inrecentworkthatexploredthe distributionofadductswithinthep50subunitofNF-k B,aknowntarget foracrolein,useofMALDI-TOFMSidentifiednovelcyclicadductson twoarginineresidues,Arg230 andArg307 (Figure2) [53].Thissuggests thatlocalsequencecontextorotherfactorsmaycreatezonesofincreased pHwithinproteinmicroenvironmen tsthatenhancethereactivityof specificargininesidechains,anintriguingreminderofthedifficulties accompanyingextrapolationfromsim pleexperimentalsystemscomprisingmodelaminoacidsorpeptidesto intactproteinswithinthetissue setting.
Collectively,acroleinisanefficientelectrophilethatattacksmultiple residueswithinsusceptibleproteins,andwhilecysteinegroupsarepreferred targetsonkineticgrounds,theinstabilityofthesespeciesmayensurethat adductioneventsatotherresidues(e.g.,lys,his,andarg)maybeof comparabletoxicologicalrelevance.Atpresent,thereisadearthofqualitativeorquantitativeinformationconcerningproteinadductionwithinthe epitheliumorparenchymaofrespiratorytissuesduringeitherchronicor acuteexposuretoacrolein.Asolidunderstandingofacroleintoxicityinthe lungwouldthusrequireknowledgeofintraorganproteinadductdistributionunderdifferentexposureconditions.Toreiterate,developmentof sensitiveassaysforthevariousspeciesofproteinadductsformedbyacrolein wouldfacilitateachievementofthesegoals.
2.3.ChemistryofDNAdamagebyacrolein SincenoneofthenucleophilespresentinDNAaresufficiently‘‘soft’’to allowreactionsatratesmatchingthoseoccurringatcysteine,protein adductionisexpectedtopredominateunderconditionsoflowacrolein exposure,whileDNAadductsseemmorelikelytoformathigherlevelsof exposure.Suchassumptionsarespeculative,however,sincenostudieshave compareddose–responserelationshipsforDNAadductionversusprotein modificationinacrolein-exposedlung.
AcroleincanreactwithallfournucleobaseswithinDNA[54–56]. Guanine,themostnucleophilicDNAbase,isthemajortarget,undergoing conversiontotwohydroxy-1,N2-propanodeoxyguanosineadducts,hereafterdesignated a-and -OH-Acr-dGua[57,58](Figure3).
Figure3 AcroleinattacksguaninebasesinDNAtoformtwoisomericspecies,both ofwhichhavebeendetectedinhumantissues.Seetextfordetails.
Giventhelong-standinginterestinusingDNAadductsasmarkersof exposuretogenotoxiccarcinogens,someprogresshasbeenmadeinthe quantitationofacrolein-derivedDNAadductsinrespiratorytissuesusing either 32P-postlabelingorMS-basedtechniques.Duetothelowlevelsof DNAadductsinthehumangenome,suchapproachesaretypicallycombinedwithadductenrichmentsteps.Thesesensitiveproceduresrequire scrupulousattentiontoprotocol,witnessedbytherecentfindingthatlow levelsofacroleininwaterusedtopreparesamplebuffersmayinfluence baselinelevelsofacrolein–guanineadductsinDNA [59]
InworkconductedbyChungandassociatesusingthe 32P-postlabelingassay,comparativelyhighlevelsof -OH-Acr-dGuaweredetectedin DNAfromthelungsofcontrolratsthathadnotbeendeliberatelyexposed toacrolein [60].Levelsofthe a-isomerwerebelowassaydetectionlimits. Thesamegroupalsousedthisapproachtoexploretheeffectsofsmoking onadductlevelsinhumans [61].Consistentwiththepresenceofacroleinin tobaccosmoke, -OH-Acr-dGualevelswereelevatedthreefoldinDNA collectedfromtheoralcavitiesofasmallsampleoftobaccosmokers (N = 11)relativetononsmokers(N = 12,adductlevelswere1.36 + 0.9 intheformervs.0.46 + 0.26 mmolmol–1 guanineinthelattergroup, p = 0.003) [61].
NewerMS-basedmethodsallowmorepreciseestimationofDNA adductswithinbiologicalsamples [62].Inthesoledefinitivestudyof acrolein-derivedmacromolecularadductsinhumanlung,Hechtand associatesusedliquidchromatography–electrosprayionizationtandemMS tomeasureboth a-and -OH-Acr-dGuainlungbiopsiesfromsmokers andex-smokers [63].Thetwoadductsweredetectedatcomparableconcentrationsinallsamples [63].Moreover,nodifferencesinadductlevels wereevidentbetweensmokersandex-smokers,nordidadductlevels correlatewithurinarynicotineorcotininelevels,twocommonmarkers oftobaccousage [63].Theseobservationsarepuzzlinggiventheknown
declineinurinaryacroleinmetabolitesonsmokingcessation [64].The inabilitytoobserveaneffectofsmokingcessationonpulmonaryDNA adductlevelsmaypartlyreflectlimitedknowledgeofDNAadductdistributionswithinhumanlungortherepairkineticsfortheselesions.The impactofalternativeenvironmentalandendogenousacroleinsourceson pulmonaryDNAadductlevelsisalsounknown.Clarifyingsuchissuesis clearlycrucialtotheongoingdebateconcerningtheroleofacroleinin smoking-relatedlungcancer(seebelow).Butworkisalsoneededtoclarify theextentandsignificanceofDNAadductionduringexposuretoacrolein underconditionsotherthanthoseassociatedwithchronictobaccosmoking,suchasSII(Figure1).
2.4.Macromolecularcross-links Asabifunctionalelectrophile,initialreactionsofacroleinwithproteinor DNAgenerateaMichaeladductthatmayparticipateinsecondaryreactions withneighboringnucleophilestoformcross-linkedspecies(Figure4).The formationofinter-andintrastrandDNA–DNA,protein–DNA,and protein–proteincross-linkscanallaccompanyexposureofrespective
Figure4 Asabifunctionalelectrophile,acrolein-adductedcellmacromoleculescan reactfurthertoformDNA^DNA,DNA^protein,orprotein^proteincross-links. Seetextfordetails.
biomoleculestoacrolein,althoughcross-linkedspeciesinvolvingguanine adductsarebestcharacterizedintermsoftheirchemicalandbiological properties [58]
Giventhe‘‘crowded’’natureofthecytosoliccompartmentacroleinis likelytoelicitextensiveproteincross-linkingwithinthissubcellularenvironment,buttodatethechemistryofthisprocessispoorlycharacterized. AspecieslikelyformedviareactionofMichael-adductedhistidineadducts withthe e-NH2 groupoflysinewasrecentlyidentifiedasacontributorto protein–proteincross-linkingwithinamodelexperimentalsystem [46]. Recentworkinourlaboratoryestablishedthatacroleinreadilygenerates intramolecularprotein–proteincross-linksinvolvingtwocommontargets forelectrophiles,heatshockprotein-90(hsp90)andtheintermediate filament(IF)vimentin(seebelow)[42,65].Althoughtheformationof proteinaggregateswaspotentiallyinhibitedbycytoprotectiveelectrophile scavengers,therelevanceofprotein–proteincross-linkingtothe invivo pulmonarytoxicityofacroleinisunknown.
