Office of Research and Development

Office of Research and Development 

# The Use of AOP Framework

to Focus Research Efforts on 

# Critical Uncertainties in Assessing Hazards

Kevin Crofton, Ph.D. Office of Research and Development / NHEERL

U.S. EPA, RTP, NCOffice of Research and Development 

# Outline 1. 

# Thyroid hormone biology 2. 

# Development of the AOP 3. 

# Focused Research 4. 

# Conclusions

2Office of Research and Development 

# Research Questions 1) Determining the relationship between alterations in circulating levels of TH and TH action in the production of adverse effects on neurodevelopment.

– High dose phenomena well characterized, lacking at low end

– Traditional notion thyroid axis model does not fit for many environmental contaminants 2) Determining the relationship between life stage, duration, dose, vulnerability to thyroid toxicants, and specific adverse consequences.

– In fetus/neonate range over which hormones can change is small as the timing of critical TH -dependent developmental events must be exquisitely maintained

– Fetus/neonate not equipped with the same compensatory responses as the adult 3) Identification of biomarkers of thyroid toxicity that reflect adverse “downstream” effects.

– Many chemical targets to disrupt thyroid system may not be reflected in changes in serum TH (e.g., TH receptors, local tissue specific deiodinases , transport proteins).

– Need for markers of TH action to reflect downstream effects rather than relying on serum hormones – will we unmask non - linearity?

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# Basic Thyroid Toxicology 101Office of Research and Development



# ORD Triclosan Research

• 

## Research Driver – OPP Needs from the 2008 Re - registration

– Critical effect: Changes in serum thyroid hormones



## – Concern: Estrogen & Androgen

• 

## Uncertainties included :

– Lack of data to inform possible species difference (extrapolation from rat to human)

– Inability to ascertain whether there are sensitivity differences across life -stages



## – Unknown mode(s) -of

-action for thyroid effects

4Office of Research and Development 

## Developing a Draft AOP

Evidence of possible thyroid pathway disruption Research Highlights • 

## Jinno et al., 1997: Increased Cyp2b and 3a1 protein and activity in rat hepatocyptes (hepatic nuclear recpectors - CAR/PXR) • 

## Hanioka et al 1997: Induction of Cyp2b, Cyp3a, and Cyp4a protein content and activity in rats (CAR/PXR) • 

## Jacobs et al., 2005: in vitro activation of human PXR by TCS in a luciferase - based reporter assay. (PXR) • 

## Veldhoen et al., 2006: Triclosan accelerated hindlimb development in Rana catesbenia and increased TR β mRNA expression – suggestive of interaction with thyroid receptor or thyroid driven development (PXR) 5Office of Research and Development 

## Developing a Draft AOP

Evidence of possible thyroid pathway disruption

6 Crofton et al. 2007 

### • 4 - day oral TCS exposure in juvenile rats
• Decreased serum thyroxin



### • Not indicative of any particular molecular initiating event, but evidence of 

### downstream effectOffice of Research and Development

Hepatic Tissue Dose Actrivate

CAR/PXR Hepatic

Phase I, II, III Decreased T4 Altered

Development Adverse

Neuro -Development 

# Proposed Rat AOP*

1. Activation of hepatic nuclear receptors (CAR/PXR) 2. Upregulation of hepatic Phase I, II, and III metabolism • Hepatic UGTs and SULTs 

### 3. Catabolism of thyroid hormones 4. Increased biliary elimination of thyroid hormones 5. Decreased circulating thyroid hormones 6. Decreased tissues levels of thyroid hormones 

### 7. Altered tissue specific mRNA and protein production 8. Altered neurodevelopment
* Modified from Crofton & Zoeller, 2005Office of Research and Development 

## Testing the Hypothesized AOP Purpose: Use in vivo and ex vivo models/tools to determine whether there is sufficient evidence to support the AOP 

## in rats Methods: In vivo exposure – hepatic mRNA, proteins & activity In vitro exposure – hepatic sandwich cultures

8 Hepatic NR activation ↓serum T4 ↑ hepatic Phase I & II mRNA & protein ↑ TH catabolism & biliary elimination 

### RatOffice of Research and Development

Phase 1 Metabolism EROD activity Ø CYP1a1 mRNA Ø PROD Activity ↑↑ CYP2B1 mRNA ↑ CYP3A1 mRNA ↑↑ Phase II Metabolism UGT-T4 Activity ↑ Ugt1a1 mRNA ↑↑ Ugt1a6 mRNA Ø Ugt2b5 mRNA Ø Sult1b1 mRNA Ø Sult1c1 mRNA ↑↑ Sult2a mRNA Ø Phase III Metabolism Mrp2 Ø Mrp3 Ø Mrp4 Ø Oatp1a4 Ø Oatp3a1 Ø



### Hepatic AhR Nuclear CAR Receptors PXR Triclosan Hepatic Tissue Phase I, Phase II & Phase III • mRNA, Proteins & Activity 

### No AhR (dioxin) like activity (this is good!) Consistent with activation of CAR and/or PXR No change in transporters Enzyme Activity Interpretation 

# Summary of ResearchOffice of Research and Development 

## Development Studies 

## Purposes: 1) Are developing rats more or less susceptible to TCS - induced hypothyroxinemia compared to juveniles? 2) Is the proposed AOP relevant for developmental effects?

