ADVERSE OUTCOME PATHWAYS:DATA FOR CHLORPYRIFOS AT VARYING LEVELS OFBIOLOGICAL ORGANIZATIONWILLIAM R. MUNDY, PH. D.RESEARCH TOXICOLOGISTUSEPA, OFFICE OF RESEARCH AND DEVELOPMENTApril 10, 2012FIFRA SAPIntroductionAdverse Outcome Pathwayconceptexampleevaluating dataAdverse Outcome Pathways for Chlorpyrifosoverviewexample for neurite outgrowthlevels of biological organizationrelationship to AChE inhibition2Adverse Outcome Pathway3An Adverse Outcome Pathway (AOP) is a conceptual framework that portraysexisting knowledge concerning the linkage between a direct molecularinitiating eventand an adverse outcome, at a level of biological organizationrelevant to risk assessment.(Chemical Safety and Sustainability Research Program, 2011; Ankley et al. 2010, Environ. Toxicol.Chem., 29(3): 730-741.)incorporates the concepts ofmechanism of action(detailedunderstanding of the molecular basis of the toxic effect) andmodeof action (set of likely key events that lead to a toxic effect)Adverse Outcome Pathway4ChemicalPropertyProfileReceptor/LigandInteractionDNA BindingProteinOxidationGene ActivationProtein ProductionAltered SignalingProtein DepletionAltered PhysiologyDisruptedHomeostasisAltered TissueDevelopmentor FunctionLethalityImpairedDevelopmentImpairedReproductionCancerToxicantMacro-MolecularInteractionsCellularResponsesOrganResponsesIndividualResponsesStructureRecruitmentExtinctionPopulationResponsesInitiating EventAdverse OutcomePropose a set of biologically plausible linkages to apical toxicityPlace data collected at different levels of organization into contextFocus on chemical mechanisms related to adverse outcomeBiologicinputs“Normal”BiologicalFunctionAdverseOutcomes(e.g.,mortality,ReproductiveImpairment)Cellinury,InabilitytoregulateAdaptiveResponsesEarlycellularchangesExposureUptake‐DeliverytoTargetTissuesPerturbationCellularresponsepathwayMolecularinitiatingeventPerturbedcellularresponsepathwayAdverseoutcomerelevanttoriskassessmentToxicityPathwayAdverseOutcomePathwayII.Adverse Outcome Pathways –definition and exampleModified From NRC 20076Molecularinitiating eventKey events or predictiverelationships spanninglevels of biologicalorganizationAdverse outcomerelevant torisk assessmentToxicantMacro-MolecularInteractionsCellularResponsesOrganResponsesIndividualResponsesglutamate (kainate)receptor activationhippocampalcell deathintracellular Ca2+dysregulationdomoic acid(EAA)memory lossAdverse Outcome Pathway:Domoic Acid7ToxicantMacro-MolecularInteractionsCellularResponsesOrganResponsesIndividualResponsesglutamate (kainate)receptor activationhippocampalcell deathintracellular Ca2+dysregulationdomoic acid(EAA)memory lossPlausibility: theoretical basis for biological connectionsTemporal relationship: effects at lower level must precede the nextDose-response: dose for effect at lower level should be≤the nextExperimental verification: altering effect at one level alters the nextAdverse Outcome Pathway:evaluation of dataPotential Adverse Outcome Pathways forChlorpyrifos8DomainChemicalMacro-MolecularInteractionsCellularResponsesOrganResponsesIndividualResponsesAChE asmorphogenchlorpyrifos,chlorpyrifos oxon,ortrichloropyridinolIn VitroIn VitroIn VivoIn Vivo(zebrafish)CholinergicreceptorsIn VitroIn VitroEndocannabinoidsystemIn VitroIn VivoROSIn VitroIn VivoIn VivoSerotonergicsystemIn VitroIn VivoIn VivoIn VivoTubulin, MAPsIn VitroIn VivoIn VitroIn VivoPotential Adverse Outcome Pathway:Morphogenic site9Molecularinitiating eventKey events or predictiverelationships spanninglevels of biologicalorganizationAdverse outcomerelevant torisk assessmentToxicantMacro-MolecularInteractionsCellularResponsesOrganResponsesIndividualResponsesBinding to AChE(morph site)Neural ConnectivityNeurite GrowthCPF, CPO, TCPCognitionCPF-esterase (morph?)CPO-esterase (morph?)CPO-morphdecreasein vitro(± AChE inhibition)decreasein vivo(+ AChE inhibition)CPF (in vitro, in vivo)CPO (in vitro, in vivo)TCP (in vitro)10DomainChemicalMacroMolecularInteractionsCellularResponsesOrganResponsesIndividualResponsesAChE asmorphogenchlorpyrifos,chlorpyrifosoxon, ortrichloropyridinolmorphogenicsite of AChEneurite growthaxon lengthCholinergicreceptorsM2 receptorcAMPEndocannabinoid systemCB1 receptorFAAHROSoxidative stressoxidative stressresponse genesSerotonergicsystemchanges in levelof serotonin,serotoninturnover,receptors, etc.changes in levelof serotonin,serotoninturnover,receptors, etc.,by brain regionchanges inbehavior, bothbasal and afterdrug challengeTubulin, MAPsbinding totubulin andMAPsaxonal transportPotential Adverse Outcome Pathways for ChlorpyrifosInitiating Event11Assessed AChE activity concurrentlyConcentration/dose-responseSignificant response in absence of significant AChE inhibitionAdverse Outcome Pathways forChlorpyrifos: Relationship to AChE InhibitionExperimental modelResponsePrimary neuron culturedecrease neurite outgrowth(in vitro)Ratincrease oxidative stress genes in CNS(developmental exposure)Primary neuron cultureinhibition of axonal transport(in vitro)Rat*change in serotonergic tone of adult(developmental exposure)CNS* AChE measured in separate experimentSummary12Plausibilitymultiple biologically plausible initiating/downstream eventsfew AOPs explicitly outlined/studiedTemporal relationshipdata show temporal relationship between exposure and effectDose-responselimited data across levels of biological organizationsome evidence of possible effects (neurite outgrowth, binding to muscarinicreceptors, axonal transport) which are similarly sensitive or more sensitivethan AChE inhibitionExperimental verificationlimited data verifying connection between levels of biological organizationQuestions?13