3.CellularToxicologyofAcrolein 3.1.Proteintargets Identifyingproteinsthatincuradductionduringthebiotransformationof xenobioticstoreactiveintermediatesisakeyobjectiveinmoderntoxicologicalresearch,drivenlargelybytheexpecta tionthatsuchknowledge canclarifythecellularpathwaysthataredisruptedduringtheonsetofany organ-directedpathology [66] .Asareactiveelectrophile,acroleinattacks numerouscellproteins,buttodate theidentitiesofthosesustaining damageinthelungduringinhalationalexposuretoacroleinorsmoke arepoorlydefined.Ashighlightedabove,inoneofthefeweffortsto characterizeproteindamageduringpulmonaryacroleinexposure,Conklinandassociatesestablishedthatahandfuloflungproteinsinmice sustainedmodificationduringa5hinhalationalexposureto5ppm airborneacrolein [50].Sincetheseresearcherswereprimarilyassessing thecardiovascularimpactofinhalationalacroleinexposure,neitherthe proteinidentitiesnortheextentoflunginjuryaccompanyingacrolein exposurewasestablished.
3.1.1.NF-k Bpathway Incontrasttothelackofinformationconcerning invivo pulmonarytargets, anumberofdamagedproteinshavebeenidentifiedinacrolein-exposed lungcells.Duringastudyofitseffectoninflammatoryprocessesin immortalizedhumanbronchialepithelial(HBE-1)cells,Valacchiand
associatesfoundthat,inkeepingwithpriorobservationswithotherreactive carbonyls,acroleinsuppressedtheactivationofNF-k Bsignalingbythe proinflammatorycytokineTNFa [67].Activatedinresponsetonumerous noxiousstimuli,thetranscriptionfactorNF-k Bdrivestheproductionof inflammatorycytokinesinpulmonaryepithelialcells.Themostcommon formofNF-k Bexistsasaheterodimerbetweenp50andp65,withthis complexmainlylocalizedincytoplasminassociationwithI-k B(the bindingofwhichmasksnuclearlocalizationsequenceswithinp65).During exposuretoproinflammatorystimuli,I-k Bundergoesphosphorylation, ubiquitination,andproteolyticdegradation,allowingNF-k Bmigration tothenucleuswhereitinteractswith k Bmotifsintargetgenes [68].One suchgeneistheproinflammatorycytokineinterleukin-8(IL-8),apowerful chemoattractantforneutrophilsandotherimmunecells [69].InHBE-1 cells,a30minexposuretoacroleinpriortochallengewithTNFa reduced cellularIL-8secretionoverthefollowing24hwhilealsosuppressingIL-8 genetranscripts [67].Useofanelectrophoreticmobilityshiftassayrevealed thatacroleininhibitedTNFa-inducedrelocationofNF-k Btonuclear extracts,aneffectthatwasaccompaniedbyincreasedcellularlevelsofits bindingpartner,I-k Ba [67].Themechanismunderlyingtheseeffects appearedtoinvolveacroleinadductiononIKKb,thekinasethat regulatesthephosphorylationstateandcellularpersistenceoftheNF-k B/ I-k Bcomplex [67].Thisfindingisintriguinggiventhesusceptibilityofp50 toacroleinadductiondiscussedinSection 2.2.3[53].SinceotherthiolreactiveelectrophilesareknowntomodulateIKKb activityviareactions withanactivesitecysteineintheactivationloop [70],thesefindings highlightamechanismwherebythereactivityofacroleindisruptsafundamentalbiologicalprocess(immunesignaling)viaactionsatspecificcell targets.Whiletheseconclusionsarebroadlyconsistentwithfindingsin otherlaboratoriesconcerningdisruptionofNF-k Bsignalingduringacroleinexposureincellsofpulmonaryorigin[71,72],futureworkisneededto confirmthe invivo relevanceoftheseobservations.
3.1.2.Intermediatefilaments Inrecentpreliminaryworkconductedwithaviewtoidentifyingnovel targetsforacroleininhumanlungcells,weusedcellularfractionation, Westernblotting,andpeptidemassfingerprintingtoidentifyseveralIFs astargetsforacroleininA549lungcells[65].Amongthemostsusceptible familymemberswasvimentin,anIFthatparticipatesinmanycellular processesandisaknowntargetfordiverseelectrophiles[65,73–75]. Othertargetsincludedvariouskeratins,withthelowabundanceIFkeratin-7provingespeciallyvulnerabletoacrolein,aswellasthemoreabundant andstructurallyimportantIFkeratins-8and-18[65].Damagetothemost abundantkeratinswasaccompaniedbyalossofcellularadhesivestrengthin
A549cellmonolayers,afindingthatisconsistentwiththemechanical functionsofIFinprovidingtensilestrengthwithincellularandtissue networks[76,77].TodeterminewhetherIFsustaindamageinother epithelialcellsofpulmonaryorigin,wecomparedpatternsofprotein carbonylationinA549cells(alveolarorigin)toCalu-3cells(bronchiolar origin)followinga60minexposuretotwoconcentrationsofacrolein (Figure5).Thelowerconcentrationofacrolein(25 mM)hadnoeffect uponcellATPlevelsduringa4hincubationineithercelllines,whereas thehigherconcentration(150 mM)diminishedcellATPinamanner consistentwiththeonsetofovertcelldeath(Figure5A andB).While expressionofthetwomostvulnerableIFseemedtodifferbetweenthetwo celllines(relativetoA549cellsvimentinwaslessabundantinCalu-3cells whilekeratin-7wasmoreabundant),theextentofadductionatthesetwo targetswasconsistentwithitslevelofexpressionintherespectivecelltypes (Figure5CandD).GiventhatIFappeartobereliabletargetsforacroleinin epithelialcellsoriginatingfromdifferentzonesoftherespiratorytract, futureworkisneededtodeterminewhethertheseproteinssustaindamage inintactlungduring invivo exposuretoacrolein.
Keratin-7
Keratin-8
Keratin-18
Keratin-7
Keratin-8
Keratin-18
Figure5 Theintermediatefilamentsvimentinandkeratin-7aretargetsforacrolein inlungcellsofalveolar(A549)andbronchiolar(Calu-3)origin.ATPmeasurements weremadeaftera4hexposuretoacrolein(panelsAandB)whileproteincarbonyls weredeterminedfollowinga30minexposure(panelsCandD).See[65]fora descriptionoftherelevantexperimentalmethods.
