Kevin M. Crofton, PhD US Environmental Protection Agency

McKim Conference Duluth MN

September 17, 2008

Thyroid Mediated CNS Dysfunction

How to use what we know about the structure and function of the thyroid system to generate data using in vitro methods

that can populate QSAR models

Outline

• Thyroid hormones and homeostatic mechanisms

• A mode-of-action for thyroid disruption and adverse outcomes on nervous system development

• Targets for disruption• Targets for screening• Summary

Thyroid Hormones- Structure and Function

• T3 and T4 are the principle hormones synthesized and released by the thyroid gland

• Development - Critical for differentiation and growth• Transient disruption = permanent effects

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

Triiodothyronine (T3)

Thyroxin (T4) OH

O

I

I

I

HO CH2

NH2 O

CH C

I

OH

O

I

I

I

HO CH2

NH2 O

CH C

I

OH

O

I

I

I

HO CH2

NH2 O

CH C

I

OH

O

I

I

I

HO CH2

NH2 O

CH C OH

O

I

I

I

HO CH2

NH2 O

CH C OH

O

I

I

I

HO CH2

NH2 O

CH C

Catabolic Enzymes

Hypothalamus

Pit

TRH

Thyroid

TSH

Blood

T3/T4

Target Tissues

Liver

Elimination from the body

+

TH binding proteins

TH binding proteins

5’-deiodinases

T4 T3

Acts as a ligand for nuclear thyroid hormone receptors (TRs)

Regulation of Thyroid Hormones

TR

TR

Cellular Acton of TH

TR XCorepressors

TR

Co Activator AAAAA

Transporter

DI

T3

T3T4

Zoeller, 2003

ThyroperoxidaseIodine Symporter

Exposure

Hepatic UDPGTs

Thyroidal

Extra-Thyroidal

Deiodinases

Thyroid Receptors

T4–TTR Binding

Targets

Cellular Transporters

EarlyBiological

Effect

TissueSpecificEffect

Altered Structure/Function

ClinicalDisease

SerumT3 & T4

Changes

TSH

TissueT3 Changes

AlteredDevelopment

ThyroidHyperplasia

ThyroidTumors

BirthDefects

EffectsCancer & Non-Cancer

Thyroid MOAs

ThyroperoxidaseIodine Symporter

Exposure

Hepatic UDPGTs

Thyroidal

Extra-Thyroidal

Deiodinases

Thyroid Receptors

T4–TTR Binding

Targets

Cellular Transporters

EarlyBiological

Effect

TissueSpecificEffect

Altered Structure/Function

ClinicalDisease

SerumT3 & T4

Changes

TSH

TissueT3 Changes

AlteredDevelopment

ThyroidHyperplasia

ThyroidTumors

BirthDefects

EffectsCancer & Non-Cancer

Toxicity Pathways

Adverse Outcome Pathways

What do we know and not know about these pathways?

Major Sequelae of Thyroid Disruption

• Adult Exposure Thyroid tumors in laboratory animals

• Not a relevant mechanism for human cancer• May increase incidence of cardiovascular disease

• Neurodevelopment Lack of THs result in adverse neurological development

(sensory, motor, cognitive) Species independent (fact) Rat is appropriate animal model for neurodevelopmental effects

These are two different outcomes that can result from the same molecular targets One is relevant for human health and one is not

Neurodevelopment and Thyroid Dysfunction

• FACT: without adequate TH the nervous fails to properly develop Iodine deficiency Congenital hypothyroidism

Dose-Response and Critical WindowDioxins, Furans and PCBs - Hearing Loss

Exposure Hepatic Phase II Enzymes

Hepatic Parent or Metabolite

SerumT4 & T3

TissueT3

Alter TR Mediated Proteins

Loss of cochlear hair cells

HearingLoss

Binding to PXR

Binding to AhR

UGTs

T4serumT3serum

DItissue

T3tissue

TRactivation

CNS Protein (1)

CNS Protein (2)

