InͲvitrosystemsandmodellingof dieselcombustiontoxicology



LuckyJoeng

Athesissubmittedforthedegreeof

DoctorofPhilosophy

ChemicalSafetyandAppliedToxicologyLaboratories

SchoolofRiskandSafetySciences

FacultyofScience

UniversityofNewSouthWales

August2011

i UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Joeng First name: Lucky Other names: Yulius Abbreviation for degree as given in the University calendar: PhD School: Risk and Safety Sciences Faculty: Science Title: In-vitro systems and modelling of diesel combustion toxicology

Abstract 350 words maximum: Combustion within diesel engines occurs at a higher temperature than petrol engines, resulting in increased fuel efficiency with lower emission of toxic gases such as CO. However, emission of other gaseous pollutants such as

NOx and particulates including ultrafine particles, may pose increased human health risks. Epidemiology studies have drawn a significant correlation between air pollution and an increase in cardiovascular/pulmonary morbidity and mortality. The precise mechanisms of such effects are not well understood. The objective of this research was to develop an in vitro test system and a mathematical analyses/modelling for toxicity assessment of diesel exhaust on human cells. A diesel engine system was utilised to generate diesel exhaust. The carbon content (organic & elemental) was determined using NIOSH5040 method while NOx levels were measured using a NOx analyser. An in vitro system was developed by utilising multiple human cell types grown on porous membranes including A549 lung cells, HepG2 liver cells and skin fibroblasts. A portable exposure system was developed for laboratory and field based studies in which cells were directly exposed to diesel exhaust at the air liquid interface. To investigate cytotoxicity, a selection of in vitro assays including: MTS (tetrazolium salt), NRU (neutral red uptake) and ATP (adenosine triphosphate) were used. Further, a Scanning electron microscopy (SEM) was utilised to study cellular morphological changes following exposure. Short term exposure to diesel exhaust significantly reduced viability of human cells even at idle settings (45.3±13.9 %; p<0.05; 15 mins; ATP). Cytotoxic effects were both time (r2=0.95; MTS) and load (r2=0.92; MTS) dependent. In addition, a significant correlation was 2 observed between cytotoxicity and NOx (r =0.98; NRU). At higher load, bleb formation and cell morphology alteration were observed using SEM. Exhaust filtration contributed no significant reduction in human cell viability (filtered: 49.5±28.8 %; unfiltered: 46.3±36.4 %; 50% load; MTS), suggesting that the gaseous component were the most responsible for inducing toxicity. Further analyses confirmed cytotoxicity was mainly 2 induced by NOx (r =0.98) rather than particulates. In vitro toxicity methods developed in this thesis for laboratory and field based studies in combination with preliminary mathematical modelling, may provide an advanced tool for diesel combustion toxicology assessments.

Declaration relating to disposition of project report/thesis I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only).

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FOR OFFICE USE ONLY Date of completion of requirements for Award Originalitystatement

I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diplomaatUNSWoranyotherleducationa institution,exceptwheredueacknowledgmentis made in the thesis. Any contribution made to the research by others, with whom I have workedatUNSWorelsewhere,isexplicitlyacknowledgedinthethesis.

Ialsodeclarethattheintellectualcontentofthisthesisistheproductofmyown work,except totheextentthatassistancefromothersintheproject’sdesignandconceptionorinstyle, presentationandlinguisticexpressionisacknowledged.

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IalsoauthoriseUniversityMicrofilmstousethe350wordabstractofmythesisinDissertation AbstractInternational (thisisapplicabletodoctoralthesesonly).

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ii  Acknowledgements

Firstly, I would like to express my sincerest gratitude to my supervisors Dr Amanda HayesandDrShahnazBakandfortheirguidancethroughoutmywholePhDprogram. Thisthesiswouldnothavebeenpossiblewithouttheirsupportandmentorship.Iam alsothankfultomypreviousandcurrentcoͲsupervisors,ProfessorChrisWinderandDr Sanghoon (Shawn) Kook for their guidance, support and encouragement during the wholethesis.

IamalsogratefultoDrAmarawanIntasiriandDrPrepramePattanamahakulfortheir technicalguidanceduringtheirstayinUNSW.Mysincerethanksgotothestaffofthe SchoolofRiskand SafetySciences,UNSW,especiallytoMsStaceyWeeks,theschool managerandAssociateProfessorRogerReadfortheirkindassistances.Iwouldliketo thank Dr Zhan He Wu from Cytogenetics Department, Westmead Hospital for supplying the human cells at the beginning of this study.I would like to thank Ms Jenny Norman of the Electron Microscopy Unit, UNSW, Mr Vince Carnevale and Dr John Olsen of School of Mechanical and Manufacturing Engineering, UNSW and Mr PatrickMcMillanofSchoolofPhysics,UNSWfortheirtechnicalguidance.

An acknowledgement goes to the Vice Chancellor’s Appeal Scholarship program, of which I was recipient in 2008Ͳ2011 and the Commercialisation Training Scheme programofwhichIwasrecipientin2009Ͳ2010.

SpecialthankstoMsJaneBeh,MrSrinivasPadalaandMsAdelineLukmantarafortheir patienceandtimeinproofreadingthisthesisandtomyfellowstudentsinbothUNSW andeTh schoolofRiskandSafetySciences,especiallyMrYiWeiXu,MsJackieChan,Mr Shane Pin Siong Koo, Mr Andrew Li, Mr Heru Sufianto, Mr Ibrahim Taib, Ms Juliana Usman,MsRachelWard,MrKarlShamlayeandMrFinanceDechsakulthornfortheir friendshipandsupportinvariousways.

Finally,mysincerestgratitudegoestomybelovedfamily,theirloveandsupporthave been instrumental for my perseverance through the difficult times throughout the research,withoutthem,Iwouldneverhavebeenabletoachievethisfar.

iii  







 

Byaskingonegoodquestion,youwillreceivethree answerssimultaneously.



iv TableofContents

Tableofcontents Originalitystatement...... ii Listoftables...... xiii Listofabbreviations...... xiv 1 Introduction...... 1 2 Reviewofliterature...... 5 2.1 Airpollution...... 5 2.1.1 AirpollutionͲhistoricalperspective...... 6 2.1.1.1 Chlorofluorocarbon(CFC)useasrefrigerant...... 6 2.1.1.2 Vehicleemissions...... 7 2.1.2 Gaseouspollutions...... 10 2.1.3 ParticulateMatters(PM)...... 13 2.1.4 Airpollutionregulations...... 15 2.2 Combustion...... 19 2.2.1 Automotivefuel...... 20 2.2.2 Automotiveengine...... 22 2.2.3 FuturedevelopmentsͲBiofuel...... 24 2.2.4 Generationofdieselexhaust...... 28 2.3 Riskassessmentandmanagement...... 31 2.3.1 Epidemiology...... 35 2.3.2 InͲvivoandinͲvitrotoxicologicalstudies...... 42 2.3.3 InͲvitrotoxicologyͲhistoricalperspective...... 46 2.3.4 InͲsilicotoxicology...... 50 2.4 Principlesoftoxicology...... 51 2.4.1 Disposition...... 51 2.4.2 Targetorgans...... 54 2.4.2.1 Respiratorysystem...... 54 2.4.2.2 Cardiovascularsystem...... 56 2.4.2.3 Liver...... 58 2.4.2.4 Skin...... 58 2.4.3 Cellinjurymechanisms...... 59 2.4.4 DoseͲresponsecurve...... 61 2.4.5 Statisticalmethods...... 62 2.5 InͲvitrotoxicologymethods...... 65 2.5.1 Primarycultureandcellline...... 65

v  TableofContents

2.5.2 Preventingcrosscontamination...... 66 2.5.3 Cellculture...... 66 2.5.4 InͲvitrocytotoxicityassays...... 68 2.5.5 InͲvitrotoxicityexposuresystemdesign...... 71 2.5.5.1 Contaminantgenerationinexposuresystems...... 75 2.5.6 Dieselexhaust...... 76 2.5.6.1 Enginesettings...... 76 2.5.6.2 Dieselexhaustgeneration/samplingmethods...... 78 3 Researchobjectives...... 81 4 Materialsandmethods...... 83 4.1 Testmaterials...... 83 4.1.1 Preparationandserialdilutionofstocksolutions...... 84 4.2 Staticgenerationoftestatmospheres...... 84 4.3 Dieselengine,fuelanddynamometer...... 85 4.3.1 Dieselfuel...... 85 4.3.2 Dieselengineforlaboratorybasedstudies...... 85 4.3.2.1 InͲcylinderpressuremonitoring...... 86 4.3.3 Apparentheatreleaseratecalculation...... 86 4.3.4 Adiabaticflametemperaturecalculation...... 87 4.3.5 Dieselengineforfieldbasedstudy...... 88 4.4 Samplingpumpandrotameters...... 88 4.5 Analyticalmethods...... 89 4.5.1 Realtimegasmonitoring...... 89 4.5.2 Carboncontentmeasurement...... 89

4.5.3 NOxmeasurement...... 90 4.6 InͲvitrotechniques...... 91 4.6.1 Safetyconsiderations...... 91 4.6.2 Mediaandsolutions...... 91 4.6.3 Cellcounting...... 91 4.6.4 Cellcultureconditions...... 92 4.6.5 Cellharvestingandsubculturing...... 92 4.6.6 Theoptimalcelldensity...... 93 4.6.7 CulturingcellsonSnapwellinserts...... 93 4.6.8 Selectedhumancells...... 94 4.6.8.1 Humanskinfibroblasts...... 94 4.6.8.2 A549–Humanlungcells...... 94 vi  TableofContents

4.6.8.3 HepG2–Humanlivercells...... 94 4.6.9 Cellretrievingandarchiving...... 95 4.6.9.1 Retrievalofcells...... 95 4.6.9.2 Cryopreservingcells...... 96 4.7 InͲvitroexposuresystems...... 96 4.7.1 Staticdirectexposuremethod...... 96 4.7.2 Dynamicdirectexposuremethod...... 96 4.8 Cytotoxicityassays...... 97 4.8.1 MTSͲtetrazoliumsaltassay...... 97 4.8.1.1 MTSprotocolfor96Ͳwellmethod...... 97 4.8.1.2 MTSprotocolforporousmembranemethod...... 97 4.8.2 NRU–neutralreduptakeassay...... 98 4.8.3 ATP–adenosinetriphosphateassay...... 99 4.8.4 Controls...... 99 4.8.4.1 Controlsfor96wellplateprotocol...... 99 4.8.4.2 Controlsforporousmembraneprotocol...... 99 4.9 Dataanalysis...... 100 4.9.1 Doseresponserelation...... 100 4.9.2 Percentagecellviability...... 100 4.10 Statisticalmethodsintoxicology...... 101 4.11 ScanningElectronMicroscopy(SEM)...... 101 5 InͲvitrotoxicityassessmentofselectednanoparticles...... 104 5.1 Introduction...... 104 5.2 Experimentaldesign...... 105 5.2.1 Testchemicals...... 105 5.2.2 Celltypesandcultureconditions...... 105 5.2.3 InͲvitrocytotoxicityassays...... 106 5.2.4 Dataanalysis...... 106 5.3 Results...... 106 5.3.1 OptimalcelldensityforinͲvitroassays...... 106 5.3.2 CytotoxicitydatausingMTSassay...... 107 5.3.2.1 ZincOxide...... 107 5.3.2.2 TitaniumDioxide...... 108 5.3.3 Cytotoxicityresults...... 109 5.4 Discussion...... 110 6 InͲvitrotoxicityassessmentofagaseousmodelcompound...... 114

vii  TableofContents

6.1 Introduction...... 114 6.2 Experimentaldesign...... 115 6.2.1 Testchemicals...... 115 6.2.2 Celltypesandcultureconditions...... 115 6.2.3 Directexposureprotocol...... 115 6.2.4 Cytotoxicityassays...... 116 6.2.4.1 MTSͲTetrazoliumsalt...... 116 6.2.4.2 NRUͲNeutralreduptake...... 116 6.2.4.3 ATPͲAdenosinetriphosphate...... 117 6.2.4.4 Controls...... 117 6.2.4.5 Dataanalysis...... 117 6.3 Results...... 117 6.3.1 OptimalcelldensityforA549lungcells...... 117 6.3.2 ConcentrationeffectcurveusingMTSassay...... 118

6.3.3 IC50ofTolueneonA549cells...... 118 6.4 Discussion...... 120 7 InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts...... 123 7.1 Introduction...... 123 7.2 Experimentaldesign...... 124 7.2.1 Celltypesandcultureconditions...... 124 7.2.2 Generationofdieselexhaust...... 124 7.2.3 Monitoringandanalysisofexhaustemission...... 125 7.2.4 Elementalandorganiccarbonmeasurement...... 125 7.2.5 Cytotoxicityendpoints...... 125 7.2.6 Controls...... 125 7.2.7 Statisticalanalysis...... 126 7.3 Results...... 126 7.3.1 Optimalairflow...... 126 7.3.2 CytotoxiceffectsofdieselexhaustonA549Ͳhumanlungcells...... 127 7.3.2.1 Laboratorybasedengine...... 127 7.3.2.2 Fieldbasedengine...... 129 7.3.3 Dieselexhaustgasmonitoring...... 131 7.3.3.1 Laboratorybaseddieselexhaustgasmonitoring...... 131 7.3.3.2 Fieldbaseddieselexhaustgasmonitoring...... 132 7.3.4 Organiccarbonandelementalcarbonanalysis...... 133 7.3.4.1 Laboratorybasedengine...... 133

viii  TableofContents

7.3.4.2 Fieldbasedengine...... 134 7.4 Discussion...... 134 8 Toxicitycomparisonbetweenfilteredandunfiltereddieselexhaust...... 138 8.1 Introduction...... 138 8.2 Experimentaldesign...... 139 8.2.1 Celltypesandcultureconditions...... 139 8.2.2 Generationofdieselexhaust...... 139 8.2.3 Monitoringandanalysisofexhaustemissions...... 140 8.2.4 Exhaustfiltration...... 140 8.2.5 Cytotoxicityendpoints...... 140 8.2.6 Controls...... 140 8.2.7 Statisticalanalysis...... 140 8.3 Results...... 141 8.3.1 OptimalcelldensityforHepG2&skinfibroblasts...... 141 8.3.2 Toxicitycomparisonbetweenfilteredandunfiltereddieselexhaust....141 8.4 Discussion...... 143 9 Toxicityassessmentofdieselexhaustacrossvariousloads...... 147 9.1 Introduction...... 147 9.2 ExperimentalDesign...... 148 9.2.1 Celltypesandcultureconditions...... 148 9.2.2 Generationofdieselexhaust...... 148 9.2.3 Monitoringandanalysisofexhaustemissions...... 149 9.2.4 Exhaustfiltration...... 149 9.2.5 Cytotoxicityendpoints...... 149 9.2.6 Controls...... 149

9.2.7 NOxmeasurements...... 149 9.2.8 Exhaustfiltration...... 150 9.2.9 Elementalandorganiccarbonmeasurement...... 150 9.2.10 Combustionflametemperature...... 150 9.2.11 Scanningelectronmicroscopy...... 150 9.2.12 Statisticalanalysis...... 150 9.3 Results...... 150

9.3.1 NOxmeasurementandflametemperaturecalculation...... 150 9.3.2 Optimisationofexposureperiod...... 152 9.3.3 Comparisonofcytotoxicityendpoints...... 153

ix  TableofContents

9.3.4 Toxicitycomparisonoffilteredandunfiltereddieselexhaust...... 154 9.3.4.1 A549lungcells...... 154 9.3.4.2 HepG2livercells...... 156 9.3.5 Elemental,organicandtotalcarbonmeasurement...... 158 9.3.6 ObservationbySEM...... 159 9.3.6.1 Morphologicalalterationofhumancellsfollowingexposure...... 159 9.3.7 Mathematicalmodelling/analysis...... 163 9.4 Discussion...... 164 10 Conclusion...... 168 Bibliography...... 175 AppendixA...... 198 AppendixB...... 201 AppendixC...... 203 AppendixD...... 204 AppendixE...... 205 

x  Listoffigures

Listoffigures

Figure2Ͳ1:ProductiontrendofCFC...... 7 Figure2Ͳ2:ProductiontrendoftetraͲethylleadfrom1920sto1980s...... 9 Figure2Ͳ3:Effectofequivalenceratioandpeakcombustiontemperatureonemission levels...... 20 Figure2Ͳ4:Combustionchamberpressureduringafourstrokecycle...... 25 Figure2Ͳ5:AgglomerateandprimaryparticlesofDEP...... 30 Figure2Ͳ6:Poisoningprocessinanimalsandhumans...... 52 Figure2Ͳ7:Pulmonarysystemandfractionaldepositionofinhaledparticles...... 57

Figure2Ͳ8:ProposedPM10mechanismsoncardiovascularsystem...... 58 Figure2Ͳ9:Stagesofcellinjury...... 60 Figure2Ͳ10:Morphologicalfeaturesofdeadcells...... 61 Figure2Ͳ11:Typicaldoseresponsecurvewithsigmoidcurvefitting...... 62 Figure2Ͳ12:Normaldistributioncurve...... 63 Figure2Ͳ13:InͲvitroassaysstructureandreactions...... 71 Figure4Ͳ1:Laboratorybaseddieselengineanddynamometersetup...... 85 Figure4Ͳ2:Laboratorybaseddieselenginecontrolconfiguration...... 86 Figure4Ͳ3:InͲcylinderpressuremeasurement...... 86 Figure4Ͳ4:Dieselexhaustsamplingsystemsschematic...... 88 Figure4Ͳ5:Samplingequipments...... 89 Figure4Ͳ6:iBrid™MX6multiͲgasmonitors...... 90

Figure4Ͳ7:NIOSH5040filtersandhighprecisionNOxanalyser...... 90 Figure4Ͳ8:GrowingandexposingcellsattheAirLiquidInterface(ALI)...... 94 Figure4Ͳ9:Layoutof96wellmicrotitreplateforinͲvitroassays...... 98 Figure4Ͳ10:Platereaders...... 100 Figure4Ͳ11:Criticalpointdryerandsputtercoater...... 103 Figure4Ͳ12:HitachiS3400ScanningElectronMicroscope...... 103 Figure5Ͳ1:Celldensityoptimisationforskinfibroblastcells...... 107 Figure5Ͳ2:CellviabilityoffibroblastcellsafterexposuretoZnO...... 108

Figure5Ͳ3:CellviabilityoffibroblastcellsafterexposuretoTiO2...... 109 Figure6Ͳ1:CytotoxicityofToluenecalculatedbyGraphPadPrismsoftware...... 119

xi  Listoffigures

Figure6Ͳ2:CytotoxicityofToluenecalculatedbyMicrosoftExcelsoftware...... 119 Figure7Ͳ1:Effectofairflowoncellviability...... 127 Figure7Ͳ2:Timecoursecytotoxicityofdieselexhaustfromlaboratorybasedstudy.128 Figure 7Ͳ3: Comparison between 0 and 24 hrs postͲexposure incubation in the laboratorybasedstudy...... 129 Figure7Ͳ4:Timecoursecytotoxicityofdieselexhaustfromfieldbasedstudy...... 130 Figure 7Ͳ5: Comparison between 0 and 24 hrs postͲexposure incubation in the field basedstudy...... 131 Figure7Ͳ6:Gasreadingsfromlaboratorybaseddieselengineexhaust....... 132 Figure7Ͳ7:Gasreadingsfromfieldbaseddieselengineexhaust....... 133 Figure8Ͳ1:CytotoxicityofdieselexhaustwithMTSinͲvitroassay...... 142 Figure8Ͳ2:CytotoxicityofdieselexhaustwithNRUinͲvitroassay...... 143

Figure9Ͳ1:NO,NO2andNOxreadings...... 151 Figure9Ͳ2:Timecoursecytotoxicityanalysisofunfiltereddieselexhaust...... 153 Figure 9Ͳ3: Cytotoxicity of diesel exhaust on A549 cells with 0 hrs postͲexposure incubation...... 154 Figure9Ͳ4:CytotoxicityofdieselexhaustonA549cellswithMTSassaywith24hrspost exposureincubation...... 155 Figure9Ͳ5:CytotoxicityofdieselexhaustonA549cellswithNRUassayat24hrspost exposureincubation...... 156 Figure9Ͳ6:CytotoxicityofdieselexhaustonHepG2cellswithMTSassayat24hrspost exposureincubation...... 157 Figure9Ͳ7:CytotoxicityofdieselexhaustonHepG2cellswithNRUassayat24hrspost exposureincubation...... 158 Figure9Ͳ8:Organic,ElementalandTotalcarboncollectedonfilter...... 159 Figure9Ͳ9:SEMmicrographsofcontrolHepG2cells...... 160 Figure9Ͳ10:Cellstreatedwithdieselexhaustat25%engineload...... 161 Figure9Ͳ11:Cellstreatedwithdieselexhaustat100%engineload...... 162 Figure9Ͳ12:Sourceofvariablesformathematicalmodel...... 163 Figure 10Ͳ1: Diesel exhaust modelling and analysis representing potential pathways linkingdieselexhaustandcytotoxicityeffects...... 172

xii  Listoftables

Listoftables

Table2Ͳ1:Greenhousegases‘relativewarmingandcontribution....... 11 Table2Ͳ2:AustralianambientairqualityNEPM...... 16 Table2Ͳ3:UKAirQualityObjectivetargets...... 17 Table2Ͳ4:Japaneseairqualitystandards...... 18 Table2Ͳ5:Dieselfuelregulationsinvariouscountries...... 21 Table2Ͳ6:Propertiesofdieselfuel...... 22 Table2Ͳ7:Factorsaffectingdieselengineemissions...... 29 Table2Ͳ8:Regulationsrequiringsafetyimprovementtoworkingconditions...... 34 Table2Ͳ9:Epidemiologystudiesonairpollution...... 39 Table2Ͳ10:InvivoexperimentsonDEPeffects...... 44 Table2Ͳ11:Comparisonbetweeninvivo,inͲvitroandinsilicomodels...... 51 Table2Ͳ12:DoseͲresponsemathematicalmodels...... 64 Table2Ͳ13:LiteraturesemployingALImethod...... 73 Table2Ͳ14:Variousenginesettingsusedforanalysingemissions...... 77 Table2Ͳ15:InͲvitrotestingondieselexhaust/particulatetoxicity...... 79 Table4Ͳ1:Physicalcharacteristicsofselectednanoparticles...... 83 Table4Ͳ2:Physicalcharacteristicsofselectedvolatileorganiccompound...... 83 Table4Ͳ3:Physicalcharacteristicsofdieselfuel...... 84 Table6Ͳ1:CorrelationbetweenA549cellnumberandassayresults...... 117

Table6Ͳ2:IC50valueofMTS,ATPandNRUassayscalculatedusingGraphPadsoftware ...... 120

Table 6Ͳ3: IC50 value of MTS, ATP and NRU assays calculated using Microsoft Excel software...... 120 Table7Ͳ1:ElementalCarbon(EC),Organiccarbon(OC)andTotalcarbon(TC)content oflaboratorybaseddieselengineexhaust...... 134 Table7Ͳ2:ElementalCarbon(EC),Organiccarbon(OC)andTotalcarbon(TC)content offieldbaseddieselengineexhaust...... 134 Table8Ͳ1:CorrelationbetweenSkinfibroblast,A549andHepG2cellnumberwithMTS andNRUassayabsorbancereadings...... 141

xiii  Listofabbreviations

Listofabbreviations

3Rs Replacement,Reductionand Refinement A549 HumanEpithelialLungCarcinomacelllines AM AlveolarMacrophage ANOVA AnalysisofVariance ATCC AmericanTypeCultureCollection ATLA AlternativestoLaboratoryAnimals ATP AdenosineTriphosphate BALF BronchoAlveolarLavageFluid BfR GermanFederalInstituteofRiskAnalysis CAAT CentreforAlternativesinAnimalTesting CAS ChemicalAbstractsService CDP Criticaldryingprocess CFC Chlorofluorocarbons CO CarbonMonoxide

CO2 CarbonDioxide COLIPA EuropeanCosmeticIndustryAssociation CPD CriticalPointDrying CSAT ChemicalSafetyandAppliedToxicology CVS CardiovascularSystem DBͲALM ECVAMDatabaseServiceonAlternativeMethodstoAnimal Experimentation DDT DichloroͲdiphenylͲtrichloroethane DEP Dieselexhaustparticulate DIMDI GermanInstituteforMedicalDocumentationandInformation DMEM/F12 Dulbecco'smodifiedEagleMedium:Ham's FͲ12nutrientmixture DMSO DimethylSulfoxide DPBS Dulbecco'sphosphatebufferedsaline ECVAM EuropeanCentreforValidationofAlternativeAssessment FCS FetalCalfSerum FRAME FundforReplacementofAnimalsinMedicalExperiments FRP Fibreglassreinforcedpolymer GMͲCSF GranulocyteMacrophageColonyStimulatingFactor HBSS Hank'sbalancedsaltsolution HC Hydrocarbon HCFC hydrochlorofluorocarbons HepG2  HumanHepatomaCellLine HEPES NͲ(2Ͳhydroxyethyl)ͲpiperazineͲNƍͲ2Ͳethanesulphonicacid ICCVAM InteragencyCoͲordinatingCommitteeontheValidationofalternative methods

xiv  Listofabbreviations

IL Interleukin INFɶ Interferonɶ

LC50 Concentrationtoinduce50% deathwithinexposedsubjects

LD50 Dose50%toinduce50% deathwithinexposedsubjects LDH LactateDehydrogenase MDDC HumanMonocyteDerivedDendriticCells MDM HumanBloodMonocyteDerivedMacrophages MEIC MulticentreEvaluationofinͲvitro Cytotoxicity MFP MelamineFacedPlywood MTS (3Ͳ(4,5ͲdimethylthiazolͲ2Ͳyl)Ͳ5Ͳ(3Ͳcarboxymethoxyphenyl)Ͳ2Ͳ(4Ͳ sulphophenyl)Ͳ2HͲtetrazoliumsalt) MTT (3Ͳ(4,5Ͳdimethylthiazol)Ͳ2Ͳyl Ͳs,5Ͳdiphenyltetrazoliumbromide NCS NewbornCalfSerum NHMRC NationalHealthandMedicalResearchCouncil,AustralianGovernment NICEATM NTPInteragencycentrefortheevaluationofalternativetoxicological methods NIEHS NationalInstituteofEnvironmentalHealthSciences NIH NationalInstituteofHealth NIOSH NationalInstituteforOccupationalSafetyandHealth NIST NationalInstitute ofStandardsandTechnology NOAEL NoObservedAdverseEffectLevel NOAEC NoObservedAdverseEffectConcentration NOEL NoObservedEffectLevel NO NitrogenOxide

NO2 NitrogenDioxide

NOx OxidesofNitrogen NRU NeutralRedUptake

O3 Ozone Pb Lead PCB PolychlorinatedBiphenyls PC Polycarbonate PE Polyethylene PP Polypropylene PMS PhenazineMethosulphate PVC PolyvinylChloride QSAR QuantitativeStructureActivityRelationship r CorrelationCoefficient R2 CoefficientofDetermination ROS ReactiveOxygenSpecies RPM RotationsPerMinute SAR StructureͲActivityRelationship

xv  Listofabbreviations

S.D StandardDeviation SEM ScanningElectronMicroscope

SO2 SulphurDioxide SRM StandardReferenceMaterial;certifiedreferencematerialbythe NationalInstituteofStandardsandTechnology(NIST) STEL ShortTermExposureLimit TEM TransverseElectronMicroscope TGA TherapeuticGoodsAdministration THC TotalHydrocarbon TLV ThresholdLimitValue TNFɲ TumourNecrosisFactorɲ TWA TimeWeightedAverage USEPA TheUnitedStatesEnvironmentalProtectionAgency UV/Vis Ultraviolet/Visible VOC VolatileOrganicCompound WHO WorldHealthOrganisation XTT Sodium3,3Ͳ[1Ͳ[(phenylamino)carbonyl]Ͳ3.4Ͳtetrazolium]Ͳbis(4ͲmethoxyͲ 6Ͳnitro)benzenesulphonicacidhydrate ZEBET CentreforDocumentationofAlternativestoAnimalExperiments 

xvi  Publicationsandawards

PublicationsandAwards

Joeng, L., Bakand, S., Olsen, J. and Hayes, A. (2009). InͲvitro toxicity assessment of dieselexhaustusingadirectdynamicexposuremethod.Posterpresentation7thWorld CongressonAlternativeandAnimaluseintheLifeSciences,Rome,Italy.

Joeng, L. (2009) Assessing Air Pollution Using InͲVitro Toxicology Methods, poster presentation in UNSW Research Showcase: Climate Change and Environmental Sustainability,18Ͳ19May2009.

Dechsakulthorn, F., Hayes, A., Bakand, S., Joeng, L., and Winder, C. (2008). InͲvitro cytotoxicity assessment of selected nanoparticles using human skin fibroblasts. AlternativestoAnimalTestingExperimentation14,397Ͳ400.

Dechsakulthorn, F., Pin Siong Koo, S., Pattanamahakul, P., Bakand, S., Joeng, L., Winder,C.,andHayes,A.(2008).AcomparisonbetweennormalͲandnanosizedzinc oxide and titanium dioxide: InͲvitro cytotoxicity assessment using human skin fibroblasts.InSETAC.5thSocietyforEnvironmentalToxicologyandChemistryWorld Congress,3Ͳ7August,Sydney,Australia.

Pin Siong Koo, S., Dechsakulthorn, F., Pattanamahakul, P., Bakand, S., Joeng, L., Winder, C., and Hayes, A. (2008). Dispersing zinc oxide and titanium dioxide nanoparticle solutions for inͲvitro toxicity assessment. In SETAC. 5th Society for Environmental Toxicology and Chemistry World Congress, 3Ͳ7 August, Sydney, Australia.

Dechsakulthorn, F., Hayes, A., Bakand, S., Joeng, L., and Winder, C. (2007). InͲvitro cytotoxicity assessment of selected nanoparticles using human skin fibroblasts. 6th WorldCongressonAlternativeandAnimaluseintheLifeSciences,Tokyo,Japan.

Papersinpreparation

Dechsakulthorn,F.,Lee,S.,Joeng,L.,Chan,C.,Styger,L.,Pattanamahakul,P.,Hayes,A. Nanotechnology: How to proceed with the lessons we have learnt from the past. SubmittedtoJournalofNanoscienceandNanotechnology.

xvii  Publicationsandawards

JoengL.,HayesA.,Kook,S.,Bakand,S.,Winder,C.InͲvitrotoxicologyindieselexhaust analysis:areview

Joeng L., Hayes A., Kook, S., Bakand, S.Application of inͲvitro method to field and laboratorybaseddieselexhausttoxicity.

JoengL.,HayesA.,Kook,S.,Bakand,S.Toxicityofdieselexhaustacrossvariousloads.

GrantsandAwards

IndustryprizeawardedbyBlackmoresLtd,forthebestperformanceinthefieldofInͲ vitroToxicologyLaboratoryScience,2011.

IndustryprizeawardedbyBlackmoresLtd,forthebestperformanceinthefieldofInͲ vitroToxicologyLaboratoryScience,2010.

ScienceFacultyResearchGrant(2010),theUniversityofNewSouthWales.

Travel grant from AIM Travel S.r.l., (Italy) to attend the 7th World Congress on AlternativesandAnimalUseintheLifeSciencesinRome,Italy,2009.

Postgraduate Research Student Support (PRSS) Grant to attend the 7th World CongressonAlternativesandAnimalUseintheLifeSciencesinRome,Italy,2009.

UniversityofNewSouthWalesCommercialisationTrainingSchemefrom2009Ͳ2010.

UniversityofNewSouthWalesViceChancellor’sAppealScholarshipofPh.D.research from2008Ͳ2011.



xviii  Chapter1:Introduction

1 Introduction

Airpollutionsuchasdieselexhaustemissionisamajorpublichealthhazard.Diesel engineshavehigherfuelefficiencythanpetrolengine(AssemblyofEngineering1982) and also emit less toxic pollutants such as Carbon Monoxide (CO). However, diesel enginesemitalargerquantityoffineparticulates(PM2.5;particulatesofaerodynamic diameter <2.5 μm)(Riedl and DiazͲSanchez 2005).The smaller size particulates of dieselengineemissioncomparedtothepetrolengine(Kocbach,Johansenetal.2005) presentshigherpotentialtoxicity.Thisisduetothesmalldiameterparticulateshaving alargersurfaceareatoweightratio(Powelland Kanarek2006)andgreatereaseto translocate into the cardiovascular system through the alveoli of the pulmonary systemhencemoredirectdeliveryoftoxicmechanismsmayoccur(Nemmar,Hoylaerts etal.2004;Oberdorster,Oberdorsteretal.2005).

Epidemiological studies have shown morbidity and mortality risks increase with increased exposure to  air pollution especially the particulate component (Pope III, Burnettetal.2002;PopeIII,Burnettetal.2004;Peden2005;HeinrichandSlama2007; Rückerl, Schneider et al. 2011).Collaborations with experimental toxicology has created better understanding of particulates’ toxic mechanisms on the human body (Samet 1995; Schlesselman 1996; Mawson 2002; Bracken 2009).In vivo toxicology experiments have provided information on the effect of pollutants on biological systems,butrecentinͲvitrotoxicologyusingisolatedhumancellshasallowedformore rapidresultsandtestingofmorespecifictoxicmechanismsofpollutantsbyisolatinga component from a more complex biological system (White 2000; Mothersill and Seymour2003).

TheaimofthethesisistoinvestigatethemeritsofinͲvitromethodsintestingdiesel exhaust emissions by utilising multiple human based cellular systems and biological endpoints.The systems were developed with applicability to hazard and risk identification especially on pulmonary, and hepatic organs.During the progress of studies, diesel exhaust sampling systems and inͲvitro methods were developed to predictobservablecytotoxiceffectsincludingtestingdifferentcelllines,inͲvitroassays

1  Chapter1:Introduction anddeterminingwhichwerethemostsensitiveandpracticalforhazardidentification andriskassessment.

Organisationalaspect

ThisresearchwasfundedbyanAustralianPostgraduateAwardͲTheViceChancellor's Appeal Scholarship, UNSW (The University of New South Wales). Research was completed at the SRSS (School of Risk and Safety Sciences), UNSW. All toxicological experiments were carried out in the Chemical Safety and Applied Toxicology (CSAT) Laboratories, SRSS, while diesel engine experiments were carried out in the Engine ResearchLaboratory,SchoolofMechanicalandManufacturingEngineering.Scanning Electron Microscopy (SEM) works were performed at the Electron Microscopy Unit, AnalyticalCentre,UNSW.Carbonanalysisofdieselexhaustsampleswereperformed byMinesRescueService,Argenton,NSW.

Thesisstructure

Thethesis'structureconsistedoftenchapters.Followingtheintroduction(Chapter1), a review of literature focusing on air pollution generation by diesel engine and its healthrisksarepresentedinChaptertwo.

The literature review explores the engine research and toxicology field and its historical perspectives.In Section 2.1, a historical perspective on air pollution and contribution by automotives are explored including historical perspective of the contributionbyleademissionfromcarsusingleadedpetrol.Section2.2providesan explanationof thegenerationofpollution dueto the nature of internal combustion enginesanddifferencesbetweenpetrolanddieselenginesincludingsomeanalysison howdifferentfuelscontributetooverallemissions.Section2.3providesanoverview on how epidemiology and experimental toxicology have significant roles in the risk assessment process which requires the elucidation of hazards and risks posed by pollutants.Section 2.4 explores principles of risk assessment and management including epidemiology, in vivo and inͲvitro toxicology collaborations to further elucidatehealthhazardsandrisksposedbyairpollution,especiallyexposuretodiesel exhaust.Finally Section 2.5 provides a review on  available literatures on toxicity

2  Chapter1:Introduction testingofdieselexhaustincludingparticulatecollectionmethodsandrecommended dieselenginesettings.

Chapter three contains the research aims and objectives.Specifically the chapter describesthesignificanceoftheresearchtothewiderfield,specificresearchquestions andthemethodologiesusedtoanswertheseaimsandobjectives.

Chapter four contains the materials and methods used in the experiments.It summarisesspecificinformationanddetailsofchemicals,equipmentsandprocedures.

Chapters five to nine contain the experiments, methodologies used, results and discussionsof theresults.Chapter five contains results from studieson particles of different chemical compounds to determine the applicability of inͲvitro methods to analyseparticulatecytotoxicity.Chaptersixcontainsresultsfromthedevelopmentof the direct exposure method by growing cells on a porous membrane and allowing exposure to gaseous pollutant such as Toluene at the apex side while receiving nutrientsfromthebasolateralside.Chapter sevencontainsresultsfrombothfieldand laboratory based studies by improving on the design of the exposure device in the formofthedynamicexposurebydirectsamplingofdieselexhaustfromapassenger car(fieldbasedstudy)andadieselenginecoupledwithadynamometer(laboratory basedstudy).Chaptereightcontainsresultsfromcomparisonofcytotoxicitybetween filtered and unfiltered diesel exhaust. This chapter investigates if the level of particulates is significant enough to contribute major cytotoxic effects on exposed cells.Chapter nine contains results from experiments utilising diesel engine run on different load settings, the experiment investigates possible doseͲresponse relationshipsofdieselengineexhaustoncellcytotoxicity.

Chaptertenpresentsthesummaryandconclusionofresearchfindings.Thesection answersthequestionsposedinchapterthreeandthecompilationofallresults,and the overall assessment inͲvitro methods and their applicability in modeling of the toxicity of diesel exhaust emissions.Other points covered include future research directiontoimproveapplicabilityofmethodbeyondlaboratorysetting,proposalsfor possiblefutureexperimentsareoutlinedaswell.Inaddition,possibleapplicationsof methodologydevelopedduringthestudyareproposed.

3  Chapter1:Introduction

Toxicology studies on health risks posed by diesel exhaust can be complex due to numerouscomponentswithintheexhaust.InͲvitromethodsutilisingvarioushuman derived cell types and diverse biological end points may offer new possibilities to determinehazardsandrisksposedespeciallytopublic health.DevelopmentofinͲvitro methodscapableofmodelingtheinvivoequivalentiscrucialforelucidatingtoxicity mechanismsofpollutantsespeciallycomplexmixturesuchasdieselexhaustemissions.



4  Chapter2:ReviewofLiterature

2Reviewofliterature

2.1 Airpollution Airpollutionisoftenpresentintwodifferentforms;gaseousandparticlecomponents. The gaseous component is in gas or vapour phase, while the particle component contains a physical form that varies in size.These two forms are produced from various sources such as wind erosion, volcanoes, fires and human activities which includecombustion,industry,agriculture,etc(Colls2002).Somekeywordstodescribe airpollutioninclude: x Aerosol:Generaltermforparticlesofsolid,liquidorsolid/liquidnature suspendedinair. x Particulatematter(PM):Smallparticlesmicrometersindiameter,subdivided furtherintovariousaerodynamicdiametersizes,forexamplediameter<10μm

isnamedPM10,while<2.5μmisPM2.5.

x Coarseparticles:OftenusedinterchangeablywithPM10.

x Fineparticles:OftenusedinterchangeablywithPM2.5.

x Ultrafineparticles:OftenusedinterchangeablywithPM0.1. x Smoke:Contaminationofairwithcarbonandmixtureofothercondensed volatileorganiccompound.Thecontaminantparticlesare<1μminsizeand oftencausedbyincompletecombustionoforganicmaterialssuchasfossil fuels. x Blacksmoke:Similartosmoke,withadditionoflargerparticlesofpoorly combustedmaterials. x Dust:Solidmaterialssuspendedinairformedbymechanicalactivitiessuchas crushingandgrindingwithparticlediameter<1μmindiameter. x Mist:Liquiddropletssuspendedinair.

Airpollutionhasbeenamajorhealthriskconcernandinconvenienceinhumanhistory. As a result, laws tas Edic  by Queen Elizabeth I banned burning of coal during parliamentary sessions to control air pollution (Boubel 1994).In modern times, epidemiological studies have shown that air pollution (especially with particulate content) poses the greatest human health risks to the young and elderly and those withchroniccardiovasculardisease,asthma orinfluenza(HeinrichandSlama;PopeIII, Verrieretal.1999;BrunekreefandHolgate2002;PopeIII,Burnettetal.2002;Green and Armstrong 2003; Englert 2004; Polichetti, Cocco et al. 2009).The heightened responseinpeoplewithpreͲexistingconditionsmaybecausedbyinducementofproͲ

5  Chapter2:ReviewofLiterature inflammatorycytokinesbycellsuponexposuretoparticulatessuchasthatfromdiesel origin(Bayram, Devaliaetal.1998; Bayram,Devalia et al. 1998; Ito, Okumuraetal. 2006).

Sources of air pollution can be divided into natural and anthropogenic sources. Natural air pollution can be caused by volcanoes (in the form of fine volcanic ash, emissionofCO2andothergaseouscompounds),whileanthropogenicairpollutionis mainly a result from emissions from mobile sources such as automobiles and stationarysourcessuchaselectricalpowerplant(Fenger2009).

2.1.1 AirpollutionǦhistoricalperspective

Although the presence of bad odour caused by the use of chemicals/chimneys in chemicalindustrieshavebeennotedsinceancientRomanandmedievaltimes(Boubel, Foxetal.1994;GaffneyandMarley2009)andevenasrecentlyasthegreatLondon smog of 1952 (Griffin 2006), air pollution and its risks  to human health have only becomeamajorfocusinthepast60years.

2.1.1.1 Chlorofluorocarbon(CFC)useasrefrigerant

One notable environmental hazard occurred from the introduction of CFC (chlorofluorocarbon).CFCs are relatively inert and sparsely soluble in water. CFCs were invented to replace toxic, corrosive and/or flammable refrigerants such as ammonia, sulphur dioxide, methyl chloride, propane and butane (Atwood 1988). However,asingleCFCmoleculecanreactwithandy destro 100,000ozonemolecules (Sher1998).CFCshavebeenblamedforthedepletionofthestratosphereozonelayer, hence causing higher amounts of ultraviolet radiation reaching the surface of the earth.This has resulted in increased skin cancer cases and impacts on agricultural output(Kenna1981).ThediscoveryofCFCwasrevolutionaryforitssaferalternative toSO2asarefrigerant,andCFCwasalsoadaptedforwideͲspreaduseinaerosolsuntil 1973butgraduallyreducedinlateryears(VanBeek1979)asillustratedinFigure2Ͳ1. AninitiativesteptowardstheMontrealProtocolin1987anditssubsequentregulatory requirements have encouraged development and testing of CFC replacements (including alternative refrigeration methods) that are nonͲdestructive to the environment(RadermacherandKim1996;McCulloch1999).

6  Chapter2:ReviewofLiterature

 Figure2Ͳ1:ProductiontrendofCFC Figurebasedondatafrom(McCulloch1999;McCulloch,Ashfordetal.2001).

