cisas proposal 28092016 · physiological and behavioral development will be evaluated by means of...

CISAS“CentroInternazionalediStudiAvanzatisuAmbiente,ecosistemaeSaluteumana”

“InternationalCentreofadvancedstudyinenvironment,ecosystemandhumanhealth”

Listofparticipants

ParticipantNo

Participantorganisationname Shortname

1 DipartimentoScienzedelSistemaTerraeTecnologieperl’Ambiente

DTA-CNR

2 Istitutoperl’AmbienteMarinoCostiero IAMC-CNR

3 IstitutodiAnalisideiSistemiedInformatica“A.Ruberti” IASI-CNR

4 IstitutodiBiomedicinaeImmunologiaMolecolare IBIM-CNR

5 IstitutodiFisiologiaClinica IFC-CNR

Collaborations

Participant Organisationname Shortname

1 DipartimentoScienzedellaTerraedelMare-UniversitàdiPalermo UniPA-DiSTeM

2 CentroRicercheAmbienteMarino_LaSpezia ENEA

3 IstitutoInquinamentoAtmosferico IIA-CNR

4 IstitutodiScienzedell’AtmosferaedelClima ISAC-CNR

5 IstitutoSuperiorediSanità ISS

6 IstitutoZooprofilatticoSperimentale(Palermo) IZS

7 UniversitàdiCatania UNICT

8 UniversitàdiMessina UNIME

9 UniversitàdiReggioCalabria UNIRC

10 UniversitàdiPisa UNIPI

11 RegioneSiciliana RegioneSiciliana

12 RegioneCalabria RegioneCalabria

13 ASPCrotone ASPCrotone

14 ASPSiracusa ASPSiracusa

15 ASPMessina ASPMessina

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TableofContents1. Excellence..................................................................................................................................................3

1.1 Objectives............................................................................................................................................41.2 Relationtootherinternational/nationalprogrammes........................................................................61.3 Conceptandapproach,qualityofthecoordinationandsupportmeasures.......................................8

2.Impact...........................................................................................................................................................9Expectedimpacts..........................................................................................................................................9Measurestomaximiseimpact....................................................................................................................10

3. Implementation.......................................................................................................................................113.1 Workplan–Workpackages,deliverablesandmilestones................................................................113.2 Managementstructureandprocedures............................................................................................47D2.12 Theworkinggroupasawhole.......................................................................................................55D2.13 Resourcestobecommitted..........................................................................................................55

4.References...................................................................................................................................................57

1. ExcellenceThe widespread development of human activities in the industrial field, since the post-war period, inadditiontothecreationofaneconomythatplacesourcountryintheeighthplaceamongtheworld’smajorindustrialized Countries has been causing an important and complex phenomenon of environmentalcontamination, with crucial effects on terrestrial and marine ecosystems and human health. A decisiveactionof theGovernmentandtheMinistries inchargeofenvironmentalprotectionallowedto identifyanumberofcontaminatedareasintheItalianterritoryinneedofanurgentandimportantrecoveryaction.Theyaretheso-calledcontaminatedSitesofNationalInterest(SIN),characterizedbysevereenvironmentaldegradationandhumanimpactphenomenaofvariousorigin,substantiallycausedbythedevelopmentofmajor industrialactivities. Inrelationtotheenvironmentalcharacterizationoftheseareas, ithasrecentlybeenconsideredcrucialandnecessarytounderstandthespecificimpactofthesefactorsonthehealthofthe people living in proximity of the above mentioned sites. The environment and human health aregenerally observed and investigatedwith a sectorial approach: rarely have synergic actions been carriedouttostudyquantitativelycause-effectrelationshipsbetweenpollutionandpopulationhealthconditions.The collection of information on the impact of pollution on the environment and on human health is amandatory step for the development ofmodern technologies in the fields of chemistry, systembiology,biotechnology,medicine,etc.Thepresent researchprogramaimsat investigatingenvironmental pollution and its connectionwith theecosystemandhumanhealth,startingfromasignificantnumberofcasestudies(SIN’sofAugusta,MilazzoandCrotone),truenaturallaboratoriessuitableformodernmultidisciplinaryinvestigation.Epidemiologic studies regarding the health effects of environmental contaminants tend to focus on themore susceptible segments of society: in the last years many efforts have thus been employed in theevaluationoftheeffectsofenvironmentalcontaminantsonchildren’shealth.Morerecently,thetheoryof“intrauterine origins of health and disease susceptibility” is gaining interest as a new approach tounderstanding the cause of many multifactorial disorders. In fact, adult diseases may have an in uteroorigin, when suboptimal intrauterine conditions - including exposure to environmental contaminants -inducesirreversiblechanges,whichmanifestthemselvesinpost-natalandadult life. Inthecontextofthepresent research project pregnant women living in the three selected contaminated Sites of NationalInterest of southern Italy, along with pregnant women living in control areas, will be recruited andfollowed-up until delivery. Maternal and umbilical cord blood and placental tissue will be sampled atdeliveryand stored for further analysis. Childrenwill be followed-up in the first three yearsof life: theirphysiological and behavioral development will be evaluated by means of regular follow-up visits andparental survey conducted via multimedia support. Such information will provide new insights on theinfluence of prenatal environmental exposure to pollutants on developmental disorders. Moreover,preservationofhigh-qualityplacental tissuespecimenswillenablethesearchfornewbiomarkersofpre-natal exposure to pollutants and promote better understanding of the mechanisms through whichpotentialdisruptorsaretransmittedfrommothertofetus,thusfocusingdirectlyduringpregnancyfortheearlypreventionofsomeadultdiseases.Thelong-termperspectiveoftheprojectandthepossiblydelayedevidenceforchildren’simpairmentsinphysical,psychological,socialandcognitivehealthrequiretheneedforthecreationofabiobankforthestorageofmaternalandfetaltissues.Furthermore,exvivoandinvitroapproacheswith human andmurine cell lineswill be employed to study these events at themolecularlevel.Invivoanalysiswillbeperformedinamurinesystem.Besides the specific focuson the interactionbetweenenvironmentandhealth, the secondcharacteristicfeature of the present research project is its reliance on biomathematical analysis of the investigtedphenomena.Mathematicalanalysisofbiomedicalproblems,particularly related to terrestrialandmarineenvironment, is not new. In fact, the very first president of the National Research Council of Italy, themathematician Prof. Vito Volterra, is still remembered for the population dynamics equations that hewrote to explain the variation of catches in the Adriatic Sea duringWorldWar I. Since then, the Lotka-Volterra equations have provided a textbook example of the use of nonlinear differential equations todescribeecologicalproblems.However,withthewidespreaduseandthedecreasingcostofeverincreasingcomputationalpower,mathematicalandstatisticalmodelshavebecomemoreandmoreimportantintheevaluation of biological issues, offering objective standards and the ability to aggregate meaningfully

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information from many heterogeneous sources. Modern modelling approach will provide innovativequantitativeapproachesat the studyofdifferent scenarios in variousareasof interest. Inparticular, theadoption of a modern model-based approach, should allow to understand the networks of interactionamongthevariousprocessesandtoidentifypossibleapproachesforanappropriate"manipulation"ofthedifferentcomponents.Thestudyofthemechanismsofinteraction,aswellasofthetransferandtransportofpollutantsinvarioussectors,includingtheassessingofthebioavailabilityand/orbioaccessibility,aretherealchallengetounderstandhowtheenvironmentcanreallyaffecttheecosystemandhumanhealth.1.1 ObjectivesTheprimaryobjectiveofthepresentresearchprogramistounderstandprocessesandmechanismsforthetransferofconventional (heavymetals,POPs, radionuclides,etc.)andemergingcontaminants (eg.,PBDE,antibiotics, pharmaceutical compounds of new generation, antitumorals intensively used in the studiedareasanddischargedatseagenerallywithoutstrictcontrol,etc.) fromtheenvironment(asacomplexofatmosphere,soil,sediment,inlandwatersandseamatrices)toecosystemandtohumans(seeFig.1foraconceptualviewoftheproject).Inparticular,wewanttostudytheimpactofthevariousspeciesofpollutants,usinganintegratedanalysisof the biochemical and biophysical mechanisms determining and regulating the interactions betweenpollutants,ecosystem(overallandatdifferentspecies/populationslevels)andhumanhealth.Theresearchwill focusing, inparticular,ontheselectionanduseofmodelsystems, toxicological testsandbiomarkerscloselyrelatedtotheinvestigatedenvironment.A highly inter- and multidisciplinary approach will be used, with a strong contribution from modelling,aimedattherepresentationandexplorationofdifferentscenariosinvariousareasofinterest.Inparticular,the adoption of amodernmodel-based approach should allow us to better understand the network ofinteractions among the various processes and to identify possible approaches for an appropriateintervention on the different components. The study of the mechanisms of interaction, as well as oftransfer and transportofpollutants in various sectors, including theassessmentofbioavailability and/orbioaccessibility, are the real challenge to understand how the environment can concretely affect theecosystemandhumanhealth.

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!!

SOURCES'Natural'vs.'Anthropogenic

''''''''PATHWAYS'Particles'interface'Diffusion Dispersion'models'Atmospheric'and'ocean''modelling'Deposition'processes'Chemical'modelling'

'''''''FATE'Accumulation'Sedimentation'Stabilty'Bio>/Availabiity'Geomicrobiology'Chemical'modelling'

BIOGEOCHEMICAL'DYNAMICS'

Speciation'Release'Fluxes'at'the'Sediments/Water/Atmosphere'Geomicrobiology'Processes'kinetic'Residence'time'Chemical'modelling'

TRANSFER'TO'ECOSYSTEM'Feeding/Drinking'Breathing'

Ecotoxicology'Toxicodynamics

IN'THE'TROPHIC'WEB'Contaminants'Flux'Bioaccumulation'BiomagniFication''

CONTAMINANTS'IN'THE'ECOSYSTEM

SYSTEMS'BIOLOGY'Biochemistry'interactions'Proteomics'Metabolomics'Gene'networks'Traslational'medicine'Internal'vs.'External'mechanisms''

EPIDEMIOLOGY'Descriptive'Predictive'Biostatistics'Biomonitoruing

TRANSFER

'BY'FO

OD'

Agricu

lture'

Breed

ing'

Fisheries'

TRANSFER'BY'ABIOTIC'PROCESSES'Particles'and'nanoparticles'Chemical'Fluxes'at'the'interfaces

INVENTORIES'BACKGROUND'

'

MAPPING'Distribution'Chronology'Geological'forcing'

TRANSFER

'BY'ABIOTIC'P

ROCESSES

CONTAMINANTS'LIFE'CYCLE

CONTAMINANTS''IN'HUMANS

MULTISYSTEMS)MONITORING)

Mul0ystem)support)Modelling:)complex)systems)

Fig.1Conceptualschemeoftheproject

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1.2 Relationtootherinternational/nationalprogrammesHealth, as defined by the Constitution of theWorldHealthOrganization (WHO), “is a complete state ofphysical, mental and social wellbeing and not merely the absence of disease (1948)“. With this simplephrase, theWHOnotonlyestablishestherightofeverycitizentohaveaccesstothebestcareavailable,but requires every State to “promote the health and welfare of citizens by acting on environment andbehaviourandbypromotingthedevelopmentofsciencesthatcouldhelppreventgeneticandphysiologicaldiseasescausedbybiologicalandenvironmentalfactors“(CharterofOttawa-InternationalConferenceforthePromotionofHealth - 1986). Todate, great strides in theunderstandingof the interactionbetweenenvironmental, behavioural, genetic and/or physiological factors have beenmade, but, above all, itwasrealizedthatthesefactorsshouldnotbestudiedindependentlyfromeachotherbutrelatedtotheharmfuleffectsthatexposurecouldhaveonthefinalreceptor.In1999inGothenburg,attheEuropeanCentreforHealth Policy, through a consultation of experts convened by the European Centre for Health Policy ofWHO,the“AssessmentofImpactonHealth”,wasdefinedasa“combinationofprocedures,methodsandtools thanks towhich it is possible to estimate the potential effects of the policy actions on health anddistributionofthoseeffectswithinapopulation”(ECHPWHO,1999).TheAssessmentofImpactonHealth(AIH) was to provide information to planners and policy makers in particular in relation to theconsequences either of productive and political activities or urban settlements. In that context, it wasestablishedthatAIHshouldbeamultidisciplinarywaythatwouldtakeaccountofthefactthatmanyplantsofpublicinterestshouldbeconsiderednotonlydamagingforthepublichealthandtheenvironment,butalsostrategicfortheprotectionofboththeemploymentandtheproductivelevels.So,itisnotpossibletosolve the problemwith the closure, remediation or securing of the area by turning a blind eye to theirpresence.Itisnecessarythatthetwothingsareabletolivewith.Thearduoustaskimposedasciencepolicybasedonthe“HarmHealthRating(HHR)notonlytoquantifytheimpact(insomesitesitdidnotexceedthe thresholds) but also to assess the exposure, the distribution, the health relevance event rate in thepopulation(epidemiologicalstudies)aswellasassessandquantifytheriskthatitisexpectedinthefuturegenerations (risk assessment). In Italy this process started in 2006, the year of approval of LegislativeDecree 152/2006 (Environmental Law Code) and then the Legislative Decree 260/2010. Within thatregulatory code it is established what a contaminated site is (a site in which the risk thresholdconcentrationvalues,determinedbyapplyingtheriskanalysisproceduresetoutinAnnex1toPartfourthofthedecreeonthebasisofthecharacterizationplanresults,havebeenpassed)andwhatthedifferenceswithanon-contaminatedsiteare(asitewherethecontaminationdetectedintheenvironmentalmatrixislower than the concentration of contamination thresholds or, whichever is higher, it is lower than thethresholdof risk threshold concentrationdetermined following theanalysisofhealthandenvironmentalrisks).Inthelastdecade,inItalyaseriesofnewstudieshaveallowedtoknowmoreindepthsomeoftheterritoriesatriskfortheenvironmentandforthehealthofthepopulation.In2013theMinistriesofHealthand Environment approved the Inter-Ministerial Decrete (24/04/2013) “rules for determining themethodological criteria useful for the preparation of the evaluation report of the health damage inimplementationofArticle1-bis,paragraph2ofthelawdecreeof3December2012,n.207,amendedwiththe Law24December 2012, n. 231 “. This decree, under continual review, laid the foundations for thecreation of amethodological protocol for the realization of a riskmap onwhich it will be necessary todefineactionstrategies.Lots of national and international projects aimed at collecting the huge volume of social health andenvironmentaldata,thatarecontinuouslycollectedandthatwillallowtoobtaintherightstartingpointtodevelop surveillance strategies, at short and long term,of exposedpopulations, havebeen carryingout.Amongtheother,therearethefollowingprojects(thelistisnotexhaustivebutonlyindicative):

- Routes:National Epidemiological Studyof the Territories and Settlements Exposed at risk fromPollution- CCM: Methods for the integrated assessment of environmental and health impact of the airpollution

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-ThehumanExposomeproject-Helix:High-EndClimateImpactsandExtremes-Heals:HealthandEnvironment-wideAssociationsbasedonLargepopulationSurveys-ESAMinistryofHealth-CCMSEpiAs:Epidemiologicalsurveillanceinareasaffectedbyenvironmentalpollutionbyarsenicfromnaturalsourcesorhumanactivities-CCMT4HIA:Healthimpactassessment(VIS):guidelinesandtoolsforevaluatorsandproponents-NRCEnvironmentandHealthInter-departmentalProject:presentknowledgeandprospectsforfutureresearch-WHOHumanHealthinAreaswithIndustrialContamination

In recent years, major efforts from the US research system (eg., NIH/NSF, NOAA) was aimed at thedevelopment of strategies aimed at the creation of thematic research institutes focused on multi- andinterdisciplinaryapproaches for the studyof issues related to the linkbetweenhealthandenvironment.Importantprojectsatinternationallevel,abovementioned,havebeendeveloped(inparticular,inEurope,sincethe5thFPcalls)andrelatedtothestudyoftheEarth'senvironmentimpact(withparticularreferencetoair,waterandsoil)onhumanhealth.More recently, thisefforthasbeendirected to thestudyof theimpact on health with particular reference to the sea. In the European scientific community, withpublicationofthepositionpaperoftheMarineBoard“LinkingOceansandHumanHealth”,theneedforintegratedapproachesandresearchfacilities,stronglyaddressedtothestudyofenvironmentalimpactonhumanhealth,hasemerged.Onceagain,itseemsthatanimportantroleinthisareaislinkedtotheimpactforcinginthemarinesector.TheMarineStrategyFrameworkDirectiveandtheWaterFrameworkDirective,representtheclearefforttoaccomplishandachievetheso-calledGoodEnvironmentalStatusofthesea.Thereisalsotheneedtostrengthenthedialoguewiththelegislativesystemstoimprovethetransferoftheresearchresultsandthuscontributetoinnovate,adaptandharmonize,bothnationallyandinternationally,theuseofindicatorsandproceduresforthemonitoringbythescientificcommunity.Onlyinthiswayitwillbepossibletoinvestigate,inanintegratedmanner,themanyenvironmental,ecological,toxicologicalandclinicalaspectsabletogiveanorganicvisionoftheexistingrelationshipbetweenenvironmentandhealth.In the last 2-3 years, the rich scientific literaturehas stressed theneed to create integratedobservationsystemson theabovementionedproblem,andan important stimulushasbeen specifically givenby theactionsincludedintheframeworkoftheEuropeanHorizon2020program.Itis,therefore,nowmatureandclear the need at national and international level of effective tools for research activities able to createsynergiesandnewmultidisciplinaryapproachesforstudiesofvitalinterestforthepropermanagementoftheenvironmentresourcesandtheprotectionoftheecosystemandhumanhealth.InMarch2010itwasheldinParmatheFifthMinisterialConferenceonEnvironmentandHealthorganizedbytheWorldHealthOrganization.Focusedontheprotectionofchildren'shealthinachangingenvironment,theConferencesettheagendaofEuropeonnewchallengesonenvironmentalhealth for theyears tocome. Itwasstressedthattheplanningand implementationofapolicyandastrategyfortheprotectionofenvironmentalandhealthcannotbepostponed.Furthermore,consideringthattheindoorenvironmentshaveaparticularroleforhealth,andthatthemanagementoftheindoorairqualityrequiresdifferentapproachesthanthosefortheevaluationofairofoutdoorenvironments,WHOpublishednewguidelinesfortheprotectionofpublichealthfromriskscausedbypollutantscommonlyfoundintheairofindoorenvironment.AtEuropeanlevelthere is an ever-growing attention to the effect of the indoor environment (in its physical and chemicalcharacteristics)onhumanhealth.InItaly,atthebeginningof2011,itwaspublishedthe“addressschemefortheschool-basedpreventionofindoorriskfactorsforallergiesandasthma”whichpaytheattentiononthe risks to health in the school environment, not only for allergic reasons, but also because of thepresence of pollutants present in indoor environments. TheNational Health Plan (NHP) 2011-2013 sets,amongthepriorityobjectivesforthelarge-scaleenvironmentandhealthissues,theneedforinclusionofhealthpriorities in thepolicies and standardson air,water,waste, soil, climate change, food safety andchemicalpollutants,withparticularreferencetothehealthprotectionofthemostvulnerablesectionsofthe population.Moreover, the NHP, as regards the chronic respiratory diseases, draws attention to theactivation of risk reduction programs. Again, the Regional Health Plan “Health Plan” 2011-2013 of the

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Sicilian Region places the environment among the determinants of health, especially in a region wherethere are three areas with high risk of environmental crisis due to human activities (in addition to thenatural causes)where the resultsof recent surveyshaveshownsomechanges inmortalityand recoveryprofilesinpopulationslivingnearthemajorindustrialcentres.Theadoptionofmeasuresfortheprotectionofairandwaterquality is, therefore,consideredapriority inorder toreducetheharmfulhealtheffects,bothatshortandlongterm,duetotheexposuretopollutants.InNovember2014,then,itwasapprovedbytheState-RegionsConference,theNationalPreventionPlan2014-2018,whichisalsocharacterizedbythe focus on the assessment of process and outcome, at every level. Interventions of prevention,promotionandhealthprotectionmustbebasedonthebestevidenceofeffectiveness,implementedfairlyandmustbeprogrammedtoreduceinequalities.Amongthemacro-objectivesofhighstrategicvaluesetby the NPP 2014-2018 there are the reducing of the load preventable and avoidable morbidity, themortality and disability of non-communicable diseases and the reduction of potentially harmfulenvironmental exposures tohealth.Along the Italian territory thereare sitesofnational interestofhighenvironmental risk due to human impact: therefore, the prevention of diseases with recognizedenvironmentaloriginrequiresaseriesoftargetedactionsnotonlyforthestudyofthehabitsandlifestyles,but also for the understanding of the connection between exposure to pollution sources and impact onhuman health through the observation and study of the cycle of pollutants and environmental andoccupationalexposures,andaccordingtotheepidemiologicalassessmentassociatedwithbio-monitoringmeasures. And in the exposure assessment and impact it can not be ignored the quality of indoorenvironment(home,school,job,etc.)thatsometimesarenotableto“protect“peopleand,inparticular,thosewhobelong to themost vulnerable groups (children, elderly, individualswithpre-existingdisease)fromthepollutionofoutdoorenvironment.

