Office Office of Research and Development 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, NC
Office of Research and Development

Outline

1. Thyroid hormone biology

2. Development of the AOP

3. Focused Research

4. Conclusions

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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 101
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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

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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 TRβ mRNA 

expression – suggestive of interaction with thyroid receptor or 

thyroid driven development (PXR)

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Developing a Draft AOP

Evidence of possible thyroid pathway disruption

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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 effect
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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, 2005
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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

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Hepatic 

NR 

activation

↓serum 

T4

↑ hepatic 

Phase I & II 

mRNA & 

protein

↑ TH 

catabolism & 

biliary 

elimination

Rat
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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 Research
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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? 

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Hepatic 

NR 

activation

↓serum 

T4

↑ hepatic 

Phase I & II 

mRNA & 

protein

↑ TH 

catabolism & 

biliary 

elimination

Rat
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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 animals
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Serum TCS concentrations suggest diminishing 

exposure to pups throughout lactation may account 

for a lack of serum T4 effects at these ages.

Percent of control T4 at 300 mg/kg/d (mean ± SE)

50

60

70

80

90

100

Percent of control T4 at 300 mg/kg/d

Animal Age

GD20D

GD20F

PND4

PND14PND21

PND22D

Total TCS in Serum at 300 mg/kg/d (ng/mL, mean ± SE)

0

10000

20000

30000

40000

Total TCS in serum at 300 mg/kg/d
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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 upregulation of markers of UGT are consistent with the proposed 

MOA and minor hypothyroxinemia observed.
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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

Human
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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 AC50 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. 

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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 

Treatment concentration (μM)

0.1 1 10 100

% Maximal DEX Induction ± SE

0

20

40

60

80

100

120

Dexamethasone (DEX)

Triclosan (TCS)

Treatment concentration (μM)

0.01 0.1 1 10 100

% Maximal RIF Induction ± SE

0

20

40

60

80

100

120

Rifampicin (RIF)

Triclosan (TCS)

hPXR rPXR
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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 Phenobarbital
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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

√
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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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