2.1.1.2 Vehicleemissions

Anothermajorcontributorofairpollutionisautomotivevehiclesinwhichpetroland diesel engines are the most popular engine types used in automobiles.The contribution by petrol engine usage was predominantly the Lead (Pb) component (until it was phased out) in fuel and emissions such as the toxic Carbon Monoxide. Dieselengineshasmorecompleteandahighercombustiontemperature,leadingto higher emissions of oxides of nitrogen (NOx) and smaller sized particulates (diesel exhaustparticulates).

Petrolfuel

Lead (in the form of tetraͲethyl lead) was used as a component of fuel for petrol enginesfrom1930sto1970s.AdditionofLeadinpetrolincreasedfuelefficiencyand reduceddamagescausedbyengineknocks(Anonymous1970;Nriagu1990)andwas rapidly adopted by the market (Gilbert and Weiss 2006) in many countries.Its inventor, Thomas Midgley, Jr was recognised and awarded the Perkin Medal of the AmericanSectionoftheSocietyofChemicalIndustryin1937(Anonymous1937). 7 Chapter2:ReviewofLiterature

AlthoughLeadpoisoningiswelldocumentedsincetheancientGreektimes(Yu2001), thedebatecontinuedonthecontributionofleadcontentintheatmospherefromcars running on leaded petrol during the 1950s to mid 1970s.The main supporters believedthathumanshadadaptedtoaconstantbackgroundleadexposure,hencethe amountofleademittedfromthecarspresentednoriskfortheexposedpopulation.

Since the 1920s, scientists such as Alice Hamilton raised concerns on the environmentalandhealthhazardsposedbytheusageofleadinpetrol(Nriagu1990). Thehealth effectsofleadwasunderscrutinyin1960whenClairPattersonshowedthe general population was exposed to Lead at 100 times higher than natural level (Patterson1965;Stikkers2002).Epidemiologicalandexperimentaltoxicologystudies suggested intellectual development in children could be impaired upon exposure to Lead.SomeoftheexposurepathwaysincludedinhalationandingestingchipsofleadͲ containing paint used in houses constructed before 1945 (Needleman 1983; Needleman,Schelletal.1990;Markowitz2000;Yu2001).Inaddition,deathoccurred aftercontinuousexposuretohighlevelsofLeadsuchasbyworkersatthetetraͲethyl leadproducingplants (RosnerandMarkowitz1985).

Due to high demand of Leaded fuel, progressive reduction of lead content was implementedinsteadofdirectbansuchastheUSwithitsregulatoryrequirementto reduceleadusageinpetrolstartingfrom1971tofullbanfrom1stJanuary1996via CleanAirActamendmentsbecominglawin1990(Stikkers2002)asseeninFigure2Ͳ2. AlthoughJapanwasthefirstcountrytoofferleadfreepetrolin1970s,inotherpartsof the world such as Africa in which leaded fuel is still heavily used (Fenger 2009).In Australia,leadedpetrolwasphasedoutin2003andleadlevelhavedroppedrapidly and have been below current NEPM standards at 0.5 μg/m3 since 1996 (Australian GovermentͲDepartmentofTransportandRegionalEconomics2005).



Dieselengineanditsrisingpopularity

Thedieselenginehasbeentheengineofchoiceforlargevehiclessuchastrucksdueto itshighertorqueoutputatlowerrotationsperminute(rpm)duetoslowercombustion

8  Chapter2:ReviewofLiterature rateofdieselfuel(Stone1985;Pulkrabek2004).Thedemandforthedieselenginehas increasedduetolimitingpetroleumsupplywithhigherpricing.Howeverlimitations suchaspassengercomfortandunacceptableexhaustsmelllimitedthedemandgrowth ofthedieselengine(Monaghan1988).

Figure 2Ͳ2:ProductiontrendoftetraͲethylleadfrom1920sto1980s Figureadaptedfrom(Nriagu1990;Stikkers2002;GerardandLave2005).

By regional comparison, the popularity of diesel engines is more obvious in Europe sincethe1970s(Verboven2002).InEuropethereislesstaxondieselcarownership andfuel(MayeresandProost2001).Inadditiontofueleconomy,thedieselengine has cleaner emissions than petrol engines since the combustion  process occurs at higherpressureandtemperature(Zelenka,Cartellierietal.1996).

Since diesel engines have to meet emission requirements such as USA’s Corporate AverageFuelEconomy(CAFE),continuousproductionofengineswithloweremissions hasbeenpredictedwithincreasedUSGDPasenginesaredomesticallyproducedand lessimportationof petroleumisneeded(Zelenka,Cartellierietal.1996).Toaddress 9 Chapter2:ReviewofLiterature thedemandsoflegislation,engineeringimprovementsonenginetechnologiessuchas electronic controls (Monaghan 1988) direct injection of fuel also improve fuel consumptionby5Ͳ25%overthepreviousindirectfuelinjectionmechanism(Tindaland Uyehara1988).Additionally,similartothedevelopmentoftheethanol/petrolblend, blended bio diesel/petro diesel is an alternative which does not require further modificationofthedieselengine(Canakci2007).Althoughunblendedbiodieselsuch as thatofpeanut oil may causeperformance problems due to higher viscosity and lower volatility (Wang, Lyons et al. 2000) whereas palm oil based biodiesel may be morecorrosiveoncopperandaluminiummetals(Fazal,Haseebetal.2010).

2.1.2 Gaseouspollutions

CarbonMonoxide(CO)

CarbonMonoxide(CO)isahighlytoxicgaswhichisodourless,colourlessandtasteless (Hodgson, Mailman et al. 1998) and is produced by the incomplete combustion of carboncontainingmaterials.ApersonwithCOpoisoningwillexperiencesymptoms suchasheadache,mentaldullness,nauseaanddeathifnottreatedsoon.DeathbyCO iscausedfromtoxicasphyxiasincehaemoglobinhastheaffinityupto250timeshigher forCOthanoxygen(O2),henceCObindseasiertohaemoglobinwhilepreventingthe binding of O2 for delivery to organs (Budavari 1996).Although CO is a natural gas produced and metabolised by plants, anthropogenic activities have contributed to dangerous levels of CO.Sources of CO include combustion such as in automobile engines,forexampleupto90%ofUK’sCOemissionwerecontributedbyautomobile emissions(Colls2002).

CarbonDioxide(CO2)

CO2 isanonͲflammableandodourlessgas.CO2isnaturallyproducedbyplantsand animalsduringgasexchangeandisamajorgreenhousegasalongsideothergasessuch asMethane(NH3),NitrousOxide(N2O)andOzone(O3).AlthoughCO2isnotthemost destructivegreenhousegas,itisthelargestcontributor(Table2Ͳ1)duetocombusting fossilfuels.WithintheatmosphereCO2canreactwithH2Otocreateamildlyacidic

Carbonicacid(H2CO3).

10  Chapter2:ReviewofLiterature

CO2 overexposure symptoms include: hyperventilation, sweating, headache, loss of consciousness, or even death in serious cases.Hyperventilation is triggered by the receptorswithinthecardiovascularsystemwherehypoxemia(dangerouslylowlevelof

O2)andhypercapnia(dangerouslyhighlevelofCO2)canbedetected(Sherwoodand Sherwood1997).

Table2Ͳ1:Greenhousegases‘relativewarmingandcontribution.

Gas Relativewarming Relativecontributionto effectivenesspermole thegreenhouseeffect(%)

CarbonDioxide(CO2) 1 50

Methane(CH4) 23 20

NitrousOxide(NOx) 270 5

CFCͲ11(CFCl3) 14000 5

CFCͲ12(CF2Cl2) 19500 10

TroposphericOzone(O3)Ͳ 10 Modifiedfrom(Sher1998).

SulphurDioxide(SO2)

SO2 is colourless and a nonͲflammable gas with a strong suffocating odour at concentrations >0.5 ppm. SO2 gas is produced from the burning of the sulphur component inorganicmaterials suchas fossilfuels.However, theemissionlevel of

SO2 has decreased significantly since commercial usage has switched to natural gas whichcontainsnegligibleamountsofsulphur.Inaddition,SO2hasbeenusedasanair pollutionindicatorbuthasnowbeenreplacedwithmeasurementsofNOx(Colls2002).

OxidesofNitrogen(NOx)

NOx is a collective term for oxides of nitrogen including nitrogen dioxide (NO2) and nitrogenoxide(NO).NOxisformedbythermaldecompositionprocessessuchasthe combustioninenginesNitrogengas (N2) in air isdecomposed into N molecules and bindingtoO2moleculeswhichinturncanbindwithH2OtocreateamildlyacidicNitric acid(HNO3).Incombustion,NOxformationisdependentonseveralvariablessuchas airͲfuelratio,combustiontemperatureandresidencetimeofN2andO2moleculesin theflame(Reis2005).NOxisusedasacommonindicatorofairpollutionreplacingSO2

11  Chapter2:ReviewofLiterature especiallyduringthe1990sforitseaseinconversionfromNOtoNO.Bothcompounds aremeasuredasindicatorsofairpollution(Colls2002).

NO2isareddishbrowncolouredgaswithanirritatingodour.NO2isinsolubleandthis allows it to reach the pulmonary (bronchioles and alveoli) part of the respiratory system.Atlowconcentration,itcancausemildupperrespiratorytractirritation,while at high concentration it can cause respiratory oedema and eventually death (Carel

1998).Exposure to the combination of SO2 and NO2 enhances airway response to inhaledallergens(Devalia,Rusznaketal.1994).

Hydrocarbons(HC)

HCareageneraltermdescribingmoleculesthatconsistsofonlyhydrogenandcarbon atoms.Thehydrogenandcarbonatomscanthencombinewithdifferentcomponents such as alcohols, aldehydes and aromatics.The health effects of HC such as 1,3 butadiene include irritation, odorants and even cancer while benzene is associated with leukaemia (Schnatter, Rosamilia et al. 2005).In addition, hydrocarbon componentsreactwithatmospheregasesformingphotochemicalsmogs(Carel1998).

Ozone(O3)

O3isastrongoxidantcapableofincreasingthereactivityofairwaysespeciallyforthe asthmatic.Although stratospheric O3 is commonly known as the gas that helps preventUVradiationfromreachingtheearthsurface,theTroposphericozonecanbe toxic.OverexposuretoO3cancausecoughing,drynessofthroatandchestdiscomfort atconcentrationsaslowas0.3–0.9ppm.Chronicexposuretolowconcentrationof

O3couldleadtoemphysema,whilechronicexposuretohighconcentrationcouldlead torespiratoryedema(Carel1998).

Lead(Pb)

Pbisatoxicelementandhasbeenknownforitsadversehealtheffectssinceancient civilizationtimesuchasAncientRomeduetoitswidespreaduse(Nriagu1983).The main exposure to childrenis by inhalation of Lead contaminated dust and ingestion paintchipscontainingLeadinhousesconstructedpriorto1975.HealtheffectsofLead includeneurologicalandreproductivedisorders(Needleman1983;Needleman,Schell 12  Chapter2:ReviewofLiterature etal.1990;Markowitz2000;Yu2001)orevendeathtocontinuoushighlevelexposure (RosnerandMarkowitz1985).

2.1.3 ParticulateMatters(PM)

Particulate matters (PM) are conventionally measured gravimetrically especially in regulations(Griffin2006)

ൊ ʹͶǤͶͷ Equation1:Concentrationofcontaminantper ܹܯ כ Ɋ‰Τ ݉͵ ൌ ݌݌ܾ volumeofmediumintermsofweight Where: x μ‰݉Τ ͵=totalweightofcontaminantper1cubicmetervolume. x ppb=ratioofcontaminanttototalnumberofparticulates(inpartsperbillion) x MW=molecularweightofcontaminant. x 24.45=molarvolumeofgasatstandardtemperatureandpressure.

Although weight measurement of PM10 is well established,particle sizes are more toxicologicallyrelevantsincesmallersizeparticlesindicatelargersurfaceareahence higher reactivity (Oberdorster, Oberdorster et al. 2005).Therefore it has been suggestedPMshouldbemeasuredinnumberofparticles(Peters,Wichmannetal. 1997;Penttinen,Timonenetal.2001)orsurfacearea (MaynardandMaynard2002; Moshammer and Neuberger 2003) as an addition of more conventional gravimetric terms (Lison, Lardot et al. 1997) and mean/median size (Nygaard, Samuelsen et al. 2004).

Particulates with the same gravimetric weight (for example 50 μg/m3) may have differenttoxicities.Asparticlesofsimilaraerodynamicdiameter(maybeofdifferent physicalshapes)exhibitsimilarcharacteristics(Wilson1990),anaerodynamicdiameter is used by calculating particles’ size in its spherical shape equivalent with specified equivalentdensity(Equation2)(MausandUmhauer1997).

Equation 2: Aerodynamic ߩ௣ ݀ ൌ݀ ඨ  ௔௘ ௣ ͳ݃ܿ݉ିଷ diametercalculation

Where:

x dae=aerodynamicdiameteroftheparticulates.

x dg=geometric(physical)diameter.

x ʌp=particledensity.

13  Chapter2:ReviewofLiterature

Particulatematterswithaerodynamicsdiameter<10μm(PM10)

PM can be in simple or agglomerated form at ambient atmosphere.Initially, particulatesexistinnucleationmodeasitsstructurehasundergonelittleornochange sinceitsformation.Aftersomeresidence timeinambient atmosphere, particulates exist in accumulation mode in which individual particulates form a long chain of primary particles called “spherules”.After further residence time and reaction, particulatesexistincoarsemodeinwhichithasdensersolidcorewithanouterlayer ofvolatilematerials(Eastwood2008).

DeterminingsourcesofPM10 canbedifficultasPM10aremainlyfoundfarfromtheir source as PM10 are transportable for long distance range such as from continental

EuropetotheMediterraneanSea.Inaddition,PM10levelsareinfluencedbyweather factors such as temperature, wind direction and stagnating air masses (Vardoulakis andKassomenos2008;Koçak,Mihalopoulosetal.2009).

PM10hasbeeninvestigatedforitshealthrisksviatheinhalationrouteduetopossible heightened response in humans with allergy to airborne bacteria (Alexis, Lay et al. 2006)andlongtermeffectsonbothpulmonaryandcardiovascularsystems(Zanobetti, Schwartzetal.2003).The10μmdiametersizeistheesiz limitforparticulatestobe respirable whereas particulates >10 μm would be blocked by pulmonary defence system.The pulmonary defence system include mucociliary mechanisms of the bronchial epithelium cells (BruskeͲHohlfeld and Peters 2008).In environmental studies, PM10 can be categorised into primary and secondary components.The primary PM10 has undergone little or no structural change since its formation while secondaryPM10hasmorestructuralchanges,forexamplesulphatesareformedfrom

SO2afterresidencetimeintheatmosphereandreactingwithH2Otoformacidrain (PryorandBarthelmie1996).



Particulatematterswithaerodynamicsdiameterlessthan2.5μm(PM2.5)

PM2.5iscategorisedasoneoftheenvironmentalpollutantsbecauseofitshazardssuch asdeeperpenetrationintotherespiratorysystemandfurthertranslocationintothe

14  Chapter2:ReviewofLiterature

cardiovascularsystem(Nemmar,Hoylaertsetal.2004).PM2.5hashigherreactivitydue to its larger surface area to mass ratio as compared to PM10 (Powell and Kanarek

2006).Apportionment studies have found sources of PM2.5 to be similar to

PM10,however PM2.5is mainly emitted from anthropogenic activities such as coal combustion,motorvehiclesexhaustandnonͲcombustionsourcessuchasdustsfrom constructionsites,laserprintersandsoildust(Ho,Caoetal.2006;Yue,Lietal.2006; He,Morawskaetal.2010;Mooibroek,Schaapetal.2011;Thurston,Itoet. al 2011).

CurrentlyPM10 standardsarecommonwhilePM2.5standardsareunderconsideration for implementation in various regions.Regulators in Australia, UK, EU and Japan currently set PM10 limits with optional monitoring of PM2.5.Only the US regulators have set a limit on PM2.5 emission since 1997 (Meyer 1998).The main challenges associated in applying PM2.5 standard are the sampling and analytical technology limitations as PM contains a wide range of chemicals that is difficult to detect individually.The technology limitations include possible errors during weighting, difficultyinprocessingPM2.5filters.Inaddition,impactorshaveitslimitationofitsnon trulyisokineticnatureandcyclonesamplersareunablesamplePMwithexactcutͲsize (SlossandSmith2000).

2.1.4 Airpollutionregulations

Australia

Theambientairqualitynationalenvironmentprotectionmeasure(AmbientAirQuality NEPM) is the main standard for air pollution level (Table 2Ͳ2).The Environment Protection and Heritage Council (EPHC) (formerly known as National Environmental ProtectionCouncil;NEPC)istheentityinwhichitsmembers(withtwoͲthirdsmajority) take partindraftingtheNEPMandallowpublicconsultationforatleasttwomonths (NEPC2010).AlthoughtheNEPM’saimistocreateaconsistentapproachtomonitor air pollutants, implementation of legislation is made by individual state or territory jurisdictionsbypassingactsandregulations.Examplesofpassedactsandregulations includeNewSouthWales’ProtectionoftheEnvironmentOperationsAct1997andthe ProtectionoftheEnvironmentOperations(CleanAir)Regulation2002.OtherNEPM createdbyNEPCincludetheairtoxicsNEPMandtheDieselVehicleEmissionsNEPM.

15  Chapter2:ReviewofLiterature

The air toxics NEPM define “air toxics” as pollutants which may be in low concentrations but poses risk to human health such as combustion products from vehiclesandwoods.TheaimofDieselVehicleEmissionsNEPMistoreduceemission levelsfromdieselvehiclesby facilitatingcompliancewithserviceemissionsstandards (NEPC2010).

Table2Ͳ2:AustralianambientairqualityNEPM

Pollutant Concentration Averagingperiod CO 9ppm

NO2 0.12ppm 1hrs 0.03ppm 1year

O3 0.10ppm 1hrs 0.08ppm 4hrs

SO2 0.20ppm 1hrs 0.08ppm 24hrs 0.02ppm 1year Pb 0.5μg/m3 1year 3 PM10 50μg/m 24hrs * 3 PM2.5 25μg/m 1day 8μg/m3 1year *Includedsince2003asadvisoryreportingstandard;Source:(AustralianGovermentͲ DepartmentofSustainability2010)page19.

EuropeanUnion

The air quality framework directive (96/62/EC) on air quality assessment and management and followed by subsequent directives in July 1999 (99/30/EC), 2000 (2000/69/EC),2002(2002/3/EC)and2004(2004/107/EC)(Eionet2011)setsthelimit for its member states,.In addition, the EU directive 96/91/EC introduced the integratedpollutionpreventionandcontrol(IPCC)inwhichasingleregulatorhavethe authoritytosetthelimitsandharmonisationofimplementationacrosstheEuropean Unionmemberstate(O'Malley1999).

UnitedKingdom

IntheUK,theCleanAir6Actof195 wasinresponsetothegreatLondonsmogof1952. ThePollutionPreventionandControlAct(PPCA)1999legislationbroughtEUdirective

16  Chapter2:ReviewofLiterature

96/91/EContointegratedpollutionpreventionandcontrol(IPPC)(Colls2002).TheUK AirQualityObjectivetargetsareoutlinedinTable2Ͳ3.

Table2Ͳ3:UKAirQualityObjectivetargets

Pollutant Concentration(μg/m3)Datetoachieve Benzene(all) 16.25ʅg/m3 (runningannualmean) 31/12/2003 Benzene(E,W) 5ʅg/m3(annualmean) 31/12/2010 Benzene(S,NI) 3.25ʅg/m3(runningannualmean) 31/12/2010 1,3butadiene 2.25ʅg/m3 (runningannualmean) 31/12/2003 CO(all) 10,000ʅg/m3(runningannualmean) 31/12/2003 Pb(all) 0.5ʅg/m3(annualmean) 31/12/2004 0.25ʅg/m3(annualmean) 31/12/2008 3 NO2(all) 200ʅg/m no more than 18 times a year (24 hrs 31/12/2005 mean) 31/12/2005 40ʅg/m3(annualmean)  3 PM10 (all) 50ʅg/m nomorethan35timesayear(24hrs mean) 31/12/2004 40ʅg/m3(annualmean) 31/12/2004 3 PM10(E,W,NI) 50ʅg/m nomorethan7timesayear(24hrs mean) 31/12/2010 20ʅg/m3(annualmean) 31/12/2010 3 PM10(London) 50ʅg/m nomorethan10timesayear(24hrs mean) 31/12/2010 23ʅg/m3(annualmean) 31/12/2010 3 PM10(S) 50ʅg/m nomorethan7timesayear(24hrs mean) 31/12/2010 18ʅg/m3(annualmean) 31/12/2010 3 SO2(all) 350ʅg/m nomorethan24timesayear(1hrs mean) 31/12/2004 125ʅg/m3nomorethan3timesayear(24hrsmean) 31/12/2004 266ʅg/m3 no more than 35 times a year(15 mins 31/12/2005 mean) Abbreviations:E=England,W=Wales,S=Scotland,NI=NorthernIreland. Adaptedfrom(Longhurst,Beattieetal.2006).  Japan

TheJapaneseBasicLawforEnvironmentalPollutionControlwasimplementedtoset upenvironmentalqualitystandards(EQS)in1967withthegoalofreducingpollution causedbythe rapidindustrialisationofJapan.Thebasiclawwasthensupersededby theBasicEnvironmentalLaw1993(Kawamoto,Phametal.).ThesummaryofJapanese airqualitystandardsareoutlinedinTable2Ͳ4.



17  Chapter2:ReviewofLiterature

Table2Ͳ4:Japaneseairqualitystandards

Substance Environmentalconditions Implementation date Sulphurdioxide x Dailyaverageforhourlyvalues:ч0.04ppm 16/5/1973 x Hourlyvalues:ч0.1ppm Carbonmonoxide x Dailyaverageforhourlyvaluesч10ppm 8/5/1973 x Averagehourlyvaluesforanyconsecutive8hrs period:ч20ppm Suspended Definedasairborneparticleswithadiameter <10ʅm 8/5/1973 particulatematter x Dailyaverageforhourlyvalues:ч0.10mg/m3 x Hourlyvalues:ч0.20mg/m3 Nitrogendioxide Dailyaverageforhourlyvaluesshallbewithin0.04Ͳ0.06 11/7/1978 ppmrangeorbelow Photochemical Definedasoxidisingsubstancesnitrateproducedby 8/5/1973 oxidants photochemicalreactionssuchasozoneandperoxiacetyl (onlythosecapableofisolatingiodinefromneutral potassiumiodide,excludingnitrogendioxide) x Hourlyvalues:ч0.06ppm Benzene x Annualaverage:ч0.003mg/m3 4/2/1997 Trichloroethylene x Annualaverage:ч0.2mg/m3 4/2/1997 Tetrachloroethylene x Annualaverage:ч0.2mg/m3 4/2/1997 Dichloromethane x Annualaverage:ч0.15mg/m3 20/4/2001 Dioxins IncludesPCDDs,PCDFsandcoplanarPCBs 27/12/1999 x Annualaverageч0.6pgͲtoxicTEQ/m3 FineParticulate DefinedasairborneparticlesthatpassthroughasizeͲ 9/9/2009

Matter(PM2.5) selectiveinletwitha50%efficiencycutͲoffat2.5ʅm aerodynamicdiameter. x Annualstandard:<15.0ʅg/m3 x 24hrsstandard(annual98thpercentilevalues)at designatedmonitoringsitesinanarea:ч35ʅg/m3

Adaptedfrom(MinistryoftheEnvironment).

 UnitedStates

TheUSCleanAirAct(CAA)consistsofmultipleactsdesignedtocontrolairpollution. The CAA includes Air Pollution Control Act of 1955, which was replaced by CAA of 1963, with Clean Air Act amendments (CAAA) in 1967, 1970, 1977 and 1990 where each amendment includes more pollutants to be controlled and management approaches(Vandenberg2005;SueyoshiandGoto2009).CAAhasinitiatedreduction

18  Chapter2:ReviewofLiterature air pollution from motor vehicles via the CAA 1963 and 1970 amendment.These regulatory requirements resulted in the invention of the catalytic converter in 1975 and the threeͲway catalyst in 1981, both reduced HC, CO and NOx emission by convertingthemintolessharmfulgasessuchasCOintoCO2(GerardandLave2005)

ImplementationofCAAA1990aimedatreducingPM10levelsbetweenyear1990Ͳ2005 andhasreducedmortalityby23,000in2010(Vandenberg2005;Auffhammer,Bento et al. 2009).Although some pollutants emission has been reduced, there has been doubtswhetherpollutantssuchasSO2wasreducedbecauseofCAAAimplementations thereisscarcityofreliableevidenceforCAAA’sdirectcostandbenefits(Greenstone 2004).

Corporate Average Fuel Economy (CAFE), also known as Federal Automotive Fuel Economy Standard is a US standard required cars and light trucks to improve fuel efficiency.CAFEwasestablishedUS EnergyPolicyandConservationActof1975(PL94Ͳ 163)inresponsetofuelpricehikeduring1973Araboilembargo.Initiallytherewas debateonCAFE’seffectivenessanditspotentialcompromiseonsafety,howeverthe regulationhasresultedinloweremissionbyvehicles(Teotia,Vyasetal.1999).

2.2 Combustion Combustion is a chemical reaction between fuel and oxidiser in which energy is releasedintheformofheator light with other residues.Combustionperformance dependsonsomevariablesincluding:

x Fueltoairratio(FAact):ratioofmassͲofͲfueltomassͲofͲairinputintoengine.

x IdealorstoichiometricfuelͲairratio(FAstoich):fueltoairratioinwhichtheairis justsufficientenoughtoburnthefuelcompletely. x Equivalenceratio(Ɍ):istheratiooffuelͲtoͲairͲratiotoidealͲorͲstoichiometricͲ

fuelͲairͲratio.MathematicallyexpressedasɌ=FAact/FAstoich.

Theequivalenceratioisanindicationofavailabilityofoxidiser(mainlyair)tofuel.At “lean” conditions (Ɍ<1), more air molecules than fuel molecules are available for combustion,whereas“rich”conditions(Ɍ>1)morefuelmoleculesthanairmolecules are available.A lean combustion characteristic is preferable as rich  combustion condition reduces combustion efficiency.The combustion temperature also has a

19  Chapter2:ReviewofLiterature

strong influence on emission formation, for example, NOx emission formation is correlatedwithhigherflametemperaturesduetothemoreefficientcombinationof

N2 and O2 during combustion (Figure 2Ͳ3 (a)), while CO emission is reduced as the highertemperatureallowsittobindtomoreoxygenmoleculestoformCO2(Figure2Ͳ3 (b)).Inaddition,asdieselenginesoperatesathighercompressionratioswithleaner fuelͲair mixture, diesel engines have higher fuel efficiency than petrol engines (AssemblyofEngineering1982).

(a)  (b)  Figure2Ͳ3:Effectofequivalenceratioandpeakcombustiontemperatureonemission levels (a) Effect of equivalence ratio on emission level; (b) Effect of peak combustion temperatureonemissionlevel. Adaptedfrom(Pulkrabek2004)page333and(Boubel,Foxetal.1994)page81.

2.2.1 Automotivefuel

Quantifyingfuelquality

Fuelqualityquantificationwasperformedbycomparingeaseofdetonationwiththe referencemixturecompound.Inthe1920’s,thereferencemixturecompoundwasa toluene/heptanesmixturenamed“toluenenumber”inwhichpuretoluene’signition quality was rated at 100 while pure heptane’s ignition quality was rated at 0 (Monaghan1988).

Currently, research octane number (RON) is used to determine the quality of spark ignition(SI)enginefuelsuchaspetrol.Thetestfueliscomparedtoamixtureoftwo

20  Chapter2:ReviewofLiterature reference fuels: isoͲoctane and heptanes (Zvirin, Gutman et al. 1998), RON is then calculatedusingEquation3.

RON=%isoͲoctane+%nͲheptane. Equation3:Researchoctanenumber  calculation

Cetane number (CN) is used to determine quality of diesel fuel.The test fuel is compared to a mixture of nͲcetane and heptamethyl nonane (Zvirin, Gutman et al. 1998).CN is then calculated using Equation 4 where nͲcetane is a compound with highͲignitionqualitywithCNof100whereasheptamethylnonaneisahighlybranched compoundwithverylowignitionqualitywithCNnumberof15.AlthoughCNprovides a general indication of fuel quality and is often used by regulations as a quality indicatoraslistedinTable2Ͳ5,otherpropertiesincludingsulphurcontentandenergy densityalsodescribethequalityofdieselfuelasoutlinedinTable2Ͳ6.

CN=%nͲcetane+%heptamethylnonane Equation4:Cetanenumber calculation 

Table2Ͳ5:Dieselfuelregulationsinvariouscountries

Country Standard Cetane Reference(s) Number

Australia AS3570Ͳ1998 >49 (StandardsAustralia1998) European EN590 >49 (Rashid,Anwaretal.2008; Union EN14214(biodiesel) >51 TorresͲJimenez,Jermanetal. 2011) Germany DINEN590:1993 >49 (Krahl,Knotheetal.2009) DINV51605(Rapeseedoil) >39 United ASTMD975(1994) >40 (Demirbas2007) States ASTMD6751(Biodiesel) >47 



21  Chapter2:ReviewofLiterature



Table2Ͳ6:Propertiesofdieselfuel

Property Explanation Cetane Measurementofignitionquality,highercetaneindicateeasier Number ignition,improvedcoldstarting,reducedsmokeemissionduring warmͲup,reducednoise,reducedfuelconsumptionandreduced exhaustemission Volatility Highvolatilitymaycauseenginemisfire(vapourlock)andfailureto restartaftershutdowninhotcondition Density Aquickandgeneralindicationofotherpropertiessuchasignition quality,poweroutput,lowtemperaturecharacteristicandsmoking tendencies. Viscosity Higherviscosityincreasesfueldropletsize,affectingfuelinjection timing. Low Sinceupto20%ofdieselfuelcontentisheavyparaffinhydrocarbons, temperature itwilldepositoutaswaxwhenadequatelycooled,causingpotential characteristic problemsinoperationifwaxisdepositedinthevehiclefuelsystem. Sincewaxhashighercetanenumber,itreducestheignitionquality andintroducesotherproblems(asdescribedabove). Stabilityof Indicationofdegradationinqualityduringlongtermstorage. dieselfuel Sulphur Lowersulphurcontentcanhavereducedparticulateemission,less content conversionintosulphurdioxideorsulphurtrioxideanddecreased wearinpistonringsandcylinderlinerssincesulphurcandepositsand inducecorrosion. Aromatic Loweraromaticcontentlowersemissionofparticulatesandlower content emissionofpolyaromatichydrocarbons(Nelson,Tibbettetal.2008) Waterand Waterallowgrowthoforganismssuchasfungiandbacteria,causing sediment filterplugging,corrosion,wearinengineandwearinfuelinjection content system Source:(Zvirin,Gutmanetal.1998).

2.2.2 Automotiveengine

Inpetrolengines,fuelandoxidiser(suchasoxygeninair)arepremixedintheintake manifold.The fuelͲair mixture enters the combustion chamber and is then compressedbythepistonoftheenginetoraisethepressureandtemperaturetomake it more ignitable.The spark plug initiates combustion of the fuelͲair mixture by

22  Chapter2:ReviewofLiterature introducinganelectricsparkandignitingthemixture.Thecombustionthenproduces mechanicalwork by rapid expansion of gas that pushes the piston down (Pulkrabek 2004).

DieselenginesdonotuseanelectricsparktoignitethefuelͲoxidisermixture.Theairis letintothecombustionchamberandcompressedbythepistonstoraisebothpressure andtemperatureoftheair.Justbeforereachingmaximumcompression,thediesel fuelisinjectedintothecombustionchamber.Theinjectionmixeshotairanddiesel fuel and initiates ignition due to the high temperature and pressure inside the chamber.Thecombustionproducesmechanicalworkintheformofrapidexpansion ofgasthatpushesthepistondown(Pulkrabek2004).Asthedieselengine’sfuelͲair mixtureisnonpremixed,theresultantdiffusivecombustionresultsinanincreaseof particulatesandgaseousemissions(TindalandUyehara1988).

FourͲStrokeCompressionignitionengine

FourͲstrokecyclecomprisesoffourseparatelinearstrokesofpiston.Thefirststroke (intakestroke)istheopeningofintakevalveandmotionofpistonfromitstopͲdeadͲ centre(TDC)positionintobottomͲdeadͲcentre(BDC)position,loweringpressureinside combustionchamberthereforeinducingair in.Duringthesecondstroke(compression stroke) both intake and exhaust valves are closed and the piston rises from its BDC position to TDC position therefore raising pressure and temperature of the air. The dieselfuelistheninjectedasthepistonapproachestheTDCposition,initiatingauto ignitiondueto thehighpressureandhightemperatureofairinsidethecombustion chamber.Thethirdstroke(powerstroke)occursasaresultofhigherpressurefrom rapidexpansionofgasfromthecombustionprocess,whichpushesthepistondown intotheBDCposition.Thefourthstroke(exhauststroke)theninvolvesopeningofthe exhaustvalvesandejectionofexhaustgasfromthecombustionchamberasthepiston returnstoitsTDCposition

The pressure inside the combustion chamber varies during different phases of combustionasillustratedinFigure2Ͳ4.Combustionchamberpressureduringafour stroke cycleyieldsa pressure curve in which the peak pressure is reached after the completionofthecompressionstroke.Withcombustionhowever,theextrapressure

23  Chapter2:ReviewofLiterature isgeneratedbytherapidexpansionofgasfromthecombustion.Inthecaseofdiesel engines, fuel is injected a moment before the piston reaches the TDC position to compensateforignitiondelayofair/fuelmixture.

FourͲstrokeandTwoͲStrokeengine–acomparison

Twostrokeenginesdonothaveseparateintakeandexhauststrokeasinfourstroke engines,theblowͲoutissimultaneouslyperformedduringthepowerstrokewhilethe intake is simultaneously performed with the compression stroke.In comparison to fourstrokeengines,twostrokeenginesaremorepowerfulastherearemorepower strokesperunitoftime(Stone1985).Duetothelessairpressuredifferencebetween atmosphereandcombustionchambersduringtheblowͲoutprocess,theresiduegas reduces volumetric efficiency for the next combustion, causing a decrease in fuel efficiencyandincreasesHCemission(Pulkrabek2004).

The  four stroke engine is more popular than the two stroke engine, although the outputlevelisaround75%ofitstwostrokeequivalent(ChallenandBaranescu1999). Someoftheadvantagesincludeeasierlubricationconditions,lowerthermalloadingof the engine, higher thermal efficiency and higher volumetric efficiency due to  longer periodsavailableforthegasexchangeprocess.

2.2.3 FuturedevelopmentsǦBiofuel

Biofuel is a general term describing liquid, gas or solid fuels derived from biomass sources (Demirbas 2008).Biofuel are blended with petroleum derived fuels (petro fuels)forimprovedcompatibilitywithcurrentengines.Addingbiofuelsuchasethanol topetroldecreasesemissionssuchasCO2fromtheenginebutvehiclemileageaswell asethanolhasalowerenergydensitythandieselandpetrol.Asacomparison,petrol has an energy density of 115,000 BTU/gallon and CO2 emission of 315 g/mile while ethanolhas76,000BTU/gallonandCO2emissionof243g/mile(GaffneyandMarley 2009).

24  Chapter2:ReviewofLiterature

 Figure2Ͳ4:Combustionchamberpressureduringafourstrokecycle Adaptedfrom(Stone1985)page65.

Biofuelgarneredpublicandgovernmentinterestforitsmoreenvironmentalfriendly nature,reductioninemissionsanditsabilitytobeproducedlocally,allowingnationsto be more energy independent from imported crude oil and its price volatility (Wiesenthal, Leduc et al. 2009). Since the environmental and economical cost associatedwithproductionofbiodieselisstillrelativelyhigh(IEA(InternationalEnergy Agency)2004),governmentpoliciesareoftenimplementedtoencourageresearchand theproductionofsecondgenerationbiofuel(FrondelandPeters2007;Szklo,Schaeffer etal.2007;PetersandThielmann2008).Asconsumers(ingeneral)havebeenshown tobemoreconcernedwith pricethanenvironmentalcosts,governmentalsupportis essentialtogenerateinterestforresearchonbiofuel(Bomb,McCormicketal.2007) For example, the European Union is encouraging the use of biofuel by partial tax exemptionsuntilitreachesthegoalof>10%biofuelsubstitutioninitstransportation fuelusage(Wiesenthal,.Leducetal 2009).

Totransformfeedstockintobiofuel,transesterificationisperformedtolowerviscosity levels(Murugesan,Umaranietal.2009),improvecetanenumberandimproveheating value(Hasimoglu,Cinivizetal.2008).Inchoosingbiofuelfeedstock,growerstendto choosefeedstockmoresuitabletothelocalenvironmentandyieldgreaterquantities. ExamplesincluderapeseedoilintheEUandpalmoilsinSouthEastAsiancountries suchasMalaysiaandIndonesia(KojimaandJohnson2006).

25  Chapter2:ReviewofLiterature

Compatibilitywithcurrentengines

Current petrol engines can use ~10% ethanol blend without any modification, but >10% higher ethanol content may require modification to the engine (IEA (International Energy Agency) 2004; Biofuels Taskforce 2005; Kojima and Johnson 2006).Forpetrolenginestobecompatiblewithbothpetrolandhigherethanolblend fuel,aflexfuelsystemcanbeutilisedtodetecttheethanolblendlevelandadjustthe engine operating parameters such as the airͲfuel ratio (Delgado, Araujo et al. 2007; Szklo,Schaefferetal.2007).Similarlyfordieselengines,nomodificationtotheengine isrequiredwhenusing<20%biodieselblend(Balat2005;Demirbas2007).

Amongthebiofuel,bioalcohols(suchasethanol,propenolandbutanol)aregrowingin popularity for their soot reduction owing to its additional oxygen atoms in the chemical structure.Ethanol is the most popular amongst the bioalcohols and is produced by fermenting sugars from vegetable materials such as corn, sugar cane, molasses (Rakopoulos, Rakopoulos et al. 2008), household wastes, wood and straw (Demirbas2008).Fordieselengines,10Ͳ30%ethanolͲbiodieselblendsarecompatible for use in diesel engines (Hansen, Zhang et al. 2005; Rakopoulos, Rakopoulos et al. 2008;Huang,Wang.etal 2009),although,fewstudieshaveinvestigatedtheeffectof mixingethanolwithpetrodieseltoincreasedieselengineperformance.

Althoughpureplantoilscanbedirectlyusedindieselengines,theirhigherviscosity (10Ͳ17 times higher than petrodiesel fuel) and lower cetane number requires modificationtodieselengines(Harwood1984;Demirbas2007).

Benefitsofbiofuel

InvestigationshaveshownbiodieselhasalowerlevelofemissionofCO,SO2,PM,total HCandbenzeneduringproductionandcombustionofbiodiesel(JoshiandPegg2007; RIRDC2007;Ferreira,dosSantosetal.2008).Biofuelcontainmoreoxygenmolecules hence allowing a more complete combustion therefore reducing emitted pollution.

The nitrogen molecules combine with oxygen in air within the biofuel to form NOx hence,increasingemissionsofNOx.

26  Chapter2:ReviewofLiterature

Biofuelcropscanbeproducedfromagriculturalplotsandsupplythedomesticmarket suchasisoccurringinBrazil(Pousa,Santosetal.2007;Coronado,deCarvalhoJretal. 2009),howeverothercountriesmayfindmoreeconomicalandagriculturalbenefitto import biofuel especially if the necessary infrastructures are not available and produciblecropsyieldlowamountofbiofuel(ThamsirirojandMurphy2009).

BiodieselcanbemorecosteffectiveduringproductionsincethebyͲproduct(glycerol) canbeconvertedintoethanol(Jegannathan,Chanetal.2009).Governmentssuchas the European Union support development  of biofuel to increase economy competitiveness,energysupplysecurityandenvironmentalprotection(Jansen2003).

Costsofbiofuel

Biofuelproductionrequiresvastarableland,1%ofarablelandisneededtosubstitute 1% oftransport fuel usage (IEA (International Energy Agency) 2004; Thamsiriroj and Murphy2009).Decreasingtheamountofavailableland forfoodproductionleadsto increaseinfoodandglobalcommodityprices,includingcornandtortillasinUSA,milk inChinaandcanolaoilintheEU(RIRDC2007;Mueller,Andersonetal.2011).

As different crops yield different amount of biofuel, choice of biofuel crop to grow affecttotal amountofarablelandneededforbiofuelproduction.Currently,palmoilis inhighdemandforitshighbiodieselyieldsthanothercrops.However,countriessuch asGreece(Panoutsou,Namatovetal.2008),Ireland(ThamsirirojandMurphy2009), India and Tanzania (Peters and Thielmann 2008) have native crops only capable of yieldinglowamountofbiofuel.Therefore,thesecountriesmayfindmoreeconomic efficiency to import palm oils instead of growing it themselves (Thamsiriroj and Murphy2009).Thishasenvironmentalimpactsinpalmoilproducingcountriesaswell, as the high demand for palm oil encourages growers to clear forests for  palm oil plantations(Fitzherbert,Struebigetal.2008).

The Emission reduction benefit by substituting petroleum fuel to biofuel is also questioned.Lower biofuel blend ratio has been criticised to have minimal if not negligible greenhouse gas emission reduction (Niven 2005; RIRDC 2007; Szklo, Schaefferetal.2007).Inaddition,current biofuelproductionmethodsarerelatively

27  Chapter2:ReviewofLiterature inefficient since not all parts of the crop is used during biofuel production (Moore 2008).Inresponsetothischallenge,secondgenerationbiofuelproductionmethods are under development to utilise more parts of the crop, therefore increasing productionefficiency(KojimaandJohnson2006)and poseslessthreattofoodsupply level(Deurwaarder2005;Peters andThielmann2008).However secondgeneration biofuel crops are relatively recent and still under development, therefore requiring new infrastructures, governmental support and subsidies (Charles, Ryan et al. 2007; Moore2008;TimilsinaandShrestha2011).

2.2.4 Generationofdieselexhaust

Engineexhaustcontainsnumerouscompoundsasnonfuelcomponentssuchasengine oil which may seep through and combust together with the diesel fuel.The componentsandtheirsourcesinclude(Eastwood2008): x Volatileorsoluble 2Ͳ o Sulphatefraction:H2SO4.nH2O(i.e.:SO4 )+metalsulphatesfrom enginefuelandlubricant. Ͳ o Nitratefraction:HNO3(i.e.NO3 )+metalnitratesfromreaction

betweenH2OandNO2.

o Organicfraction:ͲCH2+N,OandSfromenginefuelandlubricant. x NonͲvolatileorinsoluble

o Carbonaceousfraction(soot):ͲC8H,C9H+N,OandSfromenginefuel, lubricant,airandmaterialdisintegration. o Ashfraction:Numerousmetals(e.g.:Fe,Cr,Cu,Zn,Ca)andafewnonͲ metals(Si,P,S,Cl)fromenginefuelandlubricant.

