Development of a Biologically Based Development of a Biologically Based Pharmacokinetic (BBPK) Model for the Pharmacokinetic (BBPK) Model for the HypothalamicHypothalamic--Pituitary Thyroid Axis in Pituitary Thyroid Axis in

the Maturing Rat for the Dose Response the Maturing Rat for the Dose Response Assessment of Developmental Assessment of Developmental

NeurotoxicityNeurotoxicity

RD-83213401-0

Jeffrey Fisher, Ph.D., Duncan Jeffrey Fisher, Ph.D., Duncan Ferguson, V.M.D., Ph.D and John Ferguson, V.M.D., Ph.D and John

Wagner, Ph.D.Wagner, Ph.D.University of GeorgiaUniversity of Georgia

Interdisciplinary Toxicology ProgramInterdisciplinary Toxicology Program

Multifaceted Objectives of Research

Modeling (Jeff Fisher, Eva McLanahan, Libby Myers)1) To better understand relationships between administered dose and HPT axis

disturbances in the immature rat and neurodevelopmental toxicity:A. Biologically based models of the HPT axis are under development for different

reproductive states of rats, adult male rats, and human. B. PBPK models for thyroid active chemicals will be linked to the HPT axis models

to predict neuro-developmental toxicity endpoints.

Experimental (Duncan Ferguson, John Wagner, Matthew Taylor,Michael Stramiello,Nadia Paolino)2) Using gestational/neonatal exposure of rats to thyroid disruptive compounds, to:

A. Examine the sensitivity, capacity and development of compensatory mechanisms of thyroid hormone secretion/metabolism by the thyroid, brain, and liver.

B. Develop quantitative ‘dose-response’ relationships of serum and tissue markersof thyroid status and correlate with developmental neurotoxicity endpoints.

ApproachApproach-- Cooperative Cooperative agreementsagreements

• Develop team of interdisciplinary scientists; while working independently, are aiding each other in experimental design, sharing samples and data.

-- UGA, UMass (Tom Zoeller) and USEPA (Kevin Crofton, Mike DeVito, and Mary Gilbert)

PBPK

Dose of Toxicant

BBPK Thyroid axis

Describes the kinetics of the toxicant and itsMOA for disturbing the HPT axis.

Describes the HPT axis and perturbations in the HPT axis from chemical insult.

CNS responses in the brain of the pup or fetus.

Project Concept for Computational Modeling ofDose-Response in the Fetal/Neonatal Rat

% C

NS

Toxi

city

Dose-Response

Internal Dosimetrics

Approach for Computational Approach for Computational ModelingModeling

…Fill fundamental D-R data gap (HPT disruption hypothyroidism developmental neurotoxicty)

• Use propylthiouracil (PTU), as a probe to establish high to low dose quantitative relationships between disruption of the HPT axis leading to hypothyroidism in the developing rat and neurotoxicity.

…Select two thyroid active chemicals with substantial data• Perchlorate (iodide blocking at thyroid) and PCB 126

(increased hepatic T4 metabolism). Both environmental chemicals cause hypothyroidism in rats.

Sub models _____BBPK- HPT axis_________

DietaryIodideToxicant

T4TSH T3PBPK

Linking the Sub ModelsLinking the Sub Models-- I see I see the lightthe light…………..

•PBPK models•PBPK dosimetry model with MOA (perchlorate and PCB 126)--adopt aspects of perchlorate PBPK models

•Pup growth PBPK model using growth equations for organs--adopt in-house research from deltamethrin•Utilize in-house PCB 126 kinetic data sets

HPT axis models•Recalibrate radiolabeled iodide submodel, calibrate radiolabeled T4and T3, calibrate endogenous TSH submodel

•Develop endogenous 127I, T3, T4 and link with TSH (feedback)

•Articulate compensatory mechanisms for HPT axis such as T3/T4 shift in thyroid hormone production, D2 induction in brain, NIS induction in thyroid, extra-thyroidal D1 decrease

Current Status of ModelsCurrent Status of Models(two Ph.D. students)(two Ph.D. students)

• Develop radiolabel sub-models for iodide and T4 in PND 13 pup and adult rat.

• Examine feedback equations for endogenous serum TSH and T4 concentrations.

• Dietary iodide model for adult human

Thyroid TissueBound Free

Thyroid Blood

Plasma

ivdose

Kel*AP_i

GI Contents

GI Tissue

GI Blood

Liver

Body

PAGC

QG*Ca_i

QL*Ca_i

QB*Ca_i

QB*Cvbody_i

QG+QL*Cvl_i

QC*Ca_i

QT*Ca_iQT*Cvt_i

QG*Cvg_i

Kthprod*AIB

urine

RGNIS

RTNIS

PAGT

Total Iodide

PAT

Oral dose

125125I Model I Model StructureStructure

