2009 Crofton DNT Thyroid Disruptors NBTS Meeting

Developmental Neurotoxicity and Thyroid Hormone Disruptors: Past,

Present and Future

Kevin M. Crofton, PhDSystems Biology Branch

Integrated Systems Toxicology DivisionNational Health and Environmental Effects Research Laboratory

Office of Research and Development Research Triangle Park, NC

June 28, 2009Neurobehavioral Teratology Society

Roi Grande PR

The views expressed in this presentation are those of the presenter and do not necessarily reflect the views or policies of the U.S. EPA

Developmental Neurotoxicity and Thyroid Hormone Disruptors: Past,

Present and Future

Kevin M. Crofton, PhDSystems Biology Branch

Integrated Systems Toxicology DivisionNational Health and Environmental Effects Research Laboratory

Office of Research and Development Research Triangle Park, NC

Wadsworth Center, Albany NYMay 14, 2009

Mode-of-action, Source to Outcome, Toxicity Pathway: No Matter What You Call It Behavioral Effects Are Critical and Behavioral Toxicologists

are Needed

Outline• Mode-of-Action:

What is it and what is it good for

• MOA Examples: Thyroid hormone disruption, thyroid cancer and developmental neurotoxicity Thyroid 101: Hormones, homeostasis and signaling Sites-of-action for TDCs

• Relevancy of animal data to humans• How can MOA help identify research needs

Developmental neurotoxicity of TDCs Acute neurotoxicity of pyrethroids

• How can thinking of MOA help with screening for toxicity

What is a Mode-of-Action?

• Mode of action is defined as the sequence of key cellular and biochemical events (measurable parameters) that result in a adverse health outcome.

• Critical to MOAs are causative relationships, not just correlations (Bradford Hill’s criteria for etiologic significance in epidemiologic investigations of causality)

Mode of ActionMode of ActionExposure-Dose-Response, Toxicity Pathway, Exposure-Dose-Response, Toxicity Pathway,

Adverse Outcome PathwayAdverse Outcome Pathway

Mode-of-action Framework by:

Schulte, Environ Res. 48:129-44 1989

KineticsHow does it get to where you don’t want it to be?• uptake• distribution• metabolism• elimination

DynamicsWhat does it do when it gets there?• initial key target• consequent effects at molecular,

cellular, organismal levels• adverse outcomes

TDCs - Multiple Targets

T4 -Gluc

T4 TTR/TBG

Hypothalamus

TRH

TSH

+

T3 & T4

T3 & T4

T4 > T3

Ah-Receptor

T4

UDPGTs

CAR/PXR

T4 T3Thyroperoxidase

I + tyrosine

T3 & T4

_

_Liver

T4 -Gluc

Biliary Excretion

Blood

Free- TH

Bound- TH -

Hypothalamus

Pituitary

Thyroid +

+

Ah-Receptor

T4

CAR/PXR

Iodine

Perchlorate Thiocyanate

T3

I + tyrosine

T3 & T4PTU

MancozebPronamide

PTU Octyl-methoxycinnamate

HO-PCBsEMD 49209

Dioxins

PBDEsPCBs

Peripheral Tissues

_

_

Deiodinases

TH TTR/TBG

UDPGTs

Tran

spor

ters

NISNISNISNIS

Thyroperoxidase Transporters

PBDEs

CoR

TH

TH CoA

AAAAA

TRE

TRTR

TDCs – Multiple Targets Thyroid Hormone Receptors

TRE

TR TR

CoR

XBPA

Ho-PCB

CoA

99

Thyroid Hyperplasia

Rat Thyroid Tumors

Health Risk

SerumT4 & T3

Modes of Action - ExamplesModes of Action - ExamplesCancer and Neurodevelopment in RatsCancer and Neurodevelopment in Rats

Altered Neuroanatomy

DevelopmentalDefects

HealthRisk

ThyroperoxidaseIodine Symporter

ExposureHepatic UDPGTs

Thyroidal

Extra-Thyroidal

Deiodinases

Thyroid Receptors

T4–TTR Binding

TSH

Mode-of-action Framework by:Schulte, Environ Res. 48:129-44 1989

MOA or Pathway “Thinking”What is it good for?

• Transparent process to characterize the weight of evidence to support an animal MOA

• Allows a determination of whether there is any evidence to reasonably exclude the same MOA in humans e.g., humans lack an enzyme that generates a toxic metabolite

• Importantly, the process of working through this analysis allows for identification of data needs = focused research

• Knowledge of key events allows identification of target that can be used in screening chemicals

Adapted from Cohen 2009

MOA Framework(IPCS, EPA, Health Canada)

Boobis et al (2008)

Do you have an MOA in animals and is it relevant to humans?