Vimentin
A549Calu-3 A549Calu-3
Vimentin
Acrolein conc. (μM)
Acrolein conc. (μM)
(A) ATP - A549 cells
(B) ATP - Calu-3 cells
(C) IF protein carbonyls
(D) Coomassie blue
3.2.1.AREgenes
Cellstypicallyup-anddownregulategeneclustersinresponsetonoxious chemicals,withanalysisoftheaffectedpathwaysprovidingpredictiveinsights intothetoxicologicaleffectsthatmightaccompanyexposuretothesubstance [78–80].Interestinthetranscriptionalresponsesofcellstoacroleinhas typicallyfocusedonmembersoftheclassicantioxidantresponseelement (ARE)locus.Thiskeyadaptivepathwayiscontrolledbynuclearfactorerythroid2-relatedfactor2(Nrf2),acap‘n’collar(CNC)basic-region leucinezipper(bZIP)transcriptionfactorthatnormallyinteractswiththe intracellularredoxsensorKEAP1 [81].Underregularconditions,Nrf2 transcriptionalactivityissuppressedduetocytosolicsequestrationbyactinassociatedKEAP1,butexposuretooxidantsandelectrophilescausesNrf2 releaseanditsmigrationtothenucleus,promotingtheexpressionofupto 100cytoprotectivegenesthatcontainAREsequencesintheirpromoters [81,82].ThemechanismsunderlyingthelossofKEAP1-mediatedrestraint onNrf2activityonexposuretoelectrophilesissubjecttodebate [83] AcroleinisanespeciallyeffectiveNrf2inducerinlungcells,stronglyactivatinghallmarkAREgenessuchashemeoxygenase-1(HO-1),NAD(P)H: quinoneoxidoreductase-1(NQO1),glutathione-S-transferase(variousisoforms),andglutamate-cysteinesynthetase(GCS) [83].Recentfindingsin HBE1cellssuggestaroleforvariouskinasesinacrolein-inducedinductionof arangeofNrf2-drivengenes,includingmitogen-activatedprotein(MAP)andPI3-kinasefamilymembers [84].Ingeneral,increasedexpressionof Nrf2-drivenAREgenesislikelyto‘‘blunt’’thetoxicconsequencesaccompanyingrepeatedacroleinexposure.
3.2.2.Mucin-relatedgenes
Theroleoftranscriptionalresponsesinmediatingthedeleteriouseffectsof inhaledacroleinisalsoofinterest.Moreneedstobedoneinthisregard,buta usefullineofrecentresearchhasexploredtheroleoftranscriptionaleventsin themucoushypersecretionthatoccursintheairwaysofanimalsupon inhalationalexposuretoacrolein [85].Mucusisrichinhighlyglycosylated mucinproteinsproducedbygobletcellsliningsecretoryductsinthelarge airways.Mucinsprotecttheepitheliumagainstdamagebyinfectiousparticles andnoxiousairbornesubstances,butexcessproductionisdeleteriousin diseasessuchasCOPD.Althoughseveralmucingenesarepresentinthe humangenome,mucin5,subtypesAandC(MUC5AC)appearmost importantinhumanairwaysecretions.MUC5ACisupregulatedbynoxious substancesincludingacrolein,withstronginductionseeninthelungsofmice aftera4-weekexposureto2ppmairborneacrolein(6hperday,5daysa week) [86].LowconcentrationsofacroleinalsoupregulatedMUC5AC mRNAtranscriptsinNCI-H292humanlungepithelialcells [87].
MUC5ACupregulationwashighlysensitivetoacrolein,occurringatconcentrationstwo-tothreefoldordersofmagnitudelowerthanthosethat depletedcellularglutathione [87].MUC5ACupregulationbyacroleinproceedsviaamultistepprocessinvolvingactivationofthemitogen-activated proteinkinase(MAPK)pathwaybyepidermalgrowthfactorreceptor (EGFR)ligandsformedviatheactivityofvariousmatrixmetalloproteinases includingADAM17,MMP-9,MMP-12,andMMP-14 [88].Thispathway wasconfirmedusingMAPKinhibitors(e.g.,PD98059)andanti-EGFreceptorantibodiestosuppressacrolein-inducedMUC5ACupregulationinculturedcells [87].SinceMAPKactivationunderliesothercellularresponsesto acrolein,thispathwaywillbeexploredfurtherinSection 3.3 below.
3.2.3.Egrfamilygenes Transcriptionalresponsestohigherconcentrationsofacroleinduringthe onsetofcelldeathmightdifferfromthoseelicitingmucushypersecretionat lowerconcentrations.Toexplorethisissueweusedmicroarraystostudy mRNAprofilesinA549humanlungcellsafter1,2,or4hexposurestoa concentrationofacroleinthatelicitedapoptoticchangesduringa24h incubation(cytochrome c releaseandDNAfragmentation)butdidnot inducetheovertnecrosischaracteristicofshort-termexposuretohigh concentrationsofacrolein(e.g.,areductionincellATP) [89].Inkeeping withitspronouncedchemicalreactivity,acroleinalteredtranscriptlevels forhundredsofgenes [89].Consistentwithtranscriptionalresponsesto otherstrongstressors,theinitialcellularresponsewastodownregulatelarge numbersofgenes,withthemRNAlevelsfor478genesdiminishedby twofoldormoreafter1h(Figure1B).This‘‘knee-jerk’’reactionwasoffset byarapidrecoveryintranscriptlevelssothatafter2hjust172geneswere downregulatedrelativetocontrol [89].Thisrapidrecoverycouldreflect DNArepairwithindamagedpromoterstorestoretranscriptionofattenuatedgenes,oralternatively,spontaneousreversalofMichaeladductionof transcriptionfactorsinvolvedincontrollingtheexpressionoftargetgenes. After4h,geneupregulationhadcometodominatethetranscriptional response,with286transcriptsincreasedovercontrols(58.1%oftotal).
