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In-Vitro Systems and Modelling of Diesel Combustion Toxicology

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In-Vitro Systems and Modelling of Diesel Combustion Toxicology 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). ………………………….. …………………………… ……………………… Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. 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. Signed Date Copyrightstatement ‘I hereby grant the University of New South Wales or its agents the right to archive and to makeavailablemythesisordissertationinwholeorpartintheUniversitylibrariesinallforms ofmedia,noworhereafterknown,subjecttotheprovisionsoftheCopyrightAct1968. Iretain allproprietaryrights,suchaspatentrights.Ialsoretaintherighttouseinfutureworks(such asarticlesorbooks)allorpartofthisthesisordissertation. IalsoauthoriseUniversityMicrofilmstousethe350wordabstractofmythesisinDissertation AbstractInternational (thisisapplicabletodoctoralthesesonly). IhaveeitherusednosubstantialportionsofcopyrightmaterialinmythesisorIhaveobtained permission to use copyright material; where permission has not been granted I have applied/willapplyforapartialrestrictionofthedigitalcopyofmythesisor dissertation.' Signed Date Authenticitystatement ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approvedversionofmythesis.Noemendationofcontenthasoccurredandifthereareany minorvariationsinformatting,theyaretheresultoftheconversiontodigitalformat.’ Signed Date 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 SchoolofRisk andSafetySciences,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
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