ADVERSE OUTCOM

DATA FOR CHLORPYRIFOS AT VARY

BIOLOGICAL O

WILLIAM R. 

RESEARCH T

USEPA, OFFICE OF RESEARCH AND 

FIFRA SAP 
Introduction

 Adverse Outcome Pathway

 concept example evaluating data

 Adverse Outcome Pathways for C

 overview example for neurite outgrowth levels of biological organizatio relationship to AChE inhibition

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Adverse Outcome Pathway

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An Adverse Outcome Pathway (AOP) is a conceptual fraexisting knowledge concerning the linkage between a dirinitiating event and an adverse outcome, at a level of bio

relevant to risk assessment. 

(Chemical Safety and Sustainability Research Program, 2011; Ankley et alChem., 29(3): 730-741.)

 incorporates the concepts of mechanism of act

understanding of the molecular basis of the toof action (set of likely key events that lead to 
Adverse Outcome Pathwa

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Chemical

Property

Profile

Receptor/Ligand Interaction

DNA Binding

Protein 

Oxidation

Gene Activation

Protein Production

Altered Signaling

Protein Depletion

Altered Physiology

Disrupted 

Homeostasis

Altered Tissue 

Development

or Function

Toxicant Macro-Molecular

Interactions

Cellular 

Responses

Organ

Responses

Initiating Event 

 Propose a set of biologically plausible linkages  Place data collected at different levels of orga Focus on chemical mechanisms related to adver
Biologic

inputs

Adaptive

Responses

Early cellular

changes

Exposure

Uptake‐Delivery to Target Tissues

Perturbation

Cellular response pathway

Molecular

initiating event

Perturbed cellular

response pathway

Adve

re

risk 

Toxicity Pathway

Adverse Outcome Pathway

II. Adverse Outcome Pathways –

definition and example

M
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Molecular

initiating event

Key events or predictive

relationships spanning

levels of biological 

organization 

Toxicant Macro-Molecular

Interactions

Cellular 

Responses

Organ

Response

glutamate (kainate)

receptor activation

hippocam

cell dea

intracellular Ca2+

dysregulation

domoic acid

(EAA)

Adverse Outcome Pathway:Domoic Acid
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Toxicant Macro-Molecular

Interactions

Cellular 

Responses

Organ

Response

glutamate (kainate)

receptor activation

hippocam

cell dea

intracellular Ca2+

dysregulation

domoic acid

(EAA)

 Plausibility: theoretical basis for biological conn Temporal relationship: effects at lower level mu Dose-response: dose for effect at lower level sh Experimental verification: altering effect at one 

Adverse Outcome Pathway:evaluation of data
Potential Adverse Outcome Path

Chlorpyrifos

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Domain Chemical Macro-Molecular 

Interactions

Cellular 

Responses

AChE as 

morphogen

chlorpyrifos, 

chlorpyrifos oxon, 

or 

trichloropyridinol

In Vitro In Vitro

In Vivo

Cholinergic

receptors

In Vitro In Vitro

Endocannabinoid

system

In Vitro

In Vivo

ROS In Vitro

In Vivo

Serotonergic

system

In Vitro

In Vivo

Tubulin, MAPs In Vitro

In Vivo

In Vitro

In Vivo
Potential Adverse Outcome Pathw

Morphogenic site

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Molecular

initiating event

Key events or predictive

relationships spanning

levels of biological 

organization 

Toxicant Macro-Molecular

Interactions

Cellular 

Responses

R

Binding to AChE

(morph site)

CPF, CPO, TCP Neurite Growth Neura

CPF-esterase (morph?)CPO-esterase (morph?) CPO-morph

decrease in vitro(± AChE inhibition)decrease in vivo (+ AChE inhibition)

CPF (in vitro, in vivo)CPO (in vitro, in vivo)

TCP (in vitro)
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Domain Chemical MacroMolecular 

Interactions

Cellular 

Responses

AChE as 

morphogen

chlorpyrifos, 

chlorpyrifos 

oxon, or 

trichloropyridinol

morphogenic 

site of AChE

neurite growth a

Cholinergic

receptors

M2 receptor cAMP

Endocannabin

oid system

CB1 receptor

FAAH

ROS oxidative stress ox

re

Serotonergic

system

changes in level 

of serotonin, 

serotonin 

turnover, 

receptors, etc.

cha

o

re

by 

Tubulin, MAPs binding to 

tubulin and 

MAPs

axonal transport

Potential Adverse Outcome Pathways for 

Initiating Event
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 Assessed AChE activity concurrently Concentration/dose-response Significant response in absence of significant AChE inh

Adverse Outcome Pathways for Chlorpyrifos: Relationship to AChE 

Experimental model ResponsePrimary neuron culture decrease neurite out(in vitro)

Rat increase oxidative st

(developmental exposure)Primary neuron culture inhibition of axonal (in vitro)

Rat* change in serotonerg

(developmental exposure) CNS

* AChE me
Summary

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 Plausibility

 multiple biologically plausible initiating/downstream  few AOPs explicitly outlined/studied

 Temporal relationship

 data show temporal relationship between exposure 

 Dose-response

 limited data across levels of biological organization some evidence of possible effects (neurite outgrowth

receptors, axonal transport) which are similarly sensthan AChE inhibition

 Experimental verification

 limited data verifying connection between levels of 
13 Questions?