Transcriptsupregulatedbyacroleinbelongedtonumerousbiological pathwaysincludinggenesinvolvedinapoptosis,cellcyclecontrol,antioxidantandheatshockresponses,cytoskeletalmaintenance,and,forthe categorycontainingthelargestnumberofaffectedgenes,transcription [89].Themoststronglyinducedgenesincludedmembersoftheearly growthresponsefamily,knowntoencodeahomologousclassofDNAbindingproteinsbelongingtothezincfingerclassoftranscriptionfactors. ThemRNAtranscriptforthebestcharacterizedmemberofthisfamily, Egr1,wasstronglyupregulatedinatime-dependentmannerinA549cells duringacroleinexposure,aneffectthatwasshownbymicroarrayand
RT-PCRanalysis(Figure6).Thesefindingsconcurwiththeupregulation ofEgr1inA549cellsduringexposuretocigarettesmoke [90].Egr1is activatedbytheleucine-zippertranscriptionfactorc-fos,thetranscriptof whichwasupregulatedbyacroleininaconcentration-dependentmanner (Figure6).Acroleinalsoinducedstrongbuttransientupregulationofc-Fos expressionpriortotheincreaseinEgr1proteinlevels(Figure6).cFos
Figure6 AcroleinelicitsstrongupregulationofEgr1andc-fosatboththemRNAand proteinlevelsinA549lungcells.mRNAlevelsweremeasuredviamicroarrayanalysis (upperpanel)orRT-PCR(middlepanel)followingexposureto100 mMacroleinforthe timesindicatedinthefigurelegends.Eachdatapointrepresentsthemean – SEoffour independentdeterminations.Thelowerpaneldepictsimmunoblotsobtainedvia WesternblottingusingantibodiesagainstEgr1,c-fosandtheloadingcontrol b -actin (25 mgprotein/lane).See [89] fordetailsconcerningexperimentalprotocols.
activationbyacroleinislikelymediatedbytheclassicextracellularMAPK, ERK-1/2,atopicthatisexploredinSection 3.3.
ThefactthatEgr1isupregulatedbyarangeofstimuliincludingvarious stresses(e.g.,UVirradiation)andphysiologicalstimuli(e.g.,hormones, growthfactors)whilealsoparticipatingindiversebiologicalphenomena suchascellgrowth,differentiation,andmitogenesis,complicatesclarificationofthesignificanceofanypulmonaryEgr1upregulationelicitedby acrolein.Basedon invitro studies,anumberofEgr1-dependentpathways oftoxicologicalsignificanceseemplausible.Onepossibilityisthat Egr1upregulationdrivestheactivationofmatrixmetalloproteinases (e.g.,MMP-2)thatpromoteactivationoftheMAPKpathway [91]. AnotherpossibilityisthatacroleinaugmentstheroleofEgr1incontrolling theexpressionofrepressorsthatregulatetheexpressionoftight-junction proteins(e.g.,claudinsandcadherins) [92].
InsightintothepotentialnumberofEgr1-regulablegeneswassupplied duringrecentuseofchromatinimmunoprecipitationandtiledpromoter arraystoidentifyseveralthousandEgr1-bindingsitesinthegenomeof phorbolester-stimulatedTHP-1monocytes [93].Themostcommon Egr1-bindingsitescolocalizedwithhistoneacetylationsiteswithinGC-rich consensussequenceslocatednearthetranscriptionstartsitesofconstitutively activepromoters [93].Manyofthesegeneswereinvolvedintranscription andtranslationcascades,indicatingthatduringmonocytedifferentiationat least,Egr1isakeyinitiatorofinformationtransmission.Intriguingly,comparisonofthefindingsofKobosaki etal.withresultsobtainedduringstudyof UVirradiation-inducedpatternsofEgr1bindingtoDNAinhumanprostate M12cellsrevealedcomparativelyfewoverlappinggenes,mostofwhich wereinvolvedinregulatingtranscriptionandnucleicacidfunctions [93,94].ThesefindingsindicatethatthetranscriptionalprogramaccompanyingEgr1activationvariesamongcelltypesandaccordingtothespecific stimuli.SuchconclusionshighlighttheneedforcarefulanalysisofacroleininducedpatternsofEgr1bindingtogenetargetsinhumanlungcells.
3.3.Moleculareffectsofacrolein Knowledgeofcellularsignalingandcelldeathpathwaysthataredisrupted by a,b-unsaturatedaldehydesandotherelectrophilicspecieshasgreatly expandedinrecentdecades [68].Asapromiscuouselectrophile,acrolein disruptsmultiplesignalingpathways,butthosedemonstratedwithinmodels relevanttothepulmonarysettingwillreceivemostattentionhere.
3.3.1.MAPKsignaling Aswithcellularresponsestoothertoxicstresses,acroleinactivatesone ormoreofthethreemainMAPKpathways,namely,theextracellular
signal-regulatedkinase(ERK),c-JunNH2-terminalkinase(JNK),orthe p38kinasecascades.Activationofthesestress-activatedpathwayscommonlysignalsashifttoaproapoptoticenvironment,butwithinthelung canaccompanyachronicproinflammatorystate [95].EachofthesepathwaysconformstothecommonpatternofaMAPKkinasekinase (MAPKKK)activatingaMAPKkinase(MAPKK)whichactivatesa MAPKpathwaythatisvariouslylinkedtodownstreamtargetsthatinclude variousphosphorylation-dependenttranscriptionfactors.Phosphorylation ofthelattertypicallybooststranscriptionofvariousimmediate-earlygenes. InChinesehamsterovarycells,acroleinwasrecentlyshowntoactivate boththeERKandp38MAPKpathways,eventsthataccompaniedthe inductionofapoptosis [96].However,therelevanceofthesefindingsto lungcellsisuncertaingiventhatp38andERKinhibitorsdidnotblockthe inductionofclassicNrf2-drivengenesinHBE-1cells [84].
Whilep38andERKMAPKpathwaysmaynotparticipateinNrf2mediatedresponsestoacroleininlungcells,thesepathwaysmayparticipate inthechronicpulmonaryinflammationthatoccursinsmokerswhoare exposedtoacroleinoveranextendedtimeframe [97].Ashighlightedin Section 1.3.2,COPDisadebilitatingrespiratorydiseasethatmostcommonlyafflictssmokers.CentraltoCOPDpathogenesisisaninflammatory cascadeinvolvingproductionofcytokinesandchemokinesthatrecruit neutrophilsandmacrophagestotherespiratorytract [12].Thebarrageof freeradicaloxidantsreleaseduponactivationoftheseimmunecellsprogressivelydamagestherespiratoryepitheliumandadjacentmicrovasculature.ThepowerfulchemoattractantsIL-8andTNFa appeartobe especiallyimportantininitiatingandmaintainingachronicinflammatory stateduringCOPD [12]
AroleforacroleininCOPDemergedduringstudiesofthestimulatory effectsofcigarettesmokeextractsontheproductionofIL-8andTNFa by modelhumanmacrophages [98].NotonlyweretheIL-8-releasingeffects ofsmokeextractsinmacrophagesreproduciblewithequivalentconcentrationsofacrolein,butpriorbubblingwithnitrogengas(whichreduced aldehydecontentsoftheextractsby70%)producedcorrespondingreductionsincytokinereleasefromthecells [98].Likewise,coexposureofthe macrophagestocarbonyl-trappingreagentsbutnotantioxidantsblunted theIL-8releaseelicitedbycigarettesmokeextracts [98].Thesefindings wererecentlyextendedbyexploringtheroleofMAPKinsmoke-induced IL-8productioninlungfibroblastsandsmallairwayepithelialcells [97].In bothcelltypes,cigarettesmokeextractsandequivalentconcentrationsof acroleinelicitedIL-8releasethatwasattenuatedbytheacroleinscavenger MESNA.InlungfibroblaststheIL-8upregulationwasaccompaniedby phosphorylationofbothERK1/2andp38.Moreover,inhibitorsofthe upstreamsignalingproteinMEKkinase(MAPkinasekinase)andERK1/2 itselfinhibitedsmoke-inducedERK1/2phosphorylationandIL-8release
[97].Whileawaitingconfirmationwithinthewholeanimalsetting,these observationsimplicateacroleinandMAPKpathwaysaskeymediatorsof theneutrophiliaandpulmonaryinflammationthatplaguelong-term tobaccosmokers.