10 Hepatic NR activation ↓serum T4 ↑ hepatic Phase I & II mRNA & protein ↑ TH catabolism & biliary elimination 

### RatOffice of Research and Development



# Developmental Research Update

• 

# A series of developmental exposure were conducted



# – Oral exposure, dam only, GD6- PND21

– Serum/plasma and liver collected on GD20, PND4 (pups only), 14 (pups only), & 21 • 

# T4, T3, TSH • 

# Hepatic enzyme activity (EROD, PROD) • 

# Hepatic mRNA – Custom Taqman ® Low Density Fluidic PCR Arrays arrays for 23 biomarkers of Phase 1, 2 and 3

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## Mild hypothyroxinemia in dams, fetuses, and neonates Unique pattern of 

### effects

No change in T3 or TSH for any of these animalsOffice of Research and Development



### Serum TCS concentrations suggest diminishing exposure to pups throughout lactation may account 

### for a lack of serum T4 effects at these ages.Office of Research and Development 

## Data Summary

Support for the hypothesized MOA during



## development Metabolic Phase Marker Dams Fetuses/ PND4 pups AhR/CAR

inducers (+ con) CAR/PXR

inducers (+ con) Phase I Cyp2b activity (PROD) 3 -fold 2 -fold 30 -fold 20 -fold Phase II UGT

-T4 activity 1.5-fold (-) 2 -fold (-) Phase I Cyp2b2 expression 2.5- to 3 - fold (-) 5-fold 7 -fold Phase I Cyp3a1 expression 2 -fold (-) 5-fold 7 - to 10 -fold Phase II Ugt1a1 expression (-) (-/+) (-) 5 -fold Phase II SULT1b1 expression 1.7 -fold (ns) 1.7 -fold (ns) (+) (+) 

## Up - regulation of markers of Cyp2b and Cyp3a and minor up regulatio n of markers of UGT are consistent with the proposed MOA and minor hypothyroxinemia observed.Office of Research and Development



# Development Summary

• Thyroid Effects

– Dams: Mild hypothyroxinemia during gestation and lactation

– Fetus/Pups: Mild hypothyroxinemia during fetal gestation and early lactation, but not late lactation

– No apparent sensitivity differences in fetal, pup or maternal compartments

• Triclosan distribution

– Lack of effects on T4 in late lactation is due to decreased pup expsoure

– No apparent accumulation of triclosan in fetal or pup blood or liver compared to maternal concentrations

• Adverse Outcome Pathway

– Data suggest the AOP for juvenile rats is relevant for developing rats.
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## Species Extrapolation

In Vitro Models of Molecular Initiating Events 

## Purpose: Use model molecular models/tools to determine whether the are difference in the molecular initiating events between rats and humans Hypothesis: Based on Jacobs et al (2005) study we proposed that TCS activates hepatic PXR.

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Hepatic NR activation ↓serum T4 ↑ hepatic Phase I & II mRNA & protein ↑ TH catabolism & biliary elimination Hepatic NR activation ↓serum T4 ↑ hepatic Phase I & II mRNA & protein ↑ TH catabolism & biliary elimination 

### Rat HumanOffice of Research and Development



## ToxCast High Throughput Screening Results (Judson et al., 2010)

• 

### No PXR activation observed at 2μM triclosan in multiplexed reporter gene assays run by Attagene . • 

### Reporter gene assays for PXR run at the NCGC for both rat and human PXR also negative. • 

### Cytotoxicity possible limiting factor in these assays.

• 

### Triclosan was active in a biochemical PXR binding assay for run at 

### Novascreen which showed an AC 50 

### of 13 uM . • 

### Increases in target genes for PXR and CAR nuclear receptors in the quantitative nuclease protection assay (qNPA) run by CellzDirect on primary human hepatocytes also observed . • 

### Data suggest triclosan may have PXR and possibly CAR agonist activity but at concentrations concurrent with cytotoxicity in the in vitro cellular assays. 17Office of Research and Development



# Nuclear Receptor Activity Puracpy hDPX2 and RPXR Species -specific cell lines

– hDPX2: human PXR (parental = HepG2)

– RPXR: rat PXR (parental = FAO)

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TCS activates human PXR but not rat PXR 

### hPXR rPXROffice of Research and Development



# Ongoing CAR/PXR Reporter Cell

Assay Work

• 

## Method:

– Collaboration with Indigo Biosciences, Inc

– Chimeric receptors for human, rat, and mouse CAR and PXR - all in HEK293T cell lines (no effects of cell line or differential cytotoxicity for each receptor).



### • 

## Objectives:

– Confirm our findings for human PXR and rat PXR, and add mouse PXR.

– Test for TCS activity at all three functional splice variants of human CAR (1, 2, 3), rat 

### CAR, and mouse CAR.

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## Summary of Receptor Reporter Assays 

## Receptor TCS Activity Similar to activity of. . . hPXR Agonism Rifampacin rPXR -- 

## mPXR -- hCAR1 Inverse agonism Clotrimazole , DEHP hCAR2 Strong agonism >> DEHP (prelim data) hCAR3 Agonism DEHP, Phenobarbital rCAR Inverse agonism Clotrimazole , TCPOBOP mCAR Inverse agonism PhenobarbitalOffice of Research and Development 

# Species Extrapolation

Remaining Uncertainty

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### • Can activation of different NRs between species lead to the same outcome? 

### • Data from some positive controls ( eg.,phenobarbital ) show decreases in T4 in both rat and humans

rCAR 

### ↓ serum T4 

### ↑ biliary elimination of THs hCAR1 + hCAR2 + hCAR3 + hPXR 

### ↓ serum T4 ↑ hepatic Phase I & II mRNA & protein 

### ↑ biliary

elimination of THs 

### Rat Human 

# √ ?

√ √

↑ hepatic Phase I & II mRNA & protein 

# √Office of Research and Development



# Collaborations and Acknowledgements:

– Dr. Katie Paul, University of North Carolina 

## (now at The Hamner Institute, RTP, NC)

– Ciba Geigy/BASF



## – Office of Pesticide Programs

– Dr. Jack Van den Heuvel, Penn State Univ

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