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CasestudyonassemblingandassessingAOPinformation:AlkylationofDNAleadingtoheritablegeneticeffects
CaroleL.YaukEnvironmentalHealthScienceandResearchBureau
HealthCanadaOttawa,ON
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Outline
• Review-MIE,KE,AO,KERsandtheAOP• IntroductionandbackgroundrelevanttocasestudyAOP• Selectionandsummaryofevents• Summaryofweightofevidenceforkeyeventrelationships
• Biologicalplausibility,empiricalsupport,uncertainties/inconsistencies
• Assessmentofoverallweightofevidence• Biologicalplausibility,empiricalsupport,essentiality
• Conclusions
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HepatocyteReducedVTGproduction
OvaryImpaired
OocyteDev.
FemaleDecreased
ovulation/spawn
PopulationDecliningTrajectory
AromataseInhibition
GranulosaReducedE2synthesis
PlasmaReduced
circulatingE2
WhatareAOPs?
• Awaytoorganizeinformation• Basedonbiologicalplausibilityand/orstatisticalinference• Ahypothesisandasetofmeasurementswecanmaketotestthathypothesis
• Eventsspanlevelsofbiologicalorganisationrelevanttoriskassessment(e.g.,molecular,cellular,tissue/organ,organismand/orpopulation).
AnAdverseOutcomePathway(AOP)isaconceptualframeworkthatportraysexistingknowledge
concerningthelinkagebetweenadirectmolecularinitiatingeventandanadverseoutcome,ata
levelofbiologicalorganizationrelevanttoriskassessment(Ankleyetal.2010,Environ.Toxicol.
Chem.,29(3):730-741.)
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FivePrinciplesofAOPDevelopment
1. AOPsarenotchemicalspecific
2. AOPsaremodular
3. IndividualAOPsareapragmaticsimplificationofbiology
4. AOPnetworksarethefunctionalunitofprediction(inmostcases)
5. AOPsare“living”documents
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PrinciplesofAOPDevelopment
KeyEvents(KEs)
• Functionalunitofobservation/verification• Observable∆biologicalstate(measurable)• Essential(butnotnecessarilysufficient)
TwoPrimaryBuildingBlocks
AOMIE
KeyEventsRelationships
(KERs)• Functionalunitofinference/extrapolation• directedrelationship• StateofKEupprovidessomeabilitytopredictorinferstateofKEdown
• Supportedbyplausibilityandevidence• Quantitativeunderstanding
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Section6.
KERPages• Title• Description• Biologicalplausibility• Empiricalsupport• Inconsistenciesanduncertainties
• Quantitativeunderstanding
Plausibility• largelybasedon“normalbiology”• UnderstandingofstructuralandfunctionalrelationshipsbetweenKEs.
EmpiricalSupport• Largelybasedonperturbationstudies(e.g.,toxicologystudies)
WOEAssembly
CreatingKERs
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OverallassessmentofAOPsWeightofevidenceassessmentforcausality
BradfordHillcriteria:minimalconditionsnecessarytoprovideevidenceofacausalrelationshipbetweeneventsandtheconsequenceformingtheweightofevidenceconsiderationsforcausality&levelofconfidenceinanAOP.
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ER
Antagonism
HepatocyteReducedVTGproduction
OvaryImpaired
OocyteDev.
FemaleDecreasedovulation/
spawning
PopulationDecliningTrajectory
Aromatase
Inhibition
GranulosaReducedE2synthesis
AR
Agonism
Hypothalamic Neurons
(-)Feedback
CYP17,CYP11A
Inhibition
PlasmaReduced
circulatingE2
Reducedbonedensity
Osteoperosis
CorporaAllataReducedJHsynthesis
HemolymphReducedJH
concentrations
FatBodyReducedVTGproduction
Poornutritionalstatus
Formostreal-worldapplications,AOPnetworksarethefunctionalunitofprediction
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4.EnteryourAOP,bylinkingtoexistingcontentand/orcreatingnewpagesasappropriate.