TheprecisemechanismsunderlyingMAPKactivationduringtheprogressiontoaproinflammatorystateinsmokersareunknown.Ideally,a suitableexplanationoftheeffectsofacroleinonMAPKpathwaysshould includedemonstrablechemicalreactivitywithmemberproteinsinthis pathway.OnepossibilitycouldbethatacroleindirectlymodifiesMAPK kinases,triggeringconformationalchangesthatpromotephosphorylation ofsubstrateproteins.Althoughaprecedentforsuchanmechanismexists forother a,b-unsaturatedaldehydes [99],literaturesupportisweakforthis mechanismasageneralpathwayofkinaseactivationbyelectrophiles.More feasibly,acroleinmightattackcriticalcysteineresiduesinphosphatasesthat regulateMAPKs,amechanismthatisconsistentwiththevulnerabilityof MAPkinasephosphatasestothioloxidants [100].Thefindingthatacrolein attacksacriticalcatalyticcysteineonakeyproteintyrosinephosphatase, PTP1B,furthersupportsthismechanism [101]
3.3.2.IL-8synthesis—up-ordownregulatedbyacrolein?
ThepreviouslydiscussedfindingsconcerningupregulationofIL-8productionbyacroleinappearcontradictoryinlightofthedatadiscussedin Section 3.1 regardingitsinhibitoryeffectsonNF-k B-drivenIL-8expressioninbronchiolarepithelialcells [67].Suchreportssuggestacroleinmay exertopposingactionsonIL-8production;aninhibitoryeffectinvolving attenuationoftheNF-k Bpathway,andastimulatoryactioninvolving activatedMAPKs.Perhapsdifferencesinexperimentaldesignbetween themodelsystemsusedintheserespectivestudiesarerelevanttothis paradoxsincethevariouslaboratoriesuseddissimilarculturemediaconditionsthatarelikelytoinfluenceexperimentaloutcomes.Inrecentwork, wefoundthatthepresenceofnucleophilicacrolein-scavengingconstituentssuchascysteineorfetalbovineserum(FBS)stronglyinfluencedthe transcriptionalresponsesofA549lungcellstoacrolein [102].Thus ~10-foldhigherconcentrationsofacroleinwereneededtoupregulatethe Nrf2-drivenHO-1genewhencellswereintoxicatedinthepresenceof nucleophilicmediaconstituentscomparedtowhencellsweremaintained innucleophile-freebufferedsaline [102].Itthusseemssignificantthatthe in vitro modelsusedtodemonstrateMAPK-mediatedupregulationofIL-8 expressionbyacroleinusedcysteine-containingRPMI1640mediasupplementedwith10%FBS,anexperimentalconditionthatlikelycaused extensivescavengingofextracellularacrolein[97,98].Incontrast,the inhibitoryeffectofacroleinonTNFa-activated,NF-k B-drivensynthesis ofIL-8inbronchialepithelialcellswasseeninanucleophile-freeexposure
Figure7 Theeffectofacroleinonpulmonaryproductionoftheproinflammatory chemokineIL-8islikelyabalanceofitseffectontwoopposingpathways. Themagnitudeofacroleinexposureisalikelydeterminantoftheoverallresponse. Seetextfordetails.
medium(Hank’sbalancedsaltsolution)whichlikelyallowedmuchhigher intracellularconcentrations [67].Theapparentlycontradictoryeffectson IL-8productioncouldthusbereconciledbythelikelihoodthataMAPKdrivenupregulationofIL-8productionpredominatesunderconditionsof lowacroleinexposure,whileathigherlevelsofexposure,adductionof proteinsthatregulateNF-k BtranscriptionalactivityeffectivelyinhibitIL-8 release.Atanygivenlevelofexposure,overallIL-8productionwillreflect theneteffectofacroleinontheseopposingpathways(Figure7).Once again,theseconclusionsmustbetestedinrelevant invivo systemsthatallow clarificationofthedosedependenceoftheeffectsofacroleinoncytokine productionwithinthelung.
3.4.Mutagenicityofacrolein ThetoxicologicalsignificanceoftheDNAdamagethatisinflictedby acroleinwithinthelunghasgeneratedsignificantrecentcontroversywithin thecontextofsmoking-relatedhumanlungcancer [19].Acroleinislong knowntoinducebase-pairsubstitutionmutationsinbothbacterialand mammaliancelllines[103–106].Uponsequenceanalysisofacroleininducedmutationsinashuttlevector-basedsystem,mutationsmainly occurredatguanineresidues,consistentwiththetendencyofacroleinto targetguanineduringreactionswithDNAtoformcyclic a-and -OHAcr-dGualesions(Figure3 andSection 2.3 above).While -OH-Acr-dG
Pulmonary IL-8 production
MAPKNFκB
Acrolein
Magnitude of acrolein exposure
Phosphatase inhibition
IKKβ adduction
appearsmoreabundantwithinthegenomeofacrolein-exposedcells, molecularstudiesonthemutagenicityofthislesionduringreplicationin mammaliancellssuggeststhisadducthaslessmiscodingpotentialthan a-OH-Acr-dG [107].Anumberof invitro studieshavereportedalow incidenceofmutationaleventsduringtranslesionalbypassof -OH-AcrdG-containingDNAtemplates[108,109].
Controversyconcerningthestatusofacroleinasamediatoroflung cancerintobaccosmokersaroseuponpublicationofapaperbyFeng etal in2006[2,19].Theseresearcherspresenteddatashowingastrongsimilarity betweenthepatternsofDNAdamagewithinacrucialmutationaltarget (thep53tumorsuppressorgene)inducedbyacroleinandthespectrumof p53mutationsfoundinlungcancerinsmokers [19].Theresearchersalso notedthatacroleinreducedthecellularcapacitytorepairDNAadducts generatedbyamajorpromutagenicspeciesformedfromtobaccocombustionproducts,benzo[a]pyrenediolepoxide.Weighingthesefindings againstthefactthatacroleinconcentrationsintobaccosmokeareseveral ordersofmagnitudehigherthanthoseofbenzo[a]pyrene,theauthors suggestedacroleindeservesgreaterattentionasamediatorofcigarette smoke-relatedlungcancer [19].