Comparedtopetrolengineemission,dieselengineemissioncontainshigherNOxand particulates level but lower CO emissions level.Although a higher level of NOx is emittedduetothehigherpressureandtemperatureofthecombustionprocess,there areotherfactorsaffectingdieselexhaustemissioncontentincludinginjectionrate,age ofengine,fuelinjectornozzlegeometry(Clark,Kernetal.2002;SarviandZevenhoven 2010)andcombustionchambergeometry(asoutlinedinTable2 7).

Diesel exhaust particulates are spherically shaped carbonaceous particulates generated by the combustion within the diesel engine.The particulates are mainly measuredbytheirsizeinsteadoftheirmassduetotheinabilityofmassmeasurement devices to measure the number of particulates and their surface area.Although 28  Chapter2:ReviewofLiterature primaryparticulatestendtobesphericalinshapeatformationinsidethecombustion chamberofanengine,thereisatendencyfortheseparticulatestoagglomerateand formnewshapesandsizesbeforeexitingtheexhaustsystem(Stevenson1982).

Table2Ͳ7:Factorsaffecting dieselengineemissions

Factors HC NOx CO Unburned Smoke HC Injectiontiming 3 3 1 3 2 Injectionrate 2 2122 Sprayconeangle 1 1 1 1 1 Secondaryinjection 3 1131 Combustion 3 3 1 1 3 chambergeometry Injectorlocation 2 2111 Symbols:highernumbersindicatethegreatereffecteachfactorhasonincreasing emissions;Adaptedfrom(AssemblyofEngineering1982).

The term “size used” in describing diesel particulates should be carefully defined as various size measuring methods yield different measurements.For example, sizes measured from TEM microscopy may be similar to measurementsm fro  scanning mobility particulate sizers (Mathis, Kaegi et al. 2004) and electrical low pressure impactors (Shi, Harrison et al. 1999) but may be double the measurement from differential mobility analysers (Chandler, Teng et al. 2007).In addition, errors in measurementcouldbecausedbyinterferencesduringsampling,henceminimisation oferrorsisessentialbyutilisingappropriatesamplingmethodssuchasthermophoretic samplingforTEMobservationofdieselexhaustasitprovidestheleastinterferenceto theTEMsystem(Maynard1995).

ThesizeofprimaryparticulatesismainlyaffectedbytheenginespeedandtheairͲfuel ratio.These factors in turn affect oxygen concentration, gas temperature and residence time period of particulates within the combustion flame.Studies have shown an increase in particulate size with higher engine speed (Shi, Harrison et al. 1999).Howeverasgeneratedparticlesreachtheexhaust;theexhausttemperature has moreeffect on the particulate size.At temperature exhaust of <300oC, no size differenceofparticulatesizeisdetectablealongtheexhaustpipe(Lapuerta,Martoset

29  Chapter2:ReviewofLiterature al. 2007), However, at higher exhaust temperature (>300oC) there are conflicting resultssuchasbyZhuetal(2005)whichshowedatrendindecreaseofparticulatesize whileNeeretal(2006)showedatrendinincreaseofparticulatesize(Zhu,Leeetal. 2005;NeerandKoylu2006).

DieselExhaustParticulates(DEP)ismainlycomposedof acarbonaceouscoreinwhich various organic compounds may adsorb onto the surface or within the particulate’s core(Løvik,Høgsethetal.1997;Heo,Saxonetal.2001).Thisposeshealthhazardsas both physical properties of particulates and its adsorbed compounds may have adverse health effects (Granum and Løvik 2002).Organic compounds that could adsorbonDEPincludealiphaticandaromaticHC(Schuetzle,Leeetal.1981;Bayona, Markidesetal.1988)whichmaysuppressalveolarmacrophagefunctionsbyreducing cytokineproduction(Siegel,Saxenaetal.2004)thusrenderingthehumanpulmonary systemasatargetorgan.

 Figure2Ͳ5:AgglomerateandprimaryparticlesofDEP Agglomerate (left); Primary particle (top right); further magnified image of primary particle(bottomright);Adaptedfrom(Lapuerta,Martosetal.2007).

Incomparison,dieselenginesemitupto100timesmorePMthanpetrolengines(Riedl andDiazͲSanchez2005).Dieselengineparticulatesarealsosmallerinsizethanpetrol engineparticulates(BanͲWeiss,McLaughlinetal.2008;Wehner,Uhrneretal.2009).

Diesel engines are also capable of emitting smaller PM  sizes including PM1; its generationmechanismisnotwellunderstood.Althoughfactorssuchascombustion equivalenceratio,exhaustdilutionrate,residencetimeduringdilutionarespeculated

30  Chapter2:ReviewofLiterature

tohaveeffectsonPM1formation(AmannandSiegla1981;Shi,Harrisonetal.1999; Ning,Cheungetal.2004;NeerandKoylu2006).Ascurrentairpollutionregulatory requirements are based on larger sized PM10 and PM2.5, it may be inadequate to addressrisksposedbythesmallersizedparticulatesastodate,theserisksarenotwell characterised.

2.3 Riskassessmentandmanagement Themainaimoftoxicologyandepidemiologystudiesistoassessrisksbyexposureto contaminants.Understanding the hazards and their associated risks allows minimisationof risks.Although risk assessmentand risk management are different, bothareinterͲdependentoneachothertoreducerisks.Riskassessmentistheability to identifyhazards,elucidatepotentialrisksposedbasedonavailabledatasuchasthe numberofpeopleexposed.Riskmanagementusesinformationgeneratedfromrisk assessmenttoperformriskreductionandpredictitseffectivenessbyanalysissuchas thecostͲbenefitanalysistodecideonappropriateactionstocontroltherisks.

Thewholeriskassessmentprocesscanbeoutlinedasfollowswithsteps1Ͳ4definedas theriskassessmentphaseandsteps5Ͳ8definedastheriskmanagementphase(Van Leeuwen1995): 1. Hazardidentification:Identifyadverseeffectsofasubstance,characterise behaviourofsubstancewithinenvironmentorbiological systemsuchas(cells andorgans). 2. Effectsassessment:StudydoseͲresponserelationshipofasubstance, quantitativelyestimateseverityofeffectwithdifferentlevelsofexposure. Dataaregatheredfromexperiments,fieldstudies,etc. 3. Exposureassessment:Calculate/estimateamountofsubstancethepopulation isexposedto. 4. Riskcharacterisation:Basedonthefirstthreesteps,determinegeneralrisk andadverseeffectsofasubstanceonitstargetenvironmentandbiological system. 5. Riskclassification:Determineifrisksareatacceptablelevelandwhetherany furtherriskreductionisneeded,usuallyperformedbypolicymakerswithina consultativeframework. 6. Riskbenefitanalysis:Ifriskreductionisdeemednecessary,addressexisting issuessuchastechnicalfeasibility,economicandsocialfactorsandresearch dataavailability.

31  Chapter2:ReviewofLiterature

7. Riskreduction:Reducesriskefficientlywithadministrativesimplicityand acceptedbythegeneralpublic. 8. Monitoring:Continuallyobserveeffectivenessofriskreductionandif necessary,followupwithimprovements/enforcementsifneeded.

The risk reduction and monitoring phase are continually cyclic in which constant monitoringandenforcementofriskreductionpoliciesaremaintainedandifneeded furtherriskreductioncanbeapplied.Oneexampleofoccupationalriskmanagement istheapplicationofsixdifferentlevelsofriskcontrol,withthehighestcontroltaking prioritytobeimplementedandlowerprioritycontrolsimplementedonlyafteroptions athighercontrolhierarchyhavebeenexhausted(NewSouthWaleslegislation2001; SafeWorkSouthAustralia2007).Thesecanbedescribedindescendingpriorityorder asbelow: 1. Elimination:Ridofhazardshencenofurtherriskcontrolisneeded. 2. Substitution:Replacehazardouscompoundswithlesshazardouscompounds. 3. Isolation:Tosingleoutthehazardouscompoundsuchthatnoonewillneedto makecontactwithit. 4. Engineeringcontrols:Provideengineeringsolutionsuchassafetydevicesto protectthepersonnel. 5. Administrativecontrols:Providepersonnelwithtrainingtoensurecompetency inhandlinghazardousmaterials,dealingwithemergency,etcandcaneitherbe generalcontrol(e.g.supervisionortraining)orspecificcontrol(safeworking system). 6. Personalprotectiveequipment(PPE):Providepersonnelwithprotection devicessuchasgloves,respirators,etc.

Contributionoftoxicologyworksinriskmanagement

Toxicologicalworkandotherdisciplineshavecontributeddataandevidenceforrisk assessors to create a hazards overview and determine its corresponding risks. Disciplinesthathavecontributedtoenvironmentaldecisionsinclude(Patton1993): 1. Laboratoryfieldwork:Disciplinebasedincludingchemistry,biology,geology, toxicologyandepidemiology. 2. Riskassessment:Multiplescientificdisciplinesincludingphysicalsciences (safety),occupationalhygiene,statistics,medicineand sciencepolicy. 3. Riskmanagement:Multiplescientificdisciplinesincludingnatural,physicaland socialsciences,economics,politics,lawandothersinputssuchassocialvalues andconcerns.

32  Chapter2:ReviewofLiterature

Data from laboratory/field work are used in risk assessment within collaboration betweenscientificdisciplines.Theriskassessorsthencollaboratewithriskmanagersto strategiseinminimisingrisks.Oneexampleofsuchaprocessisepidemiologicalstudy results providing evidence for lowering exposure limit of lead from 80 μg/m3 to 2 μg/m3(GilbertandWeiss2006)henceimprovingqualityoflifeforsusceptiblegroups ofthepopulation,suchaschildren.

Theepidemiologyexamplealsohighlightstheneedforriskassessorsandmanagersto considerhowtoanalysetheinformationandstrategiseriskminimisation.Sincethe benefits may not be beneficial for the whole population as for example lowering exposurelimitofPbismorebeneficialtothosemoresusceptibletoPb(Rutter1983),

Tobalancethecostsandbenefitsandreduceunnecessarycosts,variousanalysisare essential(Lutter2001)OneexampleofawellbalancedcostͲbenefitsolutionwasthe replacementofPbcontaminatedwindowstominimiseexposuretoPbandbringother benefits such as higher energy efficiency and higher security for the house (Nevin, Jacobsetal.2008).

Anothercontributionoftoxicologytoriskassessmentandmanagementisimproving efficiency in assessing a new chemicals’ potential risk to humans (Sakai, Shoji et.  al  2001).TheUSAEnvironmentalProtectionAgency(EPA)regularlyfiltersoutpotentially hazardous materials for further screening to determine hazards and minimise their posedrisks.Conventionally,invivostudieswereutilisedtoinvestigatethetoxicityof the new compounds.As technological improvement grows exponentially, the EPA neededfastertestingand fasterapprovalprocess.Inresponsetotheseneeds,EPA has adopted various inͲvitro and in silico methods such as quantitative structureͲ activity relationship (QSAR) analysis, to increase efficiency in screening and filtering out more hazardous chemicals/drugs for further screening therefore allowing less hazardouschemicalstobepassedforlessstringentscreening(Clements,Nabholzetal. 1993).

 

33  Chapter2:ReviewofLiterature

Regulatoryrequirementofriskassessmentandmanagement

Riskassessmentandregulationhasbecomepartoflegalandregulatoryrequirements in various jurisdictions as outlined in Table 2Ͳ8.Many regulations emphasise risk minimisation of hazards for the workers who may have been exposed to hazardous compoundsforextendedperiodsoftime(SchulteandSalamancaͲBuentello2007).

Table2Ͳ8:Regulationsrequiringsafetyimprovementtoworkingconditions

Country Legislation Australia NewSouthWalesOccupationalHealthandSafetyAct(OHSA)1983instateof NewSouthWales. WorkHealthandSafetyAct2011(nationallevel;tocommenceon1stJanuary 2012). Germany Arbeitsschutzgesetz(ArbSchG)1996(OccupationalHealthandSafetyact). Chemikaliengesetz(ChemG)(Statuteconcerningchemicalsubstances). UK HealthandSafetyatWorketc.(HSW)Act1974 USA OccupationalSafetyandHealthAct. ToxicSubstancesControlAct. Japan Article65Ͳ2oftheIndustrialSafetyandHealthLaw. Article21Ͳ1oftheEnforcementOrderoftheIndustrialSafetyandHealthAct CabinetOrder. Articles25,26,26Ͳ2,and26Ͳ3oftheOrdinanceonPreventionofHazardsDueto Dust;MinistryofLabourOrdinance. European GeneralDirective89/391/EEC(ontheimprovementsinthesafetyandhealthof Union workersatwork). Directive 98/24/EC, Directive 2004/37/EC, Directive 1992/92/EC (on the protection of the health and safety of workers from risks related to chemical agents,carcinogensormutagens,andexplosiveatmospheres). Council  Directive 89/655/EEC (concerning the minimum safety and health requirementsfortheuseofworkequipmentsbyworkersatwork). Adapted from (Parliament of New South Wales ; Malich, Braun et al. 1998; Myojo, Ogamietal.2010;BhattandTripathi2011).

Currentchallengesinriskassessment

Riskassessmentcanbealaboriousandtimeconsumingprocess,thereforehavinga complete hazard and risk information may not be possible.As thousands of new chemicals are introduced into the market therefore strain on the risk assessment process,isbecomingamajorconcern(VanLeeuwen,BroͲRasmussenetal.1996).In addition,insufficientdataandincompleteassessmentmodelsmayincurmeasurement

34  Chapter2:ReviewofLiterature uncertaintyhenceextrapolatinglaboratoryresultstotherealworldmaycausefurther uncertainty(VanLeeuwen1995).

Recently,theadventofnanotechnologysuchasnanoparticles(MaynardandKuempel 2005) has raised the need to modify the current risk assessment process. Modificationsneededincludecreation ofanintelligent,tieredtestingstrategyaidedby carefullydesigneddecisiontreeapproachwhichaimstoreduceresourcesneededand increase efficiency.One potential solution is to integrate the process within the product development process as part of the manufacturer’s responsibility (Arts, Muijseretal.2008;FairbrotherandFairbrother2009;Savolainen, Aleniusetal.2010).

Althoughtheimportanceofriskassessmentisacknowledgedbypolicymakers,ithas been criticised as being nonͲrealistic, unclear for general public and has an overͲ reliance on in vivo data (Graham 1995).Extra regulation such as requirements for producers to provide more toxicity data on developed chemicals would allow risk assessorstodesignmoreefficientstudies.Inturn,thisallowsriskassessorstoscreen out unneeded studies as large amounts of data do not necessarily mean better consensus between risk assessors (Rudén 2006).Therefore, it is also important to apply risk assessment and management in a socioͲpolitical context to increase its applicability(Eduljee2000)andimprovethescientificbasisofriskassessment.This can be achieved by applying probabilistic risk analysis instead of using deterministic quotient of dose and noͲeffect level (Jager, Vermeire et al. 2001; Aven 2011).In addition, to improve risk communication, providing a proper prior understanding of theriskassessmentprocessisneeded(Montague2004).

2.3.1 Epidemiology

Epidemiologyisthefieldofstudywhichdeterminesthecauseofhumandiseasesby analysing observational data (Mawson 2002).Epidemiology as a tool is used to establishanyassociationbetweendifferentsetsofdata.Associationisdefinedashigh significancebetweentwocategoriesofevents.TheassociationscouldeitherbenonͲ statisticallyassociation(independent)orstatisticallyassociated.Statisticalassociation can further be divided into non causal (secondary) and causal (direct or indirect) association(MacMahonandTrichopoulos1970).Historically,epidemiologicalstudies

35  Chapter2:ReviewofLiterature have drawn links between cause and effects.One example is the work of Dr John Snowduring19thcenturywhoshowedcholeraaswaterͲbornediseaseinsteadofairͲ borne(asitwasacceptedin19thcenturyBritain)(Gratt1996).

Epidemiology studies need to hypothesise the population at risk, potential causes, expected effect and if possible, any doseͲresponse and timeͲresponse relationships. Since epidemiological studies usually have less variables than desired, outcome can still be deduced by applying additional constraints such as age, gender and/or occupation of population, various testing methods such as difference, agreement, concomitant variation and analogy are available to strengthen the hypothesis (MacMahonandTrichopoulos1970)including: x Methodofdifference:Searchesforavariableabsentfromonegroupbut existentinanothergroup,inwhichcomparisonbetweenbothgroupswillshow whetherthevariablecausesanyeffect. x Methodofagreement:Searchesforavariablecommontomultiplegroupsand ifsimilareffectsarefoundthenthevariablemaybethecause. x Methodconcomitantvariation:Searchesforfactorswhichfrequencyvariesat differentfrequencyofdisease.Morequantitativeanalysisthandichotomous resultofdifferenceandagreementmethods. x Methodofanalogy:Usingdeductivereasoningandavailabledata,explanation offindingcanbeperformed.Howeverfalseanalogiesarestillpossibleeven thoughlogicalreasoninghavebeenestablished.

To determine causality, epidemiologists may encounter challenges as direct experiments may not be available.Epidemiologist could strengthen causality considerationbyemployingvariousanalysestoproveanydirectorindirectcausality including(MacMahonandTrichopoulos1970):

x Timesequenceconsideration:Determineifcausativeeventprecedethe effects. x Strengthofassociation:Thestrongerthestatisticalassociationbetween differentevents,themorecausal.Forexampleexistenceofdoseresponse relationship. x Consonancewithexistingknowledge o Anyextraknowledgetosupportepidemiologyfindingwillmakeitmore reasonable:Forexample,anexistingknowledgeofcellularandsubͲ cellularmechanismsmaysupportanepidemicepidemiologyfindings. o Similarityofdistributionofcausativeeventanditseffects:Amore similardistributionindicatesstrongercausality. 36  Chapter2:ReviewofLiterature

o Evidenceobtainedthroughexclusion:Themoreunsuccessfulattempts toidentifyothernoncausalassociationofthehypothesis,thestronger thecausality.

In addition, prior to judging an association to be a causality, epidemiologists often refertotheninecriterialistedbyAustinHillin1965(Hill1965).Thelistcontainswhat iscommonlyperceivedasninedifferentrulesofcausation,althoughitwasoriginally anexampleprovidedtheauthortonotoveremphasiseusageofstatisticstoexplain possiblecausations(PhillipsandGoodman2004).

The difference between direct and indirect causality is the existence of any intermediateprocess.Directcausalityhas nointermediateprocesswhiletheindirect effect has intermediate process before any effect is observed.For example, scurvy causedbylackofVitaminCisadirectcausalitywhereasscurvycausedbylackoffruit consumption is an indirect causality since the intermediate process is the lack of vitaminCconsumed(MacMahonandTrichopoulos1970).

Epidemiology studies should be scrutinised for any biases (detection, transfer and susceptibility bias).Detection bias occurs when increased of conditions reflects changeofdetectionmethodinsteadoftheconditionitself,transferbiasoccursdueto history difference between case and control subjects and susceptibility bias occurs when possiblefactorsforaconditionisnottakenintoconsideration(Feinstein1997).

Epidemiologicalstudiesonairpollution

Epidemiologicalstudieshavecorrelatedhigherlevelofexposuretoairpollutionwith highermortality.Bycomparingdailymortalitydatawithdailyairpollutionlevels,it hasbeensuggestedaprevalenceofpulmonaryand cardiovasculardiseasesamongst thepopulationexposedtoepisodesofincreasedairpollutiondays(Dockery,Popeet al.1993;Abbey,Ostroetal.1995;Rossi,Vigottietal.1999).Inaddition,population segmentwithgreatersusceptibilitytopulmonaryandcardiovascularrelateddiseases areinfants,elderlyandthosewithpulmonaryconditionssuchaasasthm (Heinrichand Slama2007).

Althoughepidemiology hascontributed much knowledge and basisforeffectsofair pollutiononpublichealthasshowninTable2Ͳ9,itisfacingcriticisms.Onecriticismis

37  Chapter2:ReviewofLiterature thetendencybymassmedia(duetopoorunderstandingofepidemiologymethods)to treat correlation as direct causality without considering the confounding factors (Beaver 1997).Recent epidemiological studies also have been found to have inadequatelyaddressedpotentialconfoundedvariableswhichmaycausebias(Wynder 1994; Groenwold, VanDeursen et al. 2008).In addition,as it is uncommon for the studied population to be exposed to just one single pollutant (exposure to other pollutants may include asbestos and cigarette smoking), it adds to uncertainty in measuringlungcancerriskposedbyexposuretodieselexhaust(Higgins;1984 Muscat 1996).

Toxicology and epidemiology fields are complementary.While epidemiology is observationalandaimstoprovideageneraloverviewofevent,itisusuallynotfeasible toestablishadoseͲresponserelation.Meanwhile,toxicologyisexperimentalandaims topredictsubsequenteventandabletogeneratedoseͲresponserelationships(Gaffey 1985).Toimproveepidemiology’sapplicability,futurestudiesneedcollaborationwith other fields such as experimental toxicology to generate a more robust hypothesis. This can be achieved by supplementing toxicity mechanism data to epidemiological data(Samet1995;Schlesselman1996;Mawson2002;Bracken2009).



38  Chapter2:ReviewofLiterature

Table2Ͳ9:Epidemiologystudiesonairpollution

Source Method Result

(Gamble, Jones et al. Examination of 238 male workers diesel bus garage workers. Probablechroniceffectonpulmonaryhealthofworkerssuchas

1987; Gamble, Jones ComparedsymptomstoacuteNO2,respiratoryparticulatesand increasedcoughinganddifficultybreathing.Acuteeffectswere etal.1987) chronicdieselexhaustexposurelevels. noticedbutnotstatisticallysignificant.

(Dockery,Popeetal. Comparisonofdailymortalityratesandamountofparticulate FineͲparticulate air pollution may contribute to increased 1993) airpollutionindifferentUScities mortalityfromcardiovasculardisease.

(Mauderly1994) Review of toxicological and epidemiological studies of petrol Petrolanddieselexhaustexposureposeriskofcancerousand engineanddieselengineexhaust. nonͲcancerouspulmonarydisease.However,epidemiologydid notallowquantitativeestimationofrisk.

3 (Abbey, Ostro et al. Long term study of nonͲsmokers living near airports, data PM2.5 concentration > 20 μg/m  is associated with increase in 1995) gatheredusingquestionnairescontainingrespiratorysymptoms pulmonaryrelateddiseasessuchasairwayobstructivedisease, relatedquestions. bronchitisandasthma.

(Bhatia, Lopipero et Meta analysis of previous literatures on relation between MetaͲanalysis showed correlation between increased of al.1998) occupationalexposuretodieselexhaustandlungcancer. exposuretodieselexhausttoincreasedlungcancerrisk

(PopeIII,Verrieretal. Ambulatoryelectrocardiographicmonitoringonsevensubjects Change in heart rate and heart rate variability may indicate 1999) for a total of 29 days before, during, and after episodes of possible pathophysiologic mechanisms or pathways linking

elevatedpollutionespeciallyPM10. cardiovascularmortalityandPM.

(Rossi, Vigotti et al. Comparisonofdailymortalityandairpollutants(SO2,TSPand Increaseinbothpulmonaryandcardiovascularrelateddeaths.

1999) NO2).

39  Chapter2:ReviewofLiterature

(McDonnell, NishinoͲ Long term study of male nonͲsmokers recruited in 1977 and Fine fraction of PM10 (i.e.: PM2.5) is associated more with Ishikawaetal.2000) datafromquestionnairesanddeathcertificatewerecompared mortalityofthesubjectsofthestudy. withairpollutiondatacollectedatnineairports

(Schwartz and Neas Longitudinalstudyon1,844schoolchildrensampleinsixurban Fineparticulateshavestrongerassociationwithasthmarelated 2000) areas in eastern USA. Compared daily report on child’s responses compared to coarse particulates.Sulphate

respiratorysymptomstoPM2.1andPM10measurements. componentoffineparticlesisabetterproxyfortoxicparticle constituentsthantotalfinemass.

(Hoek, Brunekreef et Randomsampleof5000peoplefromfullcohortofNetherlands Cardiopulmonary deaths associated with living near major al.2002) Cohort study on Diet and Cancer study from 1986 to 1994, roads.NonͲcardiopulmonary, nonͲlung cancer deaths were LongͲterm exposure to trafficͲrelated air pollutants (soot and unrelated toairpollution.ChronictrafficͲrelatedairpollution

NO2)wereestimated. exposuremayshortenlifeexpectancy.

(Pope III, Burnett et 500,000adults’healthstatuseswerelinkedwithpollutiondata. Chronicexposuretofineparticulatescanincreaseriskfactorfor al.2002) cardiopulmonaryandlungcancermortality.

(McConnell, Berhane 175 fourth graders were recruited and with follow ups.Data Organic carbon and NO2 pollution level potentially causes etal.2003) fromchildrenwithhistoryofasthmawithbronchiticsymptoms chronicsymptomsofbronchitisinchildrenwithasthma. were compared with data from air pollution monitoring stations.

(Gauderman, Vora et Two cohorts of fourthͲgrade children were recruited in 1993 Residentialdistance<500mfromafreewayisassociatedwith al.2007) and 1996, health status of children were followed up to 8 by significantdeficitsin8Ͳyearperiodrespiratorygrowth,resulting surveys.Comparisons between survey meteorological data indeficitsinlungfunctionatage18. werethenmade.

40  Chapter2:ReviewofLiterature

(Hales, Blakely et al. RegressionstudyusingPM10meteorologicaldataandfollowup An increase of exposure to PM10 increases mortality risk 2010) fromcensusdata. especiallytothosewithlessresidentialmobility.

(Maté, Guaita et al. Statistical comparisons between circulatory system based LinearrelationshipbetweenPM2.5levelsandmortality.

2010) mortalitydataandPM2.5concentrationsinMadrid,Spainwere made.

(Gehring, Wijga et al. Statisticalcomparisonbetweenpretermweightandbirthterm Positive, statistically nonͲsignificant associations between

2011) weight with estimated maternal exposure to NO2, PM2.5 and exposure to soot during entire pregnancy and during the last soot during pregnancy using logistic and linear regression month of pregnancy and preterm birth and no indication of modelswithandwithoutadjustmentformaternalvariables. adverseeffectofairpollutionexposureonterm birthweight.

(Villeneuve,Parentet Casecontrolstudybycollectingquestionnairesfrom1681lung Positive associations between lung cancer and occupational al.2011) cancer and 2053 control subjects of >40 years old in various exposuretoasbestosandsilica.Exposuretoasbestos,silicaand jobs with exposure to asbestos, crystalline silica, petrol and diesel emissions increase risk of squamous and large cell lung dieselemissions. cancer.Petrol emissions were unrelated to lung cancer (risk maybeunderrated).

(Van Roosbroeck, 40childrenparticipatedinthestudy,fourmonitoringsessions 35%higherpersonalexposureto‘soot’forchildrenlivingnear

Wichmann et al. in which children carry portable sampler in a backpack to busy roads while NO, NO2 and NOx exposure were 14Ͳ15%

2006) monitorNO,NO2andsootlevels. lower.Resultsshowproximityfrombusyroadsagoodmeasure of exposure in epidemiological studies on effects of traffic air pollutioninchildren.

41  Chapter2:ReviewofLiterature

2.3.2 InǦvivoandinǦvitrotoxicologicalstudies

Invivomethodsinvolvetheusageofanimalsasmodelsforthehumanbody,therefore enabling the toxicity assessment of chemicals (Crone 1986).Recent in vivo studies includetoxicityassessmentofsubstancesonhumanssuchasDEP(asoutlinedinTable 2Ͳ10).

Drawbacks of in vivo studies include its reliability of results due to crossͲspecies extrapolation problem.One example of error caused by extrapolation problems is withxenobiotictransformationofparacetamolstudiesashumansaremoreresistant tohepatotoxiceffectsofparacetamolthananimals(Tee,Daviesetal.1987).Another exampleofinvivoshortcomingsasamodelfor humantoxicitystudiesisthalidomide whichwasmarketedasmorningsicknessmedicationforpregnantwomen.Meanwhile there has been a higher rate of birth defects by mothers who were prescribed thalidomide,afterfurtherstudiesthalidomidewasdeterminedtobeteratogenicand subsequentlywithdrawnfromthemarket.Claimsweremadethatin vivotestingdid notraiseanyalarmofitspotentialteratogenicityeffectsonhumans(Monamy1996)or atleastnotenoughresultswereavailabletoprovethesafetyofthedrug(Nicolland Russell 1992).Whether the thalidomide tragedy could have been avoided is still debatable and can be provocative (Critchley 1998; Dally 1998; Smithells 1998). Nevertheless, the tragedy stirred interest for more stringent and diverse testings of drugs and other possible toxins such as by US Food and drug administration (FDA) guidelinesonteratogenicitytestings(Raheja,Jordanetal.1988).

InͲvitrotoxicologyinvolvestheusageofisolatedcellsinsteadofthewholeorganismto assessthetoxicityofcompounds.Progressintechniquesandtechnologytoculture humancellshaveallowedtheestablishmentofinͲvitrotoxicologyasaformoftesting hazardoussubstances(MothersillandSeymour2003).Thisallowstoxicitytestingof compoundsatconcentrationlevelslethaltohumans (Ekwall1983).InͲvitrotestinghas thefollowingadvantages(Vickers1997): x Species(includinghuman)differencesandsusceptibility x Investigatemechanismofadverseeffects x Identifymarkersorsurrogatemarkersforclinicaluse x Determineconcentrationsatwhichtoxicityisreversibleandirreversible

42  Chapter2:ReviewofLiterature

x Addresswhetherparentcompoundormetabolite(s)isresponsiblefortoxicity x CrossͲspeciescomparisonofcompoundormetabolite(s)isresponsiblefor toxicity x Druginteractions,induction,peroxisomeproliferation x Potentialuseofprotectiveagentstocircumventorattenuatetoxicity x Investigatemechanismsandstimulitoinduceregeneration x Aidinthedesignoffurtherinvivotests InperforminginͲvitrotoxicologytesting,itisimportanttoconsidermultipleassaysand cell lines as inͲvitro toxicology methods have their own limitations such as overͲ sensitivityofsomecelltypes(Parish1986;Korting,Schindleretal.1994).AlthoughinͲ vitromethodshaveadvantagesoverinvivomethods,someoftheseadvantagesmay wellbeadisadvantage.OneexampleofsuchisinͲvitrotoxicology’srelativesimplicity andlesscrossͲspeciesextrapolationproblemencounteredbyinvivostudies.However, the simplicity of inͲvitro studies renders it unable to reflect entire organism’s more complexbiologicalsystem,thereforeinͲvitrocanneverfullysubstituteaninvivostudy (MothersillandSeymour2003;Zucco,DeAngelisetal.2004).Additionaladvantages anddisadvantagesofinͲvitrosystemsareaslistedbelow(Beaudoin1985). Advantages: x Cell,tissueandorgansreceivedirectexposuretotestedcompounds. x Effectscanbeobservedstrictly. x Underdefinedcultureconditions,differentiationisconsistentand reproducible. x Lessexpenseandtimerequired. x Fastertestingbyautomation(White2000). x Supplementsunansweredquestionsbyepidemiology(Allen2006). x Individualcomponentsoforganreactions canbestudied(Allen2006). Disadvantages: x Drugmetabolisingenzymesareusuallyabsent. x Unknowncompoundsmaybedifficulttotest. x Extrapolationofresultsdifficult. x Cannotpredictadditiveorsynergisticeffects(White2000). x Moreemphasisonacutetoxicitytestinginsteadofchronicorrepeated exposuretoxicity(Eisenbrand,Pool ͲZobeletal.2002). x ExclusionofantagonistordefenceprocessesasininͲvivoresults(Parish1986; Allen2006). x Limitedrouteofadministration(Parish1986;Allen2006).

43  Chapter2:ReviewofLiterature

Table2Ͳ10:InvivoexperimentsonDEPeffects

Ref Animal/subject Contaminant Endpointsinvestigated

(Bond, Mauderly F344/Nratsexposedfor7h/day, Filtered air (controls) or to diluted DE DNA from rats’ lungs were analysed for etal.1990) 5days/week,forupto12weeks (0.35–10mgsoot/m3) presence of DNA adducts by 32PͲ postlabeling

(Ichinose, Yajima ICR mice intratracheally injected DEPcollectedonglassfibrefilterfroma Formation of 8Ͳhydroxydeoxyguanosine in etal.1997) withDEPinsterilesolution. 2740 cc diesel engine set at 1500 rpm. lungDNA and10Nm

(Birumachi, Transgenic mice carrying the DEP collected from diesel engine set at Respiratory resistance, calculated from Suzuki et al. humanprototypecͲHaͲrasgene 1500rpmand10kg/m measured endotracheal pressure and 2001) respiratoryflow

(Sato, Onose et F344 rats, killed 24 h after 3 mg/m3 of suspended particulate Glycosaminoglycans, HematoxylinͲeosin al.2001) cessationofexposure; matter staining, Alcian blue staining and Immunohistochemicalanalysis.

44  Chapter2:ReviewofLiterature

(Dybdahl, Risom BALB/CJ mice and transgenic 90Ͳmin of DEP inhalation at 20 and 80 BAL cells number and cell viability, cell etal.2004) mice mg/m3  in one session or split into four composition (macrophages, lymphocytes  sessionof 90ͲminofDEPinhalationat5 andneutrophils) and20mg/m3

(Pourazar, Frew Healthy human subjects, nonͲ DEat300ʅg/m3 for1hinanexposure Bronchoscopy was performed to obtain etal.2004) atopicandnonͲsmoking. chamber endobronchialmucosalbiopsies

(Knudsen, Blood samples from shaleͲoil N/A DNA damage, DNAͲadduct, 32PͲ Gaskell et al. mineworkersinEstonia. postlabelling 2005)

(Shima, Koike et BALB/cmice. DE from 2740 cc diesel engine at 1500 Number of inflammatory cells and al.2006) rpmand10kg/m,exhaustwascollected neutrophils infiltrating into peritoneal onglassͲfibrefilter cavityofmice

(Hemmingsen, TimeͲmated young adult female Inhaled 20 mg/m3 of DEP SRM2975 or immunohistochemistry of testes tissue to Hougaard et al. mice (C57BL/6BomTac); Male cleanairfor1h/day determine mRNA expression and protein 2009) offspring killed 170 days after levels birth

45  Chapter2:ReviewofLiterature

2.3.3 InǦvitrotoxicologyǦhistoricalperspective

ThedemandforalternativestoanimaltestingisincreasingonnonͲessentialitems.For example,thereismoredemandforalternativetestingoncosmeticsasitisconsidered a nonͲessential item, but less demand for alternative testings on pharmaceutical productsandoverͲtheͲcounter(OTC)drugs(Garthoff2005).However,with numerous newchemicalsintroducedintothemarketeachyear,invivostudieshavebecomenon feasiblehenceinͲvitroandinsilicomethodsarebeingutilisedtoscreenouthighrisk compoundsforfurtherinvivotestingsifnecessary(ValerioJr2009).

Anotherissueassociatedwithinvivoexperimentsis theethicsofanimalexperiments, animalexperiments are often argued to becruel and no respect is given for animal rights.Toaddresstheseconcerns,the3Rprinciplewasformulatedtoreduce,refine and replace animal experimentation.As a response to public demand, various organisations have created guidelines in performing in  vivo experiments such as by ECVAM, REACH (Garthoff 2005).These guidelines have become a legal and ethical obligationforinvestigatorsinEuropesince1986(HalderandWorth2003).

The3Rprinciple

Thereplacement,reductionandrefinement(3R’s)principlewasintroducedin1959by WilliamRusselandRexBurch.The3R’sprincipleconsistsofreplacement(usingother materials such as cells instead of animals), reduction (minimise number of animals usedinanexperiment)andrefinement(minimisepainandsufferingexperiencedby animalsduringexperiments)(Russell,Humeetal.1992).Anexampleofanapplication of the 3R’s principle is the rabbit eye primary irritation test.Initially, tests were performedonliverabbitswherechemicalsamplesareappliedonitseyeandfurther observations were made to determine its irritancy effect.The method now has a validated inͲvitro alternative (3R3 NRU assay) as a replacement to the primary irritationeyeteston liverabbits.

Developmentofalternativemethodsoftoxicitytestinghasleadtoareductiononthe numberof animalsusedininͲvivoexperiments.Inthemiddleof1970s,around5.5 million animals were used but by the 1990s this had decreased to approximately 3 million animals (Broadhead and Bottrill 1997).sThi  trend of decreasing in usage of 46  Chapter2:ReviewofLiterature animals concurrently occurred with a decreasing number of procedures requiring animaltesting,whichsuggestsmoreacceptancesinalternativemethods.

FRAME

The Fund for Replacement of Animals in Medical Experiments (FRAME) was established in 1982 aimedto replace live animals with cells in measuring toxicity of chemicals(BallsandHorner1985).FRAMEpromotesthe3R’sprinciplebydeveloping andvalidatingthealternativemethodsbycollaboratingwithotherresearchcentres. FRAMErecognisedthechallengeincreatingrepeatabletestsasnostandardsexisted andlittlecollaborationexistsbetweenlaboratories.Toaddressthischallenge,FRAME emphasisedcollaborationbetweenresearchcentrestoperformblindtestsonvarious inͲvitroassaysanddeterminethesignificantbiologicalfactors(suchasoriginandtype ofcells)anditspracticality(forexample,easeofuseandstorage).In1989,FRAME launchedthepeerͲreviewedinternationaljournalAlternativesToLaboratoryAnimals (ATLA) to provide information of ongoing researches on development, validation, assessmentanduseofalternativemethodsinbiomedicalandtoxicologyresearchfield (FRAME2006).

FRAMEalsoestablished“INVITTOXdatabase”whichlistsprotocolsforinͲvitromethods andisaccessiblefromawebsite.Theaimofthedatabaseistoeliminateproblems associatedwithinͲvitro protocolssuchaslackofdetail,unreportedcriticalstepsand outdated methods (Jirova, Kejlova et al. 2003).In addition, the database provides contacts and updates on development of inͲvitro methods.Currently INVITTOX database is a part of ECVAM Database Service on Alternative Methods to Animal Experimentation(DBͲALM)whichiseaccessibl online(ECVAM2007).

ZEBET

ZentralstellezurErfassungundBewertungvonErsatzͲundErgänzungsmethodenzum Tierversuch (ZEBET) is a department within the Bundesinstitut für Risikobewertung (Bfr),whichisGermany’sfederalinstituteforriskassessment.ZEBETwasestablished in 1989 as an advisor on animal experiments for scientists and public authorities in

47  Chapter2:ReviewofLiterature compliance with legislations such as the German Animal Welfare Act (Liebsch and Spielmann2002;ZEBET2007).

ThemainroleofZEBETistobethesourceofinformationonalternativemethods.The role includes advising publicauthorities in complying with animal welfare provisions (suchastheAnimalWelfareActofGermanyandEUCouncilDirective86/609EEC)and gatherinformationrelatedtoalternative methods (ZEBET2007).Inaddition, ZEBET maintainsadatabaseonalternativemethodsAnimAltͲZEBETwhichisaccessiblefreeͲ ofͲcharge online through the German Institute for Medical Documentation and Information(DIMDI)(ZEBET2005).

For validation and testing purposes, EBET coͲordinates interͲlaboratory work (for example,withFRAMEandCOLIPA)inresearch,developmentandvalidationofinͲvitro methods (Spielmann, Gerner et al. 1991; Spielmann, Balls et al. 1994; Spielmann, Liebschetal.1995;Scholz,Genschowetal.1999).

EuropeanCentrefortheValidationofAlternativeMethods(ECVAM)

TheEuropeanUnionestablishedECVAMin1991toaddresstherequirementsofthe directive 86/609/EEC which requires animal experiments to apply the 3R principle (Fentem,Archeretal.1998).SincethenECVAMhavecollaboratedwithbothZEBET andFRAMEtoestablishthe3Rprinciplein Europe(Spielmann2009).ECVAM'sduties include coͲordinating validation tests of alternative methods and encourage communication between the stakeholders on development of alternative methods (Marafante,Smyrniotisetal.1994).Inaddition,ECVAMalsomaintainsadatabaseof alternative methods (accessible online) and run workshops on InͲvitro models and testingmethodologiessuchasofinhalationtoxicology(Lambre,Aufderheidetal.1996; BéruBé,Balharryetal.2007)andgenotoxicity(Kirkland,Pfuhleretal.2007;Pfuhler, Kirkland et al. 2009).Working closely with ECVAM is the ECVAM scientific advisory committee (ESAC), which consists of representatives from European member countries,academics,institutions,industryorganisations.ThemaintaskofESACisto adviseECVAMonalternativemethodsdevelopmentandpromotethemtoEUmember states(ECVAM;Balls1995).

48  Chapter2:ReviewofLiterature

Interagency Coordinating Committee on the validation of alternative methods (ICCVAM)

ICCCVAMhassimilarrolestoECVAMbut isactive intheUSA.ICCVAM was initially establishedin1994asanadhoccommitteetoaddressrequirementsbytheNational Institute of Health (NIH) revitalisation 3act of 199  (public law 103Ͳ43).The act requiredNIHtocreateavalidationsystemforalternativemethods(ZeigerandStokes 1998).The ICCVAM committee consists of 15 US federal agencies and compiled a reportwhichwaspublishedin1997(NICEATM/ICCVAM).

Intheyear2000,ICCVAMbecameapermanentorganisation asapartofNIEHSbythe ICCVAM authorisation act 2000 (public law 106Ͳ545, 42 U.S.C.2851Ͳ3).At the same time,NICEATMwasestablishedtoassistICCVAMbyperformingexperiments,organise workshops and encourage communications between stakeholder on progress of alternative methods in the US and internationally (NICEATM/ICCVAM ; Zeiger and Stokes1998;HattanandKahl2002).

MulticentreEvaluationforInͲvitroCytotoxicity(MEIC)

The aim of the MEIC program was to find application of inͲvitro toxicology data to determinehumantoxicity.TheprogramwasinitiatedbytheScandinavianSocietyof CellToxicologyin1989andbytheendoftheprogramin1996,50chemicalshavebeen testedbyvariouslaboratoriesinternationally.The50chemicalswerechosenforitsin vivodataavailabilitysuchasitslethaldosage,LD50(inratsandmice)andtoxicokinetics (inhumans)(Ekwall,GomezͲLechonetal.1990).