3.IdentifyexistingcontentyourAOPcanlinkto.
2.Search/browsethewikiforrelatedAOPs/KEs/KERsbeforeenteringnewAOP.
1.MapoutyourAOPaheadoftime
RecommendedWorkflowforAOPCreation
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IMPORTANT BECAUSE: Germline mutations are deleterious and can result in death of the developing embryo. May result in any type of genetic disease in an offspring. Can potentially contribute to the population gene pool.
CASESTUDY:AlkylationofDNAleadingtoheritablegeneticeffects
Principle:Germlinegeneticdamageoccursinspermoreggandcanbetransmittedtoanoffspring
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AOP:AlkylationofDNAleadingtoheritablemutations
Motivation:• Oneofthebestcharacterizedmodesofactioningenetic
toxicology.• Providecontextofusefornewmethods/technologiestodetect
somaticandgermcellmutations.• Emphasizegapsinresearchinthisfield.• Denovomutationsareincreasinglyrecognizedascontributing
toalargearrayofhumangeneticdiseases.Proposedchangesinthewayheritablehazardsareassessedarebeingconsidered.
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BackgroundonbiologyofthisAOP
• Alkylatingagents:chemicalsthataddalkylgroups(e.g.,methyl,ethyl,propyl)tocellularmolecules.
• OccursatvarioussitesinDNA.Eachsitehasadifferentdegreeofstability.
© 2013 Nay SL, O'Connor TR. Published in [short citation] under CC BY 3.0 license. Available from: http://dx.doi.org/10.5772/54449
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Prototypealkylatingagent
• AlthoughAOPsarenotchemical-specific,thedatabaseonalkylationofDNAisheavilybiasedtowardsafewprototypeagents.
• E.g.,N-ethyl-N-nitrosourea(ENU)
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Alkyladductscanberepaired
• PrimaryrepairproteinisAGT:Alkylguaninetransferase.• AGTirreversiblybindsthealkylgroupandisinactivated.• VeryefficientrepairatLOWdoses.• AGToverwhelmedatHIGHdoses=alkyladductsretained.• Notverygoodatrepairingalladducttypes.
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ReplicationofalkylatedDNAcausesmutations
• ReplicationoveranalkyladductcancauseinsertionofanincorrectbaseintheDNAduplex.
• Mutationbecomes‘fixed’andcanpropagatetodaughtercells.
O4thyminealkylation=AT-GCtransitionsO6guaninealkylation=GC-ATtransitions
Note:someadductsnotmutagenic.N-alkyladductstendtobebypassederror-free
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TARGETCELLS:Spermatogonialstemcells
• Mutationsinpre-meiotic,replicating,spermatogonialstemcellscanpersistinanorganismandclonallyexpand.
• Spermderivedfromthesestemcellswillcarrythesemutations.• Fertilizationofaneggwithspermcarryingmutationscanresultinanoffspringwiththesemutations.
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Selectionofmeasurableendpoints
* Adduct✔
* Notrepaired✔
Repaired✔
Mutations(somatic)✔Mutations(sperm)✔
Replication✗Spermwithmutation✗ fertilizingegg
Offspringwithmutations✔
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MolecularInitiatingEvent
CellularResponse
OrganismResponse
AlkylationofDNA
(Malepre-meioticgerm
cells)
Offspring:Increasednumbersof
mutationsinalltissues
Mutations
InsufficientorincorrectDNArepair
Inheritedmutations
KER1 KER2 KER3
KER4KER5
MIE AOKE1 KE2
AOP:AlkylationofDNAleadingtoheritablemutations
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EntryofinformationintoOECDknowledgebase
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KEs
KERs
Networkview
EntryofinformationintoOECDknowledgebase
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WeightofEvidenceforKeyEventRelationships