YES

NO

NO

YESMOA notRelevant

YESMOA notRelevant

NOProceed with

risk assessment

Proceed withrisk assessment

Is the weight of evidence sufficient to establish a mode of action (MOA) in animals?

Can human relevance be reasonably excluded on the basis of qualitative differences between animals and humans?

Can human relevance be reasonably excluded on the basis of quantitative differences between animals and humans?

1212

Thyroid Hyperplasia

Rat Thyroid Tumors

Health Risk

SerumT4 & T3

Modes of Action for TDCs - RatModes of Action for TDCs - Rat

Altered Neuro-Development


HealthRisk



Thyroidal

Extra-Thyroidal

Deiodinases

Thyroid Receptors

T4–TTR Binding

TSH


Do these pathways apply to humans?

1313

Thyroid Hyperplasia

Rat Thyroid Tumors

Health Risk

SerumT4 & T3

Modes of Action for TDCs - RatModes of Action for TDCs - Rat



HealthRisk



Thyroidal

Extra-Thyroidal

Deiodinases

Thyroid Receptors

T4–TTR Binding

TSH


Do these pathways apply to humans?

1414

Thyroid Hyperplasia

Rat Thyroid Tumors

Health Risk

SerumT4 & T3

Modes of Action for TDCs – HumanModes of Action for TDCs – HumanExample CarbamazepineExample Carbamazepine



HealthRisk



Thyroidal

Extra-Thyroidal

Deiodinases

Thyroid Receptors

T4–TTR Binding

TSH


Subjects TT4 FT4 TSH Reference Healthy Volunteers Vainionpaa et al (2004) Epileptic children Liewendahl et al (1978) Epileptic girls Bensten et al (1983) Epileptic adults Fischel et al (1978) Epileptic adults Liewendahl et al (1980) Epileptic adults Connell et al (1984) Epileptic adults Rootwelt et al (1978)

= decrease; = no effect

Effects of carbamazepine on thyroid function in humansSubjects TT4 FT4 TSH Reference Healthy Volunteers Vainionpaa et al (2004) Epileptic children Liewendahl et al (1978) Epileptic girls Bensten et al (1983) Epileptic adults Fischel et al (1978) Epileptic adults Liewendahl et al (1980) Epileptic adults Connell et al (1984) Epileptic adults Rootwelt et al (1978)


Effects of carbamazepine on thyroid function in humansSubjects TT4 FT4 TSH Reference Healthy Volunteers Vainionpaa et al (2004) Epileptic children Liewendahl et al (1978) Epileptic girls Bensten et al (1983) Epileptic adults Fischel et al (1978) Epileptic adults Liewendahl et al (1980) Epileptic adults Connell et al (1984) Epileptic adults Rootwelt et al (1978)


Effects of carbamazepine on thyroid function in humans

1515

No Thyroid Hyperplasia

No Thyroid Tumors

No Health Risk

SerumT4 & T3

MOA Thyroid Tumors – HumanMOA Thyroid Tumors – HumanExample - CarbamazepineExample - Carbamazepine



HealthRisk



Thyroidal

Extra-Thyroidal

Deiodinases

Thyroid Receptors

T4–TTR Binding

No TSH


Is the Developmental MOA in Rats Relevant to Humans? YES!

• Normal thyroid hormone levels and receptor function are CRITICAL for normal development of the structure and function of the brain in both rats and humans• Humans:

• Over 100 years of data for iodine deficiency• Congenital hypothyroidism

• Animals:• Lots of analogous animal models

1717

SerumT4 & T3

MOA Thyroid Tumors – HumanMOA Thyroid Tumors – HumanExample – Iodine DeficiencyExample – Iodine Deficiency



HealthRisk


DietaryIodine

Hepatic UDPGTs

Thyroidal

Extra-Thyroidal

Deiodinases

Thyroid Receptors

T4–TTR Binding


“High dose” = Cretinism“Low dose” = IQ losss

Research NeedsHow MOA ‘thinking” can focus research

1. One if MOA in animals is judged to be incomplete where are the data gaps Example pyrethroid acute neurotoxicity

2. In Human relevancy analysis – where are the data gaps• Lack of data translates to assumed relevance.• Example interspecies sensitivity to TDCs

3. Screening for prioritization If you know the molecular target – then develop low-cost

and high-throughput methods for that target

MOA Ex #1 - Pyrethroids

Exposure AbsorbedDose

Target Tissue Dose

Binding Molecular

Target

In Vitro HumanNeurons

OrganLevelEffect

AdverseClinicalOutcome

Exposure AbsorbedDose

Target Tissue Dose

MolecularTarget

In Vitro RatNeuron

Firing Rate

OrganLevelEffect

AdverseClinical

Outcome

Human

Exposure & Kinetics Modeling

Effects Modeling

Too many unknowns for to call this an established MOA

Binding Molecular

Target

In Vitro HumanNeurons

OrganLevelEffect

AdverseClinicalOutcome

MolecularTarget

In Vitro RatNeuron

Firing Rate

OrganLevelEffect

AdverseClinical

Outcome

Human

Na Channel work almost all in vitroNeed a good in vivo biomarker

Mostly in vitro firing ratesNeed in vivo data

Rat

MOA for Acute Pyrethroid Neurotoxicity?