CNS malformation

Functional Loss (eg. IQ)

Increasing Dose and/or Time

Res

pon

se

PXR Binding

PHAHs and Ototoxicity

BirthConception Weaning

Th

yroid

Ho

rmo

ne

Fe

tal/P

os

tnat

al P

CB

Ex

po

su

re

Cochlear Development

Critical-Period Model for Chemical-Induced Postnatal Hypothyroxinemia and Ototoxicity

High -- Frequency -- Low

Low-frequencyHearing loss

BirthConception Weaning

Th

yroid

Ho

rmo

ne

Fe

tal/P

os

tnat

al P

CB

Ex

po

su

re

Cochlear Development

Critical-Period Model for Chemical-Induced Postnatal Hypothyroxinemia and Ototoxicity

High -- Frequency -- Low

Low-frequencyHearing loss

Log Thyroxine Concentration, % Control

3103060100

Hea

ring

Loss

, dB

SP

L (

diffe

ren

ce fr

om

con

trol

)

0

10

20

30

40

50

60

y = -33.68*logX + 68.72 r ² = 0.8519

Log Thyroxine Concentration, % Control

10.030.060.0100.0

He

ari

ng

Lo

ss

, d

B S

PL

(d

iffe

ren

ce

fro

m c

on

tro

l)

0

10

20

30

40

Dose-Response Perchlorate, Propylthiouracil and Hippocampal

Physiology

Exposure SerumT4 & T3Thyroid

Perchlorate

HippocampalT3

Alter TR Mediated Proteins

Synaptic Malformation

LearningImpariment

Binding to TPO

Inhibition Of TPO

ThyroidPTU

Altered SynapticFunction

Dam T4 vs BL EPSP Max - % Control

Dam T4 Percent of Control0 10 20 30 40 50 60 70 80 90 100 110

BL

EPS

P M

ax %

Con

trol

30

40

50

60

70

80

90

100

110

120

M0805 PTUM0703 PERCM0102 PTU

r2=0.77

Normalized Spike vs EPSP

Normalized EPSP

0 10 20 30 40 50 60 70 80 90 100

Nor

mal

ized

Pop

ulat

ion

Spik

e

0

10

20

30

40

50

60

70

80

90

100

0 ppm3 ppm10 ppm

Hippocampal physiology

Day

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Cue

Lat

ency

in S

econ

ds

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

0ppm (n=11)3ppm (n=15)10ppm (n=10)*

Water maze learning

Dose-Response Perchlorate, Propylthiouracil and Hippocampal

Physiology

What do we not know?

• Dose-response relationships• Critical windows• Sensitive biomarkers (T4?)

• However, we can’t get lost in the need to understand everything in the pathway Causative and predictive is minimum Quantitative models are the holy grail

What can we do to inform QSAR models

• Develop in vitro test methods for known targets

In Vitro Models for Thyroid Disruptors

• Iodine Symporter (NIS)

• Thyroperoxidase (TPO)

• Deiodinases

• Transporters – Blood

• Transporters – Cellular

• Thyroid Receptors

• Hepatic Nuclear Receptors

GeneBLAzer TR-UAS-bla HEK293 Cell Line

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

LigandLigand

T3 Stimulation of TR

T3 (M)

**

****

**

****

**** Substrate

Courtesy of Keith Houck, NCCT

In Vitro Models – Thyroid Receptor Beta

Human TR Reporter Gene Assay Heat Map of AC50’s

1456 chemicals; 14 concentrations; agonist and antagonist modes

Levothyroxine

T3

ACTIVES 62

SELECTIVE 0

ACTIVES 65

SELECTIVE 2

Courtesy of Keith Houck, NCCT

TH Action Assay – T-Screen (Gutleb et al. EnvToxPharm 2005)- measures TH dependent cell proliferation in GH3 cells- 96 well plate assay

Summary

• Thyroid pathways are known

• Multiple targets involved

• In vitro models are available for many of the targets

• Need to begin testing chemicals

Thank You