1.3 Conceptandapproach,qualityofthecoordinationandsupportmeasuresThe CISAS offers a flexible and concrete “complex andmultidimensional tool” and organization schemesuitable to investigate with a modern vision and approaches , the effects environmental pollution onhumanhealthandecosystems.Thesteeringcommittee(SC)worksasthehighestdecisionmakingbodyoftheworkinggroup,monitoringtheprogressoftheproject,withreferencetothetimetable,outputsandexpectedimpacts.WP1 will create the habitat and provide tools for managing the machinery of the project, including: i)coordination; ii)governingtheproject; iii)supporttoconnectingactivitiesoftheWPs; iv)communicationanddissemination.WP2 isaboutEnvironment focusedonthestudyofphysico-chemicalprocessesof themainconventionaland emerging pollutants identified in the different environmental matrices and at the air-water-sol-sedimentinterfaces,specificallyinthethreestudyareaThisWPhasalsotwoadditionaltasksdedicatedtoi)thetransferofpollutantsintheterrestrialandmarinetrophicwebwithconsequentialeffectsonthefoodcompartmentandii)thegenerationofanetworkofmodernearly-warningsensorsformonitoringspecificclassesofpollutantsinthedifferentenvironmentalcompartments.WP3willprovidenewinsightsonmechanismsoftoxicity inmarineanimals,exertedbyconventionalandemergingpollutants(whichcanenterinthehumanfoodchain)identifiedinthreestudyareasaccordinglytothepurposeofCISAS.Differenttaskswerethusideatedinordertoaddressthefollowingissues:

i) dynamicofbiodiversityincontaminatedareas;ii) alteration of transcriptional expression of genes in selected species associated to chemical

“defensome”iii) transcriptomeandepigeneticmodificationsinresponsetopollutants.

Different approaches, including in vitro, in vivo and in silico experiments,will be considered in order tounveilmolecularmechanismsinvolvedinemergentdiseases.Theseinformationswillbeachievedbymeansofmarineanimalmodelsystems.

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WP4aimstodevelopknowledgeabout theassociationbetweenprioritypollutantsspecific foreachareaand selected health conditions, identified on the basis of environmental and epidemiological knowledgeandrelevanceforresearchandinnovation.Becausehumanplacentaisposedattheinterfacebetweenmaternal/externalenvironmentandembryo,itcanbetakenasanenvironmentalmonitoringsystem.Followingthetheoryofthe“intrauterineoriginsofhealthanddiseasesusceptibility”,WP5willinvestigatetheeffectsofenvironmentalpollutantsonplacentalphysiologyandpregnancyoutcomes. Theeffectsofenvironmentalpollutantsonplacental transcriptomeandtheirroleonthepredispositiontolateonsetdiseaseswillbeevaluatedinpopulationsampleslivinginhighlypollutedareascomparedtosubjectslivingincontrolareas.Furthermore,WP5willfocusitsactivitiesontheeffectofenvironmentalpollutantsininvitroandexvivoassayswithhumanandmurinecelllinesontheattempt toevaluate the influenceofsuchpollutantsoncellular response.Finally, suchadatawillbematchedwiththosegeneratedbyfunctionalstudiesinamurinesystem.WP6 will serve, transversally, the scientific investigational WP’s from 2 to 5. In WP6 the informationframework for data exchange and cooperative work among temas will be deployed; data explorationtechniqueswill be used to generate hypotheses from existing data collections; experimental design andstatistical analysis of experiments will be conducted; mathematical models will be built, representingenvironmental phenomena, toxicant diffusion, distribution of compounds in the food chain, exposure ofhuman subjects, and manifestation of diseases due to toxicodynamic effects. Furthermore, in WP6simulations of potential interventions in scenarios of interest will be performed, in order to supportdecisionmakersintheevaluationofalternatives.2.ImpactExpectedimpactsAsynthesisofthemostimportantscientificandtechnologicalimpactsofthisprojectarereportedbelow:

• knowledgeimprovementonthestabilityanddynamicsoftraditionalandemergingpollutantsinalltheenvironmentalcompartments(terrestrialandmarine);

• development of dispersalmodels of contaminants in the different environmental compartmentsandestimateofinventoriesatsmallandlargescale;

• developmentofnumericalmodelsformalizingthebio-geochemicalcycleofcontaminants,togetherwiththestudyofthequalitativeandstructuralpropertiesofsuchmodelsandtheiruseforscenariosimulations.

• improvement of knowledge about transfer mechanisms within the marine and terrestrialecosystems;

• developmentofunderstandingaboutmechanismsand interactionsatbiochemicalandmolecularlevelofcontaminantsinmodelspecies(terrestrialandmarine);

• Improvement of knowledge on ecotoxicology and ecotoxicodynamics of selected groups ofpollutants;

• developmentofepidemiologicalknowledgeinthestudiedareas;• modelspecieswithecotoxicologicalapproachesfocusedonearly-warningapproaches;

• improvementofknowledgeaboutmechanismsoftransferofcontaminantsfromtheenvironmenttohumansbyfood,drinking,breathing;

• building of integrated and innovative systems for monitoring In real time all the environmentalmatrices;

• development of conceptual models oriented to a modern management of landscapes andecosystems;

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• Compilationofreports,whitepapersandscientificarticles,tobepublishedinhigh-impactscientificjournals,soastodisseminatetothewidestpossiblepublictheresultsoftheproject.

• Developmentofdecisionsupporttoolsbasedonscenariosimulations,aimedatsupplyingregionaldecision-makerswithprojectionsastotheexpectedresultsofhypothesizedinterventions.

The CISAS project will mainly contribute to the assessment of the contaminant effects by developingmethods for inferring toxicity via the integrationof in vivo/in vitro studies and in silicomodels. It is thecombinationofthesetwoelementsthatwillprovidethelong-termbasisofapredictiveframeworkthatwillguide decision making in environmental matters. The expected results of the project will have specificimpactinthefollowingareas:

• The characterization of molecular structure and physico-chemical properties of the differentpollutants will be useful for predicting relevant contaminant properties and consequently toestimatetheirpossibleeffectonenvironmentandhumanhealth.

• Theinsilicocharacterizationoftheeffectsproducedbypollutantsonbiologicalsystemswillimpactthescientificcommunitybyprovidingacomprehensivedynamicrepositoryofcontaminanttoxicityinformation,includingbothtoxicologicalinformationbutalsomechanisticinformationondifferentlevelofinteractionswiththemanyotherfactorsatplay.

• The statistical analysis of the gathered information will provide the basis for in silico profiling(parameterdetermination)ofmechanismsofactionandtoxicityrelationshipaswellasthemodelpredictions describing the biological activity and the toxicity behaviour of the toxicants in thehumanbody and in the environment.Moreover the same information could be used to identifysimilaritiesindistributionandbiologicalactivityforthecategorizationoftoxicantsinclassesandfortheestablishmentoftherelationsbetweenexperimentalandcomputedproperties.

• Theconsiderationofplausibleenvironmentalexposurescenarios,willgivethepossibilitytoexploredifferent situations also involving very low concentrations of the different species of toxicants,insufficienttogenerateacutetoxicityresponsesbutabletoproducelong-term(chronic)effectsoreffects that can be transmitted to the progeny. The in vitro experiments will facilitate theestablishmentofearlydetectionofthesepotentiallong-termeffectsmechanisms.

Measurestomaximiseimpacta)DisseminationandexploitationofresultsArelevantactionofCISAS(specificallytheWP1)isstronglyfocusedoncapillarydisseminationfromlocaltointernational level towards different stakeholders and through multiple channels. Considering thebroadnessofthethemesandtheirimpactsonthecommunityofpolicymakers,theprivatesectorandthecivilsociety.A correct and effective information is therefore of the uttermost importance in order to increase theconsciousnessofthecommunityatlargeofthestrategicopportunityofferedinthelongtermbyCISAS.Awareness,understanding,andparticipationarekeydriversofadedicateddisseminationandexploitationplanthatwillbedevelopedasmilestoneoftheprojectandconstantlyupdated, intightcooperationwiththecoordinationandmanagementactivities.Theplanwillbearticulatedinageneralcomponenttargetingmeasuresformultipleusersandtailoredeventsrelyingmainlyondirectinteractionwithtargetedusers.Itseffectiveness will rely on working group network and leverage on the pivotal role of the involvedstakeholders.§ TheWebsite is theproject’s showcase for abroadaudience to get informationandupdates.On the

basis of inputs from the working group, professionals will work in order to guarantee an appealinggraphics,nicepicturesandcleartext,aswellasaneasy-to-navigatestructure.Timing:updatedallalongtheproject

§ Socialmediachannels(e.g.Facebook,Twitter,andInstagram)willtransmitcatchingmessagesforrapiddissemination purposes. They will also allow a virtual dialogue with the same channels of relevant

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stakeholders, including relevant projects/initiatives, and the wider public to feed into the project.Timing:allalongtheproject

§ The project’s Newsletter targets interested subscribers who will increase also thanks to theparticipation of partners in other networks. Timing: delivered every six months till the end of theproject

§ Pressreleases/promotionalarticleswillbetimelyrelatedtorelevant/newsworthyactivities,eventsandresults.Professionalswillbe inchargeofwritingalso tohelp turning facts intostories,e.g. thepressofficesofpartnerInstitutionwhosecontactswillbealsousedforacleverdistribution,includingtonon-specializedmedias.Timing:releasedtimelyallalongtheproject

§ Dedicated material for the exploitation of results, including tutorials, demo for training courses,databaseheritagewillbedesignedanddistributed.Timing:finalizedbeforetheclosingoftheprojectinordertoguaranteetheusageafterit.

Theorganizationofandtheparticipationtoadvantageouseventssuchasconferences,workshopswillbeexploredduringthewholedurationoftheprojectandafteritsend.Someofthemwillbeco-organisedwithrelevantplayers(publicauthorities,privatecompanies,NGOs)toimpactondecision-makingprocesses.Data, metadata and information used by all partners during the project to support milestones anddeliverableswillbeorganized,storedandmadeavailable.Publications(reportsandpeer-reviewedarticles)presentingresultsobtainedwillbeaccessibleeitherthrough“green”and“gold”openaccess(GuidelinesonOpen Access to Scientific Publications and Research Data in Horizon 2020, October 2015). In case of“green”openaccess,theembargoperiodwillbekeptasshortaspossible.3. Implementation3.1 Workplan–Workpackages,deliverablesandmilestonesOverallstructureTheprojectisorganisedaroundsixworkpackages(WP)closelyinterlinkedandstronglyinterconnected.

1) Project’sGanttincludingworkpackagesandtasks.M=MilestoneandD=Deliverablecorresponds

tothedeliverymonth.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

WP1.Projectcoordination,management,andcommunicationTask1.1–Coordinationandmanagement D1.1.1 D1.1.2 D1.1.3

Task1.2–Governance M1.2.1D1.2.1/M1.2.2

Task1.3–Communication,disseminationandengagement D1.3.1

WP2.ContaminantsintheenvironmentTask1.Stateoftheart M2.1 D2.1.1 D2.1.2Task2.Monitoring M2.2 D2.2Task3.Radionuclidesintheenvironment D2.3 M2.3

Task4.TheageofcontaminationM2.4/D2

.4

Task5.Biogeochemicaldynamicsofcontaminants D2.5.1D2.5.2/D2.5.3 M2.5

Task6.Spatialdistributionofcontaminants:vulnerabilityandriskmapanalysis D2.6Task7.Contaminantsintheatmosphere:dynamicsandprocesses D2.7Task8.Contaminantsintheatmosphericdeposition:quantificationanddispersionpatterns D2.8Task9.Contaminantsinthetrophicwebsandfood D2.9Task10.Contaminantsintheenvironmentandeffectsofclimatechange

D2.10.1/D2.10.2

D2.10.3/2.10.4

Task11.Newgenerationsensorsandobservatorynets D2.11

WP3.EcosystemandcontaminantsTask3.1–Evaluationofthestatusofbiodiversityintheselectedareas:Priolo-Augusta,MilazzoandCrotone D3.1.1 M3.1 D3.1.2Task3.2Ecotoxicologicalandbiochemicalanalysesinvitro D3.2 M3.2TASK3.3–INVIVOEXPERIMENTS D3.3.3 D3.3.1 D3.3.5 M3.3TASK3.4–GENEEXPRESSIONPATTERNANALYSISANDEVALUATIONOFEPIGENETICMODIFICATIONSINTARGETSPECIES D3.4.1/D3.4.2 D3.4.3

WP4.EtiologicalepidemiologyTask1.Coordination,analysisandmonitoring D4.1. M4.1Task2.RiskoflivercancerinthePrioloarea M4.2.1 M4.2.2 D4.2Task3.RiskofthyroidcancerintheMilazzoarea M4.3.1 M4.3.2 D4.3Task4.PilotstudyonrenalfunctionimpairmentandearlykidneydamageintheCrotonearea M4.4.1 M4.4.2 D4.4.1/D4.4.2Task5.Disseminationandscientificnetworking D4.5

WP5.MolecularEpidemiologyTask5.1-Bio-accumulationofenvironmentalcontaminantsinplacenta,maternalandnewborntissuesandtheirassociationwithplacentaltranscriptome,pregnancyandlong-terminfantoutcomes

D5.1.1 M5.1.1 M5.1.2 D5.1.2 D5.1.3 M5.1.3 D5.1.4

D5.1.5

Task5.2-Cellularmodelsandenvironmentalpollutantsinbiochemicalandbiomolecularmechanismsofairwaydiseases

M5.2.1 M5.2.2

D5.2.1 D5.2.2 D5.2.3

Task5.3-Invitroandinvivostudiesofmechanismsofimmunomodulationandimmunotoxicity

M5.3.1 M5.3.2 D5.3.1 D5.3.2

Task5.4-Biomolecularmarkersrelatedtoexposureinvitroandcancer

M5.4.1 D5.4.1

WP6.Biomathematics

Task1.DataframeworkM6.1/D6.1.1

Task2.UserandfunctionalrequirementsM6.2.1/D6.2.1

Task3.Referencecollection M6.3Task4.Modelblueprints D6.4.1 M6.4 D6.4.2Task5.Modelformalization D6.5.1Task6.DataanalysisTask7.Reporting D6.7.1Task8.Simulationanddecisionsupport D6.8.1

M4.5

Workpackagedescription

Workpackagenumber 1 StartDateorStartingEvent 1

Workpackagetitle Projectcoordination,management,andcommunication

Shortnameofparticipant DTA IAMC

Person/monthsperparticipant:

Objectives

The Work Package 1 provides tools for creating the CISAS environment, managing and monitoring itsevolution.Tothisend,theWP1implementsallmeasurestoensuretheproperfunctioningoftheprojectinsideandoutsidetheworkinggroup.Itoperatestheadequategovernancemechanismsandsupportsthefull deployment of its potential, including the implementation of communication and disseminationactivities,andthesystematicinformationmanagementanddatasharing.

Descriptionofwork

Task1.1–Coordinationandmanagement(DTA)ThisTaskistheproject’scontrolroomdealingwithallrelevantorganisationandadministrativeaspects.

OperationalworkflowmanagementThisTaskwilldrivetheactivitiesofthepartnership,handlingthedaily information-streambothinternallyand externally. It will promote internal communication, producing effective synergies among WorkPackagesandCoordinator.This Taskwill develop an integratedmonitoring, reporting and internal communication systemaimed tovouchsupportthatisflexible,well-timed,andefficient.Under this Task, the Project Coordinator will promote exchanges among partners and prevent lack ofinvolvementthroughregularcontacts.

Financialandcontractualadministration.TheTaskwillalso:- managetheproject’sresources,distributingworkinggroupcontribution,assistingpartnersonfinancial

issues,monitoringtheexpenditures;- keeprecordsandfinancialaccountsingoodordersothat,atanytime,itwillbepossibletodetermine

financialcontributionamountswhichhavebeentransferredtoeachpartner;- deliver periodic activity reports including an overview of the activities carried out, milestones and

deliverablesachieved;- deliverthefinalreportwithasummarydescriptionoftheprojectcontextandobjectives,adescription

of the main results, the potential impact and the main dissemination activities as well as theexploitationofresults/foregrounds,andtheuseofresources.

Task1.2–Governance(DTA+All)Inviewof thestrategic impactof theCISAS, thedecisionmakingprocessneeds tobeduly supported, inordertostructureasolidgoverningmechanismabletoensurethesustainabilityoftheproject.ThisTaskoperatesthemanagementand interactionof internalandexternalgoverningandsupportingbranchesoftheprojectbeing:

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§ theSteeringCommittee(SC)>>highestleveldecision-makingbody;§ theAdvisoryBoard>>officialadvisorybody.

Adetaileddescriptionofthegoverningbodies’compositionandfunctioningispresentedinparagraph3.2‘Managementstructureandprocedures.Inparticular,thisTaskembracestheaspectsreportedbelow.

Officialmeetings - Preparing, coordinating, executing and providing themeetings, including the kick-off.Thiswillincludetodealwithtechnicalandlogisticissues,draftinganddistributionofagendas,invitations,minutes,andotherrelevantmaterial.

The Final Conference - Organizing the Final Conference, ensuring its smooth running. A report includingmajor results and achievements as well as proposal for the next steps to sustain the strategy will bepresented.

Task1.3–Communication,disseminationandengagement(DTA,IAMC)

InternalcommunicationThe activities will be carried out through the development and management of internal processes ofinformation, communication, sharing of operational programs, activities, data,within the entireworkinggroupoperatingatvariouslevels,fromthestrategictothemanagementone,fromthemonitoringtotheoperationalone.Theworkinggroupwillbemanagedusingstructuredformsofcommunication, includingtheuseof an internalwebplatform,whichwill allow the sharing andexchangingof project documents,publications and articles, as well as any other useful material useful to carry out the activities (slides,periodicreports,summarydocuments,etc.).

ExternalcommunicationactivitiesThisactionprovidesthecreationofanadhoccommunicationplanaimingatspreadingtheresultsoftheprojecttodifferenttarget:universities,researchinstitutes,stakeholders,industriesandthegeneralpublic.Inparticular,focusedcommunicationactionswillbecarriedoutthroughtheorganizationof:

-Akickoffmeetingandthepresentationoftheprojecttomedia;- Seminars andworkshops, also international,where the results of the projectwill be presentedanddiscussed;-Communicationsonsocialcommunities,websitesandotherchannels,toattracttheattentionofawideraudience;- A final conference open to international experts, where the results of the activities will bepresentedtotheresearchcommunity.

Information,orientingandtrainingTheactivitieswillbe finalisedtoprogramsof information,educationandorientingofa largeaudience inorder to spread the knowledge on the environmental protection and its impact on human health. Theprogramwillallowtotrainanewgenerationofactors,fromdifferentsectorsofsocietytofacemultiandinterdisciplinary aspects related to the effect of environment on human health and to drive modernapproachestotheprotectionofenvironmentanditsresources.•AudienceTheaudienceoftraining,informationandorientingactivitieswillbeconstitutedbyatleast800individualsselected among NEET 18-29 population; entrepreneurs, unemployed workers, self-employed, managersandofficialsofSicilianlocalauthorities.•TypeofactionTherequestedserviceconsistsincarryingoutthefollowingactions:

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1. Training/information activities designed to discuss the problem of the environmental protection in arationalcognitiveframeworkcoherentwiththedifferentstrategicandorganizationalconditions;2. Training activities aimed to promote the socialization of the trainees and enhance the learning levelsespeciallywithregardtothemanagementmodel,theleadership,andtheteamwork;3.Technicalandscientificandoperationalcoordination,planninginthemaking;4.Evaluationoftheresults.