ThecollaboratinglaboratoriesusedinͲvitromethodstoinvestigatedifferentendpoints and compare the results with available human acute toxicity data to determine any correlations (Ekwall, GomezͲLechon et al. 1990; Walum, Clemedson et al. 1994; ClemedsonandEkwall1999;Ekwall1999).ResultsoftheMEICprojectwerepublished in 8 parts in the journal ATLA, which enables comparison of results between investigators such as by Malich et al (1997) and Lestari et al (2005) who compared theirresultswithMEICdata(Malich,Markovicetal.1997; Lestari,Hayesetal.2005).

49  Chapter2:ReviewofLiterature

AftertheMEICproject,EDITprojectwasinitiatedasa6yearprojectin1998withthe aimofdevelopinginͲvitrotestbatteries.Themainrequirementofthetestbatteriesis tohavehighaccuracyinpredictingtoxicityofunknownchemicals(Ekwall,Clemedson etal.1999).

2.3.4 InǦsilicotoxicology

In silico toxicology uses computers for simulating and creating physical models for assessingtoxicityofcompounds.Althoughcomputersusageformodellingpurposesis notnovel,onlyrecentlypersonalcomputersarebothaffordableandhaveadequate processingpowertoperformcomplexcalculations.Oneexampleofinsilicotoxicology modelling include conversion ofdCT an  MRI scans into a digital lung model (Annapragada and Mishchiy 2007), modelling of air flow and particle transport/deposition in pulmonary airways (Kleinstreuer, Zhang et al. 2008), more complexphysiologicallyͲbasedpharmacokinetic(PBPK)(Yang,ElͲMasrietal.2004)and three dimensional computational fluid dynamics simulation of particle deposition in thelungs(LongestandHolbrook,InpressͲcorrectedproof).

OneofthemostcommonlyusedinsilicotoolisQuantitativeStructureActivityRelation (QSAR).QSAR is a mathematically based tool to predict toxicity of compounds by analysingitsstructure.QSARhasbeenusedbytheUSEPA(Clements,Nabholz.etal  1993) and the Danish EPA (Vedani, Dobler et al. 2005) for quicker screening and determiningtypesofhazardsforfasteroperation.

The advantage of in silico toxicology includes the ease of performing numerous experiments.Asnophysicalmaterialsoranimalsareused,itsavesbothcostandtime while simultaneouslyavoidingethicalissuesrelatedwithanimalusage.However,in silicotoxicologyisnotadefinitivehazardidentifieryethencedatafromotherstudies havetobeconsideredaswell(ValerioJr2009;Mesens,Steemansetal.2010).

Although in vivo, inͲvitro and in silico techniques have their own  advantages and disadvantages (as outlined in Table 2Ͳ11).Each technique is not designed to completely replace each other but more as complementary techniques for more efficientriskassessment.Thiscanbeachievedbycombiningresultsfromthethree

50  Chapter2:ReviewofLiterature techniques, which allows better detection and elucidation of possible hazard and associatedrisksposedbythechemicals.

 Table2Ͳ11:Comparisonbetweeninvivo,inͲvitroandinsilicomodels

 Invivomodels InͲvitromodels Insilicomodels Pros Humanandanimaltests Acceptedas Applicabletoany possible;extensivehistory pharmaceuticaltool, geometry/species/patien indrugdevelopment. manycommercialdrugs t;parametricstudies havebeendeveloped possible. usingthesemodels. Cons Radioactivetracersand Castsbased:plastic Accuracydemands concernsaboutexposure castsmadeare imagingͲbased toionizingradiation; destroyedafterusein geometry; parametricstudiesare experimentsandcanbe computationallyhighly technicallyfeasiblebutnot highlyinaccurate intensive. practicalowingto CTbased:potentially radiationconcerns. applicabletohuman Modelpreparationis veryinvolved. Parametricstudiesare notpossible. Bestuse Recentdevelopmentsin Earlystagefeasibility WhatͲif(hypothetical) ofmodel PETtracerssynthesisallow testingofdrug scenariosareeasyto labelingofactualdrug preparations; address;parametric moleculesinreal comparative,headͲtoͲ variationsaresimple; formulation. headstudiesofmultiple excellentasadesign preparations. tool. Access Viasophisticatedmedical Widelyavailablewith CTimagingrequired; centers. routinelaboratory highperformance facilityrequirements. computationrequired. Relevant None. None. None. patents Source:(AnnapragadaandMishchiy2007). 

2.4 Principlesoftoxicology

2.4.1 Disposition

Whenabiologicalsystemisexposedtotoxins,thetoxinsundergothroughdisposition. Disposition describes the fate of toxins within biological systems which includes absorption (via uptake), transport, storage, metabolism, action and excretion

51  Chapter2:ReviewofLiterature processes(asoutlinedinFigure2Ͳ6).Thestudyofdispositionoftoxinsprovidesan overviewofthemechanismsoftoxinswithinorganisms.Thedispositionprocesscan befurtherelucidatedwithresultsfrominvivo,inͲvitroandinsilicostudies.Studiesof dispositionofchemicalshaveenabledmoreefficientdrugdelivery(MeijerandSwart 1997)andbioavailabilityofcompoundsincontaminatedwater(PedlarandKlaverkamp 2002).

 Figure2Ͳ6:Poisoningprocessinanimalsandhumans Source:(Yu2001)page35.

Absorption/uptake

Absorptionisthemainuptakemechanismoftoxinsintobiologicalsystems.Inhalation isoneofthemainexposureroutesespeciallyforgaseouspollutantsandsmallersize particulatematterduetothelargesurfaceareainthegasexchangeareainthealveoli ofthepulmonarysystem.Alveoligasexchangearea facilitatesexchangeofbetween

O2andCO2moleculesinandoutoftheblood.

Anothermainuptakerouteisthroughingestionduetoitslargeavailablesurfacearea in the Gastric Intestinal Tract (GIT).The large surface area for absorption allows

52  Chapter2:ReviewofLiterature relatively high absorption of toxins into the body although at lesser rate than inhalation(GangolliandPhillips1990).

Theuptakeprocessinvolvespermeationoftoxinsthroughbiologicalmembranes.The permeation can be either through passive, facilitated, active or phagocytosis/pinocytosisprocesses(GangolliandPhillips1990;.Yu2001)  x The passive uptake process transfers toxins from higher into lower concentration areas by diffusion especially for hydrophilic, nonͲionic compoundsandgasescapableofpenetratingthroughwaterfilledpores(~0.4 nm). x The facilitated uptake process transfers toxins from higher to lower concentration areas but also involve proteins or other carriers, this allow hydrophiliccompoundstobetransferredacrossthemembrane. x Theactiveuptakeprocessrequiresbothcarrierandenergytotransfertoxins, hence allowing it to permeate membranes regardless of concentration difference, thus its main uptake limit is primarily the energy availability to facilitate the uptake.Other limiting factors for the active uptake process includecompetitionwithothersubstratesandmetabolicinhibitors.

Transportandstorage

Transportationoftoxinsmainlyoccursviathecardiovascularsystemwithbloodasthe maincarrier.Thisoccursafteruptakeoftoxinsthroughuptakeareassuchasthegas exchangeareaofthealveoliortheliningsofthegastroͲintestinaltract.

Storageoftoxinscanoccurinvariousorgansincludinglungs,adiposetissuesandliver. The storage period is usually for a nonͲfixed period of time and not released until further action.One example is the dichlorodiphenyl trichloroethane (DDT) and polychlorinated biphenyls (PCB) chemicals which are stored mainly within adipose tissuesandreleasedintotheliverduringstarvation(Yu2001).

Metabolism

Themainaimofmetabolismistheconversionoftoxinsintolessharmfulsubstancesto aidexcretion.Themetabolismprocesscanbedividedintotwophases,PhaseIand phase II.Although both phases mainly occur sequentially, in some cases especially withmoresolubletoxins,theymayonlyundergooneofthephases(Aldridge1990;Yu 2001).Bothphasesperformdifferentdetoxificationprocessesasdescribedbelow.

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x PhaseI o MainlydoneviacytochromePͲ450monoͲoxygenasesystemofenzymes toproducesubstratesmorewatersolubleforphaseIIconjugation

o AppendstoxinswithadditionalfunctionsgroupssuchasOH,NH2,COOH orSHgroup)andcouldbeinformof ƒ Oxidationprocess(hydroxylation,dealkylation,deaminationand Sulfoxidetransformation) ƒ Reductionprocess(azoreduction,additionofhydrogen) ƒ Hydrolyticprocess(esterandamidebondssplitting)catalysedby esterases,amidases x PhaseII o Conjugateorsyntheticreactions,mainlydoneusing endogenous substanceincludingglucuronicacid,sulphate,glutathione,glycine, cysteine,GSH,etc) o Outputismorewatersolubleforsecretionbyurineorfurtherprocessif needed

Althoughmostmetabolicactivitiesaimtoincreasesolubilitytoaidexcretionoftoxins bymakingtoxinsmorehydrophilic,sometoxinscouldbecomemoretoxic asaresultof metabolism.Oneexampleisthetransformationofcarbontetrachloridebyliverinto chloroform,whichisatoxicandcarcinogenicsubstance(PentzandStrubelt1983).

Excretion

Depending on their solubility, toxins are excreted via different pathways.For hydrophilic compounds, the main excretion pathway is through the kidneys and subsequenttransferintourine.Forlesshydrophiliccompounds,themainexcretion pathwayisthebiliaryexcretesandsubsequenttransferintofaeces.Otherexcretion pathwaysmayincludesweatandpulmonarysystem(tolimitedextendbybreathing outthegaseoustoxins)(GangolliandPhillips1990).

2.4.2 Targetorgans

2.4.2.1 Respiratorysystem

TherespiratorysystemdeliversO2gasintothebodyandCO2gasoutofthebody.The respiratorysystemisamajorrouteofexposureforatmosphericcontaminantsdueto the large available surface area for toxins uptake (Griffin 2006).The respiratory systemconsistsof threedifferent regions:thenasopharyngeal, tracheobronchia and 54  Chapter2:ReviewofLiterature

alveolar.ThealveolicontainthegasexchangesiteinwhichO2gasenterstheblood andCO2leavesthebloodandexhaledout,thegasexchangeisfacilitatedthrougha thinbarrier(0.5 μm)between thealveoli and respiratorycapillaries.Althougheach alveolusisverysmall(300μmdiameter),theirhighdensity(400millionalveoliintotal) providesaround75m2surfaceareaforgasexchange(Yu2001).

Thealveoli’smaindefenceagainstorganismsincludesTypeI,TypeIIalveolarcelland macrophages.TypeIalveolarcellsaresquamousepitheliuminstructureandarethin anddelicate.TypeIIalveolarcellarescatteredalongTypeIalveolarcellsandsecrete phospholipidrespiratorysurfactantstoreducesurfacetensioninalveoliandprevents thealveolifromcollapsing(Martini,Timmonsetal.2003).Alveolarmacrophagesare large tissueͲbound phagocytes which engulf foreign organisms such as bacteria. However, Insoluble particulates may reside within the alveoli and cause pneumoconiosis due to particulates’ inflammatory stimulation which is capable of changingthefibrousproliferationwithinthealveoli(Myojo,Ogamietal.2010).

The respiratory system’s defence mechanisms are divided into physical and physiologicalmechanism(Carel1998).Thephysicaldefenceofthepulmonarysystem reliesonphysicalpropertiesofthematerialandnoactionfromtherespiratorysystem is required.The main physical defences include solubilisation and deposition. Solubilisation process depends on the solubility of contaminants in which highly soluble contaminants are absorbed by the aqueous lining of mucosa in the upper airways.

Deposition involves the particles to make contact with the mucus layer of the epitheliumoftheairways.nDepositio locationofparticlesisheavilydependentonthe particlesizesasshowninFigure2Ͳ7.Depositioncanoccureitherbyinertialimpaction, sedimentationordiffusiondependingontheparticlesizes(Carel1998). x Inertialimpactionsoccurforparticles7–10ʅminsize,particlesofthissizehave moremomentumhenceunabletochangedirection,thereforecrashingintothe airwaywallsandembeddedinthemucouslayeroftheairways. x Sedimentationoccursforparticles0.5–0.7ʅminsize;particlesofthissizeare pulled by earth’s gravity and make contact with the mucous layer of the airways.

55  Chapter2:ReviewofLiterature

x Diffusionoccursforparticles0.3–0.5ʅminsize,particlesofthissizeareeasily suspendedinair,thereforeheavilydependentontheairflowdirectiontomake contactwithmucouslayeroftheairwaywalls.

Physiologicalmechanismdefencereliesonreflexoftherespiratorysystemtoremove contaminants.Examples of such physiological mechanism include airway reflexes, bronchoconstrictionandantioxidantactivity(Carel1998). x Airway reflexes occur when receptors detect particles; the receptors then induce reflex actions such as coughing and sneezing to expel the detected particles. x Bronchoconstriction decreases diameter of airway to reduce amount of particles>5mmdiameterreachingthealveoli.

x Antioxidant activity mitigates formation of oxidisers such as NO2 and O3 by secretionofantiͲoxidantsfromthetracheobronchialtree.

Alveolarmacrophages

Alveolar macrophages (AM) reside within the alveoli and have the main task of neutralising foreign organisms such as bacteria.The neutralisation process involves engulfing of foreign organisms through the process of phagocytosis (for solid materials)orpinocytosis(forliquidmaterials).AM’shaveamajorroleinrespiratory defencemechanismsbutsincePM2.5canreachthealveoliandpotentiallyimpairtheir phagocytosismechanisms,itisalsoregardedasatarget(HadnagyandSeemayer1994; Renwick,Donaldsonetal.2001).Contaminantssuchasultrafineparticlesanddiesel exhaust particles can inhibit phagocytosis activities.This inhibition of phagocytosis mayleadtoanincreasedsusceptibilitytoinfection,asthmaandChronicObstructive PulmonaryDisease(Lundborg,Dahlénetal.2006).

2.4.2.2 Cardiovascularsystem

The cardiovascular system consists of the heart and the systemic circulation.The heart’s pumping action powers the blood flow throughout the blood vessels, meanwhilethebloodcarriesnutrients,O2andCO2gases.Theheartisanalogousto twosynchronouspumpsinwhichonesideoftheheartpumpsoxygenatedbloodinto the arteries (therefore delivering O2 gas to organs), the other of the heart pumps deoxygenatedbloodintogasexchangearea,whereO2andCO2gasexchangeoccurs betweenalveoliandpulmonarycapillaries(Martini,Timmonsetal.2003).

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Figure2Ͳ7:Pulmonarysystemandfractionaldepositionofinhaledparticles Adapted from (Oberdorster, Oberdorster et al. 2005).

Thecardiovascularsystemisconsideredatargetorganassmallparticulates(suchas

PM2.5)cantranslocatefromtherespiratorysystemintothecardiovascularsystemand subsequentlythroughoutthebody(Kreyling,SemmlerͲBehnkeetal.2006;Simkhovich, Kleinmanetal.2008).Inaddition,particulatescan: x Stimulate systemic and alveolar inflammatory response due to deposition of particulatesinlungs. x Cause direct insult to systemic  circulation especially by the smaller size particulatesduetotranslocatingparticulatesfromlungstosystemiccirculation (asillustratedinFigure2Ͳ8). x Affect autonomic nervous system hence affecting heart mechanisms (Neas 2000;Polichetti,Coccoetal.2009). x Damagetobloodvessellinings,acceleratingatherosclerosis(Kunzli,Jerrettet al.2005).

57  Chapter2:ReviewofLiterature

Figure2Ͳ8:ProposedPM10mechanismsoncardiovascularsystem Source:(Bai,Khazaeietal.2007).

2.4.2.3 Liver Liver is the detoxification and metabolic centre of the body, therefore high concentrations of toxins tend to accumulate around the portal veins (Balazs 1981). Thisexposureoflivertohighconcentrationoftoxinsrenderstheliveratargetorgan. Structurally,thecellsoftheliverconsistof: x 70%(60%byvolume)hepatocytesandparenchymalcells. x 30Ͳ35% (20% by volume) non parenchymal cells and sinusoidal cells (Kupffer cells,endothelialcells,fatstoragecells,pitcellsandbileductepithelialcells). x Remaining20%ofcellsconsistofextracellularspacesandextracellularmatrices (Rojkind1994citedin(Vickers1997)).

2.4.2.4 Skin

Skinprovidesabarrierbetweeninternalorgansandtheexternalenvironmentwhich allowsmaintenance of homeostasis.Although skin provides high protection against contaminants, the degree of protection a skin region offers depends heavily on the

58  Chapter2:ReviewofLiterature region’sstructuresuchasitsthickness,hydration,vascularityandstructuralintegrity (Wilson1990).

2.4.3 Cellinjurymechanisms

Cell injury classifications include basal, organͲspecific and organisational cytotoxicity (Barile1994): x Basal cytotoxicity affect functions common all cells, this includes cell membraneintegrity,mitochondrialactivityandproteinsynthesis(Barile,Arjun etal.1993). x OrganͲspecificcytotoxicityaffectcellfunctionsspecifictoacelltype. x Organisationaltoxicityaffects theoutputofcellsbutdoesnotnecessarilyaffect thecellsthemselves.

Cell response to injury can either be reversible (recovery is possible) or irreversible (recovery is not possible).For irreversible injury, the resultant cell death includes necrosis,apoptosisandautophagosis(asillustratedinFigure2Ͳ9).

Necrosisisa nonͲprogrammedcelldeathinducedbyexternalfactorssuchasexposure totoxins.Necrosisinvolveschangestocellstructurewhicharelistedbelow(Sparks 1972; Meyers and Hendricks 1985; Trump and Berezesky 1994; Newman and Unger 2003). x Compartmental volume morphology changes: Disintegration of the cell membrane, formation of dense aggregates, calcification of cytoplasm, formationofblebsonnucleus. x Pyknosis: Chromation distribution in nucleus changes and condenses into stronglystainingmassandirregularinshape. x Coagulation: Extensive coagulation of cytoplasmic protein, making cells appearsopaque. x Liquefactive(cytolytic):Rapidbreakdownofcellasaconsequenceofreleaseof cellularenzymes.Mayproducenecroticspacesintissue. x Caseous:Cellsdisintegratetoformamassoffatandprotein. x Gangrenous:Combinationofcoagulationandliquefactivenecrosis,often followedbypuncture(lackofbloodsupplytodamagedtissue)andsubsequent infectionbyspecificgangrenouscausingmicroorganisms. x Fat:Depositionofsaponifiedfatsindeadfatcells.

Although necrosis is conventionally known as nonͲprogrammed/unintentional cell death,ithasbeenhypothesisedthatnecrosisactsasabackuptoprogramcelldeath.

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Necrosismayresultwhenapoptosisisnotpossiblesuchasuponexposuretoviruses withantiͲapoptoticproteins(EdingerandThompson2004).

Inautophagy,cellsconsumetheirowncontenttoproducemoreATP.Currentlyitis unclearwhetherautophagyisasurvivalorcellͲdeath function,althoughitisknownto beasurvivalfunctionofmammaliancellswhennotenoughATPisgenerated(Edinger andThompson2004).

Apoptosis is a programmed cell death induced upon receiving the apoptosis signal fromothercells.Incomparisontonecrosis,apoptosisislessinflammatoryinresponse as during apoptosis, the membrane of the cell is still intact (as illustrated in Figure 2Ͳ10).The intact membrane allows macrophages and other cells to perform phagocytosisonthedeadcells.Inaddition,anintactmembranepreventcontentsof apoptoticcellspillsoutsideandtriggeringinflammatoryresponse(Kroemer,Petitetal. 1995; Lelli,Becksetal.1998).Foracelldeathtobeconsideredasapoptosis,some observable occurrence must be made such as cleavage of chromosomal DNA into internucleosomalfragments(EdingerandThompson2004).

 Figure2Ͳ9:Stagesofcellinjury (a) Normal steady state; (b) subͲlethal injury; (c, c’, c’’) cell recovery and (d) lethal injury;Imagesource:(TrumpandBerezesky1994).  60 Chapter2:ReviewofLiterature

 Figure2Ͳ10:Morphologicalfeaturesofdeadcells (a)Normal;(b)autophagic;(c)apoptotic;and(d)necroticcells. Imagesource:(EdingerandThompson2004).

2.4.4 DoseǦresponsecurve

CentraltotoxicologyisthecalculationofthedoseͲresponsecurvewheretoxicitycan bedetermined.ThedoseͲresponsecurveistypicallyasigmoidalshape(Figure2Ͳ11) andhasthefollowingparameterswhichdeterminethetoxicresponseofcells(Gratt 1996): x Noobservedadverseeffectlevel(NOAEL):Minimumlevel inwhichnoeffects areobserved. x Safetyoruncertaintyfactor:Usedinsafetyregulationstoprovidesafemargin duetouncertaintyfromextrapolationofnonͲhumandatainformofdivisionof

NOAEL/LD50/TLClevels.

x Lethaldose50%(LD50):Doselevelinwhich50%ofexposedsubjectsdie

x Lethalconcentration50%(LC50):Concentrationlevelofcontaminantresulting in50%deathofexposedsubjects. x TotalLethalConcentration(TLC):ConcentrationLevelinwhichallsubjectsare dead.

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Figure2Ͳ11:Typicaldoseresponsecurvewithsigmoidcurvefitting PlottingdoseͲresponserelationbasedon(a)cellsurvivability;(b)celldeath.

Interactionbetweenpollutants

Withinamixedcompound,therearepossibleinteractionsbetweencompoundswhich canbeeitheradditive,synergistic,potentiatingorantagonistic(Yu2001). x Additive response involves a combined toxic response as a sum of both substances’individualtoxicityresponses. x Synergistic response is a higher toxicity response than the sum of individual substances’ toxic response.For example is exposure toluene and acetylsalicyclic acid mixture capable of inducing greater auditory impairment (Côté,Davisetal.1998).Anotherexampleiscombinedasbestosexposurewith tobaccosmokingininducinghigherdamagetoalveolarepithelialcells(Kamp, Greenbergeretal.1998). x Potentiationresponseoccurswhenasubstancehasnegligibletoxicitybyitself butinducestoxiceffectonlyinpresenceofanothersubstance.Forexample, carbon tetrachloride with presence of methanol causes liver damage as methanolincreasesrateoflivermetabolism(Allis,Brownetal.1996). x Antagonistic response is a lower toxic response than the sum of both substances.Onesuchexampleismelatonin(apinealhormone)whichprotect exposedsubjectsagainstcyanideͲinducedneurotoxicitysincemelatoninhasa freeradicalscavengingeffects(YamamotoandTang1996).

2.4.5 Statisticalmethods

Assumptionofnormaldistribution

Intoxicology,normaldistributionisthemostcommonassumptionondistributionof toxicresponses.Inanormaldistribution’sdensitygraph,themeanIC50valueisthe

IC50formajorityofpopulationwhilesmallpartofpopulationareeitherverysensitive or not sensitive (Stine and Brown 1996).With normal distribution, the cumulative

62  Chapter2:ReviewofLiterature distributiongraphwillbesigmoidinshape,allowingcalculationofthedoseͲresponse curve.

 Figure2Ͳ12:Normaldistributioncurve

(a)IC50Probabilitydensitycurve;(b)Cumulativedistributioncurve.

Althoughthesigmoidalcurveisanidealmathematicalmodelforcumulativefrequency ofcellcytotoxicitydata,itismoredifficulttoimplementassigmoidalcurvesrequire multipleparameters(aslistedinTable2Ͳ12).Astherearemultiplewaystocalculate thedoseresponsecurve,atransparencyinmathematicalassumptionis neededforthe riskassessmentteam(Buchanan,Smithetal.2000).

Statisticaltestings

The most commonly used statistical test to compare results includes Analysis of Variance(ANOVA)andStudent’stͲtest.TheANOVAtestisconsideredsuperiortotͲ tests as ANOVA prevents type I error by making simultaneous comparison in one mathematicaloperation(PeatandBarton2005).

Thetwoerrortypesinstatisticaltestscausehypothesistobeeitherfalselyrejectedor falselyaccepted. x TypeIerrorgeneratesfalsepositiveresultssincenullhypothesisisrejectedin error,whichoccurswhen a statistical significant difference between group is foundbutnoclinicallyimportantdifferenceexists. x Type II error false gives negative results since null hypothesis is accepted in errorwhichoccurswhenastatisticalsignificantdifferencebetweengroupexist but does not reach statistical significance.Type II error is usually caused by smallsamplesize(PeatandBarton2005).

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Statistical tests can be performed either as two tailed or one tailed test.The main differencebetweenbothtestsisthedatatobeconsideredforrejection.Onetailed test only can reject data on one extreme end, while two tail test can reject both extremeends(Adler1964).

Table2Ͳ12:DoseͲresponsemathematicalmodels

Model Annotations Fourvariablesigmoidal y(x)=responseatdosex;s=dose;A=responseat ࡭ െࡰ zerodose;B=exponentor“slopefactor”;C=dose ࢟ሺ࢞ሻ ൌ ࡰ ൅  ૚൅ሺ࢞࡯Τሻ࡮ giving 50% of the response; D = response produced byasupramaximaldose

Fourvariablesigmoidal y(x)=responseatdosex;s=dose;A=responseat ࡭ െࡰ zerodose;B=exponentor“slopefactor”;C=dose ࢟ሺ࢞ሻ ൌ ࡰ ൅  ࡮ giving 50% of the response; D = response producedכ૚൅૚૙ሺ࢒࢕ࢍ࡯ି࢞ሻ byasupramaximaldose

Exponential Pi(d) = probability of infection at dose (d); d =

ࡼ࢏ሺࢊሻ ൌ૚െ܍ܠܘሺെ࢘ࢊሻ dose(colony forming unit); r = model parameter specificforeachpathogen

BetaͲPoisson Pi(d) = probability of infection at dose (d); d = ିࢻ ࡼ࢏ሺࢊሻ ൌ૚െሺ૚൅ࢊΤሻ ࢼ  dose(CFU);ɲ= model (infectivity) parameter;ɴ= model(shape)parameter

WeibullͲGamma Pi(d) = probability of infection at dose (d); d = ࢈ Ȃࢻ ࡼ࢏ሺࢊሻ ൌ૚െൣ૚൅൫ࢊ ൯ࢼΤ ൧  dose(CFU);b=model(shape)parameter;ɲ=model (infectivity) parameter;ɴ= model (infectivity) parameter

Weibull Pi(d) = probability of infection at dose (d); d = ࢈ ࡼ࢏ሺࢊሻ ൌ૚െ܍ܠܘሺെࢇࢊ ሻ dose(CFU); a = model (infectivity) parameter; b = model(shape)parameter

Gompertz Pi(d) = probability of infection at dose (d); d =

ࡼ࢏ሺࢊሻ ൌ૚െ܍ܠܘൣെ܍ܠܘ൫܉ dose(CFU); a = model (intercept) parameter; b = ൅܊܎ሺܠሻ൯൧ model(slope)parameter;f(x)=functionofdose Sources: Rodbard and Hutt (1974) cited in (Gomeni and Gomeni 1980); (Niklas, Noor et al. 2009);Haas (1983), Farber et al (1996), Krewski and van Ryzin (1980), Coleman and Marks (1998)citedin(Buchanan,Smithetal.2000).



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2.5 InǦvitrotoxicologymethods

2.5.1 Primarycultureandcellline

Primary cell cultures have morphological and biochemical features similar to the originaltissue,henceallowingcomparativestudiesofspecialisedtissuesfromdifferent species(Barile1994).Primaryculturesaremoresensitivetotoxiceffectsofchemicals ascellsarestilladaptingtonewenvironment.Thisconditionrenderscellstoresemble morecloselytothesourceanimalandmayprovideamorerealistictoxicologicalresult although,itismoredifficulttodevelop(AustinandMothersill2003).

Acelllineisaprimaryculturedcelllinewhichhasbeentransferredintonewvessels, since various cells within the primary culture have different survivability.The transferredcellsintimebecamemorehomogenousascellswithlowersurvivabilitydo not survivethe transfer.Although similar to a finite cell line, itis able to undergo divisionsperpetuallywhereasfinitecelllinesareonlyabletodividefor50timesbefore degrading in quality (Barile 1994).Cell lines have the advantage of being readily available such as from ATCC, cell lines are easier to grow since methods have been establishedanditscharacteristicswellunderstoodalthoughdisadvantageincludethe furtherresemblancefromitssourceanimalandresultsmaynotextrapolatetosource animaldata(AustinandMothersill2003).

Sincecytotoxicitycanbedividedintobasal,organͲspecificandorganisationaltoxicity, thechoiceofcellsbecomecrucialinexperiments.Basalcytotoxicitycanbededuced byusingvariouscelllinestodetermineanycommonoruniqueeffectbetweenthecell lines,commoneffectsthencanbeconsideredasabasalcytotoxicity whereasunique effects can be considered as organͲspecific cytotoxicity and even species specific cytotoxicityifusingcellsofdifferentspecies(EkwallandEkwall1988).Inaddition,cell lineswhichexhibitpoorsurvivabilityininͲvitrosettingcanbescreenedouttosimplify study processes (Yang, Cardona et al. 2002).Organisational toxicity meanwhile is determinedbyexaminingsubstrateorproductofcellmetabolism(Ekwall1980;Barile 1994).

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2.5.2 Preventingcrosscontamination

Crosscontaminationofcellcultureshasbeenanunderratedissueeventhoughithas severeconsequences(Freshney2000).Crosscontaminationofculturesincludesyeast and cross cell line contamination and their sources can include the serum used in media or unclean water in the water bath (Ryan 2007).However most often, contaminationsisaccidentlyintroducedbytheoperator,thusvariousprecautionare neededincludingonlyworkingwithonetypeofcelllinewithinoneexperimentperiod (Freshney2000).

2.5.3 Cellculture

In designing inͲvitro experiments, both cell culture conditions and cell line specificationsaretobenotedtoallowstandardisationofmethodsforinterͲlaboratory collaborations (Purchase, Botham et al. 1998; Liebsch and Spielmann 2002).In addition,anydifferenceinspecificationandconditioncouldaffecttheresults(Austin andMothersill2003;Veranth,Cutleretal.2008).

ThechoiceofmediatypeforuseininͲvitrotoxicologyisvitaltoensureoptimalcells’ health.Culture media consists of basal nutrients that include growth factors, mitogens,hormonesandcytokines(Karmiol2000).Duringculturingofcells,CO2can be injected into the incubator to act as buffer to neutralise byͲproducts of cell metabolism(Freshney2000;Ryan2007).

Serumcanbeaddedtomediaat5Ͳ10%ratiotoaidcellgrowthandiscollectedfrom animals such as fetal bovine or newborn calf (Ryan 2007).However since sera are collectedfromananimal,theremaybevariationinitsperformancebetweenbatches. In addition, serum has been considered a major source of contamination in cell cultures therefore there has been a development for serum free media although serumisstillcommonlyusedtoimprovetheculture’shealth(LéryandFédière1990; Stoll,Mühlethaleretal.1996;Kim,Leeetal.2006).

Toincreaseresistancetowardsinfections,antibioticscanbeaddedtoculturemedia. Althoughconventionallyused,therehasbeenconcernbythemedicalfieldonoveruse inantibioticsasitmaypromotegrowthofantibioticresistantstrains(Ryan2007).

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To harvest cells from flasks, a physical or enzymatic process can be utilised.The physical process involves a cell scraper to physically detach the cells from the flask surface.Theenzymaticprocessutilisesenzymessuchastrypsintodetachthecells.By comparison,theenzymaticprocessyieldshighercellnumberthancellscraping.

Thecellgrowthcurve can bemodelled witha sigmoidalcurve, inwhich thegrowth phase is the optimal time to harvest cells.The curve reflects a biological growth patterninwhichgrowthisinitiallyslowerduetolowcellnumbers.As thenumberof cellsincrease,growthbecomesexponentialuntilcellsrunoutofareaforgrowth.This slowingdownofgrowthrateisknownastheplateauphase.Toensureoptimalhealth of cells, cells should be harvested during its growth phase to avoid any issues associatedwithcellsoverpopulating.

AnotherpossibleadditiontotheculturemediaincludesphenolredandHEPES.Phenol redisusedasapHindicatorincellculturemedia.HEPEShasbeenusedforbuffering. CareisneededinusingHEPESandphenolredeachmaybetoxictosomecelltypes (Karmiol2000).

Longterm storageofcells

Forlongertermstorage,freezingthecellscanbeperformedatvarioustemperatures aslistedbelow: x AtͲ70oCbymechanicalfreezer o x AtͲ135 CbyLiquidNitrogen(LN2)vapour x AtͲ197oCbyimmersioninliquidnitrogen.

The lower the temperature, the longer the possible storage time period is.The freezingofcellsmaydamagethecellsduetofreezingofthewatercontentofcells.To prevent the water content of cells from freezing, a cryoprotective agents such as dimethyl SulfoxideO) (DMS  is added to the cell suspension solution(Ryan 2007).

Amongstthefreezingtemperatures,freezingbyLN2vapourispreferredforlowerrisk of LN2 liquid sipping into the cell vials.LN2 liquid in cell vials introduce a risk of explosionduetorapidexpansionofLN2intoitsgaseousphase(Hay,MirandaͲCleland etal.2000).

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2.5.4 InǦvitrocytotoxicityassays

Toquantifythecytotoxicityofsubstancesoncells,variousinͲvitroassaysareavailable. InͲvitro assays enable investigation of various biological endpoints such as cell metabolismandATPcontent(RissandMoravec2004).

Tetrazoliumsaltbasedassays(MTT,XTTandMTS)

The tetrazolium salt based assays measure Dehydrogenase enzyme contents of cell  (Slater,Sawyeretal.1963).OneexampleofatetrazoliumsaltassayistheMTTassay whichiswidelyusedtoinvestigatetoxicityofsubstancesinmonolayercultures.Inan MTT assay, the tetrazolium salt MTT (3Ͳ(4,5ͲdimethylthiazolͲ2Ͳyl)Ͳ2,5Ͳdiphenyl tetrazolium bromide) is reduced by mitochondrial Dehydrogenase enzyme into a purple coloured MTTͲformazan product, in which the absorbance are recorded (Mosmann1983).

XTTassayisanimprovementtotheMTTassayasitdoesnotrequirethesolubilisation oftheformazanproduct.HoweverXTTrequireslongerincubationperiod(upto7Ͳ8 hrs)beforereadingeofth absorbance(Roehm,Rodgersetal.1991).Inaddition,after additionofabioreducer,crystalsmayformwithintheculturemediaandtheinstability of mixed XTT/PMS solution may inhibit formazan production to provide lower absorbancereadings(Goodwin,Holtetal.1995).

MTS assay measures conversion of MTS (3Ͳ(4,5ͲdimethylthiazolͲ2Ͳyl)Ͳ5Ͳ(3Ͳ carboxymethoxyphenyl)Ͳ2Ͳ(4Ͳsulphophenyl)Ͳ2HͲtetrazolium salt into a formazan productasshowninFigure2Ͳ13(b).TheMTSassayissuitablefortestingtoxicityof chemicals inͲvitro (Malich, Markovic et al. 1997) and is simpler to implement than othertetrazoliumbasedassays suchasMTTandXTT.

Incomparison,theMTSassayrequireslesspreparationandprovidesfasterresultsas compared to the MTT assay.The MTT assay requires more preparation and longer incubationperiods(Promega2001).IncomparisontoMTTassay,MTSassaysdonot require a solubilisation step MTS assay’s formazan product is already water soluble (Gieni,Lietal.1995).However,MTSassaysrequireanintermediateelectronacceptor such as PMS (Phenazine methosulphate) for faster bioreduction and hence faster

68  Chapter2:ReviewofLiterature productionoftheformazanproduct(Goodwin,Holtetal.1995).Anotheradvantageof theMTSassayisthewiderrangeofusablelaserwavelengthforplatereading.Unlike MTT and XTT assays which require different laser wavelengths for response and reference absorbances.MTS can accept a laser wavelength with range of 515Ͳ580 nm).Thisacceptanceoflargerrangeoflasersenabledusageofabsorbancereaders withwiderbandpasslaserfilter(Buttke,McCubreyetal.1993).

ThecomparisonbetweenMTT,XTTandMTSassaysareaslistedbelow(Gieni,Lietal. 1995),(Goodwin,Holtetal.1995),(Garn,Krauseetal.1994),(Buttke,McCubreyetal. 1993),(Roehm,Rodgersetal.1991),(vandeSandt,Ruttenetal.1993):

x MTTassay: o Bioreducer:Nobioreducernecessary,althoughmenadionecanbeused toacceleratebioreductionofMTT. o Solubilisationofformazanproduct:insoluble formazanproductare dissolvedusingorganicsolventssuchasSDSͲDMF o Storagestability:MTTassaycanlastuptoamonthafterpreparation o Laserrange:Responseat560Ͳ570nm,referencewavelengthat690nm o Colourdevelopment:Requires4Ͳ5hrsofincubationat37ºthenadd solubilisationsolution x XTTassay o Bioreducer:PMSisusedasbioreducer,butcrystalsmayform immediatelyafteradditionofbioreducer o Solubilisationofformazanproduct:Nosolubilisationsolutionisneeded asformazanproductisalreadywatersoluble o Storagestability:Unstablesolution,solutionneedstobepreparedjust beforeexperiment o Laserrange:Responseat450nm,referencewavelengthat650nm o Colourdevelopment:requires7Ͳ8hrsofincubationat37oC x MTSassay o Bioreducer:PMSisusedasbioreducer o Solubilisationofformazanproduct:Nosolubilisationsolutionisneeded asformazanproductisalreadywatersoluble o Storagestability:MTSassaycanlastuptoamonthafterpreparation o Laserrange:Maxresponseat515nm, fallingtoashoulderat580nm o Colourdevelopment:Minimumof1hrsincubationat37º.Best responseafter2Ͳ3hrsofincubationat37oC

69  Chapter2:ReviewofLiterature

Neutralreduptake(NRU)

TheNRUassaymeasurescells’abilitytoincorporateandbindaneutralredsolution(a supravital and weakly cationic dye)(Borenfreund and Puerner 1985).The cationic neutralreddyebindstotheanionicsitesinthelysosomesandaccumulateasthedye ischargedandcannotpassoutintothecytoplasm(Filman,Brawnetal.1975).

NRUassaywasdevelopedbyBorenfreundetalin1984asanalternativemethodto thedraizerabbiteyetest(Borenfreund and Puerner 1985)and since then has been validated by ECVAM to test for phototoxicity.UNR  assay has been used determine toxicityofvariouschemicalsonvariouscelltypessuchaslivercells(HepG2)(Lestari, Hayesetal.2005),A549(Bakand,Winderetal.2006),Humanskinfibroblasts(Lee,Kim et al. 2000) and 3T3 mouse fibroblast cells (Earl, Jones et al. 1995; Popiolkiewicz, Polkowski etal.2005).However,NRUassaymaynotbesuitableforassessingchemical toxicityonskinorgancultures(vandeSandt,Ruttenetal.1993).

NRU can be used alongside MTS to confirm toxicity data as NRU assay’s results are comparablewithMTSassay’sresults(Borenfreund,Babichetal.1988).Incomparison toMTSassay,theNRUassaymaybemoresensitiveandcheapertoperform(Repetto, delPesoetal.2008).

ATPassay

TheATPassaydeterminestheenergymetabolismofcellsbymeasuringtheamountof ATPincells.AsshowninFigure2Ͳ13(c),theATPassay monoͲoxygenationofluciferin is catalysed bythe luciferase in presence of Mg2+, ATP and molecular oxygen which results in the production of light, which is then recorded using a luminometer (Promega2007).

The ATP assay offers advantages in lower required number of cells and for its experimentalreproducibility.Thisenablesdeterminationofcellviabilitywhichhasthe lowest number of cells available (Russell D. Petty 1995; Cree and Andreotti 1997). While ATP assay is more sensitive than the LDH, MTT and NRU assay (Weyermann, Lochmannetal.2005),itismoreexpensive.(RussellD.Petty1995).Inaddition,the

70  Chapter2:ReviewofLiterature

ATP assay’s LuminescentͲreadout is influenced by the quenching sideͲeffects of the sample(Weyermann,Lochmannetal.2005).

 Figure2Ͳ13:InͲvitroassaysstructureandreactions (a)StructureoftetrazoliumsaltsMTT,XTTandMTS;(b)conversionofMTSinto formazanproduct;(c)luciferasereactioninATPassay Adaptedfrom:(Barltrop,Owenetal.1991),(Promega2001)and(Promega2007).

2.5.5 InǦvitrotoxicityexposuresystemdesign

Criteria for an ideal inͲvitro test system includes reproducible endpoints, ability to metabolisesubstancesasinhumans,rapidandeasytoperformwhileabletoanalyse alltypesofcompoundswithoutachangeintheprotocol(Beaudoin1985).Tomeet withthisrequirements,variousexposuremethodshavebeendevelopedandcanbe grouped into either direct and indirect exposure methods and its subͲtypes (Hayes, Bakandetal.2007).

71  Chapter2:ReviewofLiterature

x Indirectexposuremethods o Exposuretothetestchemicalitself:Cellsareexposedtotestchemicals solubilisedorsuspendedinculturemedia. o Exposuretocollectedairsamples:Cellsareexposedtoairsamples collectedbyfiltrationorimpingementmethods. x Directexposuremethods o Submergedexposureconditions:Testgasisintroducedtocell suspensionundersubmergedconditionsusingimpingeorvacuumtubes o Imminentexposure:Cellsareperiodicallyexposedtogaseous compoundandculturemediumatregularintervalsusingvariationof techniquessuchasrockerplatformsandrollingbottles. o Continuous directexposureattheairͲliquidinterface:Cellsare continuouslyexposedtoairbornecontaminantsduringtheexposure time,usuallyontheirapicalside,whilebeingnourishedfromtheir basolateralsideusingcollagencoatedorporousmembranespermeable toculturemedia(Hoess,Teuscheretal.2007).

GrowingcellsonanAir LiquidInterface(ALI)allowsamorerealisticmodellingofcells' actual condition within the body in testing complex gaseous/aerosol mixtures (Aufderheide2008).Thismethodofgrowingcellshasbeenutilisedinstudiestoassess toxicityofcontaminantssuchasDEPandgoldnanoparticles(asoutlinedinTable2Ͳ13).



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Table2Ͳ13:LiteraturesemployingALImethod Reference Exposure Celllines Endpointsinvestigated

(Wallaert, Gosset et O3andNO2between0.05–1.4 AMandalveolarepithelial LowlevelofO3andNO2exposuredonotdecreasecell al.1996) ppmconcentration. cells(humanandrodent) viabilitybutproinflammatorymediatorswerereleased

(Ritter,Knebeletal. O3andNO2at25ml/min Human lung fibroblasts (WSTͲ1 assay, cell count, protein content, intracellular 2001) (Lk004), human bronchial ATP/ADPcontentandIntracellularglutathionecontentof epithelialcells,(HFBEͲ21) cells.