TheabovereportinspiredasubsequenteffortbyBesaratinia etal.to characterizethemutagenicityofacroleininBigBluemouseembryonic murinefibroblasts [110].Thisexperimentalsystemallowsscreeningfor chemicallyinducedmutationswithinthechromosomallyincorporated cII mutationaltargetgene.Aswithothercomparableattemptsincell-based models,thepronouncedcytotoxicityofacroleintowardtheembryonic fibroblastsconfoundedthedetectionofmutations,necessitatingstudyof mutationfrequenciesatlowacroleinconcentrations [110].Underthese conditions,nosignificantincreaseinmutationfrequenciesovercontrols accompaniedacroleinexposure [110].Followingthisreport,theworkers responsiblefortheinitialassociationofacroleinwithp53mutationsinlung cancerconductedarenewedanalysisofacroleinmutagenicityinashuttle vector-basedassaysystem [111].Suchmodelsfacilitatestudyofthegenotoxicityofelectrophilicchemicalsbypermittingdirectexposureofplasmid DNAtoreactivesubstancespriortocellulartransfection.Thisapproach allowsremovalofunreactedtoxicantsviaplasmidextractionprotocolsand thereforeminimizesdirectcellularexposuretotoxiccompounds [112]. Usingthismethodalongwithcarefulattentiontodeterminingadductlevels intreatedplasmids,theseworkersfoundaclosecorrelationbetweenmutationfrequenciesinviralprogenyandthelevelsofacrolein–guanineadducts [111].Theselatterfindingsprovokedastrongexchangeofviewsbetween thechiefinvestigatorsinvolvedintheseapparentlyconflictingstudies [113,114].Thisongoingdebatehighlightsthattheroleofacroleinin tobacco-associatedpulmonarytumorigenesisisacontroversialtopicthat challengeslong-standingassumptionsconcerningthecentralrolesofsuch
tobaccocarcinogensasnitrosamines,polycyclicaromatichydrocarbons, andaromaticamines.
3.5.Celldeathpathways Aswithothercellularresponsestoacrolein,thecelldeathpathwaysit initiatesincellsofpulmonaryoriginhavebeenmostcommonlydefined invitro.Incellsofnonpulmonaryorigin,acroleinhasbeenshowntoinduce bothnecroticandapoptoticformsofcelldeath,withevidencepresentedto suggestthelatterproceedsviabothmitochondrial(intrinsic)anddeathreceptor-mediated(extrinsic)pathways[41,115–117].InrecentworkconductedinA549lungcells,acroleininitiatedcellularchangesthatare characteristicofbothearly-andlate-stageintrinsicapoptosis,including Baxrelocationtothemitochondria,collapseinthemitochondrialmembranepotential,cytochrome c release,activationofcaspases-3and-7,and redistributionofapoptosis-inducingfactor-1tothenucleus [118]
OurrecentmicroarraystudyaddedtothecomplexityofacroleininducedcelldeathpathwaysinA549cellsbyidentifyingchangesinboth mitochondrialanddeathreceptorpathways [89].Forexample,acrolein stronglyinducedTNFSF10,animportantregulatoroftheextrinsicapoptoticpathway [89].WealsoidentifiedthenuclearreceptorNur77asa possiblepluripotentmediatorofA549celldeathbyacrolein.Nur77has analogousactionstop53,helpingmaintaincellsurvivalundernormal conditionswhileundergoingactivationbyproapoptoticstimulitoapotent pro-deatheffectorthattranslocatestomitochondriawhereitinteractswith Bcl-2toelicitcytochrome c release [119].Nur77mayalsotranslocatetothe endoplasmicreticulumtoactivateanovelnonmitochondrialpathwayof intrinsicapoptosis [120].Moreover,Nur77canactivatereceptor-mediated apoptosisinsomecells[121,122].
Clearly,moreworkisneededtodefinethemechanismsoflungcell deathprevailingunderconditionsofbothchronicandacuteacrolein exposurewithinthe invivo settingwherelittleisknownconcerningthe moleculareventsthatmediatetheepithelialdamageelicitedbysmokeborneacrolein.
4.RespiratoryEffectsofAcuteInhaled Asapotentirritanttomucosalmembranes,inhalationalexposureto airborneacroleinproducesdose-relatedsymptomsincludingnoseand throatirritation,andimportantacutereflexresponsesincludingreduced
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Paleogeography. When geologists began to perceive the vast significance of Hutton’s doctrine that “the ruins of an earlier world lie beneath the secondary strata,” and that great masses of bedded rocks are separated from one another by periods of mountain making and by erosion intervals, it was natural for them to look for the lands that had furnished the debris of the accumulated sediments. In this way paleogeography had its origin, but it was at first of a descriptive and not of a cartographic nature.
The word paleogeography was proposed by T. Sterry Hunt in 1872 in a paper entitled “The Paleogeography of the North American Continent,” and published in the Journal of the American Geographical Society for that year. It has to do, he says, with the “geographical history of these ancient geological periods.” It was again prominently used by Robert Etheridge in his presidential address before the Geological Society of London in 1881. Since Canu’s use of the term in 1896, it has been frequently seen in print, and now is generally adopted to signify the geography of geologic time.
The French were the first to make paleogeographic maps, and Jules Marcou relates in 1866 that Elie de Beaumont, as early as March, 1831, in his course in the College of France and at the Paris School of Mines, used to outline the relation of the lands and the seas in the center of Europe at the different great geologic periods. His first printed paleogeographic map appeared in 1833, and was of early Tertiary time. Other maps by Beaumont were published by Beudant in 1841–1842. The Sicilian geologist Gemmellaro published six maps of his country in 1834, and the Englishman De La Beche had one in the same year. In America the first to show such maps was Arnold Guyot in his Lowell lectures of 1848. James D. Dana published three in the 1863 edition of his Manual of Geology. Of world paleogeographic maps, Jules Marcou produced the first of Jurassic time, publishing it in France in 1866, but the most celebrated of these early attempts was the one by Neumayr published in 1883 in
connection with his Ueber klimatische Zonen während der Jura- und Kreidezeit.