Thetrainingwillconsistof:

•frontallecturesinclassroomholdbyexpertsofthedifferentsubjects,spacedoutwithdiscussionsbetweenteachersandtutors;•follow-upmeetingsandeducationalsimulationsingroupcoordinatedbytheTutor;•exercisesandresearchesinthecomputerlabunderthedirectcoordinationofateacherexpertincomputerscienceandtheTutor;•speechesandseminarsorganizedbyqualifiedexperts;• study visits to be carried out in local contexts, under the direct responsibility of the ProjectCoordinatorandtheTutor;•sessionsofevaluationofthedegreeoflearninginthepipelinethatwillcloseeachofthethematicsteps(trainingstepsandforms);• internships in enterprises dealing with the planning and implementation of measures ofprotectionoftheenvironmentandhealth.

Modeloftraining/information/orientingactivitiesThecourseisorientedtothelearningoftheoreticalconceptsthatwillsupportthepracticeandapplicationactivities. The teaching and training methodologies that will be favoured during the execution of theTraining-Information Course will be interactive, to facilitate the acquisition of operational tools andcompetences. The chosenmethodswill allow developing a systematic approach for themanagement ofproblems, through the three phases of analysis, diagnosis and solution. These activities will allow thetrainees to interactwith the rest of the group in order to promote the discussion and develop a betterunderstandingoftheenvironment.Thetrainingmethodologiesthatwillbecarriedoutarelistedbelow:

•frontallectures•studyanddiscussionofadhoccases•seminars•workinggroups•educationalsimulations•practicalexercises.•creationofwebsitesandinformationportals•workshops

Thedetailedstudyofsomespecifictopicswillberealizedthroughdistinctphaseslinkedwitheachotherinanongoingprocessofexchangeandintegration:

•duringthetheoretical-generalphase,theteacherswillprovidestudentswiththekeycontentonthespecifictopicsofthecourse,thebibliographicmaterial,thenecessaryreferencesfortheresearch,thebasic elements for the knowledge of the most interesting companies in the field, the possibleguidelinesforthebestperformanceoftheprofessionalandoperationalactivitiesintheframeworkoftheLocalAuthority;•duringthespecializedphase,thestudentswillhavetheopportunitytodeepenspecifictopicswithvisitsandinternshipsatpublicinstitutesand,wherepossible,qualifiedprivateenterprises,underthe

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supervisionofteachers/tutorsandexperts;•duringtheexperimental-applicationphase,thestudentswill learnmanagementtechniquesaswellas instrumental and organizational methods necessary to better manage the future professionalactivity;• during the practical phase of the project work on the simulation of a case study operationalmomentswillbeidentifiedinoccasionofwhichthetraineeswilldevelopatechnicaldossierrelatedtoarepresentativeexamplesuitablychosen;• during the implementation phase of project work, the students coordinated by professors andexperts,andunderthedirectguidanceofthetutor,willbeabletoengageeffectivelyinissuesrelatedtothesimulationoftheactivitiesthatwillplayafterthecourseinordertogaindirectexperienceandoperational skills useful to get in touch with the labour market. The case study will be chosen infunction of its uniqueness and problematic situations as to enable diversified and concrete didacticexamplesanalysis.

LogisticalresourcesandequipmentDuringthecoursethemoretraditionaltechnicalapproachtothedifferentmatterswillbeintegratedwithapplicationsoftwarecoveringthetopicsinthecourse;furthermore,informationusefulforthenavigationinthewebandforanefficientuseofinternetforfuturegoalswillbeprovided.Theywillalsobeshownandusedinexercisesandsimulationstechnologiesandtelematicstools,whicharealsoe-commerceandaccessnetworks.

•managementofthetrainingprojectASecretariatOffice(SO)willbeactivatea inasuitableareaofPalermo,properlyequippedtohandlethedifferentorganizationalphasesofthetrainingproject.Inrelationtothemanagementproceduresprovidedfortheoperationalcoordination(CO),thetechnicalstaffwillbeconstitutedby:

• No.1generalcoordinatorofthetraining,informationandorientationactionsinallitsphases.Hewillbeincharge for the implementationof thestrategicandoperationalobjectives, coordinating the trainingwiththenon-training(re-design,placement,follow-upandevaluationofresults)action.No.1coordinatoroftheeducationalarea.Thecoordinatoristhereferencepointfortheimplementationofthe training activity. He translates the guidelines of the training activities of the project in choices andoperationalactions.

• Tutor.For thewholedurationof theproject,classroomtutorswhoarepredominantlycompetentonthemainthemesoftheprojectwillbeemployed.Thefunctionofthetutorfigureintegratesandenrichestheeducational process, making the link between trainers and trainees, among coordinator and classroomactivities, including training and non-training experiences, among the educational structure and those inwhichtheeducationvisitsandthe internshipwillbeperformed.Withrespecttothe interactionbetweentrainers and trainees, the tutor has the function to support the classroom activities and the learningprocessesingeneral,topromotethecorrectandpositiveinterpersonalrelationshipsamongstudentsandteachers, to encourage the "workgroup" and the "team play". The tutor, therefore, must: ensure themethodology, the teaching continuity, contribute to the organizational coordination, the control andconsistencyof theprocessof training / information /orientation,promote the identificationof learning,ensuringthecorrespondencebetweenthetrainingactivitiesandtheobjectivesdefinedatthedesignandplanningofthecourse,assistintheorganizationofeducationalvisitsandinternships.TheCOisentrustedwiththedidacticorganizationofthetraining,informationandorientationactivitiesandwith the development of appropriate educational and organizationalmeasures necessary to ensure theconsistentandfullimplementationofthetraining,informationandorientationactivities.ThetrainingactivitieswillbecarriedouteitheratthedetachedunitofCapoGranitolaoftheIAMC-CNR,atthenewRooseveltInstitute,andatadditionallocationsthatwillbeidentified.Theminimumfacilitiesthatmustbeensuredineverycentrearethefollowing:

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Classroom equipped with slide projector and screen, interactive multimedia board, sound system, TV,telephoneconnection,foratleast50students;2computer labsequippedwithat least13"PCstations",mastercomputer,connectedtothe intranet, tohost classes of 25 students (2 students per station) and lecturer / tutor, equipped for Internet, printconnection,MS-DOSWindowsoperatingsystem,basicOfficeprograms.

Itmust also be also assured the access and the ordinary use, even in periods of interruption of schoolactivities:Inparticular:the technical and scientific coordination will develop actions of management and control, both at thebeginningandongoing,of:

o thecoherencyoftheeducationalfrontalactivities,theseminars,theeducationalvisitsandthestage,withthecontentsandtheeducationalobjectivesfixedtheapprovedfinalproject;o the relevance of the contents developed in the course with the emerging problems in the localcontextinwhichitoperates;o thequalityofthecourseactivitiesandlevelsoflearning;byindicating,whereappropriate,andfromtimetotime,thecorrectiveinterventions.

The Operational Coordination will develop, both in terms of educational profile and of technical andorganizationalaspect,appropriateactions:

§ implementationofcorrectiveactionsindicatedbytheTechnicalandScientificCoordinator;§ organization and direct control of the learning experience control, the learning conditions of thestudentsandtheirwelfare;§ organization and assessment of the operational procedures related to the organization and theeffectiveimplementationoflectures,seminars,educationalvisitsandthestage;§ organizationandverificationof the implementation,ontimeandwithinplannedways,of teachingsimulationactivities,projectworkandperiodicassessmentoflearninglevels;§ organizationandassessmentoftheeducationalandoperationaleffectivenessoftheassistanceandtutoringactivities;§ organization and assessment of the educational and operational effectiveness, of the distancelearningactivities;§ efficiencyassessment, inthevariousteachingsites,oftheequipmentandtechnical, ITandlogisticssupport.

§

Partofthebudgetforthe“Information,orientingandtraining”activitieswillbeallocatedforsubcontractingtheexternalsupport.

Listofdeliverables

D1.1.1Firstmid-termreport(M18)

D1.1.2Secondmid-termreport(M24)D1.1.3Finalreport(M36)D1.2.1ReportoftheFinalConference(M36)D1.3.1Website(M3)

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Listofmilestones

M1.2.1Kick-offmeeting(M1)

M1.2.2FinalConference(M36)M1.3.1DisseminationandExploitationPlan(M2)


Workpackagetitle Contaminantsintheenvironment

Shortnameofparticipant IAMC IZS IIA ISAC ENEA DISTEM


Objectives

ThisWPwillbefocusedonthestudyofphysico-chemicalprocessesofthemainconventionalandemergingpollutants identified inthedifferentenvironmentalmatricesandattheair-water-sol-sediment interfaces,specificallyinthethreestudyareaofAugusta,MilazzoandCrotone.Particularly,analysisofcontaminationmechanisms for a selected number of priority pollutants (e.g., behaviour of the chemical species in thevarious compartments, distribution, dispersion modes, kinetics of transformation, etc.) in the selectedstudyareaswithdirectandindirecteffectsontheecosystemandhumanhealthwillofferimportantfieldsof interdisciplinary studiesand researches.Applicationofmodelling toexploreeffectsof synergicand/orantagonisticeffectsofmixtureof contaminantswill representa cuttingedgepartof thisWPgoal.Also,dynamicsofpollutantsdrivenbypreferentialgeospatialsystemsandconsequentpotentialrisksassociatedto the population health will be also investigated along with potential impacts of climate changes onchemicalreactivityandpotentialamplificationofcontaminantseffectsontheecosystemandhumans.ThisWPhasalsotwoadditionaltasksdedicatedtoi)thetransferofcontaminantsintheterrestrialandmarinetrophicwebwith consequential effectson the foodcompartmentand ii) thegenerationofanetworkofmodern early-warning sensors for monitoring specific classes of contaminants in the differentenvironmentalcompartments.

Descriptionofwork

Task2.1.Stateoftheart(IAMC)Thistaskwillbeorientedtoacompletedatacollectionofavailable(intheclassicinternationalandnationalscientificliteratureandinthegreyliterature)informationonthecontaminantsandtheirdistributioninthethreestudyareas(Augusta,MilazzoandCrotone).Datasetforalltheenvironmentalcompartments(soils,sediments, waters, biota, etc.) will be initially assembled in GIS platforms useful as primary drivers androbust startingpoint for thenext research steps, but also immediately valuable fordecisionmakers andscientistsandwillrepresentaforthe.

Task2.Monitoring(IAMC)This task aims at monitoring and analysing distribution patterns of pollutants of relevant impact onecosystem and human health in the three study areas. In particular, for the Augusta area, a specificattentionwill be paid at the distribution of Hg, Cd, Zn , Pb, THP, Dioxins and furans, Esachlorobenzene,PolycyclicAromaticHydrocarbons,PCB,BTEXwellknownfrompreviousinvestigation,tosignificantlyaffect

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thedifferentenvironmentalcompartments(e.g.,Cundyetal.,1997,DiLeonardoetal.,2007,Ausilietal.,2008, ICRAM,2008,Bellucci et a., 2012,Bagnatoet al., 2013,Bonsignoreet al., 2013,Bonsignoreet al.,2016,SalvagioMantaetal.,2016).FortheMilazzoarea,investigationwillstartfromanalysisofPb,Hg,Ni,Cr,Cd,PAHandBTEX,againreportedasprimarycontaminantsinthisarea(e.g.,Pepeetal.,2010Duplayaetal,2014,Cosenzaetal.,2015,GrechiandBiggeri,2016).Finally,Cd,Zn,As,Pb,CuandHgwillbe initiallyinvestigated in the Crotone area (e.g.,Molinari et al., 2002, Di Stefano et al., 2005, Renzi et al., 2005).However, a complete screening of priority contaminants reported in theWater Framework andMarineStrategyFrameworkDirectivesandNationaldirectiveswillbecarriedoutforthedifferentmatricesalongwith a statistically significant analysis of emergent contaminants (specifically, PBDE, TBT and a selectednumbers among the 50 pharmaceuticals reported in thewatch list of theWFD) and considered prioritypollutantsfortheenvironmentandecosystem).Geostatisticalanalysisofthedatasetwillprovideareliable“image”ofspatialdistributionoftheselectedcontaminantsinthedifferentenvironmentalmatrices(soils,sediments,drinkingwater,biota,etc.)forthethreeselectedstudiedarea.An appropriate sampling strategy will be statistically oriented and specifically assessed defined for thedifferentcompartments.

Task2.3.Radionuclidesintheenvironment(IAMC,ENEA)Anassessmentwillbemadeofgammaemittingradionuclidesconcentrationsandinventoriesinsedimentsofthethreestudyareas.Thesurveywillbeusedtodefinebackgroundlevelsandidentifythemostsuitableareas to collect sediment cores for the reconstruction of pollution trends in the last century (Task 2.4).Radionuclidesare infactpowerfultoolstodatesedimentcoresandtheyallowthedefinitionofpollutionhistoryinareaswhereregularmonitoringprogrammeshavenotbeencarriedoutinthepast.Theaccuracyof the method however is strictly influenced by the selection of appropriate sampling points. A goodmappingofradionuclidesconcentrationisabasicinformationtoselectthemostsuitablecores.

In the Augusta and Crotone areas particular attention will be devoted to the Naturally OccurringRadioactive Materials (NORM).NORMare found everywhere in the environment: they represent anintegralpartoftheplanet.Ontheotherhand,processingofsomenaturalresourcesconcentratestheNORMtoadegreethattheymayposeriskstohumansandtheenvironment.Inparticular,theuseofphosphogypsumfortheproductionofphosphoricacidbythefertiliserindustryproduceslargequantitiesofwasteswithenhancedlevelsofNORM.IntheindustrialplantsinAugustaandCrotone,residuesfromtheproduction of phosphoric acid of the last century were stored near the shore. The survey will clarifywhether this practice produced enhanced NORM levels in the marine environment (seawater andsediment).

Task2.4.Theageofcontamination(IAMC,ENEA)A number of selected marine sedimentary cores dated by 210Pb and 137Cs and analysed for differentpollutantswillprovideanaccuratehistoryofthecontaminationforthelast~100yearsinthestudiedareasand a preliminary discrimination of relatively old and recent contamination sources and pathways.Depositionandaccumulationmodelsofcontaminantsinsoilsandsedimentswillbecalculatedalongwiththeinventoriesforthedifferentcontaminantsandthebackgroundvaluesforeachsinglepollutant.

Task2.5.Biogeochemicaldynamicsofcontaminants(IAMC)A number of specific activities will e carried out to investigate and reconstruct the biogeochemicaldynamicsandkineticsoftheselectedcontaminantsforthedifferentstudyarea: i)Generationofcoupledmodels atmosphere-sea-sediments for the different pollutants combined to numerical dispersionsimulations driven by circulation dynamics, speciation and reactivity of the of contaminants; Ii)Investigation of kinetics of sorbing/desorbing of contaminants at the sediments/soil-water-atmosphereinterfaces calculated for accurate forecasting and provisionalmodelling; iii) Estimates of availability andbioavailability of contaminants in the terrestrial and marine ecosystems; iv)) Analysis of sources andpathwaysofcontaminantsbyisotopetracers,specificallyisotopesofHg,Cd,PbandZn,inallthedifferent

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environmental matrices; v) Modelling focused on deeper understanding of effects of mixture ofcontaminants and relevant patterns of these cocktails in terms of spatial and kinetic distribution; vi)Analysis of impacts of microbiological communities on the kinetics and distribution patterns ofcontaminants, in terms of speciationmodels and chemical reactivity; vii) Deployments of a statisticallysignificantnumberofbenthicandevasionchamberswillofferspecificinformationanddataforcalculationofcontaminantfluxesatthedifferentinterfacesandmassbalanceforthedifferentpollutantsinthethreeenvironmentalsystems.

Task2.6.Spatialdistributionofcontaminants:vulnerabilityandriskmapanalysis(IAMC)A specific analysis of the vulnerability associated to the geospatial distribution of contaminants will beprovided.Couplingofbiogeochemicaldynamicswithpreferentialtransportmechanismsofcontaminantsinturn associated to geological and geomorphological features (channels, landslides, etc.) will provide amodernandspecificapproachtothemanagementsofcontaminantrisks in thestudiedareas.Moreover,potential impactsofgeologicalprocesses(landslides,earthquake,tsunamis,etc.)willbeevaluatedonthebasisofaspecificactivityofriskanalysisandgenerationofmapsofrisk.

Task2.7.Contaminantsintheatmosphere:dynamicsandprocesses(IIA)Distributionofsuspendedparticulateinatmosphere(PM10,PM2,5,PM1)andchemicalcompositionwithaparticularreferencetothebioavailableandresidualandorganicpollutantswillbeinvestigated.Invitroandinvivotoxicitywillbeevaluatedforthedifferentparticulatefractions.Analysisofpollutants(organics:PAH,PCB,Dioxinsandfuransandinorganics:heavymetals)inthedifferentPMfractionwillprovidethechancetoreconstructsourcesofcontamination.Bioavailableandresidualfractionswillbeestimatedbychemicalfractionationmethods.Thesampledparticulateandthedifferentcomponentswillbeinvestigatedtorevealtheir oxidative potential a and mutagenity impact on in vitro cellular determination. Genotoxicity andoxidative stress will be analysed on in vivo tests by bioindicators (gammarids of the Echinogammarusveneris(Heller1865)species).Differentmechanismsofactionwillbeverifiedbyanalysisoftheinsolubleandsolublephases).

Task 2.8. Contaminants in the atmospheric deposition: quantification and dispersion patterns (IAMC,DISTEM)Amongthevariousspeciesemittedbyanthropogenicsources,traceelementsplayanimportantroleduetotheir potential toxicity. Gases and particulates emitted by industrial activities are involved in manychemical-physical processes and removed from the atmosphere by wet and dry deposition (rain andadsorption on the surface of particulate, respectively). Upon deposition, the adsorbed materials onparticulate can be dispersed into the environment, where they have the potential to injure vegetation,livestockandpeople.Onthisbasis,theUNIPAtaskaimstoreportadetailedcharacterizationofmajorandminor trace elements in anthropogenic emissions (gases and aerosols) and atmospheric deposition (rainandparticulateasscavengerandcarrierofatmosphericpollutants).Moreover,chemistryoftherainwateranditseffectontheenvironmentsurroundingtheindustrialareaswillbeinvestigated.

Theraingaugeswillbeusedforsamplingbothrain-watersandparticulate.Chemicalcompositionofrain-waters will be determined by IC, ICP-OES, ICP-MS, CVAFS for major, minor and trace elementsdetermination. Besides, the study of the mineralogy of the salt sublimates formed onto particulate’ssurfaceswillbeperformedbyXRD(Ob1).Amongwith rainwaters, characterization of bio-indicators as proxies for anthropogenic pollutionwill beperformed.Theaimofthispartwillbeaimedtosampleandanalyzeenvironmentalbio-indicators(plantsand soil) to investigate and estimate the impact of pollutant through atmospheric deposition on Earth’ssurface.Allthelivingorganismscanbeconsideredpotentialbioindicators,fortheirbeing“livingcreatures”,sensitive to external stresses. The biomonitoring offers a key for original literature, not only based onsimplequantitativeapproachesbutalso,andmostly,onaqualitativeapproach,consideredasanessentialaspectinordertoevaluatetheconsequencesofthedeviationfromthenormalcompositionoftheair,the

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soil,andthewateronthelivingbeings.Theuptakeofelementsfromtheenvironmentbyplantsmayoccurviatheroots,foliageandstem.Elementsmaybesubsequentlyredistributedbytransportprocesseswithintheplant.Thedominantuptakerouteformostelements isthroughthesoilalthoughabove-groundplantorgans may accumulate airborne pollutants. The applied methodology will be based on passivebiomonitoring, foreseeingtostudythe localorganisms (samplingandanalysis)present in thestudyarea.Thesamplingstrategywillbeaspatialinvestigation,tovalidatetheeventualanomalousbioaccumulationoftoxicelements(heavymetals),andtherelatedvariationswiththedistanceoftheanthropogenicsources.Sampleswillbecollectedduringseveralcampaignsinthestudyareas,followingthemethodologyproposedby the international agencies involved in environmental monitoring. Several monitoring stations whichrepresentenhancedalterationwill bedistinguished foreacharea, andoneor severalubiquitous vegetalspecies will be studied in more detail. Several campaigns will be organized to sample plants and theirrhizosphere,thethinstrataofsoilcoveringtheroots.Theanalyseswillbecarriedoutbyspectrometry(ICP-MS,ICP-OES),methodspermittingtodetectextremelylowconcentrations.