(Knebel,Ritteretal. Direct sampling of diesel Human bronchial WSTͲ1assay,cellcountingbyelectroniccellcounterand 2002) exhaustrunatdifferentspeed epithelial cell line, HFBE analyser andload 21

(Cheng, Malone et Petrolanddieselexhaust A549 and TNFͲɲ primed ILͲ8production al.2003) A549

(Aufderheide, Cigarette smoke, NO2, diesel Human bronchial WSTͲ1assay,ATP/ADPratio,Intracellularglutathioneof Knebeletal.2003) fume.Diluted and sampled epithelialcells cells, intracellular redox ratio of oxidised and reduced using flow controller and glutathione(GSSG/GSH) vacuumpump

73  Chapter2:ReviewofLiterature

(Shimizu,Endoetal. Field sampled PM, loaded A549,HepG2 Acid phosphatase content, cytochrome P4501A1/2 2004) directlyontoA549cells inducibility

(Lestari,Hayesetal. Combustion products of PE, A549, HepG2, skin Tetrazoliumsalttoformazanconversion 2005;Lestari,Green PP,PC,PVC,FRPandMFP. fibroblast etal.2006)

(Bakand, Winder et Xylene, toluene, NO2; Diluted A549, HepG2, skin Tetrazolium salt to formazan conversion, ATP content, al. 2006; Bakand, and sampled using flow fibroblast Neutralreduptake Winderetal.2006) controllerandvacuumpump

(Brandenberger, Aqueous 15 nm colloidal gold A549, triple coͲculture mRNA induction of proͲinflammatory and oxidative RothenͲRutishauser particles, introduced as (A549,MDM,MDC) stress markers; protein induction of proͲ and antiͲ etal.2010) aerosolbyaerosolgenerator inflammatorycytokines;Particledepositionanduptake

Abbreviations:

WSTͲ1assay=cleavageoftetrazoliumdye(WSTͲ1assay)toitsformazanviamitochondrialdehydrogenases

Polyethylene (PE), polypropylene (PP), polycarbonate (PC), polyvinyl chloride (PVC), fibre glass reinforced polymer (FRP), melamineͲfaced plywood(MFP),humanbloodmonocytederivedmacrophages(MDM),humanmonocytederiveddendriticcells(MDDC),Interleukin(IL).



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2.5.5.1 Contaminantgenerationinexposuresystems

The static exposure system involves generating a test atmosphere without any replenishment.Thestaticexposuresystemallowsdirectexposureofcellsgrownon ALI.Thestaticdirectexposuresystemhasbeenusedtoassesstoxicityvolatileorganic compounds(VOC)includingToluene,XyleneandChlorobenzene(Bakand,Winderetal. 2006;Lehmann,Röder ͲStolinskietal.2008).

TogenerategaseousphaseVOC’s,liquidphaseVOC’sareintroducedintoachamber containingcellsamples.Thechamberisthensealedandtemperaturemaintainedat 37oC,theVOCisconvertedfromitsliquidphasetogaseousphasebyevaporation.The concentration of gaseous phase VOC within the chamber can be calculated using Equation5.

ͳͲ଺ Equation5כ ൗܹ  ܹܯ ݐ݅݋݊ሺ݌݌݉ሻ ൌܽݎ݋݊ܿ݁݊ݐܥ ܸ ൗܸ݉ 

Where: x W=Weightofvolatileliquidintroduced(ingrams). x MW=Molecularweightofvolatileliquid(ingrams). x Vm=Molargasvolume(inlitres)ofairͲcontaminantmixtureunderambient condition.

ThemolargasvolumecanbecalculatedusingEquation6.

Equation6

  Where: x 24.45=molarvolumeofidealgasinlitrespermoleundernormaltemperature andpressure(NTP;definedas760mmHgand25oCwhichis298.15oK). x P=Ambientpressure(inmmHg). x T=Ambienttemperature(inoC). 

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2.5.6 Dieselexhaust

2.5.6.1 Enginesettings

There is little agreement on the operating settings of the diesel engine for exhaust samplingasoutlinedinTable2Ͳ14.Sincecompositionofexhaustvariesatdifferent settings,resultsmaybeinconsistent(Saitoh,Seraetal.2003),therefore,anagreement is needed on both load and rotations per min settings  for the diesel engine so comparison of results are possible between each investigation. However, deciding valuesforloadandrotationsperminisdifficultsinceatypicalworkloadofavehicle involvesnumerousvariablessuchasbraking,acceleratingandrunningidleduringits operation.PreviouslyforinͲvitrosampling,theengineistypicallyatidlesettingorone fixed setting, providing limited insight into engine operating characteristic to cell cytotoxicity.

Automotive engines can be coupled with a dynamometer to enable the setting of various engine operating parameters including rotations per min, torque and power output (Plint and Martyr 1995).The dynamometer provides a more controlled environment that eliminates complicating variables although less an accurate representation of a typical journey due to more random nature of traffic.Other studies have emulated traffic conditions by mounting coupling a diesel vehicle on a dynamometerandsimulateatypicaljourneyincludingacceleration,brakingandidle timeattrafficlights(Saitoh,Seraetal.2003;Ito,Okumuraetal.2006).

Duetotheeffectoffuelcompositiontooveralldieselexhaust,fuelcharacteristicsare importanttorecordsuchassulphurcontent.Manystudiescurrentlydonotrecordnor analysethefuelcharacteristicsalthoughmostpurchasetheirfuels frompetrol/diesel stations which follow a strict regulation set by the local regulators.One main characteristic of interest in fuel properties is the sulphur content as it is the main componentofSO2exhaustcontentandparticulatecreation.



76  Chapter2:ReviewofLiterature

Table2Ͳ14:Variousenginesettingsusedforanalysingemissions

Reference Enginespec/settings Fuel Results (Schumach Displacement:9050cm3 Tested:B10,B20,B30,B40 OptimumblendatB20 er, Borgelt Setting: 1200 and 2100 rpm; 50%, 100% Control:LowSulphurdiesel#2,Essodiesel Biodiesel fuel emits less PM, THC etal.1996) loadfor10mins #1 butmoreNOx

3 (Dorado, Displacement: 2500 cm , four stroke Tested:Filteredwasteoliveoilmethylester Lower emission of CO, CO2, NO,

Ballesteros directinjection Control: European FAME standard draft SO2 etal.2003) Setting:600–2390rpm;80–560Nmrange prEN14105

(LinandLin Displacement: 3856 cm3 four cylinder Tested: Soybean based biodiesel with BiodieselhasloweremissionofCO,

2006) directinjectionandnaturallyaspirated peroxidation CO2, brake specific fuel

Setting:800–2500rpmrangeat10KgF Control:ATSM#2diesel consumptionandNOx

(Yang, Displacement: 2835 cm3, turbocharged Tested: B20 diesel/methyl ester of waste B20hasloweremissionthandiesel Chienetal. withindirectinjection cookingoil atstart 2007) Setting:Totalrunof500hrs;80,000km Control:Standarddiesel

(Devanand Displacement:661cm3,onecylinderDI Tested: Poon oil based biodiesel with 20% poon oil / 80% diesel blend Mahalaksh Setting:Fixed1500rpmat0–100%load variousblend provides best balance of various mi2009) range Control:Standarddiesel emission

(Lin,Yinget Engine: Single cylinder, 6kW, max 3600 Tested:Ricebrainoilbasedbiodiesel biodiesel soot is smaller indicating al.2009) rpm Control:Standarddiesel improved burning andcombustion Load:10–100%range reactionofbiodiesel

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2.5.6.2 Dieselexhaustgeneration/samplingmethods

Forparticulatesamples,studieshaveusedfilterssuchasofglassfibreinstalledwithin adilutionchamberoftheexhaustsystemwithapumpsamplingtheexhaustatfixed flowrate(Bayona,Markidesetal.1988;Bünger,Krahletal.1998;Song,Zhouetal. 2007; Omura, Koike et al. 2009).Prior to experiments, preparation of particulates usually includes extracting specific diesel exhaust component using solvents and resuspendingtheparticulatesinsolventsusingsonicationtopromotedisaggregation of particles as performed in studies outlined in Table 2Ͳ15.Other than particulate samples,airpollutionresearcherssampleambient pollution in set locationsinstead. Althoughthismayprovideamorerealisticrepresentationonairpollutiontoxicity,the sourcesof thepollutantsarenumerous and difficult todetermine.Newer methods include sampling DEPs from the vehicle in a bag during its journey (Jamriska, Morawskaetal.2004).

Althoughdieselparticulatesareusuallysampleddirectlyfromldiese exhaust,itisalso availableasapurchasablestandardreferencematerial(SRM)1650fromthenational institute of standards & technology.SRM 1650 represents particulates from heavy dutydieselengines(thecertificateofanalysisisavailableathttp://www.nist.gov)and werecollectedfromheatexchangerofadilutiontubefacilityfollowing200enginehrs ofparticleaccumulationfrommultipleenginesoperatingindifferentsettingstoreflect generalexhaustgeneratedbyheavydutydiesel.

SRM 1650 was intended for evaluating analytical methods in determining various chemical concentrations in diesel particulates, however it can used to study toxicity mechanismsofDEP(Boland,Baeza ͲSquibanetal.2001)suchasitsgenotoxicityeffect onA549cells(Dybdahl,Risometal.2004)andinflammatoryresponse(Patel,Eoetal. 2011).



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Table2Ͳ15:InͲvitrotestingondieselexhaust/particulatetoxicity

Ref Contaminant Celllinesandexposure Endpoints

(Bayram, Engineandsetting:unspecified Cultured bronchial epithelial cells Ciliary beat frequency, ILͲ8, GMͲCSF, Devalia et Collection: glass fibre filter at end of dilution from bronchial biopsies of asthmatic RANTES,andsICAMͲ1 al.1998) tunnel andnonͲasthmaticsubjects Preparation:suspendedincellmedia 

(Bünger, Car:VWVento1.9lTDI Cellline:S.typhimuriumTA97a,TA98, Mutagenicity by ames assay; Cell Krahl et al. Fuel: petrodiesel, rapeseed oil methyl ester TA100, and TA102 strains for ames viabilityusingNRUassay 1998) (RME) test;ratfibrocytesLͲ929cells Collection: PTFE coated glass fibre filter in <50oCexhaust

(Ito, Engine:2982cccommonrailDI Cell line: L2 cells exposed to mRNA expressions of ICAMͲ1, LDL Okumura Setting:2040rpmat35Nm suspensionsandsonicatedDEPincell receptor, PAF receptor, HOͲ1 and etal.2006) Collection: from accumulation in dilution media GAPDHusingPCRtechniques tunnel,storedatͲ80oC

(Rumelhar Source: PM collected from outdoor sites, Human bronchial epithelial cell line AmphiregulinreleaseusingELISA d, storedatͲ20oCuntilpreparation 16HBE14oͲ; normal human nasal Ramgolam Preparation: organic parts, extracted by epithelialprimarycellsculture etal.2007) sonicationandchemicaltreatments

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(Song, Engineandsetting:5785ccDIsixcylinderin S. typhimurium strains TA98 and Mutagenicity by ames test; Zhou et al. ECER49Ͳ13mode TA100forAmestest;ratfibrocytesLͲ genotoxicitybycometassay 2007) Fuel: standard light diesel, and dieselͲethanol 929cellsforcometassay blends Collection and preparation: 70 mm filter in <52oC exhaust; soluble organic fractions extracted,concentratedandstoredatͲ20oC

(Hirota, Preparation: DEP resuspended in cell media HLͲ60clone15 Cytokineslevels,proteincontent,NFͲ Akimaru et and sonicated to induce particle  ʃB,MCPͲ1andP38MAPkinase al.2008) disaggregation

(Bonetta, Source:Collectedfromoutdoorsites A549 exposed to serial diluted PM Cell count by trypan blue; DNA Gianotti et Preparation: organic parts were extracted by suspendedinmedia damagebyFpgͲmodifiedcometassay al.2009) sonicationandchemicaltreatments

(Omura, Source: 4Ͳcylinder, lightͲduty (2740 cm3 Cellline:RatalveolartypeIIepithelial Cellcountbytrypanblue Koikeetal. exhaustvolume),4JB1Ͳtypedieselengine celllineSV40T2 MicroarraysignalsfromRNA 2009) Collection: GlassͲfibre filter fitted to end of a Exposure: Extractedd an  fractioned  stainlesssteeldilutiontunnel DEPsuspendedinculturemedia 

(Patel, Eo Source:SRM1650 A549 ILͲ8 by ELISA; ROS level by H2DCF etal.2011) Preparation: Resuspended and sonicated in assay and total protein content by steriledeionisedwater BCAtotalproteinassay

80  Chapter3:ResearchObjectives

3Researchobjectives

Researchintoelucidatingthetoxicitymechanismsofdieselexhaustisincreasingdueto the popularity of diesel passenger vehicles offering higher fuel efficiency and some cleaner emissions compared to petrol engines.With an increase in diesel exhaust level, there are additional health risks to the exposed population.Therefore these risks need to be characterised and elucidated further.One of the difficulties in analysing the toxic effects of diesel exhaust is its complex gaseous and particulate composition.

This thesis explores several inͲvitro toxicology methods to analyse diesel engine exhaustusinghumanbasedcells.InͲvitrotoxicologyoffersthepotentialtoelucidate toxicity mechanisms and generate toxicity data with eliminating interͲspecies extrapolation.To achieve the main research aim and objective, research questions weredevelopedaslistedbelow.

1. Whatarethemainchemicalandgaseouscomponentsofdieselexhaust?

2. Whichhumancellsshouldbeusedtodeterminetheeffectsofdiesel exhaust on various human organs? Is there any statistical significant difference in

toxicity(suchasIC50measurements)betweencelltypes?

3. What inͲvitro assays would be suitable for determining toxicity of diesel exhaustandwhatstatisticalsignificantdifferenceexistsbetweeneachassays’ result?

4. What methods are available to determine carbon content of diesel exhaust includingtotal,organicandelementalcarboncontent?

5. Is it possible to reduce the number of steps in the toxicity testing of diesel exhaust,includingeliminatingsolventswhichmayaffectfinaltoxicityeffects?

6. Canthedirectdynamicmethodbeusedtoanalysethetoxicityofexhaustfrom dieselengines?

7. What diesel engine settings can be used to provide a  most reliable and reproducibleresult?

8. Withinthedieselengineexhaustcomponent,whatisthemajorcontributorto overallcytotoxicity?

81  Chapter3:ResearchObjectives

9. Is there much information available on in vivo testing or epidemiological studies?Ifthereis,howdothedatafromtheinͲvitrostudiescorrelatetothese dataandhowcanitbeusedforriskassessment?

10. On comparison of chemical analysis and inͲvitro toxicology, what additional informationcanbeobtained?Especiallyinrelationtocytotoxicity?

Several methodologies and experiments were developed to address the above researchquestions

1. Determine suitability of conventional methods such as reͲsuspension of contaminantsinculturemediaindeterminingtoxicityofparticlesandconclude its applicability to  determine toxicity of particulate components of diesel exhaust.

2. Develop and determine suitability of a direct exposure method in testing toxicity of gaseous contaminants such as volatile organic compounds and determiningtheirability todetermine thetoxicity of gaseous components of dieselexhaust.

3. Develop and refine a direct and dynamic exposure method in field and laboratory based studies to allow direct sampling of diesel engine exhaust, allowingtoxicityanalysisusingvariousinͲvitroassaysincludingMTS,NRUand ATP.

4. Comparetoxicitybetweenfilteredandunfiltereddieselexhausttodetermine the overall toxicity contribution of diesel exhaust particulates to overall toxicity.

5. Refinemethodsintoxicitytestingofdieselengineexhaustwhenengineisrun throughvariousloads.Thisincludesanalysingcarbonandgaseouscontentof exhaust to determine whether gaseous or particulate component of the exhaustcontributesgreatesttotoxicity

Thethesismainlyfocusedondevelopmentandtestingprocedures ofinͲvitromethods inanalysingdieselengineexhaust.Theinformationgeneratedwillbeusefulforfuture developmentandmodelingofdieselengineexhausttoxicologyanalysis.



82  Chapter4:MaterialsandMethods

4 Materialsandmethods

4.1 Testmaterials Inthepreliminaryresearches,testchemicalsincludingnanoparticles(ZincOxideand Titanium Dioxide) and volatile organic compound (Toluene) were used.All test chemicalswereatthehighestgradeavailablefromSigmaͲAldrichandAPSFinechem Australia respectively.The nanoparticles and toluene were chosen as the model particles and gaseous components of diesel exhaust.Diesel exhaust was generated usingdieselenginesasperSections4.3.2and4.3.5.Inaddition,foralllaboratoryand field based experiments with diesel engines, Australian Standards 3570 compliant dieselfuelwasusedasperSection4.3.1.

Table4Ͳ1:Physicalcharacteristicsofselectednanoparticles

 ZincOxide TitaniumDioxide

Chemicalformula ZnO TiO2

CASnumber 1314Ͳ13Ͳ2 13463Ͳ67Ͳ7

Particlesize <100nm <100nm

Molecularweight 91.39 79.87

Surfacearea 15Ͳ25m2/gram N/A



Table4Ͳ2:Physicalcharacteristicsofselectedvolatileorganiccompound

 Toluene

Molecularformula C6H5CH3

CASnumber 108Ͳ88Ͳ3

Molarmass 92.14grams/mole

Density 0.86g/ml

TLVͲTWA 50ppm

TLVbasiscriticaleffect Centralnervoussystem

Acutetoxicitydata(InhalationͲratLC50) 13,000ppm

83  Chapter4:MaterialsandMethods

Table4Ͳ3:Physicalcharacteristicsofdieselfuel

 CALTEXVortex AS3570requirement PremiumDiesel

Densityat15oC 0.82Ͳ0.87kg/L 0.83kg/L

MinimumCetaneNumber 50 45

MaximumSulphurcontent(bymass) 0.5% <10ppm

Energydensity N/A 35.9 megajoules/litre or 43.3megajoules/kg

4.1.1 Preparationandserialdilutionofstocksolutions

Inpreparingchemicalstocksolutionsforindirectexposureofparticlestocellsin96 well plates, chemicals were dissolved into 10 ml of culture media using a 10 ml volumetric flask and filter sterilised (pore size: 0.22ʅm).The stock solutions were thenusedforserialdilutionoftestchemicalsin96wellmicrotitreplates.Thehighest concentration was estimated from published toxicity data and initial trials to determinetherangeofchemicalconcentrationstoachieveafulldoseͲresponsecurve.

Stocksolutionswerepreparedpriortouseandthefollowingprotocolfollowed(layout of96wellplateprovidedinFigure.4Ͳ9) Initially,supplementedmediawereaddedto each (60 μl) well of the 96 microwell plate using an 8Ͳchannel multichannel pipette (Soccorex).Stocksolutionofchemicalswerethenaddedtothe3rdcolumnwells(20 μl)andmixed3Ͳ5timeswithoutintroducinganybubblesintothewells.Aliquotsof mixed solution (20 μl) were then transferred to the 4th column wells and mixed as previously performed.The transfer and mixing steps were repeated until the 12th columninwhichthe20μlaliquotwasremovedanddisposedof.

4.2 Staticgenerationoftestatmospheres To investigate cytotoxicity of volatile organic chemical such as toluene, a static generationmethodwasdevelopedforexposureinsideGlasschambers(322±2ml). The chambers were manufactured in the glass workshop of the UNSW.Glass was chosen as the material of choice as it has very low adsorption loses and prevented diffusion of materials through the chambers (Saltzman 1997).To generate gaseous

84  Chapter4:MaterialsandMethods contaminants, a VOC was introduced into the chamber by pipetting it onto a filter paper.ThefilterpaperassistedevaporationandmixingoftheVOCwithairtoachieve consistentcontaminantconcentration.

4.3 Dieselengine,fuelanddynamometer

4.3.1 Dieselfuel

The diesel fuel used for this study was purchased from a commercial fuel station (Vortexpremiumdiesel;CALTEX)whichcompliedwithAustralianStandard3570Ͳ1998 (Standards Australia 1998) as per Table 4Ͳ3.AS3570 requires diesel fuel to have a density of 0.82Ͳ0.87 kg/L at 15oC, minimum cetane number of 45 and maximum sulphurcontentof0.5%(bymass).TheCALTEXdieselfuelhaddensityof0.83kg/Lat 15oC, minimum cetane number of 50 and maximum sulphur content of <10 ppm, additionallytheenergydensitywas35.9Megajoules/litreor43.3MegaJoules/kg.

4.3.2 Dieselengineforlaboratorybasedstudies

For the laboratory based studies,testswereperformed at the Engine Research Lab, SchoolofMechanicalEngineering,UNSW.A6700ccsixcylinderVolvoturbocharged engine(maxpowerof169kWat2200rpm;maxtorqueof825Nmat1600rpm)was coupled to an eddy current dynamometer (Figure 4Ͳ1).The eddy current dynamometer controller measured the amount of torque generated, allowing the settingofdesiredengineoutputtorqueandspeed.Inaddition,inͲcylinderpressure, enginecoolantthermometerandexhausttemperatureweremonitored(Figure4Ͳ2).

 Figure4Ͳ1:Laboratorybaseddieselengineanddynamometersetup (a)Eddycurrentdynamometer;(b)Volvodieselengine

85 Chapter4:MaterialsandMethods



 Figure4Ͳ2:Laboratorybaseddieselenginecontrolconfiguration

4.3.2.1 InͲcylinderpressuremonitoring InͲcylinder pressure was acquired using a KISTLER piezoelectric pressure transducer (Figure 4Ͳ3).The transducer operated using piezoelectric principle in which the pressureexertedonthetransducerinsidethecombustionchamberwastransformed into electrical charge.The electric charge was then amplified and converted into electricalvoltage,allowingviewofthecombustionchamberpressurecurvethroughan oscilloscope.

(a)(b) Figure4Ͳ3:InͲcylinderpressuremeasurement (a)Kistlerchargeamplifier;(b)Oscilloscopefordisplayingpressurecurve

4.3.3 Apparentheatreleaseratecalculation

TheinͲcylinderpressuredatawasanalysedfortheapparentheatreleaserateusingan iterative,twoͲzoneheatreleasecodeinwhichgaspropertiesweredependentonboth mixture composition and temperature. The heat transfer losses were partially accountedforbysubtractingtheapparentheatreleasecalculatedforacorresponding motoredenginecycle,aprocedurewhichisalsoexpectedtocompensatetoalarge degree the enthalpy losses associated with crevice flows. Consequently, the net 86 Chapter4:MaterialsandMethods

apparentheatreleaserate(Qapp )isapproximatelyequaltotherateofchemicalheat

releaseminusthedifferencebetweentheheattransferlosseswithcombustion( Q ht,comb

)andthemotoredlosses(Qht,motored)asinEquation7.

  QQQQ   QQ  app chem ht,comb ,motoredht chem hl  Equation7:Apparentheatreleaserate

4.3.4 Adiabaticflametemperaturecalculation

To clarify the effects flame temperature has on engine emissions and its toxicity, several parameters were correlated below with the maximum adiabatic flame temperature.Thismethodologyhasbeenshownasanacceptableapproachtoanalyse engineemissions(Plee,Ahmadetal.1981;Musculus2004).

Theadiabaticflametemperaturewascalculatedfor astoichiometricmixtureofdiesel fuel and diluted intake gas using the STANJAN chemical equilibrium code (Reynolds 1986). The peak adiabatic flame temperature was defined as the adiabatic flame temperature achieved by combustion of a stoichiometric mixture at the peak gas temperature. The peak gas temperatures were estimated from the initial intake gas temperature and pressure with the assumption that the core gases are compressed isentropically.Theestimationaccountedforthegascompositionandfortemperature dependent specific heats. For the flame temperature calculation, a single heat of combustion was used for the multiͲcomponent diesel fuel, calculated from the net heatofcombustionandknownH/Cratio.

From the extended Zeldovich mechanism of NO formation, adopting a partial equilibrium assumption (Bowman 1975), an approximate NO formation rate can be written as Equation 8.This theoretical basis is the premise that engineͲout NOx emissionswillbeproportionaltothepeakflame temperature.

d  2/1 2/1 Ea / RT 3 2 2 ][][][ eNOTANO mol / cm ˜ s dt  Equation8:NOformationrate

WhereAisaconstant;Tisthelocalgastemperature;Ristheuniversalgasconstant; andEaisoverallactivationenergy.

87  Chapter4:MaterialsandMethods

4.3.5 Dieselengineforfieldbasedstudy

Forthefieldbasedstudy,adieselenginepoweredpassengercar(VolkswagenPassat; model2006)wasused.Similarly,beforeexposureofdieselexhausttotargetcells,the engine was switched on, warmedͲup for 15 mins and sampling performed at idle setting.

4.4 Samplingpumpandrotameters Asamplepump(Aircheck©2000;SKC;USA)wasusedforsamplingdieselexhaustand carboncontentsampling.Dieselexhaustsamplingswereperformedatalowsample flowof37.5ml/minbyconnectinginseriesaconstantpressurecontroller(CPC;SKC; USA)andarotameter(asillustratedinFigure4Ͳ4).Forcarboncontentsampling,the pumpwassetat2L/minandconnectedinseriestoPolycarbonatemembranefilters, 37mmindiameterand0.4μmporesizeinathreepiececlearstyrenecassette(SKC, USA)asperSection4.5.2.

Thepumpwascalibratedusing the soap bubble method in which the sample pump was connected with flexible tubing to a soap bubble meter (NIOSH 1998).Three readingsofairflow(bytimingtherateofriseofsoapfilms)weretaken,averagedand usedtoconfirmthesettingofthesamplepump.

 Figure4Ͳ4:Dieselexhaustsamplingsystemsschematic (1) Sampled )exhaust; (2  mixing chamber; (3) distribution channels; (4) exposure chamber;(5)rotameters;(6)negativepressurepump. 

88  Chapter4:MaterialsandMethods

  Figure4Ͳ5:Samplingequipments Airsamplingpump(a),rotameters(b),dynamicexposurechamber

4.5 Analyticalmethods

4.5.1 Realtimegasmonitoring

The exhaust gas from the diesel engine were analysed using iBrid™ MX6 multiͲgas monitors.ThemonitorswerecapableofanalysingCO,CO2,NO,NO2,O2,SO2,Cl2,NH3,

C3H8.During exposure time, exhaust emissions were continuously monitored using

MX6iBridgasmonitors(IndustrialScientific,USA)capableofmeasuringCO,CO2,NO,

NO2,O2andClgases(Figure4Ͳ6).

4.5.2 Carboncontentmeasurement

Carbon content was measured by thermal optical organic carbon/elemental carbon using the principles of the National Institute for Occupational Safety and Health (NIOSH) 5040 method which was conducted by Coal Services Pty Ltd.NIOSH is an organisationresponsibleforestablishingairstandardsforcoalminesandUSEPAfor ambientairsstandard affectedbydieselexhaustfromvehicles(Hinners,Burkartetal. 1981).ExhaustwascollectedonPolycarbonatemembranefilters37mmindiameter and0.4μmporesinathreepiececlearstyrenecassette(SKC,USA)(Figure4Ͳ7aandb) Thesamplingrequiredtheenginestobewarmedrupfo atleast5mins,cassetteswere connectedtothesamplingportoftheengineexhaustandsampledat2L/minfor15 min interval (same interval as sampling exhaust to cells).Afterwards the cassettes weresealed,packagedcarefullyandsenttoCoalServicesPtyLtdtobeanalysed.The NIOSH5040analysisyieldboththeorganicandelementalcarboncontentsampledon thefilter.

89 Chapter4:MaterialsandMethods

4.5.3 NOxmeasurement

TheNOxmeasurementwasperformedusingahighprecisionNitrogenOxideanalyser (EC9841AS; Ecotech, Australia).The sampling was performed by a vacuum pump capableof1standardlitreperminute(slpm)at67kPa,anO3scrubberwasconnected toremoveexcessO3andpreventdamagetothepump.

The analyser featured an inbuilt 5 μm inlet filter, fulfilling US EPA requirements for samplestobeparticulatefiltered(<5μm)anddry(nonͲcondensing).PVCtubingwas usedforconnectingtheanalyseranditsdedicatedpump.NOxemissionsofdieselthe engine were recorded after 1 min of engine operation at the desired load to allow stabilisationofexhaust.Theanalyserhadadetectionrangeof0Ͳ1000ppmrangewith 1 ppb resolution and precision of 0.5 ppb or 1% of the concentration reading (whichever was greater) with noise (root mean square) of 2 ppb or 0.1% of the concentration reading (whichever is greater).The exhaust was sampled at a 370 ml/minflowrate.

 Figure4Ͳ6:iBrid™MX6multiͲgasmonitors 

  Figure4Ͳ7:NIOSH5040filtersandhighprecisionNOxanalyser Polycarbonate membrane for NIOSH5040 organic carbon and elemental carbon measurements(a)unusedfilter;(b)filterwithsampledcarbon;(c)highprecisionNOx analyser.

90 Chapter4:MaterialsandMethods

4.6 InǦvitrotechniques

4.6.1 Safetyconsiderations

Safetyisaprioritywithinthelaboratory;thereforeidentificationandcorrecthandling of hazardous substances such as chemicals and human cells were essential.Before commencing work, a risk assessment was performed in accordance with the UNSW OccupationalHealthandSafetyGuidelines.Theriskassessmentincludedidentification of hazardous substances and their  associated risks and application of different risk mitigation strategiesto reducerisks to acceptable levels.Human cell culture works were performed inside a class II biosafety cabinet (BSC 2000 series, Australia) for protectionof operators.In addition, various personnel protective equipments were used where necessary including: laboratory gown, gloves, face shield and cryogenic gloves(whenretrievingcellsfromcryogenicstorage).

4.6.2 Mediaandsolutions

For cell culture purposes, phenol red free culture medium DMEM/F12 (Dulbecco's modifiedEagleMedium:Ham'sFͲ12nutrientmixture;Gibco,USA)wassupplemented withantibioticsintheformofpenicillinstreptomycinglutamine(29.2mgLͲglutamine, 10,000 U Penicillin and 10 mg Streptomycin per ml; Gibco, USA), the media to antibioticss ratio wa  100:1.The media and antibiotic mixture solution was supplementedwhenneededwithNewbornCalfSerum(NCS;Gibco,USA)atamediato NCSratioof20:1forimprovedcellgrowth.

For washing cells of wastes and other possible contaminants, Hank’s balanced salt solution(HBSS,Gibco,USA)wasused.

4.6.3 Cellcounting

CellcountingwasperformedusingaCountess®AutomatedCellCounter(Invitrogen, USA).To perform cell counting, trypan blue stain (0.4% w/v; Invitrogen, USA) was mixed with cells at 1:1 ratio and then pipetted into both counting chambers of the Countess®CellCountingslide.Countingwasperformedonbothcountingchambers and both readings were compared to ensure cell suspensions had a uniform

91  Chapter4:MaterialsandMethods distribution of healthy cells. Cell samples were considered healthy when >90% cells werealiveduringcounting.

4.6.4 Cellcultureconditions

Cells were cultured in sterile, vented 75 cm2 cell culture flasks.The cells were incubated in a supplemented culture medium as per Section 4.6.2.When culturing underoptimumconditions(37oCandhumidified),cellnumbersfollowedasigmoidal growthcurve.Culturedcellsgrewasamonolayertoaplasticorglasssubstrateuntil no more surface area was available for the cells to adhere to (plateau phase).To ensure cells were in the optimal phase of growth, cells were harvested prior to the plateauphasetoavoidoverconfluence.Newlyconfluentcelllayerswereenzymatically removedusingTrypsin/EDTA(Gibco,USA)andresuspendedinsupplementedculture medium.Cell viability was assessed by vital staining with trypan blue (0.4% (w/v); Sigma,USA),andcellnumberwasdeterminedbyaCountess®AutomatedCellCounter (Invitrogen,USA).

4.6.5 Cellharvestingandsubculturing

Dislodging the cell monolayer from flask wall was performed either by enzymatic dissociation(whichyieldedhighercellnumberswithlesscelldamage)orbyphysical dissociation(suchasscrapping).Carewastakennottoincubatethecellswithenzymes longer than necessary to avoid damaging cell membranes.Before harvesting, cell cultures (in 75 cm2 flask) were examined under a light microscope to assess for confluence.Culturemediawasthendecantedoutofthechosenflasksandwashed using HBSS (10Ͳ15 ml; Gibco, USA).Thawed Trypsin/EDTA was added into the cell cultureflasks(2ml)andincubatedat37oCfor3mins.Theenzymaticreactionwasthen neutralizedbytheimmediateadditionofsupplementedDMEM/F12culturemedium (10 ml).Cells were then harvested by pipetting the solution into a sterile 15 ml centrifugetubeandcentrifugedfor5minsat1000xg.

To achieve the desired cell number concentration, the supernatant solution was discarded and the cell pellet was resuspended with 5 ml of fresh supplemented DMEM/F12culturemedium.Theconcentrationofcellsinthesuspensionsolutionwas calculated by the vital staining technique using trypan blue (0.4% (w/v); Invitrogen,

92  Chapter4:MaterialsandMethods

USA),andcountedusingtheCountess®automatedcellcounter.Afterdeterminingthe cell concentration, supplemented culture media was added to modify the cell concentrationofcellsinthesolutionasneeded.

ForsubͲculturing,thecellsuspensionsolution(3Ͳ5ml)wastransferredintothe75cm2 flaskandsupplementedculturemediawasadded(10ml).Theflaskswerethenstored o at37 Cinahumidified5%CO2incubator.

4.6.6 Theoptimalcelldensity

Optimalcelldensitywasdeterminedtoprovidearangeforlinearresponseduringthe recording of absorbance or luminosity.Confluent cell layers were enzymatically harvested,resuspendedandadjustedtotargetcellconcentrationasperSection4.6.5, Serialdilutionofcellswerepreparedin96wellflatbottomedmicrotitretissueculture plates (Falcon,USA)orporousmembrane(clearpolyestersnapwellTMinsert,Corning) with replicates.Various inͲvitro assays were then performed and absorbance or luminescence data points were plotted and linear regression was performed to determine cell range which provided a linear response between cell number and absorbanceorluminescence.

4.6.7 CulturingcellsonSnapwellinserts

Human cells were grown on porous membrane (0.4μm) in Snapwell inserts (clear polyester SnapwellTM inserts, Corning).Snapwell inserts are a modified Transwell cultureinsertwitha12mmdiameterwhichprovidesagrowthsurfaceareaof1.12 cm2supportedbyadetachableringplacedinasixwellcultureplate.Togrowcells, supplementedmediawasaddedtothebottomandtoppartsofthemembranewells (bottom: 2 ml, top: 0.5 ml) and plates incubated for 1 hr at 37oC to improve cell attachment.CellswereharvestedasperSection4.6.5toobtainacellnumberdensity of 5 × 105 cells/ml which provided the best linear response of absorbance or luminescenceasdeterminedinChapters6and8.Theculturemediaonthetoppartof membranes was replaced with the cell solution (500 μl) except background control wells), plates were then incubated for 24 hrs to allow cell attachment to the membrane.

93  Chapter4:MaterialsandMethods

 Figure4Ͳ8:GrowingandexposingcellsattheAirLiquidInterface(ALI) (Left)GrowingcellsonSnapwellTMinserts;(Right)Exposingcellstocontaminants

4.6.8 Selectedhumancells

4.6.8.1 Humanskinfibroblasts

Thehumanskinfibroblastswereisolatedfromfreshskinbiopsies(takefromarmsof healthyindividualsage28Ͳ45atthecytogeneticsdepartment,TheChildren’sHospital Westmead,Sydney,Australia).

4.6.8.2 A549–Humanlungcells

The continuous cell line derived from human pulmonary adenocarcinoma epithelial cell line (A549; ATCC number: CCLͲ185) originated from a 58 yr old Caucasian male whoselunghadcarcinomicdisease.Thesecellshaveahumankaryotypeandcontain multimellar cytoplasmic inclusion bodies typically found in type II alveolar epithelial lung cells (Lieber, Todaro et al. 1976).Structurally, A549 cell line has epithelial morphology and adherent growth properties and is suitable for metabolismͲrelated toxicitystudiesbutmayproducedifferentresultsongeneexpressionofVOCsandPoly Aromatic Hydrocarbons (PAH) metabolising enzymes as compared to alveolar macrophages(AM)isolatedfrombronchoalveolardlavageflui (BALF)(SaintͲGeorges, Abbasetal.2008).Othersuitablestudiesincludesurfactantbiosynthesis,asamodel for isolated type II pneumocyte, testing organometallic fatty acid analog since fatty acidmetabolismisdistinctiveinA549cells(NardoneandAndrews1979).

4.6.8.3 HepG2–Humanlivercells

HepG2 (ATCC number: HB 8065) originated from a 15 yr old Caucasian male whose liverhadhepatocellularcarcinomadisease.HepG2cellsaremorphologicallyepithelial andable toproduce most plasma proteins with biosynthesisfunctionality of normal

94 Chapter4:MaterialsandMethods human hepatocytes (Knowles, Howe et al. 1980).Epithelial cells have microvilli, a columnar structure on its apical side which increases the rate of absorption of nutrientsanditsinteractionwithothercellsviatight,anchoringandcommunicating junctions.As a tissue (layer of epithelial cells), tight junctions provide a barrier to prevent molecules penetrating through epithelial tissue while anchoring provides structuralsupportandcommunicatingjunctionsprovidearouteforchemicalsignals betweencells(Goodman1998).

HepG2cellshavemanyliverspecificfunctionsandhavebeenusedtorepresentthe humanliverforitsdetoxifyingfunctions.eTh liverisatargetorganandoccasionally bioactivationofxenobioticsbythelivercangeneratemoreharmfulmetabolites(Okey, Robertsetal.1986)suchastheconversionofcarbontetrachlorideintochloroform,a toxicandcarcinogenicsubstance(Griffin2006).

HepG2 cells have been used in determining the toxicity of compounds such as enantiomersofhexabromocyclododecanes,aflameretardantmainlyusedinplastics and textiles (Zhang, Yang et al. 2008), antiͲcancer effects of flavonoids such as baicaleinandsilymarin(Chen,Huangetal.2009)anddoseͲresponseofglucoseandits oxidativestresswhichinducesapoptosis(Chandrasekaran,Swaminathanetal. 2010). Nanoparticles such as those from polystyrene of 20nm in diameter may undergo uptakebyhumanhepatocyteandHepG2cells(Johnston,SemmlerͲBehnkeetal.2010).

4.6.9 Cellretrievingandarchiving

Whencellswereabundant,archivingwasanoptionasalongertermstoragesolution. o o Cells were stored at 77 K (Ͳ196 C) using liquid nitrogen (LN2) and stored inside a cryogenicDewar(LS750,TaylorͲWharton,USA).

4.6.9.1 Retrievalofcells

To retrievecells,thecryogenic vials were quickly removed from the LN2 Dewar and thawedat37oCinawaterbath.TheStyrofoamholderwasusedtofloatthevialsand keeptheneckpartofthevialabovewatertoreducetheriskofcontamination.The vialswerethenswabbedwith70%ethanolbeforeplacingtheminsidethebiohazard cabinet.To wash away the DMSO solution,  cells were resuspended with warmed

95  Chapter4:MaterialsandMethods media,transferredintosterile15mltubesandcentrifugedfor5minsat1000xg.The supernatant solution was discarded and the cell pellets were resuspended with warmed fresh serum supplemented media.The cell suspension solution was then transferredinto75cm2cellcultureflasksandadditionalculturemediawasadded(10 o ml).Cellcultureflaskswerethenincubatedat37 Cinahumidified5%CO2incubator.

4.6.9.2 Cryopreservingcells

Cellswereharvestedfromthecellcultureflasks(asdescribedinSection4.6.4)untilthe removalofthesupernatantwhichleftonlythecellpelletsinsidethecentrifugetube. Culturemedia(6ml)wasmixedwithsterileDMSO(1ml)inasterile15mlcentrifuge tube; the solution was then transferred  into the centrifuge tube containing the cell pelletsandmixed.Theresultingsolutionwasaliquotedinto1.8mlvials(cryotube™; Nunc),transferredintotheͲ80oCfreezerforatleast24hrs(Rubberrackswereusedas afreezingratecontroldevice)beforebeingstoredinthecryogenicDewar.After24 hrs,cellswereretrievedfromoneofthevialsasperSection4.6.9.1andobservedfora fewdaystoensurenocontaminationhadoccurred.

4.7 InǦvitroexposuresystems

4.7.1 Staticdirectexposuremethod

Glasschamberswereusedasasealedcontainerfordirectexposureoftoluenevapours tocells.Theglasschambers(volume=322±2ml)weremanufacturedatTheUNSW glassworkshop.Smallerglasswellswereautoclavedandculturemediawasaddedto theglasswells.Thecellmembranewasplacedover theglasswellstocreateabiphasic cellculturecondition,allowingcellstosimultaneouslybedirectlyexposedtogaseous pollutantswhilstreceivingnutrientsfromthebasolateralside.

4.7.2 Dynamicdirectexposuremethod

Perspex chambers were used as a sealed container for direct exposure of diesel exhaust to cells.The chambers were manufactured in The UNSW mechanical workshop.The bottom part of the chamber was filled with serumͲfree media supplementedwithantibiotics(1%v/v),maintainedat37oCusingapreheatedblock. Themembranewasthenpositionedinsidetheblockanddieselexhaustwassampled

96  Chapter4:MaterialsandMethods asSection4.4.Afterwards,themembraneswereplacedbackinitsdetachablerings andintothe6wellplate,serumͲfreeculturemediawasthenaddedatthetop(0.5ml) andbottom(2ml)partofthemembranethenincubatedforeither0hrsor24hrs.

4.8 Cytotoxicityassays DetailsofassayreagentpreparationsareprovidedinAppendixA.

4.8.1 MTSǦtetrazoliumsaltassay

® ThePromegaCellTiter 96AQueousNonͲRadioactiveCellProliferationAssaywasusedto measuretoxicityofcontaminantsbydeterminingthenumberofviablecellsinculture (Promega2007).Thisassayhasbeenusedfortoxicitytestingofairbornecontaminants (Lestari,Hayesetal.2005;Bakand,Winderetal.2007).TheMTSmeasuredviablecells’ ability to  convert the soluble tetrazolium salt into a formazan product. The MTS reagent was mixed with electron coupling agent PMS (phenazine methosulphate; Sigma, USA) at MTS:PMS ratio of 20:1 to allow faster bioreduction and faster productionoftheformazanproduct(Goodwin,Holtetal.1995).

4.8.1.1 MTSprotocolfor96Ͳwellmethod

ToperformtheMTSassayon96wellplates,cellswereharvestedfromflasksandreͲ suspendedatthedesiredcellnumberconcentrationasSection4.6.5.Cellsuspensions werethenadded(40μl)tostandardwellsinfourreplicatesandculturemedia(40μl) wasaddedtobackgroundwellsinrfou replicates.Aftertheincubationperiodofeither 0 or 24 hrs, the MTS/PMS mixed reagent was then added to each well (20 μl) and plateswereincubatedat37oCfor4hrs.Aftertheincubationperiod,theabsorbance levels were recorded at 492 nm by a multiplate reader (Multiskan Ascent, Thermo Laboratories,Finland).