The first geologist to produce a series of maps showing the progressive geologic geography of a given area was Jukes-Brown, who in the volume entitled “The Building of the British Isles,” 1888, included fifteen such maps. Karpinsky published fourteen maps of Russia, and in 1896 Canu in his Essai de paléogéographie has fiftyseven of France and Belgium. Lapparent’s Traité of 1906 is famous for paleogeographic maps, for he has twenty-three of the world, thirty-four of Europe, twenty-five of France, and ten taken from other authors. Schuchert in 1910 published fifty-two to illustrate the paleogeography of North America, and also gave an extended list of such published maps. Another article on the subject is by Th. Arldt, “Zur Geschichte der Paläogeographischen Rekonstructionen,” published in 1914. Edgar Dacqué in 1913 also produced a list in his Paläogeographischen Karten, and two years later appeared his book of 500 pages, Grundlagen und Methoden der Paläogeographie, where the entire subject is taken up in detail.
Conclusions.—Since 1833 there have been published not less than 500 different paleogeographic maps, and of this number about 210 relate to North America. Nevertheless paleogeography is still in its infancy, and most maps embrace too much geologic time, all of them tens of thousands, and some of them millions of years. The geographic maps of the present show the conditions of the strandlines of to-day, and those made fifty years ago have to be revised again and again if they are to be of value to the mariner and merchant. Therefore in our future paleogeographic maps the tendency must ever be toward smaller amounts of geologic time, if we are to show the actual relation of water to land and the movements of the periodic floodings. Moreover, the ancient shore lines are all more or less hypothetic and are drawn in straight or sweeping curves, unlike modern strands with their bays, deltas, and headlands, and the ancient lands are featureless plains. We must also pay more attention to the distribution of brackish- and fresh-water deposits. The periodically rising mountains will be the first topographic features to be shown upon the ancient lands, and then more and more of the drainage and the general climatic conditions must be portrayed. In the seas, depth, temperature, and currents are
yet to be deciphered. Finally, other base maps than those of the geography of to-day will have to be made, allowing for the compression of the mountainous areas, if we are to show the true geographic configurations of the lands and seas of any given geologic time.
Paleometeorology. In accordance with the Laplacian theory, announced at the beginning of the nineteenth century, all of the older geologists held that the earth began as a hot star, and that in the course of time it slowly cooled and finally attained its present zonal cold to tropical climatic conditions. That the earth had very recently passed through a much colder climate, a glacial one, came into general acceptance only during the latter half of the previous century.
Rise.—Our knowledge of glacial climates had its origin in the Alps, that wonderland of mountains and glaciers. The rise of this knowledge in the Alps is told in a charming and detailed manner by that erratic French-American geologist, Jules Marcou (1824–1898), in his Life, Letters, and Works of Louis Agassiz, 1896. He relates that the Alpine chamois hunter Perraudin in 1815 directed the attention of the engineer De Charpentier to the fact “that the large boulders perched on the sides of the Alpine valleys were carried and left there by glaciers.” For a long time the latter thought the conclusion extravagant, and in the meantime Perraudin told the same thing to another engineer, Venetz. He, in 1829, convinced of the correctness of the chamois hunter’s views, presented the matter before the Swiss naturalists then meeting at St. Bernard’s. Venetz “told the Society that his observations led him to believe that the whole Valais has been formerly covered by an immense glacier and that it even extended outside of the canton, covering all the Canton de Vaud, as far as the Jura Mountains, carrying the boulders and erratic materials, which are now scattered all over the large Swiss valley.” Eight years earlier, in 1821, similar views had been presented by the same modest naturalist before the Helvetic Society, but it was not until 1833 that De Charpentier found the manuscript and had it published. Venetz’s conclusions were that all of the glaciers of the Bagnes valley “have very recognizable moraines, which are about a league from the present ice.” “The moraines ... date from an epoch which is lost in the night of time.” Then in 1834 De Charpentier read a paper before the same society, meeting at Lucerne. “Seldom, if ever, has such a small memoir so deeply excited the scientific world.
It was received at first with incredulity and even scorn and mockery, Agassiz being among its opponents.” The paper was published in 1835, first at Paris, then at Geneva, and finally in Germany. It “attracted much attention, and the smile of incredulity with which it was received when read at Lucerne soon changed into a desire to know more about it.”
Louis Agassiz (1807–1873), who had long been acquainted with his countryman, De Charpentier, spent several months with him in 1836, and together they studied the glaciers of the Alps. Agassiz was at first “adverse to the hypothesis, and did not believe in the great extension of glaciers and their transportation of boulders, but on the contrary, was a partisan of Lyell’s theory of transport by icebergs and ice-cakes ... but from being an adversary of the glacial theory, he returned to Neuchâtel an enthusiastic convert to the views of Venetz and De Charpentier.... With his power of quick perception, his unmatched memory, his perspicacity and acuteness, his way of classifying, judging and marshalling facts, Agassiz promptly learned the whole mass of irresistible arguments collected patiently during seven years by De Charpentier and Venetz, and with his insatiable appetite and that faculty of assimilation which he possessed in such a wonderful degree, he digested the whole doctrine of the glaciers in a few weeks.”
In July, 1837, Agassiz presented as his presidential address before the Helvetic Society his memorable “Discours de Neuchâtel,” which was “the starting point of all that has been written on the Ice-age,”—a term coined at the time by his friend Schimper, a botanist. The first part of this address is reprinted in French in Marcou’s book on Agassiz. The address was received with astonishment, much incredulity, and indifference. Among the listeners was the great German geologist Von Buch, who “was horrified, and with his hands raised towards the sky, and his head bowed to the distant Bernese Alps, exclaimed: ‘O Sancte de Saussure, ora pro nobis!’” Even De Charpentier “was not gratified to see his glacial theory mixed with rather uncalled for biological problems, the connection of which with the glacial age was more than problematic.” Agassiz was then a Cuvierian catastrophist and creationist, and advanced the idea of a series of glacial ages to explain the destruction of the geologic
succession of faunas! Curiously, this theory was at once accepted by the American paleontologist T. A. Conrad (35, 239, 1839).
The classics in glacial geology are Agassiz’s Etudes sur les Glaciers, 1840, and De Charpentier’s Essai sur les Glaciers, 1841. Of the latter book, Marcou states that it has been said: “It is impossible to be truly a geologist without having read and studied it.” In the English language there is Tyndall’s Glaciers of the Alps, 1860.
The progress of the ideas in regard to Pleistocene glaciation is presented in the following chapter by H. E. Gregory.