Task2.9.Contaminantsinthetrophicwebsandfood(IAMC,IZS)Investigationofastatistically significantnumberofmarineandterrestrial species,covering thecompleterange of the different trophic webs will be carried out in order to verify modes and transfer times ofpollutants andNORM from the environment to the consumer stomach. Isotope tracers (light and heavyisotopes)will provide specific quantitative tools for identifying pathways of contaminants in the trophicwebsandeventuallyinfood.Quantitativeinformationwillallowanaccuratediscriminationoftheeffectsofcontaminantstransferthroughspecificdietandcrucialadviseforthestudiedhumanpopulations.

Task2.10.Contaminantsintheenvironmentandeffectsofclimatechange(ISAC)This task aims to better characterize the current and future climate conditions of Sicily and theMediterranean Basin, a region considered a “climate change Hot-spot”. This Hot-spot region is animportant crossroad for air masses having different origins and characterised by specific contaminantswhichcanstrongly influencetheatmosphericcomposition,climateandhumanhealth.At thesametime,thechangesintheatmosphericstructurecausedbyclimatechangecouldalterthefuturecompositionandvariability of the atmosphere influencing the human activities and the environment. Therefore, regionalclimate,airqualityandenvironmentinformationisneededtostudyandbetterassessimpactandclimatechange adaptation. In this contest, and in agreementwith European EnvironmentAgency, theproposedresearchactivitiesinthistaskwillsupportawin-winstrategyinthefightagainstcontaminants/airpollutionandclimatechange,consideredtwosidesofthesamecoin.

Inordertoensurecontinuousobservationsofseveralcontaminants,enclosedShortLivedClimateForcers–SLCF(ozone,blackcarbon,methane),andLongLivedClimateForcer–LLCF(carbondioxide),solarradiationalongwithchemicallyreactivegases(nitrogenoxides,sulphurdioxide,carbonmonoxide),aerosolphysical(sizedistributionfrom50nmto20μm,absorptionandscatteringcoefficients,PM-10massconcentration)and chemical properties (organic and inorganic composition of fine and coarse aerosol), long-termcontinuousmeasurementsattheCNRclimateObservatoryofCapoGranitolawillbecarriedoutby ISAC.This station is the only reference regional station of Global AtmosphericWatch –WorldMeteorologicalOrganizationGAW-WMOinthe islandofSicily.Theacquired informationwillpermittobetter investigatethedynamicvariabilityoftheatmosphere,enclosedmineraldustprocessesfromNorthAfricaandnaturaland anthropogenic pollutant emissions (from local-scale to long-range). Moreover, the large suite ofatmospherictracersmonitoredatthisObservatory,willallowtaggingtheoriginandtheageofair-massesand related contaminants. The activities carried out at the unique climatic Observatory in the island ofSicily,willsupportstudiesonSLCF/Pcontaminantstotheenvironmentthankstotheuseofregionalclimateand air quality models. In addition, the WRF-CHIMERE-AODEM modelling system, implemented oversouthern Italy during I-AMICA PON Project (2012-2015, see http://www.i-amica.it/wordpress/?page_id=1148),willbeproperlyupgradedtosimulateatmosphericgasesandaerosolsformation,diffusionandremovalprocessesonregionalscale.Suchobservationalandmodellingtaskshaveakeyroleforinvestigatingthecontaminantseffectsontheecosystemandhumansonshort,midandlong

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term.

Contaminants concentration can be influenced by the future evolution and possible changes oftemperature, precipitation and other key climate variables. Therefore, In order to assess also possiblefuture impactsofchanges incontaminantsonhumanactivitiesandtheenvironment, thepossible futureevolutionoftemperature,precipitationandotherkeyclimatevariables in thestudyarea, inparticularofclimate extremes, has to be assessed. In addition to the knowledge of the present-day atmosphericcomposition and climate variables, evaluating thepossible future evolutionof climate conditions for thenextdecadesiscrucialtobetterunderstandthepossibleeffectsofclimatechangeontheecosystemsandhumansocietiesandtosupportfutureplanningconcerning,forexample,waterresourcesortheprovisionofenvironmentalservices.Thiscanbeachievedusingnumericalmodelsoftheclimatesystemandapplyingappropriatedownscalingmethodstoobtainclimatescenarios(inparticularprecipitationandtemperature)withhighspatialandtemporalresolution.Forapplicationtoimpactstudiesinthewatersector,agricultureand ecosystems, where changes in precipitation and precipitation extremes are important, a stochasticdownscaling technique developed at ISAC-CNR (RainFARM) will bemade available which provides high-resolutionprecipitationdataandallowsamorerealisticrepresentationofsubgridprecipitationvariability(especially in regions with complex orography) and of precipitation extremes. An elevation correctionmethodwillbeappliedtodownscaletemperaturefieldsovertheregionofinterest.Wewilldownscalethecoarse scale climate scenarios provided by state-of-the-art Global Climate Models (GCMs) or RegionalClimate Models (RCMs) in the framework of the CMIP5 and CORDEX initiatives, respectively, or fromspecificsimulationsperformedwiththeEC-EarthGCMrunatISAC(www.to.isac.cnr.it/sphinx/).Descriptiveindices of extremes defined by ETCCDI (http://etccdi.pacificclimate.org/list_27_indices.shtml), measuringcharacteristicsofextremes,includingfrequency,amplitudeandpersistencewillbecomputedfortheareaofinterest.

Task2.11.Newgenerationsensorsandobservatorynets(IAMC)Adiscrete number of sensing technologies characterised byminiaturization, earlywarning systems, low-energy consume will be designed and built. A basic networks of these sensors, combined with theenvironmental observing systems already available in the study areas, will improve the potential ofdetectionofdeliveryofcontaminantsintheenvironmentalcompartmentsandasuccessfulapproachtoamodernforecastingfordecisionmakers.

Listofdeliverables

D2.1.1 Generation of complete database for literature information on contaminants reported form thethreestudyareas(M4)

D2.1.2 Generation of a GIS platform with all the available information for the three study areas, withspecificreferencetocontaminantssources,distributionpathways,populationdensity,etc.(M6)

D2.2 Generationofdatasetsoftraditionalandemergingcontaminantsinthethreestudyareas(M18)D2.3 Generationofdatasetsofanthropogenicradionuclidesinthethreestudyareas(M18)D2.4Reconstructionoftheageofcontaminationinthethreestudyareas(M24)D2.5.1 Identificationofsourcesandpathwaysoftheselectedcontaminantsinthethreestudyareas(M24)D2.5.2 Reportsforthedifferentmodellingactivitiesrelatedtotheinvestigationoftheeffectsofmixtureof

contaminantsontheecosystems(M30)D2.5.3 Reportsonmassbalanceoftheselectedcontaminantsinthethreestudyareas(M30)D2.6 Generationofmapsofrisks(fortheselectedcontaminants)forthethreestudyareas(M32)D2.7 Reportonthedistributionofcontaminantsintheatmosphereinthethreestudyareas(M18)D2.8 Reports andmodels on atmospheric deposition and dispersion patterns in the three study areas

(M18)

D2.9 Reporton thedistributionof theselectedcontaminants in the terrestrialandmarine trophicweb

CISAS-28/09/2016 23

andlocalfood(M30)D2.10.1 ReportsonthemeasurementscarriedoutattheCapoGranitolaclimaticObservatory(M24)

D2.10.2 Generationofdatasetsofnaturalandanthropogenicclimatealteringcontaminates,reactivetracegases and aerosols and simulations of atmospheric gases and aerosols formation, diffusion andremovalprocessesonregionalscale(M24)

D2.10.3 Generationofhigh-resolutionprecipitationandtemperaturefieldsovertheMediterraneanRegionunderspecificclimatechangescenarios,asaninputforimpactstudies(M30)

D2.10.4 Generation of descriptive indices of extremes for precipitation and temperature for the specificscenario(M30)

D2.11 Report on the new sensors and integrated network of sensors built for different classes ofcontaminantsinordertofacilitatemonitoringandstudiesinthethreeconsideredareas(M34)

Listofmilestones

M2.1FirstmeetingoftheWP(M2)

M2.2SecondmeetingoftheWP(M12)

M2.3ThirdmeetingoftheWP(M24)

M2.4Intermediateconferenceandproject(M24)

M2.5Finalconferenceoftheproject(M34)


Workpackagetitle Ecosystemandcontaminants

Shortnameofparticipant IAMC IFC DiSTEM

Person/monthsperparticipant: 74,2

Objectives

ThisWPisfocusedonpollutantstoxicityandrelatedmolecularresponsemechanismsinmarineecosystem.

Biological and toxicological responses to different contaminants, including emerging ones, will beinvestigatedinthethreestudyareas(Priolo-Augusta,Crotone,Milazzo),withtheaimtohighlightpossiblerelationshipswithhumandiseases.

Biologicalresponseswillbe investigatedthroughawidepanelofbio-markers inmodelmarineorganismswithdifferentcomplexity.

In order to analyze toxic effects at various levels of biological organization, a systemic ecotoxicologycalapproach will be employed. Additionally, cellular mechanisms involved in pollutants toxicity will becharacterized,soastoprovideinformationusefulfornaturalecosystemsprotectionandmanagement.

Transcriptomicandepigeneticanalyseswillbecarriedout inordertounveil responsesandmodificationsinducedbyexposuretoselectedpollutants.

Moreover, edible,benthic andnectobenthicmarine specieswill beanalyzed,with theaim to investigatepollutantsuptakeroutesbyhumansthroughseafoodconsumption.

Data obtained by the analyses of natural samples will be validated under controlled conditions inmesocosm.

CISAS-28/09/2016 24

Descriptionofwork

Task 3.1 – Evaluation of the status of biodiversity in the selected areas: Priolo-Augusta, Milazzo and

Crotone(IAMC)The loss of biodiversity linked to marine and lagoon ecosystem represents a direct effect of pollutants

presentintheenvironment.Thus,theevaluationofbiodiversityduringthe4seasonswillbecarriedoutin

theareasofPriolo-Augusta,MilazzoandCrotone.

Ecological indices, such as species richness and population density,will be analysed and comparedwith

ecologicallyintactareas,whichwillbechoseninsynergywiththoseforepidemiologicalinvestigationsand

environmentalprofiles.Theseanalyseswillprovidedataontheeffectsofthe lossofbiodiversity,which inturnaffectsthesocio-

economicbenefitsfortheareasunderinvestigation.

Thethreestudyareaswillbeinvestigatedbyvisualcensustransectswithhorizontaltechniquesaswellas

verticalandfixedpointsdependingonthetipologyoftheenvironmentandbyaccreditedsamplingmethod

for benthos and plankton communities. Similarly, the control areas, characterised by low levels of

anthropogenicactivities,willbealsomonitoredfollowingthesametemporalsurvey.Thus,anearlyassessmentfocusedontheidentificationofthekeypointsonthedifferentstudiesareawill

becarriedout.Afterwards,periodicsurveysinthesameareaswillbeperformedforthecollectionofdata

andthesubsequentanalysis.Additionally, an evaluation of the biodiversity reported by other authors for the selected areas will be

consideredfortheidentificationofecologicalrelevantspeciesrepresentativeofcontrolandpollutedareas.

SubTask3.1.1Earlysurveys,identificationandstandardizationofprotocolforbiodiversityevaluationstatusPreliminary surveywill be carriedout inorder to identify samplingpoints in the contaminated sites and

controlareas.Additionally,specificprotocols,relatedtoeachsites,willbeidentifiedinordertostandardise

theprocedurefortheevaluationofbiodiversity.SubTask3.1.2EcologicalsurveysduringthefourseasonsTo address variations in abundance, richness and evenness for each area, surveys will be carried out

throughoutthefourseasons.Moreover,photo/videodocumentarieswillbeproducted.

Task3.2Ecotoxicologicalandbiochemicalanalysesinvitro (UniPa-DiSTeM)Thehighspecies-specificityofthemainchemicalpollutantsandtheneedtoreduceinvivotrialsforethical,

scientificandeconomicreasons,needstofindalternativeway,suchasinvitrotest,inordertoeffectively

assesstherisksrelatedtothepresenceofxenobioticsintheenvironment,alsoatsublethalconcentrations.

In addition, the limited availability of data related to the sub-lethal effects of these compounds in the

modulation of expression of oncogenes, stimulates new researches aimed to evaluate the molecular

mechanisms bywhich these xenobiotics can open signal transductions pathways that lead to apoptosis/

mutagenesisevents.The role of the unit DISTEM is to evaluate, in vitro and in vivo, the combined actions of selected

contaminants (fromWP1),onbiochemicalmarkers relatedto toxicity, inflammation,oxidativestress,cell

cyclecontrol,angiogenesisandapoptosis, incancerpromotion/prevention,byanalyzing thepathwaysof

signaltransductionandactivation.UNIPAgroup isexperienced inutilizationofcancercellsmodelsystem

and biomarkers to assess mechanisms underlying toxicity, oxidative stress, apoptosis, cell cycle and

differentiation,inducedbysyntheticscompounds,byxenobioticsandenvironmentalpollutants.Thespecificobjectiveofthistaskistoconductecotoxicologicalandbiochemicalinvestigationsatmolecular

CISAS-28/09/2016 25

andcellularlevel,inordertoidentifybiomolecularmarkersusefulassensorsandearlywarningsystems.

SubTask3.2.1 Invitrotestsonfishcellcultures.Fishcelllines,(primaryandimmortalized)willbeutilized

tomonitor indicators related to toxicity, oxidative stress, integratedmetabolism, apoptosis and cancer;

synergy studies. Fish hepatoma cell line, fish fibroblasts and pre-adipocytes,will be utilized to evaluate:

antioxidant/pro-oxidantswitch,dose-timedependenttoxicity,biomarkersofoxidativestress; cellcycle

modulation;inhibitionorpromotionofapoptoticpattern,orproliferativeeffect;markersofinflammation;

markers of angiogenesis. The analyses will be done by biomolecular investigations, gene expression,

microscopy,immunoistochemistry,fluorimetry.

SubTask3.2.2 Evaluationofbiochemicalmarkersinvolvedintheresponsetostress,inorgansandtissuesofmarine "model" organisms. Herein will be evaluated levels and gene-expressions of stress proteinsmodulatedby xenobiotics (metallothioneinMTs, heat shockproteinHsp), oxidative stress related to the

presence of xenobiotic (ROS, malondialdehydeMDA, HNE hydroxynonenal, polyunsaturated fatty acids,

PUFAs),apoptosisinducedbystress-relatedpathwaysofxenobioticsandoxidativestress(p53,bcl2family

ofproteins,caspases,PARP,etc.)withmolecularresearchtools,spectro-photo-fluorimetry,microscopyand

geneexpression.

Task3.3Theuseofmodelorganismstounveilnoveltoxicitymechanisms(IFC)Based on critical issues recognized in the three study areas, possiblemechanisms involved in emergent

diseaseswillbeinvestigatedinmodelsystems(i.e.Daniorerio,Paracentrotuslividus,Dicentrarchuslabrax,Octopusvulgaris,etc.)(Schnitzleretal.2011;Campinhoetal.20\13,2014;Coudercetal.2016;Marelliet

al.2016).

Thestrategyisbasedonthewidelyrecognizedandacceptedknowledgeoftheevolutionaryconservation

of themostmolecularmechanisms involved inbasal functions, inbothembryonalandadult tissues (like

stress response, cell cycle regulation, embryodevelopment andmorphogenesis, etc.) (Piconeet al2016).

Moreover, the overlapping between many molecular mechanisms involved in tumoral and endocrine

diseasesanddevelopment/differentiationiswellknown.Thus,embryonal/larvalstagesofselectedspecies

(easytomanageandmanipulate,andwellcharacterizedatmolecularlevel)arewellsuitablebothforthe

preliminarydetectionofphenotypicaleffects(i.e.teratogenicity,etc.)(Kinchetal.2016)oftheexposureto

pollutantsandforfurtheranalysesfocusedtounveilmolecularmechanismsinvolved(Zhuoetal.2014;Guo

etal.2014).

Among animal system in biological research, zebrafish is widely used to unveil toxicological and

developmental issues. Interestingly, ithasalsobeenconsideredasmodelforthyroiddevelopment,andit

hasbeenused tocharacterizeendocrine tumour (Liuetal.2016;Duetal.2016;Houbrechtsetal.2016;

Zhangetal.2016,Xuetal.2016;Kimetal.2016).

Becauseofthezebrafishpivotalroleinmolecular,toxicological(Haggardetal.2016;Huangetal.2016),as

well inepidemiologicalanalyses,asynergicalapproachwillbecarriedoutacross theactivityproposed in

WP3andWP4.Thus,aparternshipwithIFC-CNRwillbeestablishedinordertoexploittheexpertiseofeach

counterparts.

Morphological andmolecular analyseswill be carriedoutonembryos, both inmesocosmand innatural

environment, in order to detect alterations attributable to possibly responsible of cancer and newborn

malformationsinhumans.

Genes involved in embryo development, including those of ERK and EGF pathways (involved in

skeletogenesisanddorso-ventralspecification)willbeanalyzedbyqRT-PCR,inordertodetectalterations

inbothexpressionlevelsandspatio-temporalpattern.

CISAS-28/09/2016 26

Interference of pollutants with endocrine system will be evaluated by dosing both muscle thyroid

hormoneslevelsandtheactivityofthyroid/thyroidhormonesenzymesinDaniorerioand/orotherspecies.Such analyses will be associated with hystomorphometry. Moreover, the expression levels of selected

genes including thyroid-stimulatinghormone (tshα, TSHβ) (Donget al. 2014), tyroidhormones receptors

(TRαandTRβ)andgenesinvolvedinthyroidmetabolism(i.e.tshβ,tg,tpo,dio1,dio2,ttr)(Sunetal,2015;

Miao et al. 2015) will be evaluated by qRT-PCR in animals exposed to pollutant and related controls

(Kawaguchietal.2008;Zhangetal.2016,Guoetal.2014).

Furthermore, transcriptomicanalyses (carriedoutbymeansofNextGenerationSequencing)willprovide

global transcriptional profiles, suitable to detect variations involving any RNA species, including ncRNAs,

inducedbyenvironmentalpollutantfoundinthethreestudyareas(Caoetal.2016;Chenetal.2016).

SubTask3.3.1Findingandhousingofanimalsandpollutants.Selectedanimalmodelorganismswillbemaintainedaccordingtostandardproceduresoptimizedforeach

species.Chemicalandphysicalparameterswillbemonitoredsoastoensureconstantoptimalvalues.

Additionallythecircadianrhythmwillbeguaranteed.

SubTask3.3.2Exposureofanimals(inmesocosm)toselectedpollutantsArrayofexperimentswillbeestablishedwhereanimalswillbeexposedtodifferentamountsofeach

pollutantsaswellastheirmixturesinordertoassesstheirbiologicaleffects.

SubTask3.3.3Exposureofanimalsinmesocosmtoseawaterfromcontaminatedsites.Arrayofexperimentswillbeestablishedwhereanimalswillbeexposedtocontaminatedseawaterfrom

eachstudyareasinordertoassesstheirbiologicaleffects.

SubTask3.3.4Animalhousingincontaminatedareas.Selectedanimalcollectedincontrolareaswillbetransferredineachstudyareainordertovalidatedata

obtainedinmesocosmexperiments.

SubTask3.3.5Molecularanalyses.Analysesofgeneexpression,pathwaysactivation,detoxificationmechanismswillbeinvestigatedbymeans

ofnucleicacidanalyses.

Task3.4Geneexpressionpatternanalysisandevaluationofepigeneticmodificationsintargetspecies

(IAMC)Molecular mechanisms of detoxification and homeostasis maintenance will be investigated in selected

organisms. Theevaluationof chromatin statusaswell theanalysis themRNA levelsof genes involved in

chromatin remodelling, will be investigated in order to indentify epigenetic modifications and relative

transmittance in response to pollutant(s) exposure. In order to evaluate the risks associated with the

presenceofxenobiotics in theanalizedspecies, themolecularmechanismsbywhich theycandetermine

genotoxicity, interferencewith regulatorymechanisms and cell deathwill be examined. The results will

providethebasis forsubsequentmolecularmodelling investigationstoevaluatetheputative interactions

betweencontaminantsandbiologicalmolecules.