4.8.1.2 MTSprotocolforporousmembranemethod

AfterthepostͲexposureincubationof0hrsor24hrs,serumͲfreemediawasreplaced withfreshserumsupplementedmediaatthebottom(2ml)andtoppart(0.4ml)of the membrane. The MTS/PMS reagent was then added to the top part of the membrane(0.1ml)andthe membraneswereincubatedat37oCfor1hr.Afterthe incubation period, aliquots of 100 μl were transferred to a 96 well plate with 3Ͳ4 97  Chapter4:MaterialsandMethods replicates and the absorbance levels were recorded at 492 nm using a multiplate reader(MultiskanAscent,ThermoLaboratories,Finland)againstcontrols.

 Figure4Ͳ9:Layoutof96wellmicrotitreplateforinͲvitroassays

IC100control:100%cytotoxicity;IC0control:0%cytotoxicity

4.8.2 NRU–neutralreduptakeassay

TheNRUassaymeasuredtheabilityofviablecellstoincorporateandbindneutralred (a supravital and weakly cationic dye) which penetrates all membranes and accumulatesintracellularlyinlysosomes(BorenfreundandPuerner1985).

ToperformtheNRUassayoncellsgrownonporousmembrane,NRUmediumsolution (80μg/mlmedia)waspreparedthepreviousdayandkeptat37oC.Priortouse,NRU mediumsolutionwascentrifugedat1500xganditssupernatantfiltersterilised(0.22 μm).AfterthepostͲexposureincubationperiodofeither0or24hrs,mediafromthe toppartofthemembranewasreplacedbyNRUmediasolution(0.5ml)andtheplate was incubatedfor3hrsat37oC.Afterwards,mediawasremovedfromthebottomand top parts of the membrane and fixative solution was added to the top part of the membrane(0.5ml)fornomorethan30secs.Topandbottompartsofthemembrane werethenimmediatelywashedwithHBSS(0.5mltop;2).mlbottom HBSSfromthe toppartofmembranewasreplacedwiththesolubilisationsolution(0.5ml)andplate shakenfor10minsusinganorbitalmixer(RatekInstruments,Australia).Aliquotsof 100μlweretransferredintothe96Ͳwellplatein3Ͳ4replicatesandabsorbancewas

98  Chapter4:MaterialsandMethods recorded at 540 nm with a microtitre plate reader (Multiskan Ascent, Thermo Laboratories,Finland)againstcontrols.

4.8.3 ATP–adenosinetriphosphateassay

ThecellularATPcontentwasmeasuredbytheCellTiterͲGlo©LuminescentCellViability Assay.CellTiterͲGlo©Reagentinducedcelllysisandgenerationofluminescencewas proportional to the amount of cellular ATP the cells contain, the luminescence was thenrecordedusingaluminometer(BertholdDetectionSystems,Germany).

To perform the ATP assay on cells grown on porous membranes, after the postͲ exposure incubation period of either 0 or 24 hrs, media from the top part of the membranewasreplacedwithfreshmedia(0.25ml)andCellTiterͲGlo©(0.25ml).The platewasthenleftatroomtemperaturefor10minsandshakenusinganorbitalmixer (Ratek Instruments, Australia).Aliquots of 100 μl were transferred into a 96 well opaquewalledmicrotitreplatewith3Ͳ4replicatesandluminescencewasrecordeda luminometer(BertholdDetectionSystems,Germany)

4.8.4 Controls

4.8.4.1 Controlsfor96wellplateprotocol

TwocontrolsweresetuptodetermineIC100andIC0inthefirstcolumnof96wellplate.

The IC100 (100% inhibitory concentration; no viable cells sample) were measured in wells with media only and IC0 (0% inhibitory concentration; completely viable cells sample)weremeasuredinwellscontainingcellsolutionsonly.Backgroundreadingsof mediawithvariouschemicalconcentrationswerealsomeasuredinfourreplicatesto detectanypossiblereadinginterferencecausedbyreactionbetweenmediaandtest chemicals.

4.8.4.2 Controlsforporousmembraneprotocol

Inthestaticexposuremethods,twomembraneswerededicatedforIC100andIC0.The

IC100(100%inhibitoryconcentration;noviablecellssample)wasmeasuredfrommedia inmembranewithmediaonlyandIC0(0%inhibitoryconcentration;completelyviable cells sample) was measured in wells with seeded cells only as a reference for

99  Chapter4:MaterialsandMethods percentage cell viability calculations of exposed cells (Bakand, Winder et al. 2006). Similar to exposed cells, control cells were grown on membranes and exposed to sterilecontrolairduringtheexposuretime.

Inthedynamicexposuremethod,inadditiontothetwocontrolsasmentioned above, an additional control to consider the effects of air flow rate on cells grown on membranes.Thiscontrolairsamplewasexposedtocontrolairatthesameairflow rateassamplesexposedtodieselexhaust.

Incalculatingcellviability,theIC0wasusedasareferenceforabsorbance/luminosity of 100% viable cells whereas IC100 was a control for background (0% viability) absorbance/luminosity.In the dynamic exposure method, the control air sample absorbance/luminosity was also recorded and compared with IC0 to determine any effectofairflowoncells.

  Figure4Ͳ10:Platereaders (a) Multiskan Ascent, Thermo Labsystems (Finland); (b) Orion II Microplate luminometer(BertholdDetectionSystems,Germany)

4.9 Dataanalysis

4.9.1 Doseresponserelation

AbsorbanceandluminescencewererecordedusingtheAscent(MultiskanAscent)and Simplicity(BertholdDetectionSystems)software.Doseresponsecurveswereplotted andIC50calculatedusingGraphPadprism.

4.9.2 Percentagecellviability

GraphPad Prism software was used to calculate the cell viability values from three repetitions of experiments, in which the values were the average and standard

100 Chapter4:MaterialsandMethods deviationofeachcellviabilityvalue.Thepercentageofcellviabilitywascalculatedby assumingtheabsorbanceofIC0as100%cellviability(Bakand,Winderetal.2006)and the background absorbance at IC100 (media only), results of experiments were expressedasmean±standardͲdeviation(M±S.D).

Equation9 ݏ݈݈݁ܿ ݀݁ݏ݋݂ ݁ݔ݌݋ ܾ݁ܿ݊ܽݎ݋ݏܾܣ Ψ݈݈ܿ݁ݒܾ݈݅ܽ݅݅ݐݕ ൌ  ൬ ൰ൈͳͲͲ ݏ݈݈݁ܿ ݋݈ݎ݋݊ݐܿ ݀݁ݏ݋݂ ݑ݊݁ݔ݌݋ ܾ݁ܿ݊ܽݎ݋ݏܾܣ

4.10Statisticalmethodsintoxicology Statistical analyses and graphs were generated using GraphPad prism.Several statisticaltestswereusedanddifferenceswereconsideredsignificantwhenp<0.05: x Linearregressionanalysis:todeterminecorrelationbycalculatingcoefficient ofdetermination(R2) x Confidence intervals: 95% confidence intervals were calculated in each cell lines’doseresponsecurve. x Analysis of variance (ANOVA) and Post Hoc Test: TwoͲway and oneͲway ANOVA with Bonferroni multiple comparisons were used to compare and determinesignificanceofdifference.

4.11ScanningElectronMicroscopy(SEM) CellstructuresandinteractionwithdieselparticulateswereobservedusingaHitachi S3400scanningelectronmicroscope(SEM).Thevacuummoderequiredallsamplesto becriticalpointdriedandgoldsputtercoatedpriortoobservation.SamplesofHepG2 cells were treated with 25% and 100% torque of diesel engine as they  represented approximatelyIC50andIC100values.

Specimenpreparation

After 24 hrs postͲexposure incubation, membranes were washed with HBSS (2 ml bottom; 500 μl top) and a fixative solution (2% glutaraldehyde in 0.1 M Sodium Phosphatebuffer,pH7.2)wereadded(2mlbottom;500μltop)andstoredovernight at4oC.Fixativeswerethenremovedandmembraneswerewashedthreetimeswith buffer (0.1 M Sodium Phosphate buffer, pH 7.2).Each washing was performed by addingfreshbuffer(2mlbottom;500μltop)for10minseach.

101  Chapter4:MaterialsandMethods

The membranes were then dehydrated by immersing them in ethanol solution in increasinggradeof30%(10mins),50%(10mins),70%(3hrs),80%(10mins),90%(10 mins),95%(10mins),100%(10mins),100%(10mins)and100%(10mins).Ethanol wasusedforitsmiscibilitywithliquidCO2duringspecimendrying.

Specimendryingandmounting

Thecriticalpointdryingprocessisamoregentleapproachtodryingspecimenshence avoidingstructuralchangesinducedbyconventionalairdryingasthesurfacetension ofwaterduringthephasechangemayaffectstructureofbiologicaltissues.Thecritical pointdryingprocessavoids thisphasechangehoweveraswater’scriticalpointisat 228.5barand374oC,thetemperaturerequiredwoulddestroybiologicalsamplesby cookinganddenaturingthem.Therefore,waterwasreplacedbyliquidCO2whichhad a significantly lower critical point at 73.8 bars and temperature at 31oC to avoid damagetothebiologicalsamples.

Afterthedehydrationprocess,themembranesweremountedonaholderandheld togetherwithameshfabricandinsertedintothecriticalpointdryer(BALͲTECBALͲ030 CriticalPointDryer;Figure4Ͳ11(a)).Thewholeprocesswasperformedat8Ͳ9oCby addition and flushing of ethanol using liquid CO2 in several runs until ethanol was completelyremovedfromthesamples.ToevaporateliquidCO2,thetemperaturewas o slowly increased to 40 C to evaporate the liquid CO2, resulting in a dried yet structurally intact samples.Samples were then stored in a desiccator to maintain dehydrationuntilmounting.

Specimens were mounted on 25 mm pin stubs and gold sputter coated to enable conductionofelectronsinimagingamicrographinthescanningelectronmicroscope. Afterremovingsamplesfromthedesiccator,themembranesweremountedonastub measuring12.7mmindiameterwithadoublesidedadhesiveandgoldsputtercoated using a sputter coater (Emitech K550X, UK; Figure 4Ͳ11 (b)) at 25 mA, giving 150 Angstrom thick coating on the membrane.Coated samples were then stored in a desiccatortomaintaindehydration.



102  Chapter4:MaterialsandMethods

Specimenimaging

MicrographsofcellsweretakenusingaHitachiS3400SEM(Figure4Ͳ12).TheSEMhas a magnification ability of 20Ͳ20,000X under a vacuum environment.The working distancewassetat15mm,acceleratingvoltage(electronbeam)at15kiloVoltsand probing current of 30.Micrographs of different magnifications were recorded and storedforeachsampleprepared.

(a) (b) Figure4Ͳ11:Criticalpointdryerandsputtercoater (a)BalͲTecBALͲ030CriticalPointDryer;(b)EmitechK550Xsputtercoater. 

 Figure4Ͳ12:HitachiS3400ScanningElectronMicroscope 

103 Chapter5:InͲvitrotoxicityassessmentofselectednanoparticles

5 InǦvitrotoxicityassessmentofselectednanoparticles

5.1 Introduction Nanoparticles are very small objects that are <100 nm is size. The source of these nanometre scale objects may be either intentionally or unintentionally produced.

Examplesofintentionallymanufacturednanoparticlesinclude:TitaniumDioxide(TiO2) and Zinc Oxide (ZnO) whereas unintentionally produced nanoparticles can include Combustion Derived Nanoparticles (CDNP).Due to nanoparticles’ smaller size and largersurfaceareatovolumeratio,comparedtotheirnormalsizedcounterpart,there is an exponential increase of available surface area with identical total mass of particulates(Oberdorster,Oberdorsteretal.2005).

Thesmallsizeofnanoparticles(i.e.<100nm)posesagreaterrisktohumanhealthas they may penetrate deeper into the human body with the potential to cause substantial damage.For example, nanoͲsized pigments are now commonly used in sunscreens.UnlikeconventionalsizedpigmentsofZnOandTiO2whichresultinawhite appearanceontheskin,thesenanoͲsizedpigmentsaretransparentanddonotreflect thewhitecolour.Therewereconcernswhethernanoparticlescanpenetratedeeper intotheskinwhenappliedandthepossiblehealthrisksitposes.Therapeuticgoods administration(TGA)oftheAustralianGovernment’sDepartmentofhealthandageing conducted a literature search and determined there were no scientific evidence to datethatsuggestsnanoparticlespenetrateintotheskin,buthasacknowledgedthat nanoparticlescaninducefreeradicalformationinthepresenceoflightanddamage cells(TGA2006).

InͲvitrotoxicologyisgainingpopularity asareplacementtoanimaltoxicitytestingfor itscomparativeeaseofuse,avoidanceofanimalethicalissuesandeliminatingcrossͲ speciescorrelationerrorsbyusinghumanderivedcelllines(Ekwall,Bondessonetal. 1989).96 well plates are commonly used in laboratory based studies of toxins especiallysolublesolutions.Numerousstudiesonvariouschemicalshaveusedthe96 wellmethodstodeterminetheLC50levelofvariouschemicalsusinghumancelllines

104  Chapter5:InͲvitrotoxicityassessmentofselectednanoparticles

Thisstudyaimstodeterminethesuitabilityofthe96Ͳwellmethodtotestthetoxicity ofnanoͲsizedparticles.Nanosizedparticlesaregainingpopularityforuseinvarious fields such as in sunscreens for their transparent colour benefit over conventional sunscreens’ white colour (Newman, Stotland et al. 2009) and carbon nanotubes for theirstrengthanddurability.

5.2 Experimentaldesign

5.2.1 Testchemicals

Thechemicalsusedinthisstudywerechosentocomparetoxicitybetweennanosized

ZnOnanoͲsizedpowder,<100nmparticlesize(Aldrich)(CAS:1314-13-2)andTitanium dioxide nanoͲsized powder, <100 nm particle size (BET), 99.5% trace metals basis (Aldrich) (CAS: 13463Ͳ67Ͳ7).All test chemicals were at the highest grade available. Chemicalswerepreparedbeforeeachexperimentandserialdilutionswereperformed toproviderangeofchemicalconcentrationstoexposuretohumanrcellsaspe Section 4.1.

Tocompareeffectofnanoparticles’solubilityoncellcytotoxicity,testchemicalswith addedethanol(100μl)werealsoprepared.Conventionallyinsolubletestchemicals aresolubilisedinculturemediausingalcoholororganicsolvents(Barile,Arjunetal. 1993;AlͲGhamdi,Rafteryetal.2003)aswatlo testconcentrations,itdoesnothave effectoncytotoxicity(Clothier,Robinsonetal.1985).

5.2.2 Celltypesandcultureconditions

Human skin fibroblast cells (Section 4.6.8.1) representing human skin was selected. Thecellswereculturedinsterile,vented75cm2cellcultureflasksandkeptat37oCina humidified5%CO2incubator(asperSection4.6.4).Beforeharvesting,cellattachment was checked using a light microscope by observing confluence (75Ͳ80%). Once confluencewasachievedthecellswerethenharvestedusingatrypsinmethodandreͲ suspended in culture medium (as per Section 4.6.5).Cell counting was performed usingthetrypanblue methodtodeterminecellnumberconcentrationasperSection 4.6.3.Theoptimalcellnumberrangeforlinearresponseofcellnumberconcentration againstMTSassayabsorbanceweredetermined(asperSection4.6.6)

105  Chapter5:InͲvitrotoxicityassessmentofselectednanoparticles

5.2.3 InǦvitrocytotoxicityassays

MTSassay

® TheMTSPromegaCellTiter 96AQueousNonͲRadioactiveCellProliferationassay(MTS assay)wasusedtodeterminetheamountofviablecellsremainingafterexposureto toxins(asperSection4.8.1.1)at0hrsand24hrsincubationexposureperiods.Two internalcontrolsweresetup(IC0andIC100)asperSection4.8.4.1.Absorbanceswere recorded using a microtitre plate reader (Multiskan MS Labsystems, Finland) as per Section4.8.1.1.

5.2.4 Dataanalysis

ThecellviabilityfortheMTSassayateachexposureconcentrationwascalculatedasa percentage of Section 4.8.1.1. A dose response curve was plotted using GraphPad Prism software by non linearregression (nonͲnormalised with three variables) using absorbance data, IC50 value and its 95% confidence intervalwere also determined. Statistical analyses was performed using the twoͲway and oneͲway ANOVA with Bonferroni’s multiple comparisons tests as per Section 4.10 with differences considered statistically significant at p < 0.05.SumͲofͲsquares F test of Log (IC50) valueswereperformedtocompareIC50valuesofeachdoseresponsecurves.

5.3 Results

5.3.1 OptimalcelldensityforinǦvitroassays

Todeterminelinearcorrelationresponsebetweencelldensity(innumberofcells/ml) andabsorbances,arangeofcelldensitywereintroducedtowellsofamicrotitreplate andabsorbancesforeachoftheinͲvitroassayswererecorded.Theregioninwhichthe linearincreaseincelldensitygavealinear increaseinabsorbancewasnotedforideal numberofcellstoprepareforsubsequentexperiments.Theoptimalcelldensityof skinfibroblastcellsfortheMTSassayweredeterminedandpresentedinFigure5Ͳ1.

106  Chapter5:InͲvitrotoxicityassessmentofselectednanoparticles

 Figure5Ͳ1:Celldensityoptimisationforskinfibroblastcells Eachpointisexpressedasmean±S.D.

5.3.2 CytotoxicitydatausingMTSassay

TheconcentrationeffectcurveofZnOandTiO2nanoparticlesonhumanskinfibroblast cellsusingtheMTSassayarepresentedinFigure5Ͳ2andFigure5Ͳ3respectively.The calculable dose response curve was overlayed on each graph. Those graphs without overlayeddoseͲresponsecurves(Figure5Ͳ2(a)andFigure5Ͳ3(a))werenonͲcalculable dose Ͳresponsecurves.Nosignificantreductionsincellviabilityweredetected.

5.3.2.1 ZincOxide

SumͲofͲsquaresFtestofLog(IC50)valueswereperformedtocomparedoseͲresponse curvesbetween:

x ExposuretoZnOwith24hrspostͲexposureincubation,and x ExposuretoZnOmixedwithethanoland24hrspostͲexposureincubation

No significant difference was found.No comparisons with 4 hrs exposure were performed.



107  Chapter5:InͲvitrotoxicityassessmentofselectednanoparticles

5.3.2.2 TitaniumDioxide

SumͲofͲsquaresFtestofLog(IC50)valueswereperformedtocomparedoseͲresponse curvesbetween:

x ExposuretoTiO2with24hrspostͲexposureincubationand

x ExposuretoTiO2mixedwithethanoland24hrspostͲexposureincubation

No significant difference was found.No comparisons with 4 hrs exposure were performed.

Figure5Ͳ2:CellviabilityoffibroblastcellsafterexposuretoZnO (a)ExposuretoZnOwith4hrspostͲexposureincubation;(b)ExposuretoZnOwith24 hrs  postͲexposure incubation; (c) Exposure to ZnO mixed with ethanol with 24 hrs postͲexposureincubationperiod. Eachpointisexpressedasmean±S.D. 

108  Chapter5:InͲvitrotoxicityassessmentofselectednanoparticles

Figure5Ͳ3:CellviabilityoffibroblastcellsafterexposuretoTiO2 (a)ExposuretoTiO2with4hrspostͲexposureincubation;(b)ExposuretoTiO2with24 hrs postͲexposure incubation; (c) Exposure to TiO2 mixed with ethanol with 24 hrs postͲexposureincubation. Eachpointisexpressedasmean±S.D.

5.3.3 Cytotoxicityresults

TheIC50valuesandthe95%confidenceintervalforcytotoxicityofZnOandTiO2with MTSassaywerecalculatedandsummarisedinTable5Ͳ1. 

Table5Ͳ1:CytotoxicityofZnOandTiO2

Testchemical Incubation IC50(ppm) IC50 95% confidence time(hrs) interval

ZnO 4 N/A(nonconvergent) N/A(nonconvergent)

ZnO 24 61.47 28.82–131.1

ZnO+etOH 24 54.32 26.54–111.2

TiO2 4 9.12 0.8979Ͳ92.64

TiO2 24 3632 100.7–130939

TiO2+etOH 24 786.1 40.93Ͳ15097

109  Chapter5:InͲvitrotoxicityassessmentofselectednanoparticles

ThetwoͲwayANOVAtestwithBonferroni’smultiplecomparisontestswereperformed todetermineanydifferencesusingtheadditionofethanol.TheANOVAtestresults detectednosignificantdifferencecausedbytheadditionofethanoltothechemical solution.

5.4 Discussion TheaimofthestudywastodeterminethemeritofinͲvitroexperimentsusing96well platesfordeterminingthetoxicityofnanoparticles.Thecytotoxicityoftwoselected nanoparticles was studiedusing the MTS (tetrazolium salt, Promega) inͲvitro assays.

ThenanoparticlesincludedZnOandTiO2.

AreproducibleandconsistentprocedureisessentialforoptimisinginͲvitrotoxicology assays.SincemanyinͲvitroassayreadingsdependonthenumberofviablecells,itis essentialtodeterminetheoptimalcelldensityrangeforeachassay.FortheMTSinͲ vitroassay,themaximumcelldensitythat representedalinearabsorbanceresponse withskinfibroblastwasdeterminedas3.55x105cells/ml,forA549(lungderived)cells as2.93x105cells/ml,andforHepG2(Liverderived)cellsat6.2x105cells/ml.Forthe NRUinͲvitroassaythemaximumcelldensityforlinearabsorbanceresponsewithA549 cellswasdeterminedas7.13x105cells/ml,forHepG2cellsas6.5x105cells/mlas showninAppendixB.HenceforinͲvitroassaysexperiments,theoptimalcellnumber grownineachwellwerewithintherange0Ͳ3x105cells/mlasthisprovidedalinear responsethroughoutvariouscelltypesandinͲvitroassays.

InͲvitrocytotoxicitydatawasobtainedanddoseͲresponsecurvewereplottedincluding

IC50valuesandthe95%confidenceintervalrange.Cytotoxicitydataforskinfibroblast cellsusingtheMTSassayagainstZnOandTiO2aresummarisedinTable5Ͳ1.At4hrs exposure,forbothZnOandTiO2nanoparticles,doseresponsecouldnotbeachieved andhencenoIC50valueswerecalculated.However,24hrsexposureperiodscaused significantcytotoxicity(Figure5Ͳ2andFigure5Ͳ3).

The twoͲway ANOVA test showed cytotoxicity of both test nanoparticles were concentration and time dependent.However, the addition of ethanol in the stock chemicalsolutiondidnotcauseasignificantdifferenceincytotoxicity.Otherstudies utilising nanoparticles include Park et al (2011) who exposed HaCat cells to TiO2

110  Chapter5:InͲvitrotoxicityassessmentofselectednanoparticles concentrations of 0Ͳ1000 ppm for 24 and 48 hrs period and observed no significant decrease in cell viability using the MTT assay (Park, Jeong et al.).By comparison, resultsofthiscurrentstudyshowedasignificantdecreaseincellviability>1000ppm after24hrsexposure.Onepossibilityforthedifferencewasduetothecelltypeused as HaCat cells may be more sensitive to TiO2 compared to skin fibroblast.This emphasisestheneedtousevariouscelltypetorepresentsaspecificorganortissueof interest.

ThedoseresponsecurvecalculationinthisexperimentwasperformedbyGraphPad software using a three variable sigmoidal mathematical modelling with slope factor assumedatͲ1.IC50valuesoftestnanoparticlesafter24hrsexposurerevealedthat

ZnO was more toxic than TiO2.The results from the 4 hrs exposure were not consideredastheydidnotrepresentasigmoidalcurvemodel.The24hrsexposure periodwiththeadditionofethanolwerenotconsideredsignificantlydifferent.

On comparing the cytotoxicity between nanoparticles, TiO2 exerted lower toxicity when compared to ZnO at 24 hrs exposure.Since the particle size between both chemicals was <100 nm the difference in the calculated IC50 indicated that the comparisonofcytotoxicitybasedonchemicalcompositionremainscriticalevenatthe nanometresizescale.ThehighercytotoxicityofZnOcomparedtoTiO2supportedthe availabletoxicologicalinformationinwhichTiO2wasconsiderednonͲhazardouswhile ZnOwasconsideredhazardous.InadditionstudiessuchasthosebyXiongetal(2011) showed higher toxicity for ZnO compared to TiO2 for both nanoparticles and conventionalsizeparticlesonzebrafishafter96hrsexposure(Xiong,Fangetal.2011).

AstudybyAruojaetal(2009)similarlyshowedhighertoxicityofZnOcomparedtoTiO2 usingmicroalgaePseudokirchneriellasubcapitata(Aruoja,Dubourguieretal.2009).

The low cytotoxicity at <4 hrs incubation may be caused by the insolubility of nanoparticles.From visual observations, there was a tendency for precipitation of nanoparticles at the bottom of wells hence causing interference to absorbance readings for the MTS assay as nanoparticles may absorb light (Kroll, Pillukat et al. 2009).InsolublecompoundsareoftenconsideredinertandnonͲtoxicalthoughafew exceptionsexistsuchasasbestosandcoaldustsincetheyresidewithinreceptivelung

111  Chapter5:InͲvitrotoxicityassessmentofselectednanoparticles tissues (Wilson 1990).Thus cytotoxicity testing of nanoparticles is still possible althoughprecipitationmayinterferewithabsorbancereadings.

In comparison to available inͲvivo toxicology information, TiO2 nanoparticles are considered to be a nonͲskin irritant based on results from Draize testing on white rabbits and local lymph node assay in mice (Warheit et al 2007).The rabbits were testedusingUSEPAandOECD404guidelinesinwhichultrafineTiO2wereappliedon rabbit skin for 4 hrs and Draize scores were determined at 24, 48 and 72 hrs after removalofthechemicals,similarlymicewereusedtodetermineirritancybyapplying dilutedTiO2usingN,NͲdimethylformamidetotheirears.Thisstudydidnotshowany signs of sensitisation by the rabbit nor the mice at lower levels although at higher concentration,therewasanincreaseincellproliferationinmice(Warheit,Hokeetal. 2007).At<4hrsexposure,therabbitdidnot exhibitanyskinirritancy/sensitisationas wasthecasewiththeskinfibroblastcellsinthisstudywheretherewasnosignificant decreaseincellviability.

A variety of inͲvitro cytotoxicity assays were used to assess different biological endpoints.AlthoughtheMTSassayprovidesmorerapidresults,thereisnooneassay able to provide the full spectrum of cellular responses due to chemical exposure (Andreoli, Gigante et al. 2003).Hence by applying various inͲvitro assays a more complete spectrum of cellular responses can be obtained.Future experiments will considerusingavarietyofinͲvitroassayswhichassessdifferentbiologicalendpoints (Borenfreund,Babichetal.1988;Hayes,Bakandetal.2007).

Although often used to determine cytotoxicity of soluble toxins, the 96 well plate methodby resuspendingparticulatesinculture media does not allow direct contact with contaminants.To allow uniform suspension, methods used have included sonicationand/ordiluting nanoparticleswithothersolvents,whichdoesnotallowfull directexposureofparticlestothecells.Themetabolismandeliminationfactorssuch as that from reservoirs like furrows and hair follicles may be evident in inͲvivo experiments (Prow, Grice et al. 2011) and other structures of skin such as porcine stratumcorneumanonͲlivingouterlayerofskinasthemainmodelofnanoparticles barrierfunctionofskinneedtobeconsidered(Gamer,Leiboldetal.2006).

112  Chapter5:InͲvitrotoxicityassessmentofselectednanoparticles

Resultsofthisstudysuggestedthatincubationtime<24hrsdonotproduceadoseͲ response relation for both ZnO and TiO2 nanoparticles.By comparison, TiO2 is less toxicthanZnOonSkinfibroblastshoweverprecipitationofnanoparticlesmayinterfere with inͲvitro assays results.As diesel engine exhaust contains both gaseous and particulatecomponent,themaintargetorganforgaseouspollutantsisthepulmonary system(Ahamed,AlSalhietal.2010).Futurestudieswithinthisthesiswill concentrate ondevelopmentofaninͲvitroexposuresystemwhichallowsdirectexposureofhuman lungcellstobothgaseousandparticulatecomponents.



113  Chapter6:InͲvitrotoxicityassessmentofagaseousmodelcompound

6 InǦvitro toxicity assessment of a gaseous model compound

6.1 Introduction Methodsutilising96wellplatesareconventionallyusedtodeterminethetoxicityof chemicals and particulates.However, testing gaseous contaminants requires direct exposureofcellstothecontaminantbyremovingtheculturemedialayerfromcells, yet cells require culture media to maintain viability.Growing cells on an air liquid interface  (such as a porous membranes) allows simultaneous exposure to gaseous contaminants on the apex side and culture media nourishment to cells on the basolateralside(Aufderheide, Knebel et al. 2003).The direct exposuremethodhas been utilised to analyse toxicity of various gaseous contaminants such as Volatile organiccompounds(VOCs)and firecombustionproducts(Lestari,Hayesetal.2005; Bakand,Winderetal.2006).

Diesel exhaust contains numerous components including toluene, which forms a significantproportionofthelightmonoaromaticcomponentofdieselexhaust(TurrioͲ Baldassarri,Battistellietal.2004;CorrêaandArbilla2006).Inaddition,Tolueneisless water solublehenceabletopenetratedeeperintothepulmonarysystemthusmore suitablegastomodelresponseofinnerpulmonarysystemregionuponexposureto gases(BakandandHayes2010).Inaddition,Tolueneisamajoraromatichydrocarbon andcouldformduringcombustionevenwhenthereisnotoluenecontentwithinthe dieselfuel(PeterF1989;Rudell,Wassetal.1999;Nelson,Tibbettetal.2008).The aimofthisstudywastodeterminetoxicityoftolueneinitsgaseousphaseatvarious concentrationstohumanlungcells(A549)grownonporousmembranes.A549cells werechosenasrepresentativelungscell sinceTolueneiseasilyinhalablewhileinthe gaseous form.To quantify the cell viability, the colorimetric MTS and NRU and luminescentATPassayswereused.

114  Chapter6:InͲvitrotoxicityassessmentofagaseousmodelcompound

6.2 Experimentaldesign

6.2.1 Testchemicals

Toluene(C6H5CH3;CAS#108Ͳ88Ͳ3;APSFinechem,Australia)ofhighestanalyticalgrade wasusedasperSection4.1.InͲvitroreagentswerepurchasedfromPromega(USA)as perSection4.8.

6.2.2 Celltypesandcultureconditions

Humanlungcells(A549,ATCCNo:CCLͲ185)(asperSection4.6.8.2)wereculturedin 2 o sterile, vented 75 cm  cell culture flask and kept at 37 C in a humidified 5% CO2 incubator(asperSection4.6.4).Cellsweregrownonporousmembranes(0.4μm)in Snapwellinserts(asperSection4.6.7),whichallowedcellstobeexposedtoairborne pollutantswhilesimultaneouslyreceivingnutrientsfromthebasolateralside.Before exposure, cell attachment was checked using a light microscope by observing confluence  (75Ͳ80%).The medium was then removed and the membranes washed withHank’sbalancedsaltsolution(HBSS;Gibco,USA).

GlasswellswerefilledwithserumͲfreemediasupplementedwithantibiotics(1%v/v) andtheporousmembranesweretransferredontotheglasswell(asperSection4.7.1). Theglasswellswerethentransferredintothemodifiedglassstaticexposurechambers andmaintainedat37oCinsideapreͲheatedchamber(orbitalmixer;RatekInstruments, Australia).

6.2.3 Directexposureprotocol

Toluenevapourwasgeneratedinsidethesealedglasschamber(322±2mlinvolume) asperSection4.7.1.Glasswaschosenforitslowadsorptionlosesandnodiffusionof materialsacrosstheglasswall(Saltzman1997)aspolystyrenematerialmayreactwith gaseousaromatichydrocarbons(Croute,Poinsotetal..2000) Toluenewasintroduced intoeachchamberinquantitiesof0,10,20,40,60and100μlontofilterpaperswhich induced evaporation and allowed homogenous mixing with air to the desired concentration.

115  Chapter6:InͲvitrotoxicityassessmentofagaseousmodelcompound

Followingintroductionoftolueneintotheglasschambers,thechamberswerequickly sealedusingparafilm,andtransferredintoapreͲheatedincubatorat37oCfor1hrs. Afterwards,themembraneswerereturnedtothesixwellplates,freshculturemedia wasaddedtothelowerpartofthemembraneandMTS,ATPandNRUassayswere performed.The concentration of toluene within the internal atmosphere was calculatedusingEquation5andEquation6(Ryghseter, Jenssenetal.1992)

6.2.4 Cytotoxicityassays

ThePromegaCellTiter96®AQueousNonͲRadioactiveCellProliferationAssay,Neutral RedUptakeandATPassaywereusedinthisstudyasperSection4.8.

6.2.4.1 MTSͲTetrazoliumsalt

After exposure, the MTS assay for the membrane protocol was performed as per Section4.8.1.1.Insummary,freshculturemediawasaddedtothebottom(2ml)and top(400μl)sideofthemembranethentheMTS/PMSmixturewasaddedtotopside ofthemembrane(100μl).Theplatewasthenincubatedat37oCfor1hr,aliquotsof 100 μl were then transferred from each membrane into the 96 well plates and absorbancewasrecordedat492nm.

6.2.4.2 NRUͲNeutralreduptake

After exposure, the NRU assay for the membrane protocol was performed as per Section4.8.2.Insummary,freshculturemediawasaddedtothebottom(2ml),NRU solution,prepared24hrsprior,wasaddedtothetopside(500μl)ofthemembrane andtheplatewasincubatedfor3shr at37oC.Afterwards,mediawasremovedfrom bothbottomandtopsidesofthemembraneandfixativesolutionaddedtothetop side(500μl)ofthemembranefornomorethan30secs.HBSSwasthenusedtowash thebottom(2ml)andtop(500μl)sidesofthe membranes.AfterremovalofHBSS from the top side of the membrane (whilst leaving HBSS at the bottom side), the solubilisationsolutionwasaddedtothetopsideofthemembrane(500μl).Theplate was then shaken for 10 mins, aliquots of 100 μl were then transferred into 96 well platesandabsorbanceswererecordedat540nm.

116  Chapter6:InͲvitrotoxicityassessmentofagaseousmodelcompound

6.2.4.3 ATPͲAdenosinetriphosphate

Afterexposure,theATPassayformembraneprotocolwasperformedasperSection 4.8.3.Insummary,freshculturemediawasaddedtothebottom(2ml)andtop(250 μl)sideofmembranethenATPreagentwasaddedtothetopsideofthemembrane (250μl).Theplatewas thenshakenfor2minsandleftatroomtemperaturefora further8mins.Aliquotsof100μlwerethentransferredinto96wellopaqueͲwalled platesandluminescencewasrecordedusingaluminometer.

6.2.4.4 Controls

Ineachexperiment,twocontrolsweresetupasperSection4.8.4.2

6.2.4.5 Dataanalysis

The cell viabilities for all inͲvitro assays at each exposure concentration were calculatedasapercentage(asperSection4.9.2).Fromthecalculateddoseresponse curves,IC50valuesofchemicalswerecalculated.

6.3 Results

6.3.1 OptimalcelldensityforA549lungcells

Todeterminethemostlinearcorrelationresponsebetweencellnumberconcentration and its corresponding absorbance, various cell densities were introduced to the membranesandtheabsorbancelevelswererecorded.Eachdatapointsrepresented anaverageofthreerecordingsoftheMTSassayabsorbance,ATPassayluminanceand NRU assay absorbance were expressed as mean (m) and standard deviations (S.D). The maximum cell number concentration that gave linear response of absorbances/luminescence was determined and listed in Table 6Ͳ1 (optimisation of A549cellnumberwiththeATPassayispresentedinAppendixC).

Table6Ͳ1:CorrelationbetweenA549cellnumberand assayresults Assaytype Cellnumber/mlrange Rsquarevalue

MTS 0Ͳ11.4x105 0.94 NRU 0Ͳ5.15x105 0.98 ATP 0Ͳ5.68x105 0.99

117  Chapter6:InͲvitrotoxicityassessmentofagaseousmodelcompound

6.3.2 ConcentrationeffectcurveusingMTSassay

TheconcentrationeffectcurvesoftolueneonA549cellsusingtheMTS,NRUandATP assayswerecalculatedwithGraphPadPrismprogramandarepresentedinFigure6Ͳ1. Each point on the curve represents an average of at least three repetitions.The average and S.D were calculated as percentage of control (with subtraction of backgroundcontrol).Inaddition,toenablecomparisonofconcentrationeffectcurves generated by GraphPad Prism and Microsoft Excel, the concentration effect curves generatedbyMicrosoftExcelispresentedinFigure6Ͳ2

6.3.3 IC50ofTolueneonA549cells

2 The calculated IC50, its 95% confidence interval and dose response curve R  values werecalculatedwithGraphPadPrismandarepresentedinTable6Ͳ2.TheIC50values are expressed as the total amount of toluene (in millimoles) introduced into glass chambers(322±2mlvolume)anditscorrespondinggaseousconcentrationinppm.In addition, to enable comparison of IC50 values calculated by GraphPad Prism and 2 Microsoft Excel, the IC50 and R  values calculated by Microsoft Excel is presented in Figure6Ͳ2

118  Chapter6:InͲvitrotoxicityassessmentofagaseousmodelcompound

Figure6Ͳ1:CytotoxicityofToluenecalculatedbyGraphPadPrismsoftware (a)MTS,(b)ATPand(c)NRUassay;mM=millimolesofchemicalintroducedintothe glasschamber;Eachpointisexpressedatmean±S.D 

 Figure6Ͳ2:CytotoxicityofToluenecalculatedbyMicrosoftExcelsoftware (a)MTS,(b)ATPand(c)NRUassay;mM=millimolesofchemicalintroducedintothe glasschamber;Eachpointisexpressedatmean±S.D 

119  Chapter6:InͲvitrotoxicityassessmentofagaseousmodelcompound

Table6Ͳ2:IC50valueofMTS,ATPandNRUassayscalculatedusingGraphPadsoftware

Assa IC50(mM) IC50(ppm) 95%confidence 95%confidence Respons y interval(mM) interval(ppm) ecurve R2

MTS 0.3312 26.2x103 0.2916Ͳ 0.3762 23x103 – 29.7x103 0.98

NRU 0.4889 38.6x103 0.4170Ͳ 0.5731 32.9x103 – 45.3x103 0.94

ATP 0.1534 12.1x103 0.1345Ͳ 0.1750 10.6x103 – 13.8x103 0.98



Table6Ͳ3:IC50valueofMTS,ATPandNRUassayscalculatedusingMicrosoftExcel software

Assa IC50(mM) IC50(ppm) 95%confidence 95%confidence Respons y interval(mM) interval(ppm) ecurve R2

MTS 0.2524 20x103 0.0537 – 0.1327 4.3x103 – 10.5x103 0.9586

NRU 0.0976 7.7x103 0.1476 Ͳ 0.5232 11.7x103 – 41.4x103 0.9003

ATP 0.1373 10.9x103 0.1243Ͳ 0.1479 9.8x103 – 11.7x103 0.9702

MTSassays’resultsshowedtoxiceffectsoftoluenetoA549cellswithATPasthemost sensitiveassayfollowedbyMTSandthenNRU.

6.4 Discussion Themainobjectiveofthisstudywastodevelopamethodtoallowthedirectexposure ofcellstogaseouspollutantssuchasToluene.Tolueneisacomponentoflightmono aromaticcomponentofdieselexhaust(TurrioͲBaldassarri,Battistellietal.2004;Corrêa and Arbilla 2006).Toluene is a VOC and has the ability to evaporate at room temperature.Toluenewaschosenasamodelgaseouscontaminanttodeterminethe applicabilityofthedirectexposuremethod.Theconventionalmethodsuchasthe96Ͳ wellmethodisnotabletoanalysetolueneinitsgaseousformasitrequiresremoving culturemediafor directexposure.ͲAlthoughvariousmethodsareavailabletoexpose gaseouscompoundstocellssuchasbysuspendingcellsinculturemediasolutionand

120  Chapter6:InͲvitrotoxicityassessmentofagaseousmodelcompound introducingthegasintothesolution(Croute,Poinsotetal.2002),fulldirectexposure maynotbepossible.

Inthisstudy,A549Ͳlungderivedcellsweregrownonporousmembranesandexposed todifferentconcentrationsoftoluenefor 1hr.Biologicalendpointswereinvestigated usingtheMTS,NRU,ATPandLDHassays.ThemeanIC50resultswere26200ppmfor MTSassay,38600ppmforNRUassayand12100ppmforATPassay.Theresultsalso showed an increasing sensitivity in IC50 in order of ATP>MTS>NRU while the IC50 confidence intervals showed increasing specificity in order of ATP>MTS>NRU.In comparison to similar work by Bakand et al (2006) obtained A549 IC50 (M ± S.D) of 16600±3423.1ppmforMTSassayand12100±2256.7ppmforNRUassay(Bakand,

Winderetal.2006).Bakandetal’sIC50resultswerelower;thedifferencesmaybedue to IC50 values calculation method by using exponential mathematical model in MicrosoftExcelinsteadofGraphPadsoftware.

GraphPadsoftwarewaschosenforitsabilitytocalculateIC50valuesbyusingsigmoidal mathematical models, which is more appropriate due to assumption of normal distributionofIC50ofcells(ThomasandIsaacs2009)(asoutlinedinChap2.4.4).Thisis illustrated in Figure 6Ͳ1 and Figure 6Ͳ2, comparison between results calculated between Microsoft Excel and GraphPad, it can be seen that the sigmoid curves fit better to the experimental results.In addition, concerns have been raisedh  wit  Microsoft Excel’s accuracy in computation of statistical distributions such as normal andChiͲsquaredistribution(Leo2005).

Growingcellsonaporousmembranesimulatesmorerealisticconditionsandhigher sensitivity to particles (Bitterle, Karg et al. 2006).Similar methods have been developedbyvariousresearchgroupstoanalysegaseouspollutantssuchascigarette smoke(Aufderheide,Knebeletal.2003),Xylene,Toluene(Bakand,Winderetal.2006), combustionproducts(Lestari,Greenetal.2006),Particulatematters(Cheng,Malone etal.2003;Shimizu,Endoetal.2004)andgoldnanoparticles(Brandenberger,RothenͲ Rutishauseretal.2010).

