in vivo and in vitro screening for thyroid hormone disruptors · pdf filein vivo and in vitro...

In Vivo and In Vitro Screening for Thyroid Hormone Disruptors

Kevin M. CroftonN i l H l h d E i l R h L b

Thyroid Hormone Disruptors

National Health and Environmental Research Laboratory

Office of Research and DevelopmentNational Health and Environmental Effects Research Laboratory

California Environmental Protection AgencyHuman Health Hazard Indicators Workshop

March 15-16, 2010

Outline•Thyroid Biology 101y gy•What do we know about pathways and adverse outcomesadverse outcomes

•Recent advances in screening for thyroid h di thormone disruptors

•Challenges for use of HTS in hazard gassessment

Office of Research and DevelopmentNational Health and Environmental Effects Research Laboratory 1

Thyroid Hormones

Thyroxin (T4) OH

O

I

I

I

HO CH2

NH O

CH C OH

O

I

I

I

HO CH2

NH O

CH C OH

O

I

I

I

HO CH2

NH O

CH C

Triiodothyronine (T3)

I NH2 OI I NH2 OI I NH2 OI

OO

I I

HO CH2 CH C OO

I I

HO CH2 CH C OO

I I

HO CH2 CH Cy ( ) O

HO

I

HO CH2

NH2 O

CH C OH

O

I

HO CH2

NH2 O

CH C OH

O

I

HO CH2

NH2 O

CH C

• Development - Critical for differentiation and growth• Transient disruption = permanent effects (eg., cretinism)

• Adult – Important for energy and thermoregulation•Transient disruption = transient effects


Regulation of Thyroid HormonesHypothalamus

TRH

TR

Pit

+Acts as a ligand for

l

TR

Thyroid

TSHElimination from the body

+ nuclear thyroid hormone receptors (TR )

Catabolic Enzymes

BloodT3/T4

Liver T4 T3

(TRs)

Target Tissues

LiverTH binding

proteins 5’-deiodinases

T4 T3


Linking Disruption of Up-Stream Targets to Adverse Outcome

Adverse Outcome Pathway

Toxicity Pathway

Thyroperoxidase

Thyroidal

y y

ThyroperoxidaseIodine Symporter

H ti UDPGT

Extra-Thyroidal SerumT3 & T4

TSH ThyroidHyperplasia

ThyroidTumors

ExposureHepatic UDPGTs

Deiodinases

Cellular Transporters

ChangesTissue

T3 ChangesAltered

DevelopmentBirth

Defects

T4–TTR Binding

Cellular Transporters


Thyroid Receptors

Screening Methods for Thyroid Disruptors

1. Link early key events to adverse outcomes• Qualitative – Yes• Quantitative – no yet

2 B ild efficient screens for each “target”2. Build efficient screens for each “target”• High Throughput Screens (HTS)

• Very efficient lack integrated systems• Very efficient, lack integrated systems • Low-Medium Throughput Screens

• Less efficient, but integrated systems, g y


Ongoing Activities - Examples1) Receptor based screening at EPA’s NCCT & NIH NTP/NCGC1) Receptor based screening at EPA s NCCT, & NIH NTP/NCGC

e.g. TR-beta, hepatic targets PXR, AhR, PPAR recently completed screening of 320 pesticidal chemicals, now

working on over 1000 additional chemicalso g o o e 000 add t o a c e ca s

2) TPO screening 96 well medium throughput assay for inhibition of porcine TPO g p y p

(Mike Hornung at ORD’s Duluth Lab)

3) TH signaling in transgenic X. laevis f f ( 96-well format for detecting alterations in TR signaling (Barbara

Demeneix - Environ. Sci. Technol., 2007, 41:5908–5914)

4) OECD/IPCS Advisory Group on Molecular Screening and4) OECD/IPCS Advisory Group on Molecular Screening and Toxicogenomics”.