SubTask3.4.1 Analysisof themRNAexpressionpatternsofselectedgenes (includingHSPs,SOD,GST) inresponsetopollutant(s)

CISAS-28/09/2016 27

Stressresponsetoselectedpollutantswillbeprofiledusingwellknownmarkersinvolvedinmechanismsof

detoxification,signaling,aswellasmetalscavenging.

SubTask3.4.2IdentificationofDifferentiallyExpressedmiRNAsMechanismsof regulationof geneexpressionaffectedon theactivityofmiRNAswill beanalysedon the

basisofthepresenceandrelativeabundanceofmiRNApopulation.

SubTask3.4.3IdentificationofepigeneticstatusbymethylationspecificqPCR(MS-qPCR)Selectedclusterofgeneswillbeinvestigatedonthebasisofpromotermethylationwhichinturnaffectsthe

accessibilitytobasaltranscriptionmachineryandthusrelatedmRNAexpressionlevel.

Sub Task 3.4.4 Analysis of chromatin condensation in selected genetic loci and evaluation of chromatinremodellinginresponsetopollutants.Epigenetic status of chromatin domains bearing specific genes will be profiled on the light of post-

transcriptionalmodificationaffectinghistonesandaccessibilitytochromatin.

SubTask3.4.5mRNAexpressionanalysisofgenesinvolvedinchromatinremodelling.

The mRNA levels of genes coding for chromatin remodeling machinery will be investigated in exposedanimalstorelatealterationsinchromatincondensationtochemicalexposure.

Listandnameofdeliverables(alsotimetable)

3.1.1:Identificationofsamplingareas3.1.2:Datasetontheseasonalbiodiversityamongthethreestudyareasandcomparativeanalyseswithcontrolareas.3.2.1:Definitionofmolecularsensorsandconcentrationsforearly-warning3.3.2:Datasetaboutalterationsofmolecularpathwaysinducedbychemicalsexposition.3.3.3:Datasetaboutalterationsofmolecularpathwaysinducedbyexpositiontocontaminatedseawater.3.3.5:Datasetaboutalterationsofmolecularpathwaysinanimalsfromcontaminatedareas.3.4.1:DatasetofthemRNAlevelsinstress-relatedgenes.3.4.2:DatasetonDifferentiallyExpressedmiRNAsinresponsetopollutants.

3.4.3:Datasetonepigeneticmodifications.

Listandnameofmilestones

3.1:Shortfilmonthebiodiversitystatusinthestudiedareas(M14)

3.2:Intermediatemeeting(M24)

3.3:WorkshoponEcosystemandcontaminants(M35)


Workpackagetitle Etiologicalepidemiology

ShortnameofparticipantIFC IAMC UNIME UNICT UNIRC REGIONE

SICILIANAREGIONECALABRIA

CISAS-28/09/2016 28

ASPSiracusa

ASPMessina

ASPCrotone

UNIPI


80

Objectives

Onthepollutedsitesofnationalinterestforlandreclamation(PS)areavailabledetailedknowledgeofboththe environmental characterization, especially soil, surface and groundwater water, both on the healthstatusoftheresidentpopulation.TheSENTIERIstudyproducedinitiallyanextensivereviewofthescientificliteratureandmaterialsandmethodsforevaluatingtheevidenceonenvironmentandhealthassociations(Pirastuetal.2010);subsequentlyhasprovidedastudyonmortalitydata1995-2002(Pirastuetal.2012),followedbyastudyonmortality2003-2010,cancerincidence1999-2005andhospitalization2005-2010inPScoveredbycancerregistries(Pirastuetal.2014).

ForthePSsofMilazzoandPriolodataonmortality,incidenceandhospitalizationwereprovided(Pirastuetal. 2012 e 2014). For the PS of Crotone mortality data 1995-2002 were provided (Pirastu et al 2012),upgradedbya recent analysisondata2006-2012,providingdisaggregateddata for themunicipalitiesofCrotoneandCassano-Cerchiara(ISTISAN16/9).

InthethreePSsunderstudyseveralexcessesofhealthindicatorswereobserved.

For this research project, it was decided to enhance the knowledge on selected indicators through thestudyofriskmarkersorearlydamageinpopulationgroupsatdifferentlevelsofexposure.

Theepidemiologicalcriterionwasalsojoinedwiththeresearchinterest,anddifferentorgans/systemshavebeenselectedineachPS:cardiovascularandkidneyintheCrotonePS,liverinthePrioloPS,thyroidintheMilazzoPS.

WP4isdividedinto5tasks:

Task4.1Coordination,analysisandmonitoring

Task4.2RiskoflivercancerinthePrioloarea

Task4.3RiskofthyroidcancerintheMilazzoarea

Task4.4RiskofcardiovascularbiomarkersintheCrotonearea

Task4.5PilotstudyonrenalfunctionimpairmentandearlykidneydamageintheCrotonearea

Task4.6 Disseminationandscientificnetworking

Descriptionofwork

Task4.1.Managementandcoordination(IFC)

ManagementProjectmanagementactivities,coordinationwithotherWP,epidemiologicaldesignandstatisticalanalysis,relationshipswithotherlocal,regional,nationalbodies,monitoringandevaluationofactionsdevelopment.

LiteraturereviewAcompletealiteraturereviewonenvironmentandhealthinthethreestudyareas(Augusta,MilazzoandCrotone)isthestartingpointofthefieldwork.

MonitoringandevaluationAspecific listof indicatorshave tobedefined tomonitorandevaluate the taskdevelopment,asabasic

CISAS-28/09/2016 29

functionofthemanagementactivity.

Task4.2.RiskoflivercancerinthePrioloarea(IFC,UniversitàdiCatania,RegioneSiciliana,ASPSiracusa)

InSicily,theAugusta-Prioloareaisoneofthethreeareas(withGelaandMilazzo)establishedas“areasathighriskofenvironmentalcrisis”andithasbeenidentifiedasacontaminatedsiteofnationalpriorityforremediation. In fact, this area is characterized by a diffuse environmental contamination due to thepresenceoflargeindustrialsettlementsoperatingprimarilyinpetrochemicalfieldthatinthelastdecadeshas caused a progressive contamination of the different environmental compartments. Toxic, persistentand bio-accumulable compounds, such as heavy metals, polychlorinated biphenyls (PCBs), dioxins andpolycyclic aromatic hydrocarbons (PAHs) were detected and the past and present exposure to volatileorganiccompounds(VOCs)wasascertained[1,2].Inthissetting,theincidenceofalltypeofcancersinthepopulation living in thePrioloareashowedanexcess inbothgenderswith respect to thepoolofcancerregistries of central and southern Italy; in particular neoplasms in excess included liver and pancreascancersandmesothelioma.

Intheoverviewof liverdiseases,hepatocellularcarcinoma(HCC) is themaintypeofprimary livercancerand the second causeof cancerdeathworldwideaccording to the resultsof the recentlypublishedHCCBRIDGE study [3]. Among the risk factors forHCC there are not only viral infections (HCV andHBV) andexcess alcohol but also exposure to pollutants that act as endocrine disruptors and produce metabolicalterations[4,5].ThemechanismsforthedevelopmentofHCCarestillunknownbutHCCmightbeduetoalterationsof lipidmetabolism [6].Mostof thepollutants are lipid soluble andonce they accumulate inadipose tissue and liver they might promote cell lipotoxicity, increase de novo lipogenesis (DNL) andsynthesis of saturated fatty acids, in particular palmitic acid (PALM) and other classes of lipid such asceramides,di-andtri-acylglycerols(DAGandTAG)[7,8].

All thesepathwayshavebeenrelatedto liverdamageandHCC. Inthiscontest,the identificationofearlymarkersofhepaticdysfunctionandtissuedamage/fibrosiswillhelpintheidentificationofsubjectsatriskofHCC(Fig.1).

Figure1.Thepossiblelinkbetweentheexposuretoenvironmentalpollutantsandtheinductionofliver

damage:pollutantsstimulatepathwaysresponsibleofthedevelopmentofinflammatoryprocess,apoptoticevent,fibrosisandcarcinogenesis.

Abbreviations:DNL:DeNovoLipogenesis;EDCs:endocrinedisruptingchemicals;HCC:hepatocellularcarcinoma;PAHs:polycyclicaromatichydrocarbons;PCBs:polychlorinatedbiphenyls;ROS:reactiveoxygen

species;SCD1:stearoyl-CoenzymeAdesaturase1;VOCs:volatileorganiccompounds.

AccordingtotherecentEuropeanguidelinesoftheEASL-EASD-EASO(EuropeanAssociationsfortheStudy

CISAS-28/09/2016 30

ofLiver,DiabetesandObesityrespectively)therearenon-invasivemarkersofhepaticfataccumulation(likefatty liver index), andhepatic fibrosis (e.g., FIB-4,NAFLD fibrosis score) that togetherwith liverenzymesandcanidentifysubjectsatriskofliverdiseaseandHCC.However,newserumlipidomicmarkersareundertestandmightbetteridentifythosesubjectsathigherriskofHCC.

ExperimentalDesignTheprimaryaimofthisprotocolistoidentifyifsubjectslivingintheexposedareadisplayincreasedlevelsof established risk factors andbiomarkers of liver disease andHCC. The secondary aim is to discover ifsubjectslivingintheexposedareadisplayalterationinmetabolismthatcanpromoteliverdiseaseandHCC.

Subjects:wewillstudysubjectswithoutknownliverdiseaseorprevioushistoryofviral infectionsasHBVandHCV,aged40-65years,recruitedinthePrioloareaandinanotherareawithoutcontamination.

• afirstgroupofsubjects(n=200),withoutlivercancerlivingintheexposedareaofAugusta-Priolo

• asecondgroupofsubjects(n=200),withoutlivercancerlivinginanunexposedarea

In all subjectswewill collect plasma and urine samples for further analysis, tomeasure established riskfactors(seebelow)andwewillthenidentifyifsubjectslivinginthePrioloareadisplaypotentialbiomarkersof liver disease and specific disease-related metabolic pathways and genetic signaling for HCCdevelopment.

Measurementonbiologicalsamples• Wewillmeasureseveralparametersusedtoevaluatethestateofhealthofthesubjectsinvolvedin

thestudy:clinicalandbiochemicalparameters;

• fatty liver indexFLI= (e0.953*loge (triglycerides)+0.139*BMI+0.718*loge (ggt)+0.053*waistcircumference - 15.745) / (1 + e 0.953*loge (triglycerides) + 0.139*BMI + 0.718*loge (ggt) +0.053*waistcircumference-15.745)*100;

• fibrotic liver indexes suchas,FIB-4 indexand NAFLD fibrosis score (NFS); inflammatorymarkers,such as Monocyte Chemoattractant Protein-1 (MCP-1); adipocyte hormone leptin and thepancreatichormoneinsulin.

• Wewill test thehypothesis thatexposure topollutants stimulates lipotoxicityand that increasedDNLisanimportantriskfactorforHCC.Wewillmeasure,inplasma,DNLindexescalculatedastheratio palmitic/linoleic acid (16:0/18:2). Fatty acid composition will be measured by gaschromatographymassspectrometry(GC/MS).

• Lipidomic analysis will be performed in plasma samples by liquid chromatography massspectrometryLC-MSQ-TOF, inordertoevaluatethepotentialroleinthedevelopmentofHCC,ofseverallipidclasses,suchasceramides,di-andtri-acylglycerols(DAGandTAG).

• Presence of endocrine disrupting chemicals (EDCs) analysis will be assessed in urine samples byliquidchromatographymassspectrometryLC-MSQ-TOF, inorderto identify thepotential roleofthesechemicalcompoundsinthedevelopmentofHCC.

We expect to identify classes of toxic lipids that, due to the high concentrations of environmentalpollutantsinthePrioloarea,canpromoteandstimulatethedevelopmentofHCC.Withthisapproachweexpecttoidentifypotentialbiomarkersinvolvedinspecificmetabolicpathwaysthatcouldbeusedastargettoprevent/treatHCC.

Wewilltestthehypothesisthatlipotoxicity,duetoincreasedDNLandceramidesisanimportantriskfactorforHCC.Wewillmeasure,inplasma,DNLindexescalculatedastheratiopalmitic/linoleicacid(16:0/18:2).Fattyacidandlipidcompositionwillbemeasuredbymassspectrometry(GC/MSandLC/MS).

Task4.3.RiskofthyroidcancerintheMilazzoarea(IFC,UniversityofMessina,SicilianRegion,ASPMessina)

IntheMilazzoPS,includingthemunicipalyofMilazzo,PacedelMelaeSanFilippodelMela,theincidence

CISAS-28/09/2016 31

of thyroidcancer (TC) in2003-2010hasbeenreported inexcess (notstatistically significant)amongmenand women (24% and 40% increases rispectively), and the hospitalization for TC in 2005-2010 showedstatisticallysignificanexcessof55%amongmanand24%amongwomen.(Pirastuetal.2014)

The interest on the thyroid tumor is growing in light of the excess results in several PSs in addition toMilazzo(Brescia-Caffaro,MantuaLakes,Sassuolo-Scandiano,Taranto),inbothgendersinbothdatabasesofmortalityandmorbidity.(Pirastuetal.2014)

Since long time is well known the critical role of the thyroid gland in the development / cellulardifferentiation,andfetaltissue,inthecontrolmetabolichomeostasisandfunctionofthemajorityoforgansand systems (cardiovascular and central nervous system first of all), including the remaining endocrineorgansandthereproductivesystem.

This role is emphasized by the relief, clinical and experimental, that even minimal change in thyroidfunctionwith circulating levelsofhormoneswhile still resulting in the reference rangeofnormality '(so-calledhyperorhypothyroidismpreclinical)areabletogeneratenegativeconsequences,particularlyatfetalandneurologicallevel.

The literature on thyroid-toxic effects ofmany pollutants has been progressively accumulated in recentyears. A series ofmoleculeswith a high degree of structural similaritywith the thyroxine (T4, hormoneprecursor)andtriiodothyronine(T3,biologicallyactivehormone)areabletointerferewiththyroidfunction- and therefore on the organism in its complessita'- through multiple mechanisms operating at bothreceptor / post-receptor binding that the serum protein hormone transport, of the cellular uptakeprocesses in variousorgans and tissues,mainly by changing the feedbackhypothalamicpituitary thyroidhormonemetabolismandtheperipheral.

However, it is important to stress an underlying bias in scientific findings in this area so far available,representedbythefactthatbothpopulationandexperimentalhumanstudieswereprimarilyandalmostexclusively focused on the effects of hormone-likemoleculesMimetic poly brominated biphenyl ethers,Bisphenol-and others. Differently, greatly reducedwas the attention paid to the role of so-called heavymetalssuchasmercuryleadcadmiumandothersandtheresultsreported,aswellasscarce,areveryoftencontradictoryorevenconflicting.(Boasetal.2006;PearceandBraverman2009;Dundaretal.2006;Jinetal.2006;Lambetal.2008;Robinsetal.1983;Schelletal.2008).

Occupational studies have found a significant reduction of T4/T3 levels inworkers exposed to lead as anegative association was observed between mercury levels and thyroid hormones with a high ratio ofT4/T3, especially in women of childbearing age, resulting in both the accumulation of lipid with iodineuptakeinhibitionthatmercuryinhibitoryactionondeiodasidevices(Robinsetal.1983Nishida,etal.1986;Soldinetal.2008;Tanetal.2009;Barregardetal.1994;Ellingsenetal.2000).

Incontrast,preliminarystudiessuggestedareductionofthyroidhormonelevelsinanimalsexposedtocadmium(Yoshizukaetal.1991,GuptaandKar1997,1998;Hammoudaetal.2008;),notconfirmedinpopulationstudieswhereapositiveassociationbetweencadmiumandthyroidhormoneswasobserved.Suchadiscrepancyislikelyduetodifferencesofspeciesordosesusedinanimalstudiescomparedtodosesofexposureinhumanpopulations.

Subtask3.1EpidemiologicalinvestigationIn addition, themain limitations of the few-not european-human studies reported in the literature, arerelatedbothtotheexperimentaldesign(cross-sectional)thatthelackoflongitudinaldata.

Hencetheneedtoexpandourknowledgewithstudiesthatareabletoexaminepotentialmechanismsoftoxic action on thyroid function ofmercury, lead, cadmium, and other heavymetalswith an integratedapproach that includes the use of an animal model widely used and validated for toxicological whichzebrafish (You-Je-EnvironToxicolChem2014;33:11-17).This innovativeapproach is finalized toevaluatethedose-effectsrelationshipofheavymetalsonthefunction/thyroidcarcinogenesisthatepidemiologicalstudyappropriatelydesigned.

Theprimaryaimisto identify ifsubjects living intheexposedareadisplay increasedlevelsofestablishedriskfactorsandbiomarkersoffunction/thyroidcarcinogenesis.

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Thesecondaryaimistodiscoverifsubjectslivingintheexposedareadisplayalterationinmetabolismthatcanpromotethyroidcancer.

Subjects:wewillstudysubjectswithoutknownthyroidcancer,aged18-44years,recruitedintheMilazzoareaandinanotherareawithoutcontamination.

• agroupofsubjects(n=150),withoutTClivingintheexposedareaofMilazzo

• agroupofsubjects(n=150),withoutTClivinginanunexposedarea

Inall subjectswewill collectplasmaandurinesamples,and the followingparameterswillbemeasured:TSH,FT3,FT4,urinaryorbloodlevelsofvariousheavymetalsofinterest,adjustedforcreatininuria.

Athyroidsonographicmeasurements(glandularvolume)willbedoneineachsubject.

Subjects living in the Milazzo area displaying potential biomarkers of Thyroid will be identified andinformed.

Subtask3.2ExperimentalmodelZebrafish are widely used in the toxicological researches because of its small size, easy culture, highreproductive performance, rapid organogenesis and sensitivity to the harmful chemicals.More recentlyZebrafishhasemergedalsoas auniquemodelorganism for studying cancer genetics andbiologywheretheyhavemadeimportantcontributionstounderstandingthemechanismoftumorigenesisandtodevelopalternative therapeutic strategies. Recently, zebrafish studies have led to important insights into thyroiddevelopment,andhavebeenusedtomodelendocrinecancer.Thethyroidsysteminzebrafishissimilartomammalian,whichcanprovideavaluablereferenceforhumanbeings.

Therefore,inthepresentstudy,zebrafishwillbeusedforelucidatingtheeffectsofseveralenvironmentalpullutantsidentifiedintheareaofinterestofMilazzo,ondisruptionofthyroidfunctionandexpressionandoninsurgenceofthyroidcarcinoma.

Thefollowingcontaminantswillbeinvestigated:

-Polycyclic aromatic hydrocarbons (PAHs)(in particular 7,12-dimethylbenz[a] anthracene (DMBA);benzo[a]pyrene(BAP)dibenzothiophene(DBT)).

-Heavymetals(inparticularcadmium)

-Dioxins(inparticular2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD))

Thepossible involvementof thesecompounds in thyroid toxicityandcarcinogenicityhasbeensuggestedfromdifferentapproaches indifferentmodels.A recentstudydemonstrateda roleofPAHs in increasingthyroxine (T4) and decreasing triiodothyronine (T3) production in mammals although the differentcompounds of this class of chemicals showed apparently conflicting effects (Fowles et al., 2016). Similarobservationswere obtained also in fishes (Bar et al., 2012). In zebrafish, significant decreases inwhole-body T4 andT3 levels, along with transcriptional changes of thyroid regulating genes, were observedfollowing5dayexposuretocontaminantscontaininghighpercentageofPAHs(Kimetal.,2016).TCDD,oneofthemost potent inducers of CYP1A,was reported to increase hepaticUGT and result in decreased T4(Curran,P.G.etal.,1991)

MoreoveraneffectonthyroidcellproliferationinculturehasbeenobservedafterculturetreatmentwithPAHs and some heavymetals with cadmium showing a particularly high effect (Kobayashi et al., 2004).Interestingly, exposition of zebrafish embryos and frys to DMBA induces development of neoplasias inseveraltypesoforgan/tissueincludedthyroid(Spisbergenetal.,2000).