Older Glacial Climates.—Hardly had the Pleistocene glacial climate been proved, when geologists began to point out the possibility of even earlier ones. An enthusiastic Scotch writer, Sir Andrew Ramsay, in 1855 described certain late Paleozoic conglomerates of middle England, which he said were of glacial origin, but his evidence, though never completely gainsaid, has not been generally accepted. In the following year, an Englishman, Doctor W. T. Blanford, said that the Talchir conglomerates of central and southern India were of glacial origin, and since then the evidence for a Permian glacial climate has been steadily accumulating. Africa is the land of tillites, and here in 1870 Sutherland pointed out that the conglomerates of the Karroo formation were of glacial origin. Australia also has Permian glacial deposits, and they are known widely in eastern Brazil, the Falkland Islands, the vicinity of Boston, and elsewhere. So convincing is this testimony that all geologists are now ready to accept the conclusion that a glacial climate was as wide-spread in early Permian time as was that of the Pleistocene.[3]
In South Africa, beneath the marine Lower Devonian, occurs the Table Mountain series, 5000 feet thick. The series is essentially one of quartzites, with zones of shales or slates and with striated pebbles up to 15 inches long. The latter occur in pockets and seem to be of glacial origin. There are here no typical tillites, and no striated undergrounds have so far been found. While the evidence of the deposits appears to favor the conclusion that the Table Mountain strata were laid down in cold waters with floating ice derived from glaciers, it is as yet impossible to assign these sediments a definite geologic age. They are certainly not younger than the Lower
Devonian, but it has not yet been established to what period of the early Paleozoic they belong.
In southeastern Australia occur tillites of wide distribution that lie conformably beneath, but sharply separated from the fossiliferous marine Lower Cambrian strata. David (1907), Howchin (1908), and other Australian geologists think they are of Cambrian time, but to the writer they seem more probably late Proterozoic in age. In arctic Norway Reusch discovered unmistakable tillites in 1891, and this occurrence was confirmed by Strahan in 1897. It is not yet certainly known what their age is, but it appears to be late Proterozoic rather than early Paleozoic. Other undated Proterozoic tillites occur in China (Willis and Blackwelder 1907), Africa (Schwarz 1906), India (Vredenburg 1907), Canada (Coleman 1908), and possibly in Scotland.
The oldest known tillites are described by Coleman in 1907, and occur at the base of the Lower Huronian or in early Proterozoic time. They extend across northern Ontario for 1000 miles, and from the north shore of Lake Huron northward for 750 miles.
Fossils as Climatic Indexes.—Paleontologists have long been aware that variations in the climates of the past are indicated by the fossils, and Neumayr in 1883 brought the evidence together in his study of climatic zones mentioned elsewhere. Plants, and corals, cephalopods, and foraminifers among marine animals, have long been recognized as particularly good “life thermometers.” In fact, all fossils are climatic indicators to some extent, and a good deal of evidence concerning paleometeorology has been discerned in them. This evidence is briefly stated in the paper by Schuchert already alluded to, and in W. D. Matthew’s Climate and Evolution, 1915.
Sediments as Climatic Indexes.—Johannes Walther in the third part of his Einleitung—Lithogenesis der Gegenwart, 1894—is the first one to decidedly direct attention to the fact that the sediments also have within themselves a climatic record. In America Joseph Barrell has since 1907 written much on the same subject. On the other hand, the periodic floodings of the continents by the oceans, and the making of mountains, due to the periodic shrinkage of the earth, as expressed in T. C. Chamberlin’s principle of diastrophism and in his publications since 1897, are other criteria for estimating the climates of the past.
Conclusions. —In summation of this subject Schuchert says: “The marine ‘life thermometer’ indicates vast stretches of time of mild to warm and equable temperatures, with but slight zonal differences between the equator and the poles. The great bulk of marine fossils are those of the shallow seas, and the evolutionary changes recorded in these ‘medals of creation’ are slight throughout vast lengths of time that are punctuated by short but decisive periods of cooled waters and great mortality, followed by quick evolution, and the rise of new stocks. The times of less warmth are the miotherm and those of greater heat the pliotherm periods of Ramsay.
On the land the story of the climatic changes is different, but in general the equability of the temperature simulates that of the oceanic areas. In other words, the lands also had long-enduring times of mild to warm climates. Into the problem of land climates, however, enter other factors that are absent in the oceanic regions, and these have great influence upon the climates of the continents. Most important of these is the periodic warm-water inundation of the continents by the oceans, causing insular climates that are milder and moister. With the vanishing of the floods somewhat cooler and certainly drier climates are produced. The effects of these periodic floods must not be underestimated, for the North American continent was variably submerged at least seventeen times, and over an area of from 154,000 to 4,000,000 square miles.
When to these factors is added the effect upon the climate caused by the periodic rising of mountain chains, it is at once apparent that the lands must have had constantly varying climates. In general the temperature fluctuations seem to have been slight, but geographically the climates varied between mild to warm pluvial, and mild to cool arid. The arid factor has been of the greatest import to the organic world of the lands. Further, when to all of these causes is added the fact that during emergent periods the formerly isolated lands were connected by land bridges, permitting intermigration of the land floras and faunas, with the introduction of their parasites and parasitic diseases, we learn that while the climatic environment is of fundamental importance it is not the only cause for the more rapid evolution of terrestrial life....
Briefly, then, we may conclude that the markedly varying climates of the past seem to be due primarily to periodic changes in the topographic form of the earth’s surface, plus variations in the amount of heat stored by the oceans. The causation for the warmer interglacial climates is the most difficult of all to explain, and it is here that factors other than those mentioned may enter.
Granting all this, there still seems to lie back of all these theories a greater question connected with the major changes in paleometeorology. This is: What is it that forces the earth’s topography to change with varying intensity at irregularly rhythmic intervals?... Are we not forced to conclude that the earth’s shape changes periodically in response to gravitative forces that alter the body-form?”
Evolution. Modern evolution, or the theory of life continuously descending from life with change, may be said to have had its first marked development in Comte de Buffon (1707–1788), a man of wealth and station, yet an industrious compiler, a brilliant writer, and a popularizer of science. He was not, however, a true scientific investigator, and his monument to fame is his Histoire Naturelle, in forty-four volumes, 1749–1804. A. S. Packard in his book on Lamarck, his Life and Work, 1901, concludes in regard to Buffon as follows:
“The impression left on the mind, after reading Buffon, is that even if he threw out these suggestions and then retracted them, from fear of annoyance or even persecution from the bigots of his time, he did not himself always take them seriously, but rather jotted them down as passing thoughts.... They appeared thirty-four years before Lamarck’s theory, and though not epoch-making, they are such as will render the name of Buffon memorable for all time.”
Chevalier de Lamarck (1744–1829) may justly be regarded as the founder of the doctrine of modern evolution. Previous to 1794 he was a believer in the fixity of species, but by 1800 he stood definitely in favor of evolution. Locy in his Biology and its Makers, 1908, states his theories in the following simplified form:
“Variations of organs, according to Lamarck, arise in animals mainly through use and disuse, and new organs have their origin in a physiological need. A new need felt by the animal [due to new conditions in its life, or the environment] expresses itself on the organism, stimulating growth and adaptations in a particular direction.”
To Lamarck, “inheritance was a simple, direct transmission of those superficial changes that arise in organs within the lifetime of an individual owing to use and disuse.” This part of his theory has come to be known as “the inheritance of acquired characters.”