1) Critical summary of available assays, 2) Collating list of chemicals with evidence of thyroid disruption


) g y p(update to Brucker-Davis list)

HTS In Vitro Example – Thyroid Receptor BetaGeneBLAzer TR-UAS-bla HEK293 Cell Line

LigandLigand ** Substrate

T3 Stimulation of TR

**

**** ****

T3 (M)

Cell line contains a beta lactamse reporter gene under the control of an UAS response

****

Cell line contains a beta-lactamse reporter gene under the control of an UAS responseelement stably integrated in Hek293 cells. This line also stably expresses a fusion proteinconsisting of the GAL4 DNA binding domain and the TR ligand binding domain.


Courtesy of Keith Houck, NCCT

Human TR Reporter Gene Assay Heat Map of AC50’s1456 chemicals; 14 concentrations; agonist and antagonist modes; ; g g

T3

Levothyroxine

ACTIVES 62SELECTIVE 0

ACTIVES 65SELECTIVE 2

Office of Research and DevelopmentNational Health and Environmental Effects Research Laboratory 9Courtesy of Keith Houck, NCCT

L-MTS Example - WatchFrog®

• Fluorescent transgenic X. laevis tadpoles bearing a TH/bZIP-eGFP construct• Can detect receptor agonists & antagonists, and synthesis inhibitors

96 well plates with automatic quantification of the activation of TR


Slide courtesy of B. Demeniux

WatchFrog – Positive Controls

400 ******

***

400 ******

***

400 ******

***

300

400***

******

***

U

300

400***

******

***

U

300

400***

******

***

U

100

200

RFU

100

200

RFU

100

200

RFU

0

Ctrl T3 T4TRIACGC1

M

0

Ctrl T3 T4TRIACGC1

0

Ctrl T3 T4TRIACGC1

M

5.10

-9M

10-7

M

10-8

M

10-8

M

10-6

M

10-1

0 M

5.10

-9M

10-7

M

10-8

M

10-8

M

10-6

M

10-1

0 M Fini et al. (2007)


M-LTP Example Thyroid Peroxidase Inhibition Assayy y

• Use porcine thyroid microsomes • Chemical + SubstrateChemical Substrate• Color readout

• 96 well plates• Currently screened over 100 chemicals

• One example – MBT (2-Mecaptobenozothiazole)

• Positives are followed by ex vivo thyroid explant assay• Then in vivo testing in X laevis


Courtesy of Michael Hornung

• Then in vivo testing in X. laevis

Challenges in Use of Data from S i M th dScreening Methods

• Can we use HTS data for risk assessment right now? NO–Links between target and outcomes are not all there–Issues with relating testing concentration (media) to

t t ti dtarget tissue dose–Lack of integrated developing systems–Statistical and biological models for data interpretation–Statistical and biological models for data interpretation–Many targets are enzymes

• What can we do now?– Screening for Prioritization (rather than prediction)


Accumulation of PBDE-47 in Primary Cell Cultures of Rat Cortical NeuronsCortical Neurons

1 uM in media = 100 uM in cells after 60 min

Mundy et al 2005Office of Research and DevelopmentNational Health and Environmental Effects Research Laboratory 14

Mundy et al 2005

Challenges in Use of Data from S i M th dScreening Methods

• Can we use HTS data for risk assessment right now? NO–Links between target and outcomes are not all there–Issues with relating testing concentration (media) to

t t ti dtarget tissue dose–Lack of integrated developing systems–Statistical and biological models for data interpretation–Statistical and biological models for data interpretation–Many targets are enzymes

• What can we do now?– Screening for Prioritization (rather than prediction)


The EndThe End


Application of Research to L l f O i ti B dLevels of Organization Based

on Source to Outcome Source

Environmental

Community

EnvironmentalContaminant

Exposure Individual

Population

Key Event Cellular Effects

Toxicity Pathway

Mode of Action

Adverse Outcome Pathway


Source to Outcome Pathway

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