ToinvestigatetheroleoftheindicatedpollutantsonthyroidmetabolismZebrafish embryos (60 hpf) or larvae (2weeks) will be treated by bath exposure to the contaminant inanalysis(ortomixtureofthem).Dosesrangingfrom0to25µMofPAHs,0to40µMofCd(2+)and0to5nMofTCDDwillbe tested fordifferent lengthof time.After treatments, the zebrafishembryo/larvae (n=100)perreplicatewillbehomogenized

a) in sample diluent for whole body T4 and T3 quantification by using commercial enzyme-linked

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immunosorbentassays(ELISA)kits

b)fortotalRNAisolationtoassesstranscriptionalchangesofthyroidregulatinggenesbyQuantitativeRealTimePCR.The followinggeneswillbequantified:deiodinaseDio1andDio2,THreceptorsTR-αandTR-βandrelevantgenesinvolvedinthehypothalamic-pituitary-thyroid(HPT)axissuchastsh-βgene,theiodinetransporterslc5a5gene,thegeneencodingthyroglobulin(tg)andthetransthyretin(ttr)gene.

Moreover, a recently developed transgenic zebrafish line expressing enhanced green fluorescentprotein(EGFP)undercontroloftheregulatoryelementsoftheTSHβgenewillbealsoutilizedtobetterinvestigatetheeffectofthecontaminantsontheHPTaxis.Inthistransgenicline,EGFPexpressionwithinthepituitaryglandrecapitulattheexpressionpatternsofendogenouspituitaryTSHβmRNAandisspecificallytargetedto the thyrotrope cells located in theanteriorpituitary gland. Themonitoring system isboth convenientandcheap.Thisassaydoesnotrequireeuthanizingthefish.Additionally,effectsofinvivobioaccumulationinfishcanalsobeassessedduringtheevaluationprocedure.

Amorphologicalanalysisofthecontaminantexposedfisheswillbealsoperformedatdifferenttimesaftertreatments.Thisanalysiswillbegreatlyfacilitatedbytheuseofthetransgeniclinetg(tg:mCherry),inwhichrobustandthyroid-specificexpressionofamembraneversionofmCherryenables live imagingofthyroiddevelopmentinembryosfrombuddingstagethroughoutformationoffunctionalthyroidfollicles(Opitizetal.,2012).

ToinvestigatetheroleoftheindicatedpollutantsonthyroidneoplasiaThreeexposureprotocolswillbeutilized:bathexposureofembryos,bathexposureoflarvae,anddietaryexposureofjuveniles(twomonthsagefishes).Embryosandlarvaewillbetreatedfor48hrsandsampledfor neoplasia study 9months post-treatment. Juvenile fisheswill fedwith special diet for 2months andthen fed with basal diet for an additional 5 months, for a total of 7 month delay between start ofcarcinogen exposure and final termination. For histopathological study, 10 fishes per treatment will besampledat8,12,and16weeksafterinitiationofexposure,andtheremainingfisheswillbesampledatthefinal date. At the final data fishes will be euthanized and tissues section prepared. Sections, routinelystainedwithhematoxylinandeosin(H&E)willbeexaminedforneoplasticlesions.

Sinceneoplasiaarerather rarely found inwildzebrafish,besides thewild typezebrafishAB line, the lineknockoutforthewellknowntumorsuppressorp53genewillbealsoused.Thepresenceofthismutationwhich will be the first hit in the tumorigenesis process, will result in an increase of the neoplasiafrequenciesmakingeasiertheidentificationofamutageniceffectofthepollutantsinanalysis.

Task 4.4 Cardiovascular biomarkers in the Crotone polluted site area (IFC, Università di Pisa, RegioneCalabria,ASPCrotone)

BackgroundCrotoneisanItalianPlaceofRecovery(SitoItalianodiBonifica)duetothehighpresenceof industries. Ithas been shown the presence of high concentration of heavy metals (Troisi et al 2002) and ecologicalstudiesshowed,forthisarea,anincreasedincidenceofcardiovascularevents.TheStudySENTIERIon1995-2002mortalitydata,showedanexcessforcircolatorydiseasesamongmales(+13%)(Pirastuetal.2012)

Ina separate study for themunicipalitiesofCrotoneandCassano-Cerchiara,mortality forkidney tumorswas observed in excess amongmales and females (+ 105% and + 37% rispectively), while an excess ofurinary tract, and nephrosis kidney failure emerged among females (+43%). Hospital admission due toischemic heart disease, acute ischemic diseases, nephritis, nephrotic syndrome and nephrosis showedexcessesforbothgenders(+42-68%),forkidneycancersamongmales(+49%).(ISTISAN16/9)

In recent years, great attentionhas beenplacedon the exposure to heavymetals such as arsenic, lead,cadmium, andmercury, and its relationshipwith cardiovascular disease (CVD) (Solenkova et al 2014). Infact,bothelevatedbloodandboneleadlevels(Navas-Acienetal.2004)andurinarycadmiumlevels(Tellez-Plazaetal2013)werefoundtobeassociatedwithan increasedcardiovascularmortality.Furthermore,a

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recent cohortprospective study focussingon theexposure to low tomoderatearsenic levelshas shownthatitrepresentsasignificantdeterminantforCVDincidenceandincreasedcardiovascularrisk(Moonetal.2013). Endothelial dysfunction may represent a key role, as first sign of atherosclerosis, in thecontamination-inducedcardiovasculardamagemechanisms.Inthiscontext,inflammationandvulnerabilityofbloodtothrombosismayplayapathophysiologicalroleinfavouringCVeventsinducedbyenvironmentalpollution,butmechanismsandsourcesofsusceptibilityarestillunclear(Solenkovaetal2014,Cosselmanetal 2015). In addition, recent data showed that altered bone metabolism is associated to increasedcardiovascularrisk(Vassalleetal2016,2014). Interestingly,differentheavymetalsarepollutantscapableto critically affect bone mass, although the influence of environmental pollution on bone health andoccurrenceofosteoporosisisstillunclearandcontroversial.

ThepotentialassociationbetweenexposuretoheavymetalsandCVDhasanumberofclinicalimplications,mainlyduetoincreasedoxidativestressandinflammation(Houstonetal.2011,Alissaetal.2011).Previousstudies have highlighted that the effects of mercury toxicity include hypertension (Valera et al 2012),reductioninheartratevariability(Valeraetal2008)andincreaseincarotidintimamediathickness(Choietal. 2009); similar findingshavebeen reported forexposure tohigh levelsof arsenicum (Steaetal. 2014,2016). Moreover, a recent animal study has shown that chronic exposure to Cd can lead to increasedarterial stiffness (Sangartit et al. 2014). Arterial stiffnesswhich also demonstrate to be an effective andsensitivebiomarkerofexposuretoareaofhighairpollution(Lundbäcketal.2009).Inaddition,asignificantnegative correlation between cadmium levels and endothelial function has been found in hemodialysispatients, who are at high risk for excess of toxic trace elements (Kayaet al 2012). However, theseinvestigationsconcernsingle-elementexposureandacompletecharacterizationofpossiblecardiovascularalterationsisstillmissing.

This part of the project is aimed to evaluate early alteration in cardiovascular biomarkers in subjectsexposed to different levels of multi-element environmental pollution. The complete cardiovascularcharacterizationproposed, including theanatomicaland functionalassessmentof thevascular systemaswellastheevaluationofpossibleheartratealterations,ishelpfultoimplementpreventionprogramsanddevelop novel strategies aimed at decreasing the adverse effects of air pollution on the cardiovascularsystem.

MATERIALSANDMETHODSStudydesignandSubjectThreehundredsubjectinprimaryCVprevention,aged40-70years,thatiswithoutpreviouscardiovascularandwith orwithout known conventional CV risk factors,will be enrolled. These subjectswill be sharedaccordingtotheplaceatlowmiddleandhighlevelofenvironmentalpollution.CVriskscore,Framinghamor Euro Score, will be assessed for each subject. To this aim VIRC platform will be used providing anintegrated and multipurpose computer-based program for the assessment of CV risk (Franchi 2015). Inaddition,questionnairesonlife-stylehabitsandpsychosocialparameterswillbeconducted.

Vascularcharacterization1) Carotid-femoralpulsewavevelocityandcentralbloodpressureCarotid-femoral pulsewave velocity (PWVcf)will be assessed as a surrogatemarker of regional arterialstiffness.Tothispurpose,anapplanationtonometer(PulsePen,Diatecne,Milan, Italy)willbeemployedto record pressure waveforms at the carotid and femoral artery levels and obtain PWVcf following astandard procedure (Laurent et al 2006). Carotid pressure curveswill be processed using the rescalingmethod and brachial pulse pressure values and used to estimate central blood pressure (Pc) andaugmentation index (AIx) (Salvi et al. 2004).Aortic pulsewave velocity (PWVao), Pc andAIxwill be alsoassessed with the oscillometric method (Arteriograph, Tensiomed, Budapest, Hungary) (Horváth et al.2010).

2) LocalcarotidassessmentUltrasound(US)scansofthecommoncarotidarteryonbothsideswillbeacquiredandprocessedinorderto assess anatomical and functional parameters. In particular, systolic, diastolic and mean diameter,

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intimal-media thickness (IMT) will be estimated using an automatic edge detection software (CarotidStudio,Quipu,Pisa, Italy)(Bianchinietal.2010).Asregardsfunctionalproperties,thecarotidarterywillbeevaluatedintermsofcarotidstiffness(CS)combiningtheanatomicalinformationwiththelocalcarotidpressuremeasurementsobtainedwiththetonometricdevice(seepreviouspoint).

3) CharacterizationofcarotidatheroscleroticplaquesInthosesubjectspresentingacarotidplaqueduringtheUSexamination(accordingtocurrentguidelines(Touboul et al. 2012)), a carotid plaque characterization in terms of vulnerabilitywill be performed. Inparticular,USimageswillbeprocessedinordertoassessstandardplaqueparameters,suchasthelesionlength and area, which will be completed with the calculation of functional biomarkers and a textureanalysisgivinginformationabouttheplaquecomposition(vanEngelenetal.2014).Inaddition,Dopplerimages will be collected in order to provide a complete plaque characterization including anatomical,functionalandhemodynamicinformation.

4) EndothelialfunctionBrachialartery flowmediateddilation (FMD)willbeusedtoassessendothelial functionandperformedfollowingthestandardprocedure (Thijssenetal.2011).Briefly,US imagesof thebrachialarterywillberecorded for10minutes ((1min forbaseline, 5minof ischemicperiod, 4min for assessing changes indiameterfollowingreactivehyperaemia)andtherecordedscanswillbeanalysedusinganautomaticedgedetectionsystem(FMDStudio,Quipu,Pisa Italy) (Gemignanietal.2008).FMDwillbecalculatedas themaximalpercentageincreaseindiameterabovebaseline(meanofthemeasuresobtainedduringthefirstminute).Endothelialfunctionwillbealsoevaluatedwithperipheralarterialtonometry(EndoPAT,ItamarMedical, Israel), in which the endpoint measured is the finger arterial pulse volume amplitude (PVA)(Kuvinetal.2003).Thereactivehyperaemiaindex(RHI),anditsnaturallogarithm(lnRHI),willbeassessedas the ratio between hyperaemic and baseline PVA, normalized for the same ratio in the contralateralarm.

5) Electrocardiogram(ECG)analysisIn all the participants a 12-lead ECG will be performed. Heart rate (HR), QRS interval duration andcorrected QT interval (QTc) were automatically computed. Three QRS voltage diagnostic criteria(Sokolow-Lyon, Cornell Voltage, Cornell Product) were considered to define the presence of leftventricular hypertrophy (LVH) according to international recommendations (Hancock et al. 2009); LVHwasdefinedwhenoneormoreoftheabovementionedcriteriaweresatisfied.HRVindiceswillbederivedfromtheECGtrace.InterpretationandextractionofHRVparameterswillbeperformedautomaticallybyusingcustomsoftware.Time-domainandfrequencydomainparameterswillbeevaluated.

BiohumoraltestsThefollowingbiochemicalparameterswillbemeasuredinallenrolledsubjects:

1) inflammatorymarkers,IL6-8-10,TNFalfa,PCR

2) oxidativestressmarkers(ROMSandTotalAntioxidantCapacity)

3) NT-proBNP4) bloodvulnerabilitytothrombosis:circulatingmicroparticles,tissutalplasminogenactivator(tPA)and

CD40ligand

5) bonehealthmarkers,vitaminD,Ca,P,PTH,osteocalcin,BALP,P1NP,CTx+sclerostinandFGF23

ExpectedresultsWeexpecttofindalterationsinallthecardiovascularbiomarkersidentifiedduetothehighconcentrationofenvironmentalpollutants. Inparticular,weexpect to findalterations inanatomicaland functionalandbiohumoral vascular parameters, as well as endothelial dysfunction and modifications in ECG-derivedindexes.

We will test the hypothesis that the entity of these alterations are directly related to the level of

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environmentalpollutionatwhichtheindividualsunderstudyaresubjected.

Task4.5.PilotstudyonrenalfunctionimpairmentandearlykidneydamageinindividualsexposedtoenvironmentalriskfactorsintheCrotonearea(IFC,UniversitàdiCatania,RegioneCalabria,ASPdiCrotone,UniversitàdiReggioCalabria)

BackgroundTheincidenceandprevalenceofchronickidneydisease(CKD)haverisenconstantlyoverthelast3decades,becomingnowagrowingpublichealthproblem.

There is now considerable evidence pointing at the role of environmental pollution as a prominentaetiologicalfactorofCKDofmultifactorialorunknownorigin(CKDmfo/CKDu)(Sabathetal.2012)..Severalmechanistic and experimental studies have defined the role of ions, heavymetals and fluorides as keydeterminants of early glomerular and tubular damage,which usually anticipate the frank impairment ofrenal function, leading to End-Stage Kidney Disease (ESKD). In particular, heavy metals such as lead,cadmium,mercuryandarsenicare recognized toexertdirectdetrimentaleffectsonkidney functionandstructure,evenat“normal”levels.Thephysio-pathologicalmechanismsbehindmetal-inducedkidneyinjuryare complex, and some aspects of their metabolism and damage mechanisms remain unknown.Nevertheless, because of its ability to reabsorb and accumulate divalent metals, the kidney is the firsttargetorganofheavymetal toxicity (BarbierOetal.2005).Theextentof renaldamagebyheavymetalsdependsonthenature,thedose,routeanddurationofexposure.Bothacuteandchronicintoxicationhavebeen demonstrated to cause nephropathies, with various levels of severity ranging from tubulardysfunctionstoseverekidneyfailureleadingoccasionallytodeath.

CKD is characterized by progressive destruction of the renal parenchyma and the loss of functionalnephrons, which finally lead to ESKD requiring chronic dialysis treatment or a kidney transplantation.DiagnosisofCKDdependonserumcreatininelevelandglomerularfiltrationratewhich,however,becomesdetectableonlywhenkidneyfunctionishalved.

Giventhelimitationsofserumcreatinineasamarkerofrenalfunction,differenturinaryproteinshavebeenrigorouslyinvestigatedoverthepastdecadeaspossiblebiomarkersforearlyandaccurateidentificationoftubular dysfunction or injury, which usually anticipate by years the appearance of overt CKD (Lane BR.2013).TheseincludeN-acetyl-β-d-glucosaminidase(NAG),urinaryβ2-microglobulin(β2-MG),kidneyinjurymolecule-1 (KIM-1), Neutrophil Gelatinase Associated Lipocalin (NGAL) and liver-type fatty acid bindingprotein(L-FABP).

Recognition of early renal damagemay have strong clinical rationale as it allows prompt injury-specificinterventionthatmayavertpermanentrenaldamage.

Theaimof this study is toassess theentityof renal function impairmentandearly tubulardamage inasample of subjects geographically exposed to a different gradient of environmental pollution by heavymetals inorderto1) identifytheoverallprevalenceofCKDmfo/CKDuand2)quantifytheentityofearlyrenaldamagebytubularbiomarkersinindividualswithapparentlynormalrenalfunction.

MATERIALSANDMETHODSStudypopulationThree hundred adults with no history of renal disease will be enrolled. These subjects will be sharedaccordingtotheplaceatlowmiddleandhighlevelofenvironmentalpollution.Patientswithliver,thyroidor infectious diseases, active urinary tract infections, alterations in leucocyte count or formula andtreatmentwithsteroids,antibioticsorimmunosuppressorswillbeexcludedfromthestudy.

RenalfunctionassessmentRenalfunctionwillbeestimatedasglomerularfiltrationrate(eGFR)byusingtheCockcroft-GaultandtheMDRDformulabyassessingindividualserumcreatininelevels.Inaddition,serumcystatin-Cwillbe

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analyzedinperipheralbloodsampleasanadditionalmarkerofrenalfunctionandimplementedincystatin-Cbasedformulasforimprovingestimationprecisionofrenalfunction.

LaboratorymeasurementsCommonblood andurinebiochemical parameters, includingurea, creatinine, uric acid, serum lipids andelectrolytes, albumin, haemoglobin, proteinuria, fibrinogen, C-Reactive protein, proteinuria andalbuminuria will be measured according to standard methods in the routine clinical laboratory. Tenmillilitresof freshurinewill bemixedwith1mLof10mMtrisbuffer,pH8.6with0.05%Tween20and0.01% of NaN3 containing protease inhibitors (10mM benzamidine, 10mM aminocaproic acid, 20mMethylenediaminetetracetate and aprotinin). This mixture will be centrifuged at 3000 rpm for 8min andstoredat-80°Cuntilassayed.

Biomarkersoftubulardamage,includingNAG,β2-MG,KIM-1,NGALandL-FABP,willbemeasuredinurinesamplesusingELISAcommercialavailablekits,accordingtothemanufacturer’sinstructionsandindexedbyurinarycreatinineexcretionlevels.Allmeasurementswillbemadeinatriplicateandblindedmanner.

EXPECTEDRESULTSWeexpecttofindasignificantlyincreasedproportionofsubjectspresentingwithCKDmfo/CKDuoraltered(increased) levels of tubular biomarkers of early renal damage according to a geographical gradient ofenvironmentalpollution.

Task4.6.Disseminationandscientificnetworking

The task will be developed in the framework of the WP4 activities, in coordination with the generalcommunicationplan(WP1).

Three different studies involving human subjects are planned in areas defined at risk for environmentalpollution.Eachstudy involves theadministrationofaquestionnaire to investigatepastandcurrent risks,the information received concerning environment and health, the risk perception (Coi et al. 2016). Thescientific community will receive a contribution regarding environmental health, improved by theintegration of those studies into the wider CISAS design. Public administrators will receive accurateinformation, useful to fine-tune the remediation activities and thepreventionmeasures. The resultswillbenefit thecommunity,providingabetterknowledgeof theenvironmentalpressures in thearea,andoftheexposureofthepopulation.Thesubjectsinvolvedwillreceiveinformationaboutpersonalriskprofile.

AfirstoutlineoftheDisseminationandscientificnetworkingactivitiesispresentedhere.

Themain actors directly involved are: Participating subjects (donors), Local General Practitioners (LGP),LocalHealthAuthorities(LHA)andEthicalCommittees,PublicAdministratorintheHealthandEnvironmentsectors, The main actors indirectly involved are:, The scientific community, Local media people, Localstakeholders.

DisseminationandscientificnetworkingactivitiesA-InternalcirculationofinformationThe management of theWP is committed to circulate all the information relevant to the whole CISASconstituency

B-PreparatoryactivitiesWHAT:Mappingtargetgroupsandaudience

Testtheinformationmaterial

Explorethecommunityawarenessofenvironmentalhealthissuesandneedsforinformation

Testtoolsforstudyresultsdelivery

HOW:interviews,focusgroup,collectionofsocio-economicindicators,policyanalysis

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C-LegallybindingactivitiesWHAT: The legally binding activities are linked to the authorisation requested to the LHA EthicalCommittees.Forthisrequest,theprotocol,alltheinformationmaterial,thequestionnaire,theconsensusandtheinformationformshavetobepresentedandapproved.

HOW: draft of: 1 - the study protocol; 2 – the questionnaire; 3 – the informed consent form; 4 – aninformation sheet; 5 – a form for results restitution; 6 - other required documents; establish an officialcontact;followtheproceduresrequired.

D-TrainingWHAT:trainingactivitiescanbecrucialtodisseminatetheresultsofcomplexstudiesinthehealthdomani.Whentheenvironmentandhealthinteractionisconcerned,complexity,uncertaintyandambiguityrequireanaccurateunderstanding,toavoidminimizingooverestimatingtheconnectedrisks.Beingthedonorsasample of the general population, a training activity is recommended to inform and train the LGP, thatmightbequestionedbytheirpatients.

Other specific actors can be considered for training activities, like teachers, journalists, publicadministrators.

HOW:one-dayseminars,toberepeatedaccordingtotherequests,atleastoneatthebeginningandoneattheendoftheresearchactivities,incollaborationwithNationalandRegionalbodiesinvolvedinstudyinginthethreeareas.

