# ADVERSE OUTCOME P AT HW AY S: DA TA FOR CHLORPYRIFOS ATV ARYING LEVELS OF BIOLOGICAL ORGANIZA

TION WILLIAM R. MUNDY

, PH. D. RESEARCH TO XICOLOGIST USEP A, OFFICE OF RESEARCH AND

DEVELOPMENT
April 10, 2012 FIFRA S AP

# Introduction

 

# Adverse Outcome Pathway  

# concept  

# example  

# evaluating data **2**



###  

# Adverse Outcome Pathways for Chlorpyrifos  

# overview  

# example for neurite outgrowth  

# levels of biological organization  

# relationship to AChE inhibition

# Adverse Outcome Pathway

**3**



## An **Adv erse Outcome**  **Pathw ay**  **( AOP)**  **is a conceptual framew**  **ork that**  **portray s existing know**  **ledge concerning the**  **linkage betw een**  **a direct molecular**  **initiating event**  **and an adverse outcome**  **, at a level of biological**  **organization relevant to risk assessment.**  (Chemical Safety and Sustainability Research Program, 201 1; Ankley et al. 2010, Environ. To xicol. Chem., 29(3): 730

- 741.)



###  

# incorporates the concepts of mechanism of action (detailed understanding of the molecular basis of the toxic ef fect) and mode of action (

set of likely key events that lead to a toxic ef fect)

# Adverse Outcome Pathway

**4**

Chemical Property Profile Receptor/Ligan

d Interaction DNA B inding Protein Oxidation Gene Activation Protein Production Altered Signaling Protein Altered Physiology Disrupted Homeostasis Altered Ti ssue Development or Function Lethality Impaired Development Impaired Reproduction Cancer **To xicant**  **Macro -**  **Molecular**

**Interactions Cellular** 

**Responses Organ**  **Responses Individual** 

**Responses**
Structure

Recruitme nt Extinction **Population Responses** 

Oxidation Protein Depletion Development or Function Cancer

**Initiating Event Ad**  **verse Outcome**



###  

# Propose a set of biologically plausible linkages to apical toxicity  

# Place data collected at dif ferent levels of organization into context  

# Focus on chemical mechanisms related to adverse outcome**Biologic inputs**  **“Normal” Biological**  **Function**



### Adaptive **Re sponses**  **Early cellular**

**changes Exposure**  **Uptak e**  **- Delivery**  **to T**

**arget Tissues** 
**Pe rturbation**  Cellular re sponse pathway 

### II. **Ad verse**  **Outcome Pathw ays**  **– definition**  **and example**

Adverse Outcomes (e.g., mortality

, Re pr oductive Impairment) **Cell inury, Inability to**  **regulate** 

### Re **sponses**

Molecular initiating event Pe rturbed cellular

response pathway Adverse outcome relevant to risk assessment 

### To xicity P athway

Adverse Out come P athway Modified From NRC 2007**6**

**To xicant**  **Macro -**  **Molecular Interactions**  **Cellular Responses**  **Organ Responses**  **Individual Responses**  glutamate (

kainate ) receptor activation hippocampal cell death intracellular Ca 2+ dysregulation domoic acid (EAA) memory loss 

# Adverse Outcome Pathway: Domoic Acid

**Mo lecular**  **initiating event**  **Key e**  **vents or predictive**  **relationships spanning**  **levels of biological**  **organization Ad**  **verse outcome relevant**  **to risk**  **assessment 7**  **To xicant**  **Macro -**  **Molecular Interactions**  **Cellular Responses**  **Organ Responses**  **Individual Responses**  glutamate (

kainate ) receptor activation hippocampal cell death intracellular Ca 2+ dysregulation domoic acid (EAA) memory loss 

# Adverse Outcome Pathway: evaluation of data

 

# Plausibility: theoretical basis for biological connections  

# Te mporal relationship: ef fects at lower level must precede the next  

# Dose

- response: dose for ef fect at lower level should be ≤ the next

# Potential Adverse Outcome Pathways for Chlorpyrifos
**8**

**Domain Chemical**  **Macro**   ** Mo** **lecular Interactions**  **Cellular**

**Responses Organ** 

**Responses Individual** 

**Responses**
**AC hE**  **as morphogen**  In Vitro In Vitro In Vivo In Vivo

(zebrafish) **Cholinergic** In Vitro In Vitro

chlorpyrifos, chlorpyrifos oxon, or trichloropyridino l **Cholinergic receptors**  In Vitro In Vitro
**Endocannabin oid**  **sy stem**  In Vitro In Vivo **ROS** In Vitro In Vivo In Vivo **Serotonergic sy** **stem** In Vitro In Vivo In Vivo In Vivo
**Tu bulin**  **, MA**  **Ps** In Vitro In Vivo In Vitro

In Vivo

# Potential Adverse Outcome Pathway: Morphogenic site

**9**

**To xicant**  **Macro -**  **Molecular Interactions**  **Cellular Responses**  **Organ Responses**  **Individual Responses**  Binding to AChE (morph site) Neural Connectivity

Neurite Growth CPF , CPO, TCP Cognition

**Mo lecular**  **initiating event**  **Key e**  **vents or predictive**  **relationships spanning**  **levels of biological**  **organization Ad**  **verse outcome relevant**  **to risk**  **assessment CPF**  **- esterase**  **(morph?) CPO**  **- esterase**  **(morph?) CPO**  **- morph**  **decrease in vitro**  **( ±**  **AC hE**  **inhibition) decrease**  **in vivo (+**  **AC hE**  **inhibition) CPF (**  **in vitro, in**  **vivo) CPO (**  **in vitro, in**  **vivo )**  **TCP ( in vitro** **) 10**  **Domain Chemical**  **Macro**   ** Molecular** **Interactions Cellular** 

**Responses Organ** 

**Responses Indiv**  **idual**

**Responses**
**AC hE**  **as morphogen** 

**chlorpy rifos,** 

**chlorpy rifos**  **oxon, or**  **trichloropy ridi**  morphogenic

site of AChE neurite growth axon length **Cholinergic receptors**  M2 receptor cAMP
**Endocannabi noid**  **sy stem**  CB1 receptor FAAH 

# Potential Adverse Outcome Pathways for Chlorpyrifos

**trichloropy ridi**  **nol ROS**  oxidative stress oxidative

stress response genes **Serotonergic sy** **stem** changes in level of serotonin, serotonin turnover , receptors, etc. changes in

level of serotonin, serotonin turnover , receptors, etc., by brain region changes in

behavior , both basal and after drug challenge

**Tu bulin**  **, MA**  **Ps** binding to tubulin and axonal

transport **Initiating Event** **11**  

## Assessed AChE activity concurrently

 

## Concentration/dose

- response  

## Significant response in absence of significant AChE inhibition 

# Adverse Outcome Pathways for Chlorpyrifos: Relationship to AChE Inhibition

Experimental model Response Primary neuron culture decrease neurite outgrowth ( in vitro ) Rat increase oxidative stress genes in CNS (developmental exposure) Primary neuron culture inhibition of axonal transport ( in vitro ) Rat* change in serotonergic tone of adult (developmental exposure) CNS * AChE measured in separate experiment

# Questions? **13**