1. Biologicalplausibility• Eacheventiswellcharacterizedandunderstood.Highlybiologicallyplausible.
2. Empiricalevidenceinsupportofrelationships• FindevidencetosupportthatKE1occursbeforeKE2.• FindevidencetosupportthatincidenceKE1>KE2.• Examineconcordanceofdose-andresponse–responserelationships
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KeyEventRelationshipsEvaluation
AlkylationofDNA
Offspring:Increasednumbersofmutationsinalltissues
Mutations
Inheritedmutations
KER4KER5
MIE AOKE2
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Temporalconcordance
Adducts:Peakearly,somepersistto6d
Mutationsinsperm:Exposed28d,mutationsoccur>49dayspost-exposure
Seileretal.1997.Mutationresearch385(3):205-211.
DataderivedfromO’Brienetal.2015EnvironMol.Mutagen.56(4):347-55.
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Incidenceconcordance
Adducts>Mutations
Swenbergetal.(2008)ChemResToxicol21(1):253-265.
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Incidenceconcordance:consolidatedENUdata
Adducts>>
Mutationsinsperm≥
Mutationsinoffspring
DataderivedfromYauketal.EnvironMolMutagen.2015.doi:10.1002/em.21954.
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Incidenceconcordance:graphical
DataderivedfromdatainYauketal.EnvironMolMutagen.2015.doi:10.1002/em.21954.
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Consideruncertaintiesandinconsistencies
• Datagaps–empiricalevidencecomesalmostentirelyfromthechemicalENUingermcells.
• Someinconsistenciesacrossexperimentsduetosub-standardprotocols.
• Someinconsistenciesinincidenceconcordance–notappropriatetoquantitativelycomparesomeendpoints.
• Differencesacrossexperimentsinchemicaldeliverymadecomparisonsdifficult.
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Assessmentofoverallweightofevidence
• AOPbasedon32highqualitystudiesacrossthreerodentspecies,fliesandfish.
• Nosinglestudycomparedmultipleevents,butextrapolationscouldbemadeacrossstudies.
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Assessmentofoverallweightofevidence
BiologicalPlausibility:IsthereamechanisticrelationshipbetweenKEupandKEdownconsistentwithestablishedbiologicalknowledge?”• Broadunderstandingandextensiveknowledgeforallofthekeyeventrelationships.• Generallywidelyaccepted.• Strong.
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AssessmentofoverallweightofevidenceEssentiality:AredownstreamKEsand/ortheAOpreventedifanupstreamKEisblocked?• EvidencesupportthatovercomingDNArepairisrequiredfor
mutationtooccur.• Knock-downDNArepair,mutationsincrease;over-expressDNA
repair,mutationsdecrease.• Moderate.
Figurederivedfromdatain:Allayetal.(1999)Oncogene:18(25):3783-3787.
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AssessmentofoverallweightofevidenceEmpiricalsupport:DoesempiricalevidencesupportthatachangeinKEupleadstoanappropriatechangeinKEdown?DoesKEupoccuratlowerdosesandearliertimepointsthanKEdownandistheincidenceofKEup>thanthatforKedown?• Strongprimarilyfortheindirect(non-adjacent)KERs• UseofOECDtestguidelineor‘goldstandard’approachesindatacollection.• Highqualitystudies.• Overall:moderate–strong.
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Conclusions
• CurrentlyleveragingKEsandKERstobuildotherAOPs.
• https://aopkb.org/aopwiki/index.php/Aop:15• Seealso:Yauketal.Developmentoftheadverseoutcomepathway
"alkylationofDNAinmalepremeioticgermcellsleadingtoheritablemutations"usingtheOECD'susers'handbooksupplement.EnvironMolMutagen.56(9):724-50. 2015ANDYauketal.,AdverseOutcomePathwayonAlkylationofDNAinMalePre-MeioticGermCellsLeadingtoHeritableMutations.OECDSeriesonAdverseOutcomePathwaysISSN:2415-170X.http://dx.doi.org/10.1787/2415170X