E–CirculationofresultswithintheScientificCommunityWHAT:Specificscientificcommunitiesareinterestedinprojectresults,tofurtheradvanceresearchinthefield; high education institutions are interested in project results to improve skills and for educationaltrainingpurposes.

1. Publication of scientific results in high level scientific journals is required. All the publicationswill becollectedinadedicatedroomwithinthewebsite,inorderthatinterestedpeople(researchers,companies,end-usersatlarge)canhaveallthescientificpapersataglance.

2.Presentationoffindingsinnationalandinternationalconferences.

3.OrganizationofscientificmeetingsincollaborationwithInternational,NationalandRegionalbodies.

4.NetworkingandcollaborationwiththemainsectoralscientificInstitutions,H2020andFP7projectsthatcouldcomplementprojectactivitiesandprovidesynergies,andatthesametimeenhancedisseminationoftheprojectresultstoaveryspecializedandprofessionalaudience.

(https://ec.europa.eu/programmes/horizon2020/en/h2020-section/responsible-research-innovation)

HOW:

1.Partof thebudget is specifically allocated topublishing the results inhigh level scientific journals, forgoldopenaccess.Carewillbetakentopublishinopenaccessjournals.Dependingonlegalrequirements,partners will deposit an electronic copy of or a link to the published version or the final manuscriptaccepted for publication in an institutional or subject-based repository at themomentof publication, aswellasinanappropriatesectionofthewebsite.

2.Highlevelpresentationsofthefindingsinnationalandinternationalconferencesisplannedandforeseeninthebudget.

3.Dependinguponthetimelineofthethreeresearches,twoscientificconferenceswillbeorganized,attheRegionalandNationalLevel,andacontribution

F-DisseminationofresultsWHAT:Thecommunitymustbeinformedabouttheon-goingstudyandtheresults.Publicawarenessisacrucial pre requisite to obtain the consensus of donors, to understand themeaning of the study and toproperly use the information acquired. The study results will be delivered to the single donor, andpresented during a preliminary meeting to the Local Administrators and Authorities. Immediately afterthoseaccomplishments,theyshouldbedisclosedinasimpleway,astoallowtheircomprehensionandusebyawideaudience.

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A wider public can be reached through science communicators, science journalists, science educators,whichcanactasmultipliersforgeneralpubliccommunication.

HOW: a first public event to present the study should be organized, in coordination with the LocalAdministrations,topresentthestudyandspreadgeneralinformation,withdedicatedpressrelease.Moredetailedinformationwillbeavailableontheprojectwebsite.Disseminationwillbealsoviasocialnetworks.MeetingwithdonorsandPublicAuthorities.Afinalevent,withbeorganized,withdedicatedpressrelease.

The participation to public events dedicated to science communication and/or European research isplanned,topromotecommunicationactivities(multimediaproductionandcommunicationmaterialforthegeneral public and media), in order to intercept a wider public of science communicators and sciencejournalists,whichcanactasmultipliersforgeneralpubliccommunication.(CoriL.2016)

Communicationanddisseminationwillbemonitoredandsuccesscriteriawillbedeveloped.

Listofdeliverables

D4.1.StudyProtocol(M3)

D4.2.ReportonriskoflivercancerinPriolo(M33)

D4.3.ReportonriskofthyroidcancerinMilazzo(M33)

D4.4.1ReportonriskofcardiovascularbiomarkersinCrotone(M33)

D4.4.2ReportonpilotstudyonrenalfunctioninCrotone(M33)

D4.5.Reportofthedissemination,engagementandnetworkingactivities(M36)

Listofmilestones

M4.1.EthicalCommittees’approvalofstudydesign(M12)

M4.2.1.PrioloLocalConferencetopresentthestudy(M6)

M4.2.2.PrioloLocalConferencetopresentthestudyresults(M30)

M4.3.1.MilazzoLocalConferencetopresentthestudy(M6)

M4.3.2.MilazzoLocalConferencetopresentthestudyresults(M30)

M4.4.1.CrotoneLocalConferencetopresentthestudies(M6)

M4.4.2.CrotoneLocalConferencetopresentthestudiesresults(M30)

M4.5.Trainingcoursesinthreecities(M20-M25)


Workpackagetitle MolecularEpidemiology

Participantnumber IBIM DiSTeM ISS


Objectives

In vitro,exvivo, invivomodellingofenvironmentaleffectsonbiological systemsandeffectsofprenatalexposuretopollutantsonlate-onsethealtheffects

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Descriptionofwork

Task5.1-Bio-accumulationofenvironmentalcontaminantsinplacenta,maternalandnewborntissuesandtheirassociationwithplacentaltranscriptome,pregnancyandlong-terminfantoutcomes(IBIM)

Epidemiologicstudiesregardinghealtheffectsofenvironmentalcontaminantstendtofocusonthemoresusceptible segment of society: in the last years many efforts were employed in the evaluation of theeffects of environmental contaminants on child’s health. However more recently the theory of“intrauterine origins of health and disease susceptibility” is gaining interest as a new approach tounderstanding the cause of many multifactorial disorders. In fact, adult diseases may have an in uteroorigin, when suboptimal intrauterine conditions - including exposure to environmental contaminants -induce irreversible changes, which manifest themselves in post-natal and adult life. In this context theinfluence of environmental pollution on pregnant women is of special interest: much attention is nowfocusedontheinvestigationofi)toxicantsthataretransferredfromthemother'sbloodtothedevelopingfetus; ii) the influence of toxicants on both pregnancy outcome and fetal development and late onsetconsequences.Becausehumanplacentaisposedattheinterfacebetweenmaternal/externalenvironmentandembryo,itcanbetakenasanenvironmentalmonitoringsystem.Placentaltissuecanbeconsideredasa noninvasive exposure biomarker for different organic and inorganic pollutants. This tissue, usuallydiscardedafterbirth, iseasytoobtainandmayfurnish informationontheexposureofbothmotherandfetus.Forsometoxicantstheplacentarepresentsabarrierwhileothersareabletopenetrate.Inbothcasesit is of interest to compare different compartments of both themother (blood) and newborn (umbilicalcordbloodandhairs)alongwithplacenta.Placentaevaluationmaybeuseful inassessingenvironmentalexposure to organic and inorganics pollutants and in predicting future infant health outcomes.Polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and Heavy metals (HMs) areubiquitous,persistent,bioaccumulativeand toxiccompounds.Several studies report theaccumulationofthesesubstancesinmaternalandfetaltissues, includingmaternalandumbilicalcordblood,placentaandbreastmilk,indicatingthatprenatalandneonatalexposuretothesechemicalsislikelytooccur[Shenetal.2008; Frederiksen et al. 2009; Al-Saleh et al. 2014]. A large number of epidemiological studies haveassociatedearlyexposuretoPb,Hg,As,andCdwithneurological [Lanphearetal.,2005],developmental[Gundackeretal.,2010],andendocrinedisorders [Stasenkoetal.2010].LowtomoderateprenatalPCBsexposure has been related to a decrease in psychomotor development during the first month of life[Huismanetal.1995].Further,neurodevelopmentaleffectshavebeenrelatedtocordbloodconcentrationsof PBDEs [Herbstman et al. 2010]. The humanplacentamight not be able to limit fetal exposure to theemergingcontaminantsthathavebeenproducedbymaninthelastdecades.Wehypothesizethatpeopleliving inhighlypollutedareas - includingpregnantwomen-areexposedtoHMs,PCBsandPBDEs.Thesetoxicsubstancesmaydeveloptheirtoxicactionthroughthederegulationofplacentalphysiologyand/orbyinterfering with molecular signalling between mother, placenta and fetus, essential for leadingdevelopmentalprocessandmaypredisposetolateonsetdiseases.

Subtask 5.1.1:Epidemiological data collection, concerningmaternal health status and lifestyle (includingtheassessmentofdietaryhabits),willbecollectedthroughsurveyquestionnairesprovidedtothemotherafterdelivery;

Subtask 5.1.2: At the time of the delivery placenta tissue, maternal blood, umbilical cord blood andnewbornhairswillbecollectedinordertoassesscontaminantlevels(heavymetals,PBDEsandPCBs),andtoclarifyhowheavymetalsandpersistentorganicpollutantsinteractwithplacentaldomain;

Subtask 5.1.3: A comparative estimate of genome-widemRNA expression in placenta between high andlow exposure to toxics will be performed in order to analyze potential placental trascriptome shift, inresponse to toxic substances exposure,which can reflect the adaptive pathways – promoting early fetalsurvive–thatleadtolongtermeffectonhealth.;

Subtask5.1.4:LPossiblelng-terminfantoutcomeswillbeevaluatedduringthefirstthreeyearsoflife,fromthe birth, using hospitalization database, through regular clinic evaluations, including diagnosis afterhospitalaccesscare.

Theactivitiesofthetaskareaimedat:

CISAS-28/09/2016 41

1)selectingthepopulationsamplestobeinvestigatedalongwiththecontrolpopulationsamples;

2) identifying the childbirth centers where pregnant womenwill be recruited for the study andwherewomengivingbirthwillbeattendedbytrainedpersonnelforplacentalsampling

3) evaluating the risk of heavymetals, PBDEs and/or PCBs contamination during pregnancy in acohort ofmother-childpairs resident inhighlypollutedareas characterizedbydifferent levels ofenvironmentalcontamination;

4) evaluating the bioaccumulation features and patterns of HMs, PBDEs and PCBs by examiningtheirdistributionamongmaternal,placentalandfetaltissues;

5) determining whether the bio-accumulation of heavy metals, PBDEs and/or PCBs may impactplacentalmRNAexpression;

6) understanding whether prenatal exposure to Heavy metals, PBDEs and/or PCBs may causenegativepregnancyoutcomeandlong-termeffectsonchildrenhealthanddiseasepredisposition;

7) defining the associations of placental contamination and gene expression patternswith long-term infant health outcomes, to evaluate the validity of placental analyses in predicting futureinfanthealthoutcomes.

In the contextof the followingTask5.2 the researchactivityon the selectedpopulation sampleswill bedeveloped

Task5.2 -Cellularmodelsandenvironmentalpollutants inbiochemical andbiomolecularmechanismsofairwaydiseases(IBIM)

Theactivityofthetaskwillbeinvolvedinthedevelopmentofscientificresearchtounderstandtheeffectsofenvironmentalpollutantsidentifiedinthethree-studyareasofAugusta/Priolo,Milazzo,andCrotoneaswellasemergentcontaminantsonhumanhealthconcerninginflammatoryandcancerdiseasesofthenoseandbronchi.Wewillobtainconceptualmodelsofdiseasetofacilitatetheassessmentofpollutionmixtureson human health and to integrate the effects of individual criteria pollutants found in themixtures. Invitro/exvivostudieshaveshownthatdifferentiatedcellculturesinmonolayerortwo-dimensional(2D)orthree-dimensional (3D)models are an invaluablemodel in understanding the physiological properties ofthetissueandofthepathogenesisofdiseases.Inthestudy,wewilldevelopexperimentalmodelsusingcellculturefromnormalandcancerepithelialcelllinesaswellashumanprimaryepithelialcellsfromthenoseand bronchi. Our final output will be the development of knowledge on the interaction betweenbiochemicalandmolecular cellularevents initiatedbypollutantsand involved in theairwaydisease.Theresultswill be performedby the application of biomolecular and biochemistry techniques involving Realtime PCR, Chip-immunoassay, immunoprecipitation, western blot analysis, elisa methods,immunocytochemistry,immunohistochemistry,immunofluorescence,microscopy,andfluorimetry.

In termsof innovationwill becomeavailabledataon threshold levelsof variouspollutants (fundamentalbasis for interventions at the origin of the pollution level of bio-contamination and vehicles) and a newapproach to the prevention of environmental pollution diseases by the use of chemical andpharmacologicalantagonists(selectiveinhibitorofsignalpathways).

Subtask 5.2.1: The process of setting-up of the experimental procedures of the task will be theidentification of the pathogenic mechanisms by which environmental pollutants interfere with normalcellularmechanismspromotingcarcinogenesissuchas1)inductionofoxidativestress;2)interferenceswithDNArepairmechanisms;3)dysregulationofcellproliferation;4)genotoxiceffects;5)cellularmechanismsinvolved in the activation of many genes, biomarkers, proteins and other factors including intracellularsignalpathway.

Subtask5.2.2:Theexperimentalprocedures,byincreasingtheknowledgeofbiocellularandbiomolecularevents generated in the diseases, will be of relevance to test and support potential pharmacologicalstrategiestocounteractthenegativeeffectsofpollutantsonhumanhealth.

Task5.3-Invitroandinvivostudiesofmechanismsofimmunomodulationandimmunotoxicity(IBIMand

CISAS-28/09/2016 42

ISS)

It has been shown that exposure to contaminants in the environment via different routes can causeadverseeffectsonbiologicalsystems,suchasthecentralnervoussystem,liver,reproductiveandendocrinesystems.Althoughtheinformationontheimpactontheimmunesystemaremorelimited,recentstudiesinanimal models have shown immunosuppressive and immunomodulatory effects suggesting thatcontaminantspresent intheenvironmentcandisplayapotential immunotoxiceffect.So far, there isstillinsufficientknowledgeabouttheeffectsofthesubstancesonthedifferentcellpopulationsoftheimmunesystem from both the innate and adaptive immune system and the knowledge of related toxicologicalmechanismsisstillfairy.Tofillthisgap,studiesonisolatedcelllinesofbothhumanandmurineorigincanprovide important information leading to an integrated approach. These biological systems allow thepossibilityforasystematicscreeningbasedonthedifferentcategoriesofcontaminantsalreadystudiedinthe other thematic lines of the project. Moreover, although the use of ex vivo human cells can beconsidered the first choice to conduct these studies, thedifficulties in termsof individual variability andreproducibilityoftheassayssuggesttheuseofanimalsystems(inparticularthemurinemodelforwhichisprovided the greatest amount of knowledge on the immune systemaswell as the reagents used in theassays)inwhichthesedifficultiescanbereducedorovercome.Toaccomplishthistask,itcanbeusefultoemploybothhumanandanimalsystemswhereitcanbeeasytomanipulateimmunecelllinesderivedfromdifferent tissues and organs. This ex vivo assays can give to us the opportunity to evaluate severalendpoints and biomarkers such as cell viability or the production of independent cytokines in specificexperimentalconditions.

Inaddition,accordingtotheresultsobtainedinthisphase,itwillbepossibletoevaluateandoutlineaninvitro/invivostudyinordertodefinetheimpactofexposuretocontaminantsoncompleximmunologicalfunctionssuchashumoralandcellularresponsestoimmunizationwithstandardantigens.

Descriptionofwork

Subtask 5.3.1 - Evaluation of immunotoxicity and immunomodulation in human cell lines by means ofcellularandmolecularbiologytechniques(RealtimePCR,westernblot,ROSproduction,cellproliferationassays,ELISAassaysforthedeterminationofcytokineproductionandcytofluorimetricanalysis.

Subtask 5.3.2 - Evaluation of immunotoxicity performed in a mouse model to study the impact ofenvironmentalcontaminantsontheinnateandacquiredimmunesystem.Theseassayswillbeconductedinparallelwithhumancellslookingattheadvantagesanddisadvantagesofthetwosystems;

Subtask5.3.3 -Evaluationofthecorrelationbetweentheresultsobtainedbymeansofexvivo / invitrostudiesandthosegeneratedbyfunctionalstudiesinvivo.

Task5.4-Biomolecularmarkersrelatedtoexposureinvitroandcancer(UniPA-DiSTeM)

Manydiseasesrelatedtocontaminants, includingchronicdiseases,pathologiesrelatedtooxidativestressandcancer,haveamultifactorialorigin.Thesusceptibilityofanindividualtospecificcontaminantsmaybedefined by a variety of factors such as differences in absorption, distribution, metabolism (toxification,detoxification) and excretion of the toxicant, or repair of the damage introduced into DNA, as well aschanges in genes controlling protein expression or cell growth and differentiation. Carcinogenesis is amultistep process involving the transformation, survival, proliferation, invasion, angiogenesis, andmetastasisofthetumorandmaytakeupto30years.Thepathwaysassociatedwiththisprocesshavebeenlinkedtochronicinflammation,amajormediatoroftumorprogression.

In vitro studies, non-animal test methods, and computer-based approaches, provide important tools toenhanceourunderstandingof hazardous effects by chemicals and to enhance the extrapolation from invitro to in vivo in humans (Broadhead and Combes, 2001). In vitro systems are used principally forscreeningpurposesandforgeneratingmorecomprehensivetoxicologicalprofiles.Sinceproteins,whicharedynamicallymodifiedandprocessedatmultiplelevelsduringoraftertheirmaturation,directlyexecutethebiologicalprocesses,thestateofanorganismisessentiallyreflectedinitsproteome.

Our research group is experienced in utilization of cancer cellsmodel system and biomarkers to assessmechanisms underlying toxicity, oxidative stress, apoptosis, cell cycle and differentiation, induced by

CISAS-28/09/2016 43

naturalcompounds(Baccoucheetal2016,Abbesetal.,2013,DiFioreetal.,2013,2010,2009;Pelleritoetal.,2010),byxenobioticsandenvironmentalpollutants(Messinaetal.,2014;Trocinoetal.,2012,Pucciaetal.,2005)andsyntheticsdrugs(Carlisietal.,2011;D’Anneoetal.,2010;Dragoetal.,2008;DeBlasioetal,2005).Themaingoalofthepresenttaskistheevaluation,invitro,ofthecombinedactionofcontaminantsselected fromunit inWP1, in cancer promotion by evaluating: signal transduction, biochemicalmarkersrelatedtotoxicity,inflammation,oxidativestress,cellcyclecontrol,angiogenesisandapoptosis.

DescriptionofworkWewill apply ourmodel-systems to assess the combined action among target contaminants and targetgenes.

Themethodologyinvolves,inthefirstinstance,thepossibilitytousesomeofthefollowingcelllines:

● hepatoma,

● retinoblastoma,

● osteosarcoma,

● cancer-stemcells,

● melanoma,

● fibroblast.

Thestudywillfocuson:

● antioxidant/pro-oxidantswitchandrelatedsignaltransductionspathways,

● dose-timedependenttoxicity,

● biomarkersofoxidativestress,

● cellcyclemodulation,

● inhibitionorpromotionofapoptoticpattern,orproliferativeeffect,

● markersofinflammation,

● markersofangiogenesis.

Listofdeliverables

D5.1.1:Epidemiologicsurveyinstudyandcontrolareas:evaluationofreproductivehealth(M6)

D5.1.2:“Ecologic”evaluationofselectedpollutantinmaternalandfetaltissues(M15)

D5.1.3:Comparativeestimateofgenome-widemRNAexpressioninplacentabetweenhighandlowexposureareas(M28)

D 5.1.4: Long-term comparative evaluation of child health between high and low exposure areas(M34)

D5.1.5:Identificationofexposureplacentalbiomarkersrelatedtolate-onsetdiseases(M36)

D 5.2.1: Development of ex vivo/In vitro experimental models of disease (related to exposure totargetcontaminants)(M18)

D5.2.2:Comparativeanalysisofthedata(M24)

D5.2.3:Disseminationofscientificresults(M30)

D5.3.1Comparativeanalysisofhumanandmurinedata(M23)

D5.3.2Disseminationofscientificresults(M30)

D 5.4.1 Evaluation of molecular sensors related to exposure to target contaminants, stress, signaltransductionsrelatedtocancer(M30)

CISAS-28/09/2016 44

Listofmilestones

M5.1.1.Startofsamplingactivities(M10)

M5.1.2:Settingupandmanagementofbiobankformaternalandfetaltissues(M12)

M 5.1.3: Creation of a cohort of pregnant women recruited during the 8th month of pregnancy, whendefectsoffetalgrowthareexcluded(M30)

M5.2.1:Developmentofexperimentalmodelsofdisease:Cellcultures(monolayermodels)(M11)

M5.2.2:Developmentofexperimentalmodelsofdisease:Cellcultures(2Dand3Dmodels)(M14)

M 5.3.1 Development of human andmurine experimentalmodels of immunotoxicity: Cell culturesassays(M12)

M5.3.2Developmentofexperimentalinvivoanimalmodels(M18)

M 5.4.1 definition of molecular sensors related to exposure to target contaminants, stress, signaltransductionsrelatedtocancer(M18)


Workpackagetitle Biomathematics

Shortnameofparticipant IASI


Objectives

In this workpackage the data exchange structure of the project is prepared, the scientific questions ofinterestareformalizedandquantitativeanswersareprovidedwithstatisticalandmathematicalmodellingtools.