If key initiating event is changes in VGSC kinetics, then how does this translate into changes in behavior??

Available biochem and physiologydata not correlative

Behavior data from high dose studies

Use in vitro models to compare rat and human cell lines

100 25 00

25

50

75

100

125

0

25

50

75

100

125

66 50TH Concentration

Dys

func

tion

(% C

ontro

l)

100 25 00

25

50

75

100

125

0

25

50

75

100

125

66 50TH Concentration

Dys

func

tion

(% C

ontro

l)

Rat

Human

MOA Ex. #2 – Interspecies Sensitivity

100 25 00

25

50

75

100

125

0

25

50

75

100

125

66 50TH Concentrations

Dys

func

tion

(% C

ontro

l)

Most experimental

“sledgehammer”models

Likely human environmental

exposures

Data Need: Comparative dose-response data for rats and humans

2222

Humans dose response data is very limitedHaddow et al. (1999) Even low level maternal

hypothyroxinemia (second trimester) leads to adverse outcomes (~25% T4 leads to IQ loss)

Animal dose response data: more, but limited

Comparative Sensitivity – Rat and Human

Crofton (2004)

Pup PN21 T4 vs PS LTP 60m

Pup PN21 T4 Percent of Control

0 10 20 30 40 50 60 70 80 90 100 110

PS L

TP %

Con

trol

90100110120130140150160170180190200210

M0805 PTUM0703 PERCM0102 PTUGD18-PN21 PTU

r2=0.84

Log Thyroxine Concentration, % Control3103060100

Hea

ring

Loss

, dB

SP

L (d

iffer

ence

from

con

trol)

0

10

20

30

40

50

60

y = -33.68*logX + 68.72 r ² = 0.8519

Gilbert, unpublished

Synaptic plasticity vs T4Hearing vs T4 – 50% lossWhat amount of change is adverse?

50% during critical window

??

MOA Ex #3 - Screening• Chemicals with info on thyroid endpoint is

less than 200

• Chemicals with potential for exposure and lacking any assessments has been estimated to be around ~10,000

• Regardless of all the other uncertainties, this is a high priority

We cannot test our way out of this problem starting with rodent studies, especially not using developmental exposures

Developing a New Way of Doing Business

+ If you know the key initiating event (or events) then develop efficient screens for this target

+ Use these screen to prioritize for further testing not for regulation

+ Start with 500 or 600 chemicals – identify the ‘worst actors” – and then test them in a tiered testing strategy

+ e.g., in vitro TPO inhibition assay, then test in 96 well Xenopus assay, then in mammals, higher to medium to lower throughput

Ongoing Methods DevelopmentIf you know the key initiating event (or events) – then develop efficient

screens

1) Receptor based screening at EPA’s NCCT e.g. PXR, AhR, PPAR, TRbeta recently completed screening of 320 pesticidal chemicals

2) TPO screening 96 well medium throughput assay for inhibition of porcine TPO (Mike

Hornung at ORD’s Duluth Lab)3) TH signaling in transgenic X. laevis

96-well format for detecting alterations in TR signaling (Barbara Demeneix - Environ. Sci. Technol., 2007, 41:5908–5914)

4) OECD/IPCS Advisory Group – Collating & summarizing available assays

A NEGATIVE IN THESE SCREENS DOES NOT TRANSLATE TO NO HAZARD!

Screening for Thyroid Disruptors

Current Approaches

Standard approach

• Guidelines

• ‘Triggered’ testing

• Low throughput

New Tiered Approach

Screening for Prioritization

HTP in vitro/QSAR

L-MTP - alt species?

Targeted mammalian testing

Paradigm Shift for Tox TestingNew Tiered Approach


HTP in vitro

L- MTP alt species

Targeted mammalian testing

1,000s

100s

10s


Testing for Hazard

Assessments

Summary• MOA Approach helps to focus research

• Development of MOAs requires adverse outcome data – without it the MOA is neutered.

• A number of thyroid pathways are known that if disturbed during developmental result in neurological deficiencys Multiple targets involved In vitro models are available for many of the targets

• Need to develop testing methods that can be used for Screening for Prioritization Also needed --- tiered testing strategies

• For this strategy to work the input and research from behavioral folks is critical!

Thank you for listening

2009 Crofton DNT Thyroid Disruptors NBTS Meeting

Environment

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