This configuration has been used in diesel exhaust particulates (DEP) cytotoxicity studiessuchas of Ohtoshi etal (1998) which showed cytotoxicity on human airway

121  Chapter6:InͲvitrotoxicityassessmentofagaseousmodelcompound epithelialcellsbyexposingreͲsuspendedDEPinaculturemediumontheapicalside (Ohtoshi,Takizawaetal.1998).Howevermanyofthesestudiesarelimitedshortterm exposurestudiesof<24hrs(Ponsoda,Joveretal. 1995;Zhang,Yangetal.2008)and <72hrs(Yang,Cardonaetal.2002;Calcabrini,Meschinietal.2004)exposureperiods.

Althoughstatisticalresultsshowednosignificantdifferencebetweendifferentassays, performing multiple assays is still recommended.As diesel exhaust contains particulates which size ranges within nano meters scale, using multiples assay  will detectanypotentialinterferencebysmallersizeparticulates.SomeinͲvitroassaysare knowntointerferewithnanosizedparticles(MonteiroͲRiviere,Inmanetal.2009).

The development of this inͲvitro method showed applicability of direct exposure methodstoanalysethetoxicityofthemodelgas.In addition,variousassayssuchas

MTS,NRUandATPcanbeusedtodeterminethecellviabilityandIC50value.Further, other human cell types can be grown and used to assess the cytotoxicity of contaminantsonvarioustargetorganssuchasHepG2cellswhichrepresenttheliver.

Future works will determine the applicability of the direct exposure method in analysing toxicity of diesel exhaust.As diesel exhaust contain both gaseous and particulate component, the direct exposure method may allow toxicity analysis of exhausts.In addition, comparison will be made between various postͲexposure incubationperiodstodetermineanynonͲimmediatetoxiceffectssuchasonthecell divisionmechanisms.AnynonͲimmediateeffectsmaycauserapidͲonsetanddelayed onsettoxicity,thereforecreatingdifferenceincellviabilityafterlongerpostͲexposure incubationperiod(Riddell,Panaceretal.1986).Inaddition,variousinͲvitroassayswill beusedtodetermineanybiologicalendpointsthatismoresuitabletoinvestigatethe cytotoxicitymechanismsoftheexhaust(Garle,Fentemetal.1994).



122  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts

 7 InǦvitrotoxicityassessmentoflaboratoryandfield baseddieselexhausts

7.1 Introduction Toxicologystudieshavetraditionallydependedonanimalbased(inͲvivo)experiments. InrecentyearsinͲvitrotoxicologymethodshavebeenanareaofincreasedresearch andimplementedinregulationsandriskassessments(Herzinger,Kortingetal.1995; Combes 2005).In addition, inͲvitro toxicology allows the usage of human cells for toxicitytestingwhicheliminatestheneedforcrossͲspeciescorrelationandprovidesa more ethical method to understand toxicity mechanisms of different chemicals. Besides the multi cellular nature and complex structure ofthe distal lung, the pulmonary epithelium is composed of two distinct cell types: alveolar type I and alveolar type II which demonstratediverse essential functions in the alveolar region (BakandandHayes2010).Inthisstudy,humanpulmonarytypeIIͲlikeepithelialcells (A549) were used as a model for human respiratory system since the main target organfordieselexhaustespeciallytheparticulatesis thepulmonary epithelium and macrophages(CohenandPopeIII1995).

Dieselemissioniscurrentlyapopularresearch subject asits popularity is increasing drivenbyhigherfuelefficiencyandsupposedlylowergreenhousegasemissionsuchas

CO2(duetoamorecompletecombustionprocess).Recentlymoreinteresthasbeen generated on the transformation of emissions within the atmosphere and facilities such as the European Photo reactor (EUPHORE) simulation chamber was created to studyandelucidatethesetransformations(Zielinska2005)

To date, a number of inͲvitro based studies have investigated the toxicity of diesel exhaust(Ohtoshi,Takizawaetal.1998;Liu,Keaneetal.2005;OhandChung2006). However most of these test methods were based on assessing pure individual components rather thanthe complex mixture of diesel exhaust.These methods do notallowdirectexposureofscell toairbornedieselexhaustpollutants.AnoptimalinͲ vitro exposure system for studying the cellular responses following exposure to airbornepollutantsneedstomeetseveralcriteria.Mostimportantlythereneedstobe

123  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts

 averyclosecontactbetweentargetcellsandthetestatmosphere.Recently,anumber ofpublishedstudieshaveusedbiphasiccellculturetechniqueinwhichhumancellsare directly exposed to airborne pollutants on the apicale sid  while receiving required nutrientstomaintainviabilityatthebasalside(Aufderheide2005;Bakand,Winderet al.2006).

The aim of this study was to investigate the cytotoxicity of diesel exhausts using a dynamic direct exposure method developed by the CSAT research group (Bakand, Winder et al. 2006; Potera 2007).This research exposes cells directly to airborne pollutantsattheairͲliquidinterfaceaswouldoccurinthehumanrespiratorysystem. Studies were performed in a laboratory controlled environment and a field study environmenttodemonstratethepotentialapplicationofthedynamicdirectexposure methodfortoxicitytestingofdieseltexhaus emissionsinͲvitro.Theoverallaimwasto studythetoxicityofcomplexatmospheresgeneratedbythecombustionofdieselfuel.

7.2 Experimentaldesign

7.2.1 Celltypesandcultureconditions

HumanpulmonarytypeIIͲlikeepithelialcells(A549,ATCCNo:CCLͲ185)werecultured 2 o insterile,vented75cm cellcultureflasksandkeptat37 Cinahumidified5%CO2 incubator(asperSection4.6.4).

Cellsweregrownonporousmembranes(0.4μm)inSnapwellinserts(asperSection 4.6.7).Cell attachment was confirmed using a light microscope and observing confluence (75Ͳ80%).The medium was then removed from the top section of the membrane washed with Hank’s balanced salt  solution (HBSS; Gibco, USA), and transferred into the modified dynamic exposure chambers (Harvard Apparatus, Inc, USA)fordirectexposuretodieselexhaust.

7.2.2 Generationofdieselexhaust

Thedieselfuelusedtogeneratedieselexhaustwaspurchasedfromacommercialfuel station (Vortex premium diesel; CALTEX) as per section 4.3.1 which complied with AustralianStandard3570Ͳ1998(StandardsAustralia1998).

124  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts

 For the laboratory based study, a Volvo 230 hp truck engine, running on idle mode withnoload,wasemployedasperSection4.3.2.Theenginewasswitchedonandset on idle settingfor 15 mins for warming up before exposure of target cells to diesel exhaust.Followingthestabilisationperiodtheexhaustsweredeliveredtothedynamic directexposurechambersusinganegativepressurepumps(SKCInc,USA)calibratedat averylowflowrates(ч37.5ml/min).

Forthefieldbasedstudy,adieselenginepoweredpassengercar(VolkswagenPassat; model2006)wasused.Similarly,beforeexposureoftargetcellstodieselexhaust,the enginewasswitchedonandsetonidlesettingfor15minsforwarmup.

7.2.3 Monitoringandanalysisofexhaustemission

Monitoringofexhaustemissionwasperformedusingrealtimegasmonitors(iBrid™ MX6 multiͲgas monitors) per Section 4.5.1In addition, diesel exhaust temperature were checked to ensure it is at room temperature prior to entering the exposure chambers (maintained at 37oC) to eliminate any potential physical stress such as temperatureshock.

7.2.4 Elementalandorganiccarbonmeasurement

NIOSH5040methodwasappliedtodetermineelementalandorganiccarboncontent ofdieselexhaustasoutlinedinsection4.5.2.

7.2.5 Cytotoxicityendpoints

To assess the cytotoxicity of diesel exhaust, a range of inͲvitro bioassays was used. ThisincludedMTS,NRUandATPassayswereutilisedtomeasureavarietyofbiological endpointsasperSection4.8.

7.2.6 Controls

AppropriatecontrolswerepreparedasperSection4.8.4.2.Inbrief,anIC100control (0%cellviability;mediaonly)wasusedtocompensateforbackgroundinterference.

AnIC0control(100%cellviability;cellsonly),wasusedasreferenceandincubatedat 37oCduringtheexposuretime.Inaddition,anaircontrolwasalsousedtoconsider anyreductionofcellviabilityinducedbythedynamicairflow.

125  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts

 7.2.7 Statisticalanalysis

Statistical analyses were performed as per Section 4.10, oneͲway ANOVA and Bonferroni’s multiple comparisons test.Differences were considered statistically significant at p<0.05.Dose response curves were calculated using GraphPad Prism softwareatvariableslopewiththreevariables.Its95%confidenceintervalwasalso determined.

7.3 Results

7.3.1 Optimalairflow

Variousairflowsweretestedtodetermineoptimalairflowtodeliverthemaximum contaminantsyetnotaffectingcellviabilities.CellviabilityweredeterminedusingMTS assay with results and comparison between control (0 ml/min) and other air flows wereperformedusingoneͲwayANOVAwithBonferroni’sposthoctestare shownin Figure 7Ͳ1 with p<0.05 considered as statistically significant (* indicates p<0.05), althoughthecomparisontestsshowednostatisticaldifferencebetweenallairflows. From graphical representation in Figure 7Ͳ1 shows significant visible cell viability decreasefrom67.5ml/minonwards.

126  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts



 Figure7Ͳ1:Effectofairflowoncellviability CellviabilitywasdeterminedusingtheMTSassayat0hrspostͲexposureincubation period;StatisticalcomparisonsweremadewithoneͲwayANOVAandBonferroni’spost hoctestwithcontrolat0ml/minagainstallotherairflows. Eachpointisexpressedasmean±S.D. *indicatesstatisticalsignificanceatp<0.05. 

7.3.2 CytotoxiceffectsofdieselexhaustonA549Ǧhumanlungcells

7.3.2.1 Laboratorybasedengine

Each data point represents the average of three separate samples (n = 3) at each exposure period.Cell viabilitieswere expressed aspercentageof control asM±S.D. Thecellviabilitywasreducedinadosedependentmannerastheexposureperiodof cellstodieselexhaustincreasedfrom0Ͳ120mins.

Time course studies of laboratory based diesel exhaust on A549 cells with the ATP assaysarepresentedinFigure7Ͳ2.Comparisonsweremadeagainst15minscontrol air exposure with oneͲway ANOVA with Bonferroni post hoc test with p<0.05 consideredasstatisticallysignificant(*indicatesp<0.05;**indicatesp<0.01).

127  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts



 Figure7Ͳ2:Timecoursecytotoxicityofdieselexhaustfromlaboratorybasedstudy

A549 cell viabilities were determined using the ATP assay after 1 hr exposure to diesel exhaust in a laboratory based study with 0 hr postͲexposure incubation period. Eachpointisexpressedasmean±S.D.  Comparisonofcellviabilityafter0hrsand24hrspostͲexposureincubationperiodwas made.For this analysis, 3 different inͲvitro assays (ATP, MTS, NRU) were used (as showninFigure7Ͳ3)andthefollowingcomparisonsweremadeusingtwoͲwayANOVA withBonferroni’sposthoctest(withp<0.05consideredassignificantdifference) x Comparisonbetweencellviabilityofcellsexposedtocontrolairafter0hrsand 24 hrs postͲexposure incubation periods (0 hrs postͲexposure incubation as control). x Comparisonofcellviabilityofcellsexposedtoexhaustafter0hrsand24hrs postͲexposureincubationperiods(0hrspostͲexposureincubationascontrol).

No significant differences were found in cell viability between the 0 hrs and 24 hrs postͲexposureincubationperiods.

128  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts



Figure 7Ͳ3: Comparison between 0 and 24 hrs postͲexposure incubation in the laboratorybasedstudy (a)MTSassay;(b)NRUassay;(c)ATPassay. Eachpointisexpressedatmean±S.D.

7.3.2.2 Fieldbasedengine

Each data point represented the average of three separate samples (n = 3) at each exposureperiod.CellviabilitieswereexpressedasapercentageofcontrolasM±S.D. Thecellviabilitywasreducedinadosedependentmannerastheexposureperiodof cellstodieselexhaustincreasedfrom0Ͳ120mins.

TimecoursestudyoffieldbaseddieselexhaustonA549cellswiththeMTSandATP assaysarepresentedinFigure7Ͳ4.Comparisonsweremadeagainst15minscontrol air exposure with oneͲway ANOVA with Bonferroni post hoc test with p<0.05 consideredasstatisticallysignificant(*indicates p<0.05;**indicatesp<0.01)

129  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts



Figure7Ͳ4:Timecoursecytotoxicityofdieselexhaustfromfieldbasedstudy A549cellviabilitiesweredetermined using (a)MTS assay(b) ATP assay after 1 hr exposure to diesel exhaust in a laboratory based study with 0 hrs postͲexposure incubationperiod. Eachpointisexpressedasmean±S.D.

Comparisonofcellviabilityafter0hrsand24hrspostͲexposureincubationperiodwas made.Forthisanalysis,threedifferentinͲvitroassays(ATP,MTS,NRU)wereused(as showninFigure7Ͳ5)andthefollowingcomparisonsweremadeusingtwoͲwayANOVA withBonferroni’sposthoctest(with p<0.05consideredassignificantdifference)

x Comparisonofcellviabilityofcellsexposedtocontrolairafter0hrsand24hrs postͲexposureincubationperiod(0hrspostͲexposureincubationascontrol)

x Comparisonofcellviabilityofcellsexposedtoexhaustafter0hrsand24hrs postͲexposureincubationperiods(0hrspostͲexposureincubationascontrol).

Nosignificantdifferenceswerefoundincellviabilitybetween0hrsand24hrspostͲ exposureincubationperiods.

130  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts



Figure7Ͳ5:Comparisonbetween0and24hrspostͲexposureincubationinthefield basedstudy (a)MTSassay;(b)NRUassay;(c)ATPassay. Eachpointisexpressedasmean±S.D.

7.3.3 Dieselexhaustgasmonitoring

7.3.3.1 Laboratorybaseddieselexhaustgasmonitoring

Thelevelofgaseousemissionsfromlaboratorybaseddieselengineswasmonitored andrecordedusingiBrid™MX6multiͲgasmonitors(asperSection4.5.1)todetermine theconcentrationofgasesduringtheexposureperiod.GasesincludeCO2,CO,NO2,

NOandO2,thegasreadingsweremeasuredtwiceandtheaverageresultsplottedin Figure7Ͳ6.

131  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts



Figure7Ͳ6:Gasreadingsfromlaboratorybaseddieselengineexhaust.

Recordingsfor2hrsafterenginewarmupperiod.(a)CO;(b)CO2;(c)NO;(d)NO2;(e) Cl2;(f)O2.

7.3.3.2 Fieldbaseddieselexhaustgasmonitoring

Thelevelofgaseousemissionsfromlaboratorybaseddieselengineswasmonitored andrecordedusingiBrid™MX6multiͲgasmonitors(asperSection4.5.1)todetermine theconcentrationofgasesduringtheexposureperiod.GasesincludedCO2,CO,NO2,

NOandO2,thegasreadingsweremeasuredtwiceandtheaverageresultsplottedin Figure7Ͳ7.

132  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts



Figure7Ͳ7:Gasreadingsfromfieldbaseddieselengineexhaust.

Recordingsfor2hrsafterenginewarmupperiod.(a)CO;(b)CO2;(c)NO;(d)NO2;(e) Cl2;(f)O2.

7.3.4 Organiccarbonandelementalcarbonanalysis

7.3.4.1 Laboratorybasedengine

The organic and elemental carbon readings for the laboratory based diesel engine (Table7Ͳ1)andfieldbaseddieselenginearedisplayedinTable7Ͳ2.



133  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts

 Table7Ͳ1:ElementalCarbon(EC),Organiccarbon(OC)andTotalcarbon(TC)content oflaboratorybaseddieselengineexhaust Sampling EC OC TC EC/OC  EC/TC period concentration concentration concentration ratio ratio (mins) (μg/m3) (μg/m3) (μg/m3) 15 3.639 61.225 64.863 0.059 0.056 30 2.611 37.455 40.066 0.070 0.065 60 1.133 10.981 12.114 0.103 0.094 120 1.824 16.010 17.833 0.114 0.102 

7.3.4.2 Fieldbasedengine

The organic and elemental carbon readings for the field based diesel engine are displayedinTable7Ͳ2.

 Table7Ͳ2:ElementalCarbon(EC),Organiccarbon(OC)andTotalcarbon(TC)content offieldbaseddieselengineexhaust sampling EC OC TC EC/OC EC/TC period concentration concentration concentration ratio ratio (mins) (μg/m3) (μg/m3) (μg/m3) 30 2.379 0.863 3.241 2.758 0.734 60 2.110 0.858 2.968 2.460 0.711 

7.4 Discussion Thecytotoxicityofdieselexhaustwasinvestigatedusingthedynamicdirectexposure method in which human lung cells were grown on a porous membrane, allowing simultaneous exposure of cells to exhaust whilst receiving nutrients.A laboratory baseddieseltruckengineandfieldbaseddieselpassengercarwereusedtogenerate dieselexhaustwhichwasdeliveredtohumantargetcellsthroughadynamicexposure chamberemployingahorizontaldiffusionsystem.Cytotoxicityofthedieselexhaust wasinvestigatedinA549humanpulmonarytypeIIͲlikeepithelialcellsusingMTS,NRU andATPinͲvitrocytotoxicityassays.

134  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts

 Dieselparticulateorganicandelementalcarboncomponentswereanalysedusingthe NIOSH5040method.Ahigherelementalcarboncontentindicateahigheramountof particulates within the exhaust whereas the organic carbon content indicates the amount ofmoleculeswithcarboninitschain(GrovesandCain2000).Theresultsin Table7Ͳ1showedtheexpectedincreaseintotalcarboncorrespondingwiththelonger exposureperiod,thelaboratorybasedexperimentshowedahigherelementalcarbon component content at the longer sampling period.This change of ratio became evidentafterthe30Ͳ60minsperiodofsampling,whichsuggeststhattheVolvoengine at idle setting reached equilibrium state only after 30Ͳ60 mins. With the field study shown in Table 7Ͳ2, the ratio had no significant change suggesting that the diesel particulatefiltermayhavereduced emissionofelementalcarbons.

TheresultsofbothlaboratoryandfieldstudiessuggestexposureofA549lungcellsto dieselexhaustsforaperiodof>30minsconsistentlycausedcellulardamage(Figure 7Ͳ2andFigure7Ͳ4)comparedwithcontrolcellsexposedtocontrolairviableforupto 2 hrs.ThisisalsothecaseinanotherstudybyAufderheideetal(2003)wherecellscan wereexposedtocontrolairatlowflowratesupto2hrswithoutanysignificantcell viabilityreduction(Aufderheide,Knebeletal.2003).Inotherstudiessuchasoneby Chenget al(2004),A549cellsgrownonmembraneswereexposedtodieselandpetrol exhausts(Cheng,Maloneetal.2003).AlthoughChengetalinvestigatedproductionof ILͲ8,theexposureperiodemployedintheirstudywasmuchlongeri.e.upto6hrs.

In terms of sensitivity, for both field and laboratory based studies the order was NRU>ATP>MTS.However the MTS assay results had the smallest S.D representing moreconsistentresultwhileNRUhadthelargestespeciallywhentherewasnopostͲ exposure incubation. This could be caused by the formation of crystals as was observedinastudybyHusøy etal(Husøy,Syversenetal.1993),inthisstudysteps were taken to minimise this formation by incubating the NR medium overnight, centrifugingitandfilteringitusinga0.22μmporefilterasperformedbyBorenfreund etal(1985)(BorenfreundandPuerner1985).BoththeNRUandMTS assayreported similar decreases in cell viability after 24 hrs postͲexposure incubation time which demonstratedthecorrelationofresultsbetweenthetwoassaysindeterminingbasal

135  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts

 cytotoxicityofpollutants(Borenfreund,Babichetal.1988;Eisenbrand,PoolͲZobelet al.2002).

After exposure, the cells were either incubated for a further 24 hrs or immediately usedfortheinͲvitroassaystodetermineandcomparethecytotoxiceffectsafterboth 0and24hrsincubationtimecourses.TheresultsinFigure7Ͳ4andFigure7Ͳ5suggest that some cell functions such as cellͲdivision capability and delayedͲonset toxicity (Riddell, Panacer et al. 1986) may only be obvious after further incubation.By comparison, overnight incubation is generally more reproducible although lower in viabilitybutbothimmediateand24hrspostexposureincubationcanbeconsidered usefulinstudiesdependingonthekindofresponsetobeobserved(shorttermversus longtermeffects),inwhichlongerpostͲexposureincubationreflectsamore diverse rangeofresponsesfromcellsafterexposuretodieselexhaust.

Thisstudyinvestigatedtimedependenttoxicityofdieselexhaustbysettingthediesel engineatidlesettingandexposinghumanlungtargetcellstotheexhaustforadiverse rangeofexposureperiodsof15,30and60mins. However,ascanbeseeninFigure

7Ͳ6andFigure7Ͳ7,NOemissiondecreasedwhileNO2increasedwhichiscausedbyan increase in temperature of the combustion environments (monitored by its coolant temperature),whileCOemissionwasincreased.ThistrendofincreasingNO2ratiois usually seen in diesel engines equipped with diesel particulate filter and running at higherloads(CARB2002;DRSTA2002),sincethisstudyusesidlesetting,theengine temperaturemayhaveaffectedtheratiochange.Thesevariousgaseoustrendsmake it difficult to maintain a constant generation of exhaust, hence a doseͲdependent toxicitystudymayhavemorereproducibleexhaustinwhichtheamountofpollutant willbeincreasedbyincreasingloadorrpmoftheengine(NeerandKoylu2006).

In this study, A549 was chosen as the lung is the organ of inhalation toxicology significant.Further experiments will  concentrate on assessing the toxicity of diesel exhaustonvariouscellstodeterminetoxicityonspecificorgans.Onepossibleorganof interestis the liversince ultrafine particulates may translocate from alveoli into the brainandultimatelyintothebloodstreamtotheliver(Nemmar,Hoylaertsetal.2004; Donaldson,Tranetal.2005)wherethehealthrisksarenotwellknown.Inaddition,

136  Chapter7:InͲvitrotoxicityassessmentoflaboratoryandfieldbaseddieselexhausts

 specific components of the diesel exhaust such as particulate matter needs to be investigated further as these nanoͲsized particles have major human health impacts especiallyontherespiratorysystem(Risom,Mølleretal.2005;Mazzarella, Ferraraccio etal.2007;Hirota,Akimaruetal.2008).Inaddition,anothercontinuousdifferentiated celllinecanbeusedtorepresentanotherorganoftoxicologicalsignificancesuchas liverviathehepG2celllineorprimaryculturescouldbeusedtocoverdetectmore toxic effects and may reduce the necessity of laborious systems with specific cells (Yang,Cardonaetal.2002;CastañoandGómezͲLechón2005;Tan,Wangetal.2008) althoughpreferablythesameculturemediaisusedasdifferentculturemediamight affectinͲvitrocytotoxicityassaysresultssuchasinILͲ6secretion(Veranth,Cutleretal. 2008).

ResultsofthisstudysuggestthatthedynamicdirectexposuremethodwithrealͲtime airmonitoringtechniqueshavethepotentialtobeimplementedforbothlaboratory andfieldbasedtoxicitystudiesofdieselexhaustinͲvitro.Themainadvantageofthis method is its closer approximation to realͲlife  condition in which cells continuously receive nutrients whilst being exposed to air pollutants.Although only one type of humancellswasusedinthisstudy,themethodiscapableofusingdifferentcelllines. To address the points noted above, future works will use multiple cell lines of toxicologicalsignificance (suchascellsofliver;HepG2),usemultipleinͲvitroassaysto observe various biological endpoints.These improvements will allow a study to determine any difference of cytotoxicity between filtered and unfiltered diesel exhausts.



137  Chapter8:Toxicitycomparisonbetweenfilteredandunfiltereddieselexhaust

8 Toxicity comparison between filtered and unfiltered dieselexhaust

8.1 Introduction One of the main risks posed by diesel exhaust is the particulates component. Particulates of diesel exhaust are smaller in size range to other combustion derived particles such as of wood smoke (Kocbach, Johansen et al. 2005) and smaller size particulateshaveextrapropertiessuchashighersurfaceͲareatovolumeratiohence higherreactivity(Wilson1990).Additionally,smallerparticulateshavethepotentialto translocatethroughalveolarsurfaceintothebloodcirculationsystem.Therisksare not well elucidated and established hence further research is needed (Kreyling, SemmlerͲBehnkeetal.2006;Simkhovich,Kleinmanetal.2008).

Manystudiessampleparticulates bycollectingsootdepositsinthedilutiontunnelor filterofadieselengineexhaustsetupandpurifying[articulatecomponents(Bayram, Devaliaetal.1998;Ito,Okumuraetal.2006; Song,Zhouetal.2007).Alternatively researchers can purchase a standard reference material (Patel, Eo et al. 2011) and extract specific  components of the exhaust.Although the components are purified andspecifictoxicityinformationforeachcomponentisavailable,themethoddoesnot allowdirectsamplingofexhaust which contains a complexmixture of both gaseous and particulate components (Knebel, Ritter et al. 2002) where both contribute to varying levels of toxicity.In addition, by employing the direct exposure method of growingcellsonporousmembranes,itallowsamorerealisticconditionofexposureat the air liquid interface in which the apex side of the cell culture are exposed to contaminants while simultaneously receiving nutrients from the basolateral side (Ritter,Knebel etal.2001).

DuetosmallerPMcomponentsofexhaustanditstranslocationability,thelungwould notbetheonlytargetorganofPM.Otherorganssuchaslivermaybecomeatarget sincePMcantranslocateviathegasexchangeareaofalveoliintothebloodstream and further  distribution to other organs can occur (Nemmar, Hoylaerts et al. 2004). Uponenteringtheliver(viatheportalvein),theliverthenactsasthedetoxification

138  Chapter8:Toxicitycomparisonbetweenfilteredandunfiltereddieselexhaust centre (Balazs 1981).Various enzymatic reactions and other detoxicification mechanismsmaypotentiallyincreasethetoxicityofmetabolitesasseeninexamples such as of carbon tetrachloride into chloroform, a toxic and carcinogenic substance (Griffin 2006).Hence in this study, human cells of various organs were utilised as modelofthetargetorganstodeterminepossibletoxicityuponinteractionswiththe contaminants.

Thisstudyisaimedatfindinganysignificantcytotoxicitydifferencebetweenfiltered exhaustandnonfilteredexhaustandtofurtherdevelopthemethodofgrowingcells onporousmembrane toallowamorerapidscreeningprocesswhichwillbeusefulto studymultipledieselexhaustgenerationconditions(Madden2008).

8.2 Experimentaldesign

8.2.1 Celltypesandcultureconditions

ThecelltypesusedincludeHumanpulmonarytypeIIͲlikeepithelialcells(A549,ATCC No.CCLͲ185),Humanlivercells(HepG2,ATCCNo.HBͲ8065)andhumanskinfibroblast cellsasperSection4.6wereculturedinsterile,vented75cm2cellcultureandkeptat o 37 Cinahumidified5%CO2incubator.

Cellsweregrownonporousmembranes(0.4μm)inSnapwellinsertsasperSection 4.6.7.Cellattachmentwascheckedusingalightmicroscopebyobservingconfluence (75Ͳ80%).The medium was then removed from the top part and the membrane washedwithHank’sbalancedsaltsolution(HBSS;Gibco, USA),andthentransferred intothedynamicexposurechambers(HarvardApparatus,Inc,USA)fordirectexposure todieselexhaust.

8.2.2 Generationofdieselexhaust

Volvo230hptruckengine,runningonidlemodewithnoload,wasemployed(asper Section 4.3).The engine was switched on and set on idle setting for 15 mins for warming up before exposure of target cells to diesel exhaust. Following the stabilisationperiodtheexhaustsweredeliveredto dynamicdirectexposurechambers usingnegativepressurepumps(SKCInc,USA)calibratedatverylowflowrates(ч37.5 ml/min).

139  Chapter8:Toxicitycomparisonbetweenfilteredandunfiltereddieselexhaust

8.2.3 Monitoringandanalysisofexhaustemissions

Monitoring of exhaust emission was performed using the iBrid™ MX6 multiͲgas monitors(asperSection4.5.1).

8.2.4 Exhaustfiltration

Polycarbonatemembranefilters37mmindiameterand0.4μmporesinathreepiece clear styrene cassette (SKC, USA) were used to filter the particulate component of dieselexhaust.

8.2.5 Cytotoxicityendpoints

Toassessthecytotoxicityofdieselexhaust,twoinͲvitrobioassayswereused:MTS([3Ͳ (4,5ͲdimethylthiazolͲ2Ͳyl)Ͳ5Ͳ(3Ͳcarboxymethoxyphenyl)Ͳ2Ͳ(4Ͳsulphophenyl)Ͳ2HͲ tetrazolium])andNRU(neutralreduptake)formeasuringvariousbiologicalendpoints (asperSection4.8).

8.2.6 Controls

AppropriatecontrolswerepreparedasperSection4.8.4.2,brieflyanIC100control(0% cellviability;mediaonly)wasusedforbackgroundandincubatedat37oCduringthe exposuretimeandanIC0control(100%cellviability;cellsonly),usedforreference andincubatedat37oCduringtheexposuretime.Inaddition,anaircontrolwasalso usedtoconsideranyreductionofcellviabilityinducedbythedynamicairflow.

8.2.7 Statisticalanalysis

Statistical analyses were performed (as per Section 4.10).In addition statistical analysesusedweretheoneͲwayANOVAandBonferroni’smultiplecomparisonstests as per Section 4.10 with differences considered statistically significant at p < 0.05. DoseresponsecurvewascalculatedusingGraphPadPrismsoftwarebyvariableslope onnormalisedresponseandits95%confidenceintervalwasalsodetermined.

140  Chapter8:Toxicitycomparisonbetweenfilteredandunfiltereddieselexhaust

8.3 Results

8.3.1 OptimalcelldensityforHepG2&skinfibroblasts

To determine for skin fibroblast and HepG2 cells most linear correlation response betweencelldensity(numberofcells/ml)anditsabsorbances,variouscelldensities wereintroducedtothemembranesandabsorbanceswererecorded(Table8Ͳ1).The regioninwhichthelinearincreaseincelldensitygavealinearincreasein absorbance was determined as the ideal number of cells needed to prepare for subsequent experiments.



Table8Ͳ1:CorrelationbetweenSkinfibroblast,A549andHepG2cellnumberwithMTS andNRUassayabsorbancereadings

Celltype Assay Cellnumberrange Rsquarevalue Skinfibroblast MTS 0– 2.9x105 0.9824 Skinfibroblast NRU 0– 2.9 x105 0.9780 HepG2 MTS 0– 5x105 0.9736 HepG2 NRU 0– 6x105 0.9520 

8.3.2 Toxicity comparison between filtered and unfiltered diesel exhaust

Cell viability of A549, HepG2 and skin fibroblasts was compared to determine the effect of filtering diesel exhaust on cell viability.The filtering was achieved using a polycarbonatemembranefilters(SKC,USA).Resultsoncellviabilityafterexposureto either filtered or unfiltered exhaust across various cell types, cell viabilities were determinedusing MTS (Figure 8Ͳ1) and NRU (Figure 8Ͳ2) inͲvitro assays after 24 hrs postͲexposureincubationperiod.

141  Chapter8:Toxicitycomparisonbetweenfilteredandunfiltereddieselexhaust

In comparing cytotoxicity between filtered and unfiltered exhaust, oneͲway ANOVA withBonferroni’smultiplecomparisonsweremadebetween: x Cellsexposedtocontrolairandfiltereddieselexhaust x Cellsexposedtocontrolairandunfiltereddieselexhaust

Differences were considered significant at p<0.05 (* = p<0.05).No significant differenceswerefoundbetweenfilteredandunfilteredexhaustinducedtoxicities.

 Figure8Ͳ1:CytotoxicityofdieselexhaustwithMTSinͲvitroassay (a)Skinfibroblast;(b)A549cells;(c)HepG2cells x Controlair=exposedcellstocontrolaironly,Filtered=exposedcellstofiltered exhaust;Unfiltered=exposedcellstounfilteredexhaust. x *=p<0.05and**=p<0.01comparedtocontrolairexposure. x Eachdatapointisexpressedasmean±S.D. 

142  Chapter8:Toxicitycomparisonbetweenfilteredandunfiltereddieselexhaust

 Figure8Ͳ2:CytotoxicityofdieselexhaustwithNRUinͲvitroassay (a)Skinfibroblast;(b)A549cells;(c)HepG2cells x Controlair=exposedcellstocontrolaironly,Filtered=exposedcellstofiltered exhaust;Unfiltered=exposedcellstounfilteredexhaust. x *=p<0.05and**=p<0.01comparedtocontrolairexposure. x Eachdatapointisexpressedasmean±S.D. 

8.4 Discussion Theaimofthisstudywastodeterminedifferencesincytotoxicitybetweenfilteredand unfiltereddieselexhaustusingculturedcellsonporousmembranewithinahorizontal diffusion chamber system for delivery of diesel exhaust.Diesel exhaust (produced using a diesel engine) was sampled for each exposure period, where filters were connectedinoneofthetwoparallelandidenticaldynamicdeliverysamplinglinesto enable direct comparison of cytotoxicity for filtered and unfiltered exhaust.Each exposure was set at 30 mins as determined from previous studies as this was the minimumexposureperiodtoreducecellviabilitytolessthan50%forMTS,NRUand

143  Chapter8:Toxicitycomparisonbetweenfilteredandunfiltereddieselexhaust

ATPassays(asperinChapterseven).Thecytotoxicitycomparisonswereperformed on skin fibroblasts, A549 human pulmonary type II like epithelial cells and HepG2 humanlivercellsusingtheMTS,NRUandATPinͲvitrocytotoxicityassays.

Diesel exhaustmainlyconsistofgaseousandPM2.5whichiswithintheregionofhuman healthriskconcernduetoitssmallsizeandabilityreachdeeperintothepulmonary region of the lungs including the alveoli and translocation into the cardiovascular system(Oberdörster2000;Nemmar,Hoylaertsetal.2004)andotherareasofbody.

Gaseous component such as NOx is a main concern due to its breakdown into hazardous contaminants such as binding of NOx with H2O to create a mildly acidic

Nitricacid(HNO3).Gaseouscomponentscanreachdeepintothepulmonarysystem including the bronchioles and alveoli.Health risks due to gaseous component of exhaust include mild upper respiratory tract irritation due to exposure to low concentrationsofNOx,respiratoryoedemaandeventuallydeathatexposuretohigher concentrations(Carel1998),meanwhileexposuretocombinedSO2andNO2enhances airwayresponsetoinhaledallergens(Devalia,Rusznaketal.1994).

In the study, slightly lower mean cell viability after exposure to unfiltered diesel exhaust was observed in all human cells and inͲvitro assays compared to filtered exhaust.With regards to all human cells, the difference in cytotoxicity between filteredandunfilteredexhaustswerenotstatisticallysignificantwhichindicatedthat the gaseous compounds are the major components of the diesel exhaust which my inducecytotoxicityafterashortexposuretimeperiod.Thisresultisalsoobservedin otherworkssuchasKnebeletal(2002)whodeterminedthatdecreasein cellviability isindependentofengineoperatingconditionandfiltering(Knebel,Ritteretal.2002). Holder et al (2007) also showed no significant difference in cell viability using MTT assay after 0 hrs and 20 hrs postͲexposure incubation, although there is a strong increase in ILͲ8 release after 6s hr  postͲexposure incubation period with lower ILͲ8 releaseat20hrspostͲexposureincubation(Holder,Lucasetal.2007).Tsukueetal (2010) have compared differences in cytotoxicity between exhaust with varying PM and NO2 content and have showed less cytotoxicity by low NO2 content exhaust (Tsukue,Okumuraetal.2010).Theseobservationssupporttheideathatthegaseous

144  Chapter8:Toxicitycomparisonbetweenfilteredandunfiltereddieselexhaust component causes major cytotoxic effect compared to particulates although particulatessmallerthanthefilterporesmaystillcontributetosomecytotoxiceffects.

Comparing the result of this study and epidemiological comparisons between PM2.5 andPM10effectsonhumanhealth,itprovidesanevidenceofsmallersizedPMhaving highertoxicity.Sincethefilter has porediameter of 0.4 μm thenitis assumed the majority of particulates smaller than it will pass through and exposed on cells and potentially causing adverse effects alongside with thes gaseou  component of diesel exhaust.VariousepidemiologicalstudieshaveindicatedstrongerlinkbetweenPM2.5 toadversehealtheffectscomparedtoPM>2.5μminsize(Dockery,Popeetal.1993; Abbey,Ostroetal.1995;McDonnell,NishinoͲIshikawaetal.2000;SchwartzandNeas 2000).InͲvitro studies such as by WendyͲHsiao (2000) showed DNA damage and decreaseofcellviabilityposedbyPM2.5ofambientairpollutiononrodentfibroblasts (Wendy Hsiao, Mo et al. 2000), while Pozzi et al (2003) showed inflammatory mediatorswerealsoinducedmorebythePM2.5ofurbanairpollutantinRAW264.7 cells.Monnetal(1999)showedinflammatorymediatorsinducedbyPM2.5Ͳ10instead ofPM2.5(MonnandBecker1999;Pozzi,DeBerardisetal.2003).Inthiscurrentstudy filteringoutPM>0.4μmparticulatessuggestedPM>0.4μmmaynothavesignificant effectoncellviabilityinskinfibroblast,A549andHepG2cells.Comparisonsbetween literatures showed PM may have different composition depending on PM sources (Bonetta, Gianotti et al. 2009) such as between diesel engine in this study and contaminantscollectioninairmonitoringstations.

Byusingvariouscellularsystems,comparisonofcytotoxicityondifferentcelltypescan be made.Since there is no statistically significant difference between cytotoxicity responsesofA549,HepG2dan skinfibroblast,itisthereforemostlikelytobeabasal cytotoxicityresponseinwhichthedieselexhaustaffectcommoncellmechanismsince anorganspecifictoxicitywouldinducinghighercytotoxicityinspecificcelllines(Parish 1986;Korting,Schindleretal.1994).

Diesel engines under various loads have exhausts of different composition and size distributionofparticulates(Lapuerta,Martosetal.2007)henceeventhroughfiltering the exhaust has may have no statistically significant reduction in cytotoxicity with

145  Chapter8:Toxicitycomparisonbetweenfilteredandunfiltereddieselexhaust currentstudy,theremaybeadifferenceathigherloads.Themaincauseofthehigher cytotoxicity may include the smaller size of particulates and higher NOx emission associatedwithhighertemperatureofcombustionflameduetohigherloadlthediese  engineisrunningunder.

The study has contributed data to the concept of comparing filtered and unfiltered exhausttodeterminecytotoxicitycausedbydieselparticulates.ManyinͲvitrostudies have focussed either on gaseous component or particulate component of diesel exhaust.By exposing cells to both gaseous and particulates  component simultaneously, a deeper understanding can be made in determining cytotoxicity contribution by gaseous and particulates component of diesel exhaust and if any, synergistic,potentiatingandantagonisticeffectsbetweenthegaseousandparticulate matter components of the exhaust especially in inhalation toxicology (Yu 2001; Pauluhn2005).Thefindingssuggestthegaseouscomponentofdieselexhaustcauses morecytotoxicitytocellswithshorttermexposurehenceindicatingriskassessorsto putmoreemphasisinmanagingrisksfromexposuretogaseouscomponentofdiesel exhaust,althoughtheparticulatesmaycontributemuchlesscomparedtotheoverall shorttermtoxicity,itmaycontributetolongtermtoxicity.

Results of this study showed filtration of exhaust improved cell viability but not statistically significant.Different cell types’ responded differently to diesel exhaust withHepG2hadlowestcytotoxicitywhileskinfibroblasthadthehighestcytotoxicity. Cytotoxicitycomparisonbetweenhumancellsdemonstratedtherewasnostatistically significant difference in responses, hence the response was considered as basal cytotoxicity.As diesel engines emit different size distribution of particulates and varyinglevelofgaseouscompoundsatdifferentloads,futureresearchwillconcentrate onvalidatinginͲvitromodelofthehumanpulmonarysystemwhichallowscytotoxicity comparisonofdieselexhaustrunningunder variousloadsandcorrespondinggaseous contaminant levels to compare cytotoxicity between gaseous and particulate componentofdieselexhaust.



146  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

9 Toxicity assessment of diesel exhaust across various loads

9.1 Introduction Inhalationisthemainrouteofexposurefordieselexhausttoexertcytotoxicityinthe humanbody.HoweverotherorganssuchasthelivermayalsobeaffectedbyDiesel Exhaust Particle’s (DEPs) as they may translocate from the alveoli into the cardiovascularsystem.Astheliveractsasadetoxifier ofbloodtheinteractionwith DEPsisevidentinvivo(Folkmann,Risometal.2007).Themajorityofstudiestodate have been inͲvitro and have showed adverse effects of DEP components on liver enzymesusingHepG2livercells(Belisario,Arenaetal.1991;Chen,Chouetal.2000; eLamy, Kassi etal.2004).

Diesel exhaust concentration varies under different engine loads.At higher loads dieselengineshavethetendencytoemithigherNOxlevelsduetoanincreaseinflame temperature within the combustion chamber (Boubel, Fox et al. 1994; Challen and Baranescu 1999) whilst simultaneously decreasing other gasses e.g. CO due to conversiontoCO2.Inaddition,thesizedistributionofemittedparticulatesaresmaller at higher engine speed (Shi, Harrison et al. 1999).However, during creation temperature decreases and particulates agglomerate into chains of particles (Stevenson 1982).Therefore when measured, an exhaust particulate may have a larger mean size compared to the main particulates immediately created after combustion.

Duetothevaryingcompositionofexhaustunderthenormalrangeofengineoperating settings,itisimportanttodeterminethefulleffectsthatengineloadhasonexhaust and subsequently the toxicity to human cells.Most studies to date use very few operatingcharacteristics/settings(Ichinose,Yajimaetal.1997;Yamazaki,Hatanakaet al.2000;Birumachi,Suzukietal.2001;Ito,Okumuraetal.2006;Shima,Koikeetal. 2006) which are adequate for particulate sampling and toxicity analysis of DEP componentshowever,theeffectsofengineloadarelargelyunknown.

147  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

Theaimofthisstudywastobuilduponthemethodologydevelopedincomparingthe cytotoxicity posed by filtered and unfiltered diesel exhaust (Chapter 8).A test procedure was developed to generate diesel exhaust under varying engine torque settings(withfixedspeed).Inturn,theexhaustsweredeliveredtohumantargetcells grown on porous membranes and contained in horizontal diffusion chambers that alloweddirectexposuretodieselexhaust(Chapter7).Cytotoxicitywasinvestigated usingselectedinͲvitroassaysincludingMTS,NRUandATP.

9.2 ExperimentalDesign

9.2.1 Celltypesandcultureconditions

ThecellstypesusedincludedHumanpulmonarytypeIIͲlikeepithelialcells(A549,ATCC No: CCLͲ185) and Human liver cells (HepG2, ATCC No: HBͲ8065) as per Section 4.6. Cellswereculturedinsterile,vented75cm2cellcultureflasksandkeptat37oCina humidified5%CO2incubator.