Descriptionofwork

Task6.1-DataFramework

Establishingacommonframework(hardware,softwareandstandards/templates)fordataexchangeandjointwork.Acollaborativeenvironmentwillbechosen,centralizedstorageofdocumentsanddataprovided,secureremotedataentrytoolsmadeavailabletoallprojectscientists.

Task6.2-Userandfunctionalrequirements

Identifyingtheresearchgoalsofthebiomedicalscientists,assessingthecorrespondencebetweenavailabledataandgoals,formalizinghypothesesonthedistributionandmechanismsofactionoftheseveraltoxicantsconsidered.Hypotheseswilltakeintoaccountpotentialinteractionsbetweenmeasuredvariables(physiological,biochemical,metabolic,cellularetc),aswellastheidentificationofsurrogatemarkers.Theoutputofthistaskwillconsistofthecollectionofquestionsforwhichtheprojectengagestoprovidequantitativeanswers.

Task6.3-Referencecollection

Collaboratingwiththebiomedicalscientistsinsiftingavailableliteratureforinformationrelevanttotheformulationandquantificationofthemodels,andmoregenerallyinformingtheresearchquestionstobeaddressed.Therelevantpublishedpaperswillbestoredas.pdf,cataloged,andtheindividualinformationitemswillbeinsertedinasearchableconceptdatabase,foreasyreferenceduringmodelbuildingandmanuscriptwriting.

CISAS-28/09/2016 45

Task6.4-Modelblueprints

Draftingconcisesummariesofthemechanismswhicharedeemednecessarytodescribethegeneration,distribution,fateandeffectofthetoxicantsandtheirclinicaleffects;obtainingconsensusamongtheconcernedscientistsonthedescribedmechanisms.

Task6.5–Formalization

Writingequationsthatcapturetheabovemechanismsandformulatingexperimentaldesignsdirectedatobtainingsufficientdataformodelidentification;reachingconsensusbetweenmathematicians,statisticiansandbiomedicalscientistsontheaccuracyoftheformalizationsandthecorrectnessandfeasibilityoftheexperimentaldesign;studyingthemathematicalpropertiesofthemodelequationsandimplementingthemodelsinsoftware.TheendresultofthisTaskwillbetheformaldescriptionofeachmodelateachdevelopmentversioninaModelDescriptionSheet(MDS),astructureddocumentcarryinghypotheses,equationsandparametercalibrationinformation.

Task6.6-Dataanalysis

CompilingModelAnalysisPlansandStatisticalAnalysisPlans(MAPs,SAPs)integratingthetheoreticaldesigndevelopedintheprevioustaskswiththepeculiaritiesofthedatasetseffectivelyavailable;fittingthemodelequationstoavailabledata,validatingordetectingdeficienciesinthemodels;conductingstatisticalanalyses.

Task6.7–Reporting

Writingcomprehensivereportsonthedataanalysesperformed,linkingthemwiththeoriginalquestionsandpreparingthenecessarytabularandiconographicalmaterialforpublicationoftheresultsinscientificpapers.

Task6.8-SimulationandDecisionSupport

Implementinginsilico“whatif”simulationscenarios,capturingactualsituationsandpotentialmitigationmaneuvers(control);formulatingrecommendationsforinterventionbasedonquantitativemeasuresofcostfunctionscorrespondingtotheseveralstudiedscenariosandtothehipothesizedinterventionmaneuvers.

Listandnameofdeliverables(alsotimetable)

D6.1.1:“CISASPlatformUserManual”,detailingtheuseoftheimplementeddataframework(M8)

D6.2.1:“DataAnalysisGoals”report,detailingthesetofquestionstobeansweredbydataanalysiswithintheproject’slifetime(M12)

D6.4.1:“Preliminaryanalysisblueprints”report,detailingtheassumptions,goals,andavailableinformationforthemodels/analysestobeimplemented,preliminaryversion(M18).

D6.4.2:“Analysisblueprints”report,detailingtheassumptions,goals,andavailableinformationforthemodels/analysestobeimplemented,definitiveversion(M36).

D6.5.1:“ModelDescriptions”report,thecollectionofthecompletedMDS’sforthemodelstobeimplemented(M36).

D6.7.1:“DataAnalysisReport”,thecollectionoftheModellingReportsandStatisticalAnalalysisReportsproducedinthecourseofthedataanalysisfortheproject(M36).

D6.8.1:“SimulationReport”,thecollectionofthesimulationreportsproducedforScenarioexploration(M36).

Listandnameofmilestones(alsotimetable)

6.1:DataFrameworkcompleted(M8)

6.2:DataAnalysisgoalsdefined(M12)

CISAS-28/09/2016 46

6.3:Firstmodelimplementedandresultsdelivered(M18)

6.4:CISASBiomathematicsSummerSchool(M35)

CISAS-28/09/2016 47

3.2 ManagementstructureandproceduresThenumberofinterconnectedactivitiestobecarriedoutintheCISASprojectrequiresclearworkflows,up-to-date project monitoring and transparent decision structures are core requirements. ManagementefficiencyandeffectivenesswillbenecessarytosmoothlyfulfilalltasksduringtherealizationoftheprojectandfinallyachievetheCISASobjectives.TheprojectorganizationalstructurewilloperateataStrategiclevelwith a Project responsible, a Project Coordinator (PC) and the Steering Committee (SC). Objectives: todevelop policies and plans, give scientific & technical directions, to review the project progress andresourcesandtosolvemajorconflicts.Moreover,atthestrategiclevel,theCISASAdvisoryBoard(AB)willassisttheSteeringCommitteeonscientific,technicalandpolicyissues(seebelow).

Adetaileddescriptionofrolesandfunctionsofthecomponentsofthemanagementstructuresispresentedinthefollowing.

ProjectResponsible(PR)-ThePRisinchargetheadministrativeandscientificresponsibilityoftheproject.Heisthelegalentityoftheproject.ThePRisrepresentedbytheDirectoroftheDTA-CNR.

ProjectCoordinator(PC)-ThePCisinchargeoftheconsolidationofthetechnicalandfinancialresultsatthe endof each reporting period. In addition, he has the task to strategically promote the project in allrelevantoccasions, frompolitical toproject level.He is inchargethemanagingandmonitoringofall theaspects related to the design, reset and foundation of the research centre at the Roosevelt complex(Palermo)instrongconnectionwiththeCNRofficesinRomespecificallydedicatedtothisaction.

Secretariat – Composed by a technologist full-time dedicated, It is in charge of day-to-day project’smanagementandadministration;supportingtheproject’sgovernance, includingexchangeof informationamongthegoverningbodiesandorganizationofmeetings.Particulardutiesinclude:

ü beingapermanentcontactpointforallthepartnersregardingtheirparticipationintheproject;

ü respondingtoanyrelevantrequest;

ü following and updating the project indicators (Gantt chart, manpower matrix, list of expectedresults)

ü monitoring the status of the project ensuring the proper management reporting from both theformalandinformalstandpoints;thismightalsorequireconsultationthroughinternalmeetingorinformalcheckpoint

ü activationof conflict resolutionprocedures to reduce the impactof possibledeviations from theplannedmilestonesanddeliverables.

Steering Committee (SC) - highest level and ultimate decision-making body, it is aimed at the internalgovernanceof theproject. Itwill be theplacewhere theadvancementof theproject, critical issuesandrisks,partnercontributesandmainresultswillbeshared,discussedandapproved.TheSteeringCommitteewillthereforemonitortheprogressoftheproject,withreferencetothetimetable,outputsandexpectedimpacts, suggesting also possible adaptation measures. The SC will be composed by: the ProjectCoordinator, theWorkPackage leaders. Bydefault, itmeets twiceper year inperson; in addition, othermeetingswillbescheduledifneeded.

WorkPackageLeaders(WPL)-areresponsibleforcoordinatingtheactivitiesandmonitoringtheprogressof each WP and Task, ensuring that milestones and deliverables are fulfilled, updating to the ProjectCoordinator and the Steering Committee, and contributing to regular communication and disseminationactivities.TwoWorkPackagesareco-leadinordertoshareeffortsandresponsibilitieswhileguaranteeingcomplementarity. Since the WPLs drive the project’s activities, the Coordinator can convene remotemeetingswithoneormorethemifneeded.

WorkPackage Leader/Co-leader Organization

WP1 VeronicaGiuliano/MarioSprovieri DTA

CISAS-28/09/2016 48

WorkPackage Leader/Co-leader Organization

WP2 MarioSprovieri IAMC

WP3 AngelaCuttitta IAMC

WP4 FabrizioBianchi IFC

WP5 FabioCibella IBIM

WP6 AndreaDeGaetano IASI

CISAS Advisory Board - is the official strategic advisory body of the project for scientific, technical andpolicy issues. It will be the high-level strategic mentoring board, assisting the Steering Committee,addressing the CISAS project’s achievements, its developments, priorities, new visions and integrationamong initiatives, and counselling the project all along. The components of the Advisory Board are: theProjectCoordinator,thethreedelegatefromtheSicilianpublicuniversities,adelegatefromtheResearchInstitutionsworkinginSicily(ENEA,INFN,INGV).

Table3.1a: Listofworkpackages

Workpackage

No

WorkPackageTitle LeadParticipant

No

LeadParticipantShortName

StartMonth

Endmonth

Totalmonths

1 Projectcoordination,management,andcommunication

1

DTA 1 36

2 Contaminants in the

environment

1 IAMC

2 34

3 Ecosystemand

Contaminants

1 IAMC 1 36

4 Etiologicalepidemiology 1 IFC 1 36

5 MolecularEpidemiology 1 IBIM 1 36

6 Biomathematics 1 IASI 1 36

Table3.1b: ListofDeliverables

Deliverable(number)

DeliverablenameWork

packagenumber

Shortnameoflead

participantType

Deliverydate

1.1.1 Firstmid-termreport 1 IAMC report M18

1.1.2 Secondmid-term

report

1 IAMC report M24

CISAS-28/09/2016 49

Deliverable(number) Deliverablename

Workpackagenumber

Shortnameoflead

participantType

Deliverydate

1.1.3 Finalreport 1 IAMC report M36

1.2.1 ReportoftheFinal

Conference

1 IAMC report M36

1.3.1 Website 1 IAMC website M3

2.1.1 Generationof

completedatabase

forliterature

informationon

contaminants

reportedformthe

threestudyareas

2 IAMC database M4

2.1.2 GenerationofaGIS

platformwithallthe

availableinformation

forthethreestudy

areas,withspecific

referenceto

contaminants

sources,distribution

pathways,population

density,etc.

2 IAMC GIS

platform

M6

2.2 Generationof

datasetsoftraditional

andemerging

contaminantsinthe

threestudyareas

2 IAMC dataset M18

2.3 Generationof

datasetsof

anthropogenic

radionuclidesinthe

threestudyareas

2 IAMC dataset M18

2.4 Reconstructionofthe

ageofcontamination

inthethreestudy

areas

2 IAMC report M24

2.5.1 Identificationof

sourcesandpathways

oftheselected

contaminantsinthe

threestudyareas

2 IAMC report M24

CISAS-28/09/2016 50


Workpackagenumber

Shortnameoflead

participantType

Deliverydate

2.5.2 Reportsforthe

differentmodelling

activitiesrelatedto

theinvestigationof

theeffectsofmixture

ofcontaminantson

theecosystems

2 IAMC report M30

2.5.3 Reportsonmass

balanceofthe

selected

contaminantsinthe

threestudyareas

2 IAMC report M30

2.6 Generationofmaps

ofrisks(forthe

selected

contaminants)forthe

threestudyareas

2 IAMC map M32

2.7 Reportonthe

distributionof

contaminantsinthe

atmosphereinthe

threestudyareas

2 IAMC report M18

2.8 Reportsandmodels

onatmospheric

depositionand

dispersionpatternsin

thethreestudyareas

2 IAMC report M18

2.9 Reportonthe

distributionofthe

selected

contaminantsinthe

terrestrialandmarine

trophicweband

localfood

2 IAMC report M30

2.10.1 Reportsonthe

measurements

carriedoutatthe

CapoGranitola

climaticObservatory

2 IAMC report M24

2.10.2 Generationof

datasetsofnatural

andanthropogenic

2 IAMC dataset M24

CISAS-28/09/2016 51


Workpackagenumber

Shortnameoflead

participantType

Deliverydate

climatealtering

contaminates,

reactivetracegases

andaerosolsand

simulationsof

atmosphericgases

andaerosols

formation,diffusion

andremoval

processesonregional

scale

2.10.3 Generationofhigh-

resolution

precipitationand

temperaturefields

overthe

Mediterranean

Regionunderspecific

climatechange

scenarios,asaninput

forimpactstudies

2 IAMC report

maps

M30

2.10.4 Generationof

descriptiveindicesof

extremesfor

precipitationand

temperatureforthe

specificscenario

2 IAMC report M30

2.11 Reportonthenew

sensorsand

integratednetworkof

sensorsbuiltfor

differentclassesof

contaminantsin

ordertofacilitate

monitoringand

studiesinthethree

consideredareas

2 IAMC report M34

3.1.1 Identificationof

samplingareas

3 IAMC Map M3

3.1.2 Datasetonthe

seasonalbiodiversity

amongthethree

3 IAMC Dataset M15

CISAS-28/09/2016 52


Workpackagenumber

Shortnameoflead

participantType

Deliverydate

studyareasand

comparativeanalyses

withcontrolareas

3.2.1 Definitionof

molecularsensors

andconcentrations

forearly-warning

3 DISTEM

Dataset M20

3.3.1 Datasetabout

alterationsof

molecularpathways

inducedbychemicals

exposition

3 IAMC Dataset M31

3.3.3 Datasetabout

alterationsof

molecularpathways

inducedbyexposition

tocontaminatedsea

water.

3 IAMC Dataset M13

3.3.5 Datasetabout

alterationsof

molecularpathways

inanimalsfrom

contaminatedareas

3 IAMC Dataset M32

3.4.1 DatasetofthemRNA

levelsinstress-

relatedgenes

3 IAMC Dataset M29

3.4.2 Dataseton

Differentially

ExpressedmiRNAsin

responseto

pollutants

3 IAMC Dataset M29

3.4.3 Datasetonepigenetic

modifications

3 IAMC Dataset M35

4.1 StudyProtocol 4 IFC protocol M3

4.2 ReportonriskoflivercancerinPriolo

4 IFC report M33

4.3 ReportonriskofthyroidcancerinMilazzo

4 IFC report M33

4.4.1 Reportonriskofcardiovascularbiomarkersin

4 IFC report M33

CISAS-28/09/2016 53


Workpackagenumber

Shortnameoflead

participantType

Deliverydate

Crotone

4.4.2 ReportonpilotstudyonrenalfunctioninCrotone

4 IFC report M33

4.5 Reportofthedissemination,engagementandnetworkingactivities

4 IFC report M36

5.1.1. Epidemiologicsurveyinstudyandcontrolareas:evaluationofreproductivehealth

5 IBIM survey M6

5.1.2 Ecologic”evaluationofselectedpollutantinmaternalandfetaltissues

5 IBIM data

collectionM15

5.1.3 Comparativeestimateofgenome-widemRNAexpressioninplacentabetweenhighandlowexposureareas

5 IBIM report M34

5.1.4 Long-termcomparativeevaluationofchildhealthbetweenhighandlowexposureareas

5 IBIM report M36

5.1.5 Identificationofexposureplacentalbiomarkersrelatedtolate-onsetdiseases

5 IBIM report M36

5.2.1 Developmentofexvivo/Invitroexperimentalmodelsofdisease(relatedtoexposuretotargetcontaminants)

5 IBIM report M18

5.2.2 Comparativeanalysisofthedata

5 IBIM report M24

5.2.3 Disseminationofscientificresults

5 IBIM scientificpublication

M30

5.3.1 Comparativeanalysisofhumanandmurine

5 IBIM report M23

CISAS-28/09/2016 54


Workpackagenumber

Shortnameoflead

participantType

Deliverydate

data

5.3.2 Disseminationofscientificresults

5 IBIM scientificpublication

M30

5.4.1 Evaluationofmolecularsensorsrelatedtoexposuretotargetcontaminants,stress,signaltransductionsrelatedtocancer

5 IBIM report M30

6.1.1 “CISASPlatformUserManual”,detailingtheuseoftheimplementeddataframework

6 IASI Platform M8

6.2.1 DataAnalysisGoalsreport,detailingthesetofquestionstobeansweredbydataanalysiswithintheproject’slifetime

6 IASI report M12

6.4.1 Preliminaryanalysisblueprints”report,detailingtheassumptions,goals,andavailableinformationforthemodels/analysestobeimplemented,preliminaryversion

6 IASI report M18

6.4.2 Analysisblueprintsreport,detailingtheassumptions,goals,andavailableinformationforthemodels/analysestobeimplemented,definitiveversion

6 IASI report M36

6.5.1 ModelDescriptionsreport,thecollectionofthecompletedMDS’sforthemodelstobeimplemented

6 IASI report M36

6.7.1 DataAnalysisReport,thecollectionofthe

6 IASI report M36

CISAS-28/09/2016 55


Workpackagenumber

Shortnameoflead

participantType

Deliverydate

ModellingReportsandStatisticalAnalalysisReportsproducedinthecourseofthedataanalysisfortheproject

6.8.1 SimulationReport”,thecollectionofthesimulationreportsproducedforScenarioexploration

6 IASI report M36

D2.12 TheworkinggroupasawholeThe working group is composed by five partners from CNR institutes and a number of different sub-contractors.Allofthemarefullycommittedinbringingtotheprojectnotonlytheircross-disciplinaryandcross-sectoral know-how in different environmental and health sectors. Moreover, dissemination andtrainingactivities,relatedtodifferentsectorsoftheprojectactivities,willbecarriedoutinoutsourcingbyagencieswithspecificexperienceson thesedifferent themes.Specific tenderswillbe lunched tocommitand involve this group of agencies. The strong integration of different expertisewill enable a significantreinforcementoftheworkinggroupcapabilitytosuccessfullycompletethetasks.

D2.13 ResourcestobecommittedThe overall budget for the CISAS project is of 6,500,000€ for 5 partners for 36 months, distributedaccordingtothefollowinggraphic,intermsofWPsdistribution(Fig.2).

Figure2:Budgetdistributedinthe6WP

CISAS-28/09/2016 56

Table3.4a‘Otherdirectcost’items(travel,equipment,infrastructure,goodsandservices,largeresearchinfrastructure)

1-DTA-CNR Cost(€) Justification

Personnel 240,000 2technologistsfor36months

Travel 200,000

Outsourcing 1,400,000

Overheads 160,000

Total 2,000,000

2-IAMC-CNR Cost(€) Justification

Personnel 450,000 3researchersfor36months

Travel 100,000

Equipment 120,000 Purchasing a new generation ICP-MS + electronic devices forsensorscreation

Consumables 100,000

Outsourcing 310,000 Radio-chronology, Biomonitoring, Effects of climate change,Atmosphere,Food

Overheads 120,000

Total 1,200,000

3-IAMC-CNR Cost(€) Justification

Personnel 275,000 10researcherparttime(50%)for36MTravel 11,000

Equipment 7,000 PurchasinganewqPCRConsumables 69,500

Outsourcing 245,00 ServiceandcounsulenceTotal 607,500

4-IFC-CNR Cost(€) Justification

Travel 41,000 Travel+congress

Equipment 55,500 Pulse pen, body cardio, PC, fish house supplies, etc + 30,000maintenance

Consumables 118,820 Reagents,software,kitformolecularbiology,bloodanalysis,etc.

CISAS-28/09/2016 57

Outsourcing 134,360 Disseminationsupport+training+subcontractslabservices

Overheads 81,818

Othergoodsandservices 8,500 CostsforPublications+

Total 900,002

5-IBIM-CNR Cost(€) Justification

Personnel 386.000

Travel 22.000

Equipment 20.000

Consulences 210.000

Consumables 262.000

Total 900.000

6-IASI-CNR Cost(€) Justification

Personnel 520,000 Researchcontracts(12py’s)andresearcherpositions(4py’s)

Travel 90,000

Equipment 30,000 Computingequipment

SummerSchool 50,000

Outsourcing 120,000 Informationsystem

Overheads 90,000

Total 900,000

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