Cellsweregrownonporousmembranes(0.4μm)inSnapwellinserts(asperSection 4.6.7).Cellattachmentwascheckedusingalightmicroscopebyobservingconfluence (75Ͳ80%).The medium was then removed from the top part and the membrane washed with HBSS (Gibco, USA), and then transferred into  the dynamic exposure chambers(HarvardApparatus,Inc,USA)fordirectexposuretodieselexhaust.

9.2.2 Generationofdieselexhaust

AVolvo230hptruckengine,runningonidlemodewithnoloadwasemployed(asper Section4.3.2).Theenginewasswitchedonandsetonidlesettingfor5minsfora warming up period before exposure of target cells to diesel exhaust. Following the stabilisationperiodtheexhausts weredeliveredtodynamicdirectexposurechambers using negative pressure pumps (SKC Inc, USA) and calibrated at very low flow rates (ч37.5 ml/min).The load of engines was defined as the percentage of maximum possibletorqueoftheengine.Therangeoftorquesusedwasdividedintofivepoints %,(0%,25 50%,75%and100%ofmaximumtorqueoutput)andthespeedofengine wasset1600rpmasthiswastheminimumspeedformaximumtorque.

148  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

9.2.3 Monitoringandanalysisofexhaustemissions

Monitoring of exhaust emission was performed using the iBrid™ MX6 multiͲgas monitors(asperSection4.5.1).Inaddition,dieselexhausttemperaturewerechecked to ensure it is at room temperature prior to entering the exposure chambers (maintained at 37oC) to eliminate any potential physical stress such as temperature shock.

9.2.4 Exhaustfiltration

Polycarbonatemembranefilters37mmindiameterand0.4μmporesinathreepiece clear styrene cassette (SKC, USA) were used to filter the particulate component of dieselexhaust.

9.2.5 Cytotoxicityendpoints

To assess the cytotoxicity of diesel exhaust the inͲvitro bioassays MTS ([3Ͳ(4,5Ͳ dimethylthiazolͲ2Ͳyl)Ͳ5Ͳ(3Ͳcarboxymethoxyphenyl)Ͳ2Ͳ(4Ͳsulphophenyl)Ͳ2HͲ tetrazolium])andNRU(neutralreduptake)wereutilisedtomeasurevariousbiological endpoints(asperSection4.8).

9.2.6 Controls

Appropriate controls were prepared (as per Section 4.8.4.2), briefly an IC100 control (0%cellviability;mediaonly)wasusedforabackgroundcontrolandincubatedat37oC duringtheexposuretime.AnIC0control(100%cellviability;cellsonly)wasusedfora referenceandincubatedat37oCduringtheexposuretime.Inaddition,anaircontrol was also used to consider any reduction of cell viability induced by the dynamic air flow.

9.2.7 NOxmeasurements

Gas monitoring readings were performed using a high precision NOx reader (as per Section4.5.3).Briefly,thedieselenginewasputunderasetload,andoncethe5min warmingperiodoftheenginewascompleted,NOxreadingsstabilisedandtwominsof continualgasanalysiswasrecorded.

149  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

9.2.8 Exhaustfiltration

Polycarbonatemembranefilters37mmindiameterwith0.4μmporesizeinathree piececlearstyrenecassettewereused(SKC,USA).Thesefilterswerethesamefilter usedforElementalandOrganiccarbonmeasurements(asperSection4.5.2).

9.2.9 Elementalandorganiccarbonmeasurement

The NIOSH5040 method was applied to determine elemental and organic carbon contentofdieselexhaustasoutlinedinSection4.5.2.

9.2.10 Combustionflametemperature

Peak flame temperature based on cylinder pressure wave was recorded (as per Sections4.3.3and4.3.4).

9.2.11 Scanningelectronmicroscopy

Electronmicroscopywasemployedtoobservesurfacemorphologyofcontrolcellsand exposedcells(asperSection4.11).Briefly,membraneswerefixed,dehydratedwith ethanol, critical point dried and gold sputter coated, micrographs were taken of controlcells(IC0andcontrolair),IC50andIC100.

9.2.12 Statisticalanalysis

Statistical analyses were performed (as per Section 4.10). In addition the statistical analysesusedwereoneͲwayANOVAandBonferroni’smultiplecomparisonstests(as perSection4.10)withdifferencesconsideredstatisticallysignificantatp<0.05.Dose responsecurveswerecalculatedusingGraphPadPrismsoftwarebyvariableslopeon normalisedresponseandits95%confidenceinterval.

9.3 Results

9.3.1 NOxmeasurementandflametemperaturecalculation

TheresultsofNOxmeasurementsarepresentedinFigure9Ͳ1andTable9Ͳ1and,each datapointwasanaverageoftworeadings(expressedasmean±S.D),eachreading was recorded after 1 min of the exhaust stabilisation period.Combustion flame temperature was calculated based on cylinder pressure wave (Appendix D) and

150  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

presented in Table 9Ͳ1.NOx level and the peak flame temperature were highly correlatedatR2=0.98.



Figure9Ͳ1:NO,NO2andNOxreadings

(a)NO;(b)NO2;(c)NOx Eachdatapointrepresentsmean±S.D. 

Table9Ͳ1:NOxreading,flameandgascalculation

Engineload NOxreading Peakflametemperature (%ofmaxtorque) (ppm±S.D) (oK) 0 94.5±3.818 2659

25 299.65±39.386 2700

50 631.8±71.276 2728

75 891.9±139.724 2759

151  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

100 1044.7±163.625 2788



9.3.2 Optimisationofexposureperiod

Theoptimalexposureperiodwasdefinedasthenumberofminstoinduceatleast50% of cell death due to the exposure of exhaust from the diesel engine set at 50% of maximum torque (1600 rpm).MTS, NRU and ATP inͲvitro assays with 0 hrs post exposureincubationperiod wereusetodeterminecellviability.TheresultsforA549 cellsareshowninFigure9Ͳ2andshowedsignificantcellviabilityreductionoccurred from7.5minsonwards.Cellviabilitywasreducedtobelow50%by15minshence,15 minswasdeterminedtobetheoptimalexposureperiod.

152  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

 Figure9Ͳ2:Timecoursecytotoxicityanalysisofunfiltereddieselexhaust x (a)MTSassay;(b)NRUassay;(c)ATPassay. x Eachdatapointwasexpressedasmean±S.D. x *=p<0.05;**=p<0.01.

9.3.3 Comparisonofcytotoxicityendpoints

A549cellswerechosenascytotoxicitymodeltodetermineanydifferenceinresponse betweenvariousendpointsusingMTS,NRUandATPinͲvitroassaysasshowninFigure 9Ͳ3.In comparing cytotoxicity between filtered and unfiltered exhaust, twoͲway ANOVAwithBonferroni’smultiplecomparisonsweremadebetween: x Cellsexposed tocontrolandunfiltereddieselexhaust

Differenceswereconsideredsignificantatp<0.05(*=p<0.05;**=p<0.01)

153  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

Figure 9Ͳ3: Cytotoxicity of diesel exhaust on A549 cells with 0 hrs postͲexposure incubation (a)MTSassay;(b)NRUassay x Control=exposedtocontrolair,Unfiltered=exposedtounfilteredexhaust x Eachdatapoint wasexpressedasmean±S.D. x PFT=peakflametemperature. x *=p<0.05;**=p<0.01.

9.3.4 Toxicitycomparisonoffilteredandunfiltereddieselexhaust

9.3.4.1 A549lungcells Cell viability of A549 cells was compared to determine the effect of filtering diesel exhaust on cell viability.The filtration was performed using a polycarbonate membranefilters(SKC,USA).Resultsoncellviabilityafterexposuretoeitherfiltered orunfilteredexhaustacrossvariousengineloadsareshowninFigure9Ͳ4 andFigure 9Ͳ5.CellviabilitywasdeterminedusingtheMTSandNRUinͲvitroassaysafter24hrs postͲexposure incubation period.In comparing cytotoxicity between filtered and

154  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads unfiltered exhaust, twoͲway ANOVA with Bonferroni’s multiple comparisons were madebetween: x Cellsexposedtocontrolairandfiltereddieselexhaust x Cellsexposedtocontrolairandunfiltereddieselexhaust Differenceswereconsideredsignificantatp<0.05(*=p<0.05;**=p<0.01),thefigures alsocontaincorrelationsbetween:

x CellviabilityresultswithNOxlevel. x Cellviabilityresultswithflametemperature.

 Figure9Ͳ4:CytotoxicityofdieselexhaustonA549cellswithMTSassaywith24hrs postexposureincubation x Control=exposedcellstocontrolaironly,Filtered=exposedcellstofiltered exhaust;Unfiltered=exposedcellstounfilteredexhaust. x Eachdatapointwasexpressedasmean±S.D. x PFT=peakflametemperature. x *=p<0.05;**=p<0.01. 

155  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

Figure9Ͳ5:CytotoxicityofdieselexhaustonA549cellswithNRUassayat24hrspost exposureincubation x Control=exposedcellstocontrolaironly,Filtered=exposedcellstofiltered exhaust;Unfiltered=exposedcells tounfilteredexhaust. x Eachdatapointwasexpressedasmean±S.D. x PFT=peakflametemperature. x *=p<0.05;**=p<0.01. 

9.3.4.2 HepG2livercells

CellviabilityofHepG2cellswascomparedtodeterminetheeffectoffilteringdiesel exhaust on cell viability.The filtration was performed using a polycarbonate membranefilters(SKC,USA).Resultsoncellviabilityafterexposuretoeitherfiltered orunfilteredexhaustacrossvariousengineloadsareshowninFigure9Ͳ6. Cellviability wasdeterminedusingtheMTSandNRUinͲvitroassaysafterthe24hrspostͲexposure incubationperiod.Incomparingcytotoxicitybetweenfilteredandunfilteredexhaust, twoͲwayANOVAwithBonferroni’smultiplecomparisonsweremadebetween: x Cellsexposedtocontrolairandfiltereddieselexhaust. x Cellsexposedtocontrolairandunfiltereddieselexhaust.

156  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

Differenceswereconsideredsignificantatp<0.05(*=p<0.05;**=p<0.01),thefigures alsocontainedcorrelationsbetween:

x CellviabilityresultswithNOxlevel. x Cellviabilityresultswithflametemperature.

 Figure9Ͳ6:Cytotoxicityofdieselexhauston HepG2cellswith MTSassayat24hrs postexposureincubation x Control=exposedcellstocontrolaironly,Filtered=exposedcellstofiltered exhaust;Unfiltered=exposedcellstounfilteredexhaust. x Eachdatapointwasexpressedasmean±S.D. x PFT=peakflametemperature. x *=p<0.05;**=p<0.01. 

157  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

 Figure9Ͳ7:Cytotoxicityofdieselexhaust on HepG2cellswithNRUassayat24hrs postexposureincubation x Control=exposedcellstocontrolaironly,Filtered=exposedcellstofiltered exhaust;Unfiltered=exposedcells tounfilteredexhaust. x Eachdatapointwasexpressedasmean±S.D. x PFT=peakflametemperature. x *=p<0.05;**=p<0.01. 

9.3.5 Elemental,organicandtotalcarbonmeasurement

The NIOSH5040 method was applied to determine elemental and organic carbon contentacrossdieselengineloadsandispresentedinFigure9Ͳ8.Eachdatapointisan averageofthreesamples(expressedasmean±S.D).

158  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

 Figure9Ͳ8:Organic,ElementalandTotalcarboncollectedonfilter (a)Organiccarbon;(b)ElementalCarbonand(c)Totalcarbon x Contentsweresampledat2L/minflowfor15minsasperNIOSH5040method. x Eachdatapoint wasexpressedasmean±S.D.

9.3.6 ObservationbySEM

9.3.6.1 Morphologicalalterationofhumancellsfollowingexposure

SEM micrographs of HepG2 cells were recorded at 5000x magnification to observe surface morphology of control cells (IC0 and control air) and exposed cells (IC50 and

IC100).Exposuretocontrolairdidnotinduceanymorphologicalchange(Figure9Ͳ9) whileexhaustlevelsresultinginIC50(25%engineload)producedblebformationson cell surfaces and reduction of microvilli (Figure 9Ͳ10 a).Exposure to IC100 exhaust levels created scaly morphology on cell surfaces and further reduction in microvilli (Figure9Ͳ10b).

159  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

Microvilli

(a) 

Microvilli

(b)

Figure9Ͳ9:SEMmicrographsofcontrolHepG2cells

(a)Controlcells;(b)Cellstreatedwithcontrolair;microvilliareindicated byarrows.

160 Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

Bleb Formation

(a)  Lossof microvilli

(b) 

Cell surface morphology change

(c) Figure9Ͳ10:Cellstreatedwithdieselexhaustat25%engineload

Cellstreatedwith25%engineloadforIC50with(a)blebformation;(b) lossofmicrovilli(c)cellsurfacemorphologychangeasindicatedby arrows.

161 Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

Cell surface morphology change

(a) 

Cell surface morphology change

(b) 

Cell surface morphology change

(c) Figure9Ͳ11:Cellstreatedwithdieselexhaustat100%engineload

Cellstreatedwith100%engineloadfornearIC100withmajorcellsurface morphologychangesobservedincellsurfaceasindicatedbythearrows.

162 Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads



9.3.7 Mathematicalmodelling/analysis

Peak flame temperature, NOx content, elemental and organic carbon contents were determinedandrecordedbyutilisingindependentmethods(Figure9Ͳ12).



Figure9Ͳ12:Sourceofvariablesformathematicalmodel

2 Since peak flame temperature highly correlated with NOx (R  = 0.98), peak flame temperatureandNOxreadingswereinterchangeable.ThereforeNOxlevelsandtotal carbon (TC) levels were chosen as representatives of gaseous and particulate componentsofdieselexhaustandtheircontributiontocelldeath(CD)expressedwith Equation10inwhichtheconstantsɲandɴcouldbedetermined.

[CD]=ɲ[NOx]+ɴ[TC]

Equation10:Celldeathcontributionbygaseousandparticulatecomponents

Where: x ɲandɴareconstants. x [CD],[NOx]and[TC]arevectorscontainingexperimentalresultsforcelldeath, oxidesofnitrogenandtotalcarbonrespectively. 

163  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads

The mathematical model was then tested using previously obtained sets (11) of experimentaldatausingMATLABtocalculateɲandɴbythefollowingmathematical formuladerivation

 If [CD]=ɲ[NOx]+ɴ[TC]

 Therefore [CD]=[NOx TC][ɲɴ]’ andhence

 Ͳ1 [ɲɴ]’=[NOxTC] [CD] Equation11:Determiningconstantsɲandɴ

 Fromallelevensetsofexperimentaldata,allbutoneexperimentaldatasetreturned positiveconstants;thisindicatedthatthelinearmathematicalmodeltookintoaccount contributionofeachcontaminantintothetotalcelldeath.Thecalculatedconstants weresubstitutedbackintoEquation10andcorrelationexperimentaldatacalculated whichshowedNOxlevelscorrelatedmoretothecellviabilitydata.Thesmallerthe valueofɴthehigherthecorrelationbetweencalculatedvalueandexperimentaldata

(AppendixE),suggestingthatthegaseouscomponentsofdieselexhaustsuchasNOx wasresponsibleforexertingmostofthetoxicityoncellsratherthantheparticulates component

9.4 Discussion As described in chapter 8, the aim of this study is was to compare cytotoxicity of exhaustfromadieselenginerunundervariousloads.Althoughthereisnostandard method to describe ‘load’ ofan engine, in this study load was defined by fixing the enginespeedinrotationsper)min(rpm to1600rpmasthiswastheminimumrpmto generatethegreatestamountoftorqueexpressedasNewtonMetres(Nm).Themain benefitofusingthismethodofloadmeasurementwasthesimilarityofthissettingto conventionalsettingsforengineperformancetestinginindustryhence,moresuitable determinationofcelltoxicityatgivenengineloadconditions.

Although a longer postͲexposure incubation period may show additional response (such as in Chapter 5), statistical analyses showed no significant difference in cell

164  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads viabilitybetweencellswith0hrsand24hrspostͲexposureincubationperiodwithboth MTS and NRU assays for A549 (lung) and HepG2 (liver) cells.In addition, on comparison of results between 0 and 24 hrs postͲexposure incubation periods, the higherS.D’sfor24hrspostͲexposureincubationperiodcomparedto0hrwasnoticed (Figure 9Ͳ3 and Figure 9Ͳ4).Observation under the light microscope 4 hrs after exposure to diesel exhaust showed major cell detachment indicating that the diesel exhaustcausedreductionincellviability relativelyquicklyafterexposure.Withhigher S.D’s observed after 24 hrs postͲexposure incubation period, 0 hrs postͲexposure incubation may appear more appropriate for inͲvitro testing of diesel exhaust, althoughthisobservationmayalsosupporttheviewinͲvitrotestsaremoresuitablefor shortͲtermtestswhichis desirablewhendevelopingarapidtest(Ponsoda,Joveretal. 1995;Zhang,Yangetal.2008).

In comparing the correlation between NOx emission levels and cell cytotoxicity, a significantcorrelationwasobservedbetweencytotoxicitybyfilteredexhaustandNOx levels.This result indicates that the gas component of exhaust such as NOx levels causes the greatest cytotoxicity effects instead of the particulate component of the dieselexhaust.However,sinceNOxformationdependsonpeaktemperatureandin turn depends on cylinder pressure generated, these variables may exhibit better correlation to cell cytotoxicity.A correlation analysis was performed between cell viabilityandvariablessuchascylinderpressure,maximumcombustionflameandbulk gastemperaturesandNOxreadingswerecorrelatedwithcellviabilitiestodetermine whichvariablecausedmostvariations.The correlationanalysesindicatedboth NOx and peak flame temperature have similar correlation with the experimental data, which was expected as NOx correlated highly with peak flame temperature.This observationsuggeststhatcellviabilityismainlyaffectedbyNOxasparticulatesarenot filtered; one possible reason is less emission of particulates and unburned hydrocarbonswithhighercombustiontemperatureathigherloads.Thisobservation suggeststhatthegaseouscomponentshavethemosteffectoncellviability,although otherminorcomponentsofdieselexhaustwerenotidentifiedwithinthisstudy.

Other works such as by Knebel et al (2002) showed a decrease in cell viability with increasedengineloadbutlittledecreasewithincreasingenginespeed(Knebel,Ritter

165  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads et al. 2002).Although in this research, the engine speed on cell viability was not investigated,theincreaseinengineloadcausedanoticeabledecreaseincellviability henceengineloadmaybeamorereliableindicatorofthedegreeoftoxicityfromthe engine.

Whencomparingcytotoxicitybetweenfilteredandunfilteredexhausts,nosignificant differences were observed even at higher loads.This observation complements previous findings (Chapter 8) which showed no significant difference in cell viability between filtered and unfiltered exhaust of a diesel engine running at idle.Organic carbon and elemental carbon gravimetric measurements showed approximately the same levels across the diesel engine load, this suggest the gaseous component of dieselexhaustcontributetomostoftoxiceffectandhence,posesmoreriskcompared totheparticulatescontent,atleastfollowingshorttermexposures.

The SEM images showed a similarity of cell morphology between cells treated with controlairandincubatorcontrolcellsagreeingwithinͲvitroresultswhichshowedno cellviabilityreduction.Afterexposuretodieselexhaust,featuresincludingformation of blebs on cell surface for cells and reduction of microvilli at IC50 value can be observedwhilecellsofIC0valuehavemorescaleͲlikemembranewallindicatingless integrityofthemembrane.

Electron microscopes have been used to observe shape of particulates and create a numerical description of fractal dimension thus irregularity of DEP agglomerates (Lapuerta, Martos et al. 2007).Transmission electron microscopes (TEM) are often preferredforhighermagnificationtoobservetheinnardsofcellsincludingitsincrease innucleusdensity,mitochondriaenlargementandlipiddroplets(Cameron,Neumanet al.1998)membraneintegrity(Koningsberger,Rademakersetal.1995),apoptoticcell features with cytoplasm of higher density, nuclear chromatin condensation, vesiculation of endoplasmic reticulum and bile canaliculus (Neuman, Cameron et. al  1999;Neuman,Shearetal.2001;Neuman2002)formationofbleb,condensednuclear chromatinanddarkcytoplasm(Katz,Shearetal.2001).Scanningelectronmicroscopy (SEM)allowsimagingofthecellsurfaceshenceenablingobservationsofothercellular structures such as microvilli to qualitatively observe cell condition.No other

166  Chapter9:Toxicityassessmentofdieselexhaustacrossvariousloads publicationscouldbesourcedonresearchstudyingthemorphologicalaspectsviaSEM onexposedhumancellstodieselexhaust.

Themainaimofthemathematicalmodellingwastocreateamodelforpredictingcell cytotoxicity using various engine and exhaust parameters as inputs.The model assumed toxicity interactions between contaminants to be either additive or antagonisticforlinearityofthemathematicalequations.Themodelthenunderwent optimisation/simplificationtominimisethenumberofvariablesrequired,analogousto PrincipalComponentAnalysiswhichreducesthenumberofvariablesbydetermining which variables causet the mos  variance.The mathematical modelling calculated constantvaluesforbothgaseousandparticulatelevelsbasedonexperimentaldata, correlationwasthendeterminedbetweenexperimentaldataandthemodel’svalues. The model also suggested the gaseous component to exert more toxicity effects on cells since addition of particulate toxicity values decreases correlation with experimental data.To our knowledge this work is one of the few efforts in the creationofmathematicalmodelstopredicthumancelltoxicitylevelsofcontaminant basedonobtainedexperimentaldata.

Insummary,theresultsofthestudyshowedthatthelongerpostͲexposureincubation perioddidtno causeasignificantreductionincellviability.Hence,theexperimental protocolcouldbesimplifiedbyreducingpostͲexposureincubationto0hrs.Filteringof dieselexhaustdidnotcausesignificantdifferenceincellviabilitieswhencomparedto theexposuretounfiltereddieselexhaustacrosstheloads.Inaddition,SEMimaging canbeusedtoobservecellstructure(morphologicalchanges)toobservetheeffectsof dieselexhaustonhumancellsexposedtodieselexhaustemissions.



167  Chapter10:Conclusion



10 Conclusion

Thedieselenginehasbeenapopularchoiceamongstlargervehiclesrequiringlarge amountsoftorquetomoveheavyloadsbutwithincreasingfuelpriceandtechnology. Improvements and refinement to the diesel engine, has also seen the use of diesel enginesinpassengercarsgaininpopularity.Themainappeal ofthedieselengineisits higherfuelefficiency,lessemissionsoftoxicgas(e.g.CO)andabilitytousebiodiesel withlittlemodificationtotheengine.AlthoughdieselengineemitslessCOcompared to the petrol engine, there is more emission of the irritant NOx, allergen, whilst enhancingPM2.5emissions.Epidemiologystudieshaveestablishedanincreasedriskin therateofcardiovascularandpulmonarydiseasesbyexposuretoNOx(Gamble,Jones et al. 1987; Gamble, Jones et al. 1987; McConnell, Berhane et al. 2003) and PM2.5 (Dockery,Popeetal.1993;Abbey,Ostroetal.1995;McDonnell,NishinoͲIshikawaet al.2000;SchwartzandNeas2000;PopeIII,Burnettetal.2002).InͲvitrotoxicologyhas provided an insight into PM2.5’s mechanism in causing damage to pulmonary and cardiovascularsystems.Althoughthespecificmechanismincausingdamageisstillnot wellunderstoodmodelshavebeen derivedsuch as PM2.5deposition location within thelung(Oberdorster,Oberdorsteretal.2005)anditsmechanismwithinbloodvessels (Bai,Khazaeietal.2007).

Indirectexposurestudiesofdieselexhaustarecommonlyperformedwhichrequirethe purificationand/orextractionofspecificPMcomponentsfromcollectedexhaustand reͲsuspendingtheextractedcomponentswithin culturemedia(Bayram,Devaliaetal. 1998;Rumelhard,Ramgolametal.2007;Song,Zhouetal.2007;Hirota,Akimaruetal. 2008;Omura,Koikeetal.2009;Patel,Eoetal.2011).Howevertheresultsdescribed inthisthesisshowedthatdirectexposureofdieselexhaustonvarioushumantarget cellsear possibleandhasthepotentialforassessingtoxicityofdieselexhaust.

TheaimofthisthesiswastoexplorethepotentialofinͲvitrotestmethodsinassessing thetoxicityofdieselexhaustanditsapplicabilityforassessingrisks.Inthisthesis,a dieselenginewasrunatvarious settings(speedandtorque)anditsexhaustsampled for direct exposure to cells to draw any correlation between generated exhausts’

168  Chapter10:Conclusion characteristics and cell viability of exposed cells.Multiple human cell lines derived from organs of toxicological significance and a diverse range of inͲvitro cytotoxicity assays were employed including: MTS (tetrazolium salt; Promega), NRU (neutral red uptake; Sigma) and ATP (adenosine triphosphate; Promega).Further, the cell morphologicalchangescaused by exposure to diesel exhaust were also determined. By combining inͲvitro techniques, exposure settings and other chemical analyses developed in this thesis (Figure 10Ͳ1), a comprehensive risk assessment of engine emissionusingvarioustypesoffuelscanbeperformed.

Nanoparticlesareobjectsnanometreinscaleandcaneitherbeintentionally(e.g.ZnO andTiO2)orunintentionally(e.g.CDNPandDEP)produced.Dieselengineparticulates aresmallerthanpetrolengineparticulatesandofthenanometrescale.Nanoparticles’ smallersizeandlargersurfaceareatovolumeratio(Oberdorster,Oberdorsteretal. 2005)posegreaterrisktohumanhealthastheymaypenetratedeeperintothehuman bodywiththepotentialtocausesubstantialdamage.

Inapreliminarystudy,theapplicationandmeritsofconventionalinͲvitrotechniquesin utilisingcellsuspensionsandexposuretonanoparticlesinculturemediasolutionwere evaluated (Chapter 5).In this study, the cytotoxicity of two commonly used nanoparticleswasevaluatedusing humanskinfibroblast cells at differing periods of exposure.The results showed doseͲresponse curves calculable after 24 hrs of exposure,suggestingverylowacutetoxicityofbothZnOandTiO2nanoparticles.In addition,duetotheinsolubilityofnanoparticlesthisresultedintheirprecipitationat the bottom of wells hence potentially contributing to errors in cell viability measurements.Theseresultsandobservationssuggestthattheconventionalmethod inindirectlyexposingresuspendednanoparticlesinculturemediamaynotbethemost suitable testforassessingthecytotoxicityofnanoparticles.Asdieselexhaustscontain both gases and particulates including ultrafine particles (PM0.1), the ability of an inͲ vitrosystemtotestcytotoxicityofbothgasandparticulatesincludingnanoparticlesis highlyimportantandessential.

AninͲvitrosystemwasdevelopedtoallowdirectexposureofcellstobothgaseousand PM components of diesel exhaust, therefore requiring cells to be grown on an

169  Chapter10:Conclusion interfaceinwhichsimultaneousexposureandnutrientreceivalispossible.Asystem comprising of cells grown on a porous membrane was developed (Chapter 6) and tested with a model volatile organic compound (Toluene).Toluene was introduced intotheexposurechamber,lefttoevaporateandmakedirectcontactwithcellsgrown onaporousmembrane.Thecellviability(in%)wasthendeterminedusingvariousinͲ vitroassaysincludingMTS,ATPandNRU.Theresultssuggestedaveryreliablemethod toassesscytotoxicityofgaseouscontaminantsevenafter1hrofexposure,following postͲexposureincubationstostudyeth longereffectsofdieselexhaustincells.

Thenextstepwastoapplythetechniqueofgrowingcellsonporousmembranefor analysingdieselexhaustinbothlaboratorybasedandfieldbasedstudies(Chapter7). To allow sampling of diesel exhaust, a dynamic exposure system was developed by connecting an  exposure chamber in series with a sampling system consisting of a negative pressure pump with a flow rate controller in the form of rotameters.The results from both laboratory and field based studies showed longer periods of exposureinducingmorereductionincellviability,justifyingtheapplicationofdynamic exposuresystemsasamobiledevicefortestingthecytotoxicityofdieselexhaust.The data also suggested the gaseous component of diesel exhausts exert higher toxicity than the PM component as there were similar reduction trends between both laboratoryandfieldbasedstudies.

Thedevelopeddirectdynamicexposuresystemhasbeenshowntobeapplicablewith A549lungcellsandMTS,ATPandNRUinͲvitroassays.Whileinhalationisthemain routeofexposureforgaseouspollutants,smallersizePMhastheabilitytotranslocate intothecardiovascularsystemcausingsystemicdamageswithinthebody.Therefore thedynamicexposuresystemwasdevelopedtoallowotherhumancelltypessuchas HepG2 and skin fibroblasts to be grown on porous membrane without any modificationoftheprotocol(Chapter8).

Thenextstudy(Chapter8)determinedanysignificantreductionincellviabilitycaused by PM component of the diesel exhaust.The study improves on the methodology developedin the laboratorybased study in Chapter 7 by employing more cell types (A549, HepG2 and skin fibroblast) and exposing them to filtered and unfiltered

170  Chapter10:Conclusion exhausts.ThecellviabilitiesweredeterminedusingMTSandNRUinͲvitroassaysto detect any difference in toxicity mechanisms.The results showed no significant differenceincellviabilityreductionbetweenfilteredandunfilteredexhaustemittedby the diesel engine at idle setting, therefore supporting results of Chapter 7 that the gaseous component of diesel exhaust contributes much higher cytotoxicity than the PMcomponent,atleastaftershorttermexposures.

Furthermethodrefinementwasdevelopedtoallowtoxicityanalysisofdieselengine runningunderdifferentsettings(Chapter9).Toallowcomparisonofdieselexhaust underdifferentengine loads,anoptimalexposureperiodwasdefinedastheminimum exposure period in which an engine running at 50% of maximum torque produces below50%cellviability(IC50),whichwasdeterminedtobe15mins.Inaddition,to investigating the “loadͲresponse” relationship, the engine speed was maintained at 1600 rpm as it was the minimum speed to provide maximum torque and the loads weresetat0%,25%,50%,75%and100%ofmaximumtorque.

Multiple humanͲbased cellular systems (A549 and HepG2) and inͲvitro cytotoxicity assays(MTS,NRUandATP)wereemployedtodeterminesuitabilityoftheexposure systemindetermininganycellviabilitydecreasebyincreasingtheloadofthediesel engine(Chapter9).Theconcentrationsoforganiccarbon,elementalcarbonandNOx levels was also determined to identify which pollutant correlated better to the cell cytotoxicityresponse,SEMofcells’surfacemorphologywererecordedaswelltoallow physical observation of morphological changes caused by diesel exhaust.Results showedsignificantreductionincellviabilityat>25%loadsbutnosignificantdifference in cell  viability reduction was found between filtered and unfiltered diesel exhaust.

TheresultssupporttheoverwhelmingeffectofNOxcomparedtothePMcomponent of diesel exhaust, as suggested by results from Chapters 7 and 8.The SEM images showedsignificantchangeincludingblebformationandreductionofmicrovilli.

Results from Chapter 9 provided information on the effect of engine operation parametersonexhaustgeneration.Thestudyfoundcytotoxicitytobecorrelatedwith bothloadandNOxlevel,whileNOxlevelwashighlycorrelatedwithloadsettingofthe engineindicatingthegaseouscomponentsofdieselexhausttoexertgreatercytotoxic

171  Chapter10:Conclusion effects.Further mathematical modelling and analyses by splitting experimental toxicity results into gaseous and particulates exerted toxicity components showed higher correlation between the gaseous component toxicity and whole toxicity as comparedtotheparticulatecomponents.Thisfindingsupportedfindingsofchapter8 whichshowedthegaseouscomponentofexhaustexertedthemosttoxicityoncells.

AsNOxisknowntobeanirritantgasandresultsofthisstudysuggestmoreemphasisis neededinenginedeveloperstoreduceemissionofNOxcomponentinengineexhaust.

InͲvitrotechniquesarecomparativelylowerincostandlesscomplicatedcomparedto in vivo techniques.The developed dynamic direct exposure method fulfilled requirementsforareliableinͲvitrosystemprovidingarapidandinexpensivemethod thatisabletoanalysedifferenttypesofcompoundsof dieselexhaustandtheireffect onhumancells.Inthissystem,differentadherenthumantargetcellsweregrownon porousmembranes,exposedtodieselexhaustundervariousloadsandavarietyofinͲ vitro assays could be used.The results of this study showed that the developed dynamicexposuremethodhasthepotentialapplicationinstudyingtoxiceffectsofa largevarietyofairbornecontaminantsincludingbiodiesel.

Figure10Ͳ1:Dieselexhaustmodellingandanalysisrepresentingpotentialpathways linkingdieselexhaustandcytotoxicityeffects

Futuredirections

Thisresearchprovidesabasistofurtherdevelopmethodsinanalysingthetoxicityof dieselexhaustleadingtoabetterelucidationintheinteractionbetweendieselexhaust

172  Chapter10:Conclusion exposureͲbothparticulateandgaseousformsandtheresultingtoxicmechanisms.The results of this thesis advance technical knowledge of inͲvitro toxicology to analyse dieselexhaustbythegenerationofnewdataapplicabletodieselenginesettingsand exhaustsamplingtechniques.Thesemethodsdevelopedallow doseͲresponsestudies forbetterriskcharacterisationofdieselexhaustoncellviability.Sincethedynamic exposure method uses human based cellular systems, it removes the interͲspecies extrapolation problem associated with animalͲderived data and hence more representativedatafortoxicologicalriskassessmentinhumans.

Although inͲvitro techniques have allowed a more rapid testing of chemical compounds, application of other tools can improve testings even further.One exampleistheusageofinsilicotechniquesforfaster,cheaperandsafertestingsas computermodellingallowstoxicity predictionofhazardouschemicals which are too riskytoperform.Althoughthemost commonapplicationofinsilicomethodsarethe PBPKandQSARalgorithmtopredicttoxicityofchemicals,mathematicalmodellingof inͲvitro methods can optimise methods such as in inhalation toxicology for homogenous delivery of gaseous and PM components to all cells grown on porous membrane.

Byemployingvarioushumanderivedstargetcell andinͲvitroassays,awidervarietyof biological endpoints can be monitored and more elucidation of toxicity mechanisms exertedbycontaminants.Anydifferencesinresultsbetweeneachcelltypesand/orinͲ vitroassayscanbeanalysedtodeterminewhetherobservedtoxicityisbasalororgan specific.Other inͲvitroassaystoincludeformoreinsightintotoxicitymechanismsof diesel exhaust include proͲinflammatory release assays and ROS formation.Other methods such as imaging by TEM (coupled with SEM imaging) would allow physical observation of diesel exhaust effects on the internal structure of cells, some observables characteristics includee increas  in nucleus density, enlargement of mitochondria,increaseinlipiddroplets(Cameron,Neumanetal.1998)andintegrityof membrane(Koningsberger,Rademakersetal.1995).

Future works include improving design of exposure chamber for more homogenous distributionofairbornepollutantoncellsgrownonporousmembrane,morecontrolof

173  Chapter10:Conclusion dieselenginevariablesformoreconsistentexhaustgenerationhencemoreconsistent compositionoftheexhaustcomponents.Combinationofimprovementsinexposure system design, inͲvitro techniques and diesel engine operation can provide more comprehensive toolinelucidating toxicity of diesel engine running on various diesel fuel typesincludingpetrodieselandbiodiesel.

Overall, the developed dynamic exposure method for analysing toxicity of diesel exhaust is a modular system hence various human cells can be used without any change in protocol.By using human cells, cross species correlation issues were eliminatedandbyusingcellsofspecificorgans,more toxicityinformationindetailscan be gathered.Meanwhile its portability allows for rapid toxicity analyses of air contaminant at remote sites such as traffic tunnels, urban and industrial areas.In addition as biofuel is gaining popularity as a petrodiesel alternative, the developed dynamic exposure method allows toxicity comparison of engines while consuming differentfuels.



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197  AppendixA

AppendixA

A1Cytotoxicityassayreagents

MTSreagentpreparation

MTS reagent powder (42 mg; Promega, USA) was added to a Dulbecco phosphate bufferedsalinesolution(DPBS;21ml;Sigma,USA)inalightprotectedcontainer.The solutionwasmixedatmoderatespeedusingamagneticstirreruntilMTSpowderwas completelydissolved.ThepHofthesolution wasmeasuredthenadjustedtooptimum pHof6.0Ͳ6.5thenfiltersterilisedthrougha0.2μmfilterintoasterile15mlcentrifuge tubesandstoredatͲ20oCinalightprotectedcontaineruntilrequired.

PMSreagentpreparation

PMSsolutionwaspreparedbydissolvingPMSpowder(Sigma,USA)inDPBSsolutionat 0.92 mg/ml concentration.The solution was then filter sterilised through a 0.2 μm filter into sterile microcentrifuge tubes and stored atͲ20oC in a light protected containeruntilrequired.MTS/PMSratioof20:1wasusedforMTSassay.

NeutralRed(NR)mediumpreparation

Inpreviousdayofexperiment,neutralRedmediumwaspreparedbymixing600μlof neutral red stock solution (sigma, USA) into 25 ml of culture medium in a 50 ml centrifugetubeandstoredat37oCovernight.Priortouse,thetubewascentrifugedat 1500xgfor5mins,thesupernatantwasthenfiltersterilisedthrougha0.2μmfilter andusefortheNRUassay.

NRUfixativesolution

NRUfixativesolutionwaspreparedbyadding1.3mlformaldehydeanddissolving1g CaCl2in 100mldistilledwater

NRUsolubilisationsolution

NRUsolubilisationsolutionwaspreparedbymixing1mlaceticacid,50mlethanoland 49mldistilledwater.

198  AppendixA

ATPassayreagentpreparation

Prior to use, CellTiterͲGlo® Buffer and Lyophilised CellTiterͲGlo® Substrate were thawedtoroomtemperature.Bothbufferandsubstratewerethenmixedinsidethe amber bottle (provided by manufacturer) to reconstitute the lyophilised enzyme/substrate mixture.The mixture was then aliquoted into sterile 15 ml centrifugetubesandstoreatͲ20oCuntilrequired.

A2Specificationofequipment

Autoclave

Siltex 250D autoclave was used to sterilise pipette tips and glassware.The temperaturewassetat121oCwith15lb/in2pressure(103.5kPa)for15Ͳ20minutes.

Biologicalsafetycabinet

AclassIIbiologicalsafetycabinet(BSC2000series,Australia)wasusedtocreatean aseptic environment to perform experiments involving cell cultures.The aseptic environment was maintained by preventing microorganisms or other contaminants fromenteringtheexperimentalarea.Thispreventionwasachievedbycreatinganair curtaintoactasabarrierbetweentheexternalenvironmentandasepticenvironment within the cabinet.Simultaneously, the air within the aseptic environment is continuouslyfilteredtomaintainsterility.

Centrifuge

Thecentrifugeunit(Sigma,Germany)wasregularlyusedthroughoutthisthesis.

Incubator

Anincubator(Sanyo,Japan)wasusedtocreateanidealcellculturingcondition.The o culturingenvironmentwassetat37 CwithCO2gasconcentrationat5%.Inaddition, humiditywasmaintainedbyaddingawaterreservoirinsidetheincubator.

Lightmicroscope

Alightmicroscope(LeicaWetzlar;Germany)wasusedthroughoutthisthesis.

199  AppendixA

Waterpurificationsystem

A double processed water purification system (Modulab type II; Continental Water Systems Corporation, USA) was used throughout this thesis.The filtration system removed impurities including trace metals and metabolic products of micro organisms).Filtrationwasachievedbyserialfiltrationofwaterthroughdeionisingand organicexchangecolumns.

Freezer

AͲ80oC (Sanyo, Japan) freezer was used as a part of cell archival process.After harvesting,thecells were resuspended in a culture media with 10% DMSO mixture. Thecellsuspensionwasthenaliquotedto1.8mlcryogenicvials(Nunc;Denmark)and transferred onto a rubber rack.The rubber rack acted as a temperature decrease controllertolimitrateoftemperaturedecreaseduringfreezingthereforepreventing ice formation within cells.The vials were left overnight before transfer into the o cryogenicstorageDewarforpreservationatͲ197 Cusingliquidnitrogen(LN2).

Luminometer

A luminometer (Orion II microplate luminometer; Berthold Detection Systems, Germany)wasusedthroughoutthisthesis.Theluminometermeasuredluminescence output of luminescent based inͲvitro assays.The luminescence readings were recordedbySimplicitysoftware.

Microtitreplatereader

Amicrotitreplatereader(MultiSkan;ThermoLabsystems;Finland)wasusedtorecord absorbancesfromabsorbancebasedinͲvitroassays.Theplatereaderwascapableof readingabsorbanceof400Ͳ750nmlaserrange.Theabsorbancewererecordedbya WindowsXPbasedAscentsoftware.



200  AppendixB

AppendixB

Optimisationofcelldensityfor96wellplates





201  AppendixB



202  AppendixC

AppendixC

Optimisationofcelldensityforgrowingcellsonporousmembrane

200000

150000

100000

50000

Luminescence (RLU)Luminescence (a) 0 0 5.00u10 5 1.00u10 6 1.50u10 6 Cell density (cells/ml)

150000

100000

50000

Luminescence (RLU)Luminescence (b) 0 0 2u10 05 4u10 05 6u10 05 Cell density (cells/ml) 

OptimisationofA549cellswiththeATPassay

(a) Recorded luminescence; (b) With cell density range to give most linear luminescencecurve,95%confidenceintervalcurveissuperimposedoncurve.

203  AppendixD

AppendixD

Combustionchamberpressurewaveperstrokecycle

204  AppendixE

AppendixE

Correlation between generated results from mathematical model and experimental data

Experimental ɴ=0ɴ<ɴmax/20ɴ<ɴmax/4ɴ<ɴmax/2ɴmax data

A549MTS 0.8341 0.8190 0.7220 0.6310 0.6310 filtered

A549MTS 0.8180 0.8367 0.8678 0.8536 0.8536 unfiltered

A549NRU 0.8216 0.8347 0.8426 0.7990 0.7889 filtered

A549NRU 0.9784 0.9698 0.9175 0.8833 0.8833 unfiltered

HepG2MTS 0.7020 0.7094 0.7004 0.6809 0.6809 filtered

HepG2MTS 0.8825 0.8477 0.6936 0.5269 0.4004 unfiltered

HepG2NRU 0.9812 0.9747 0.9222 0.9029 0.9029 filtered

HepG2NRU 0.9644 0.9520 0.8658 0.7384 0.6129 unfiltered

A5490hNRU 0.8745 0.8714 0.8714 0.8714 0.8714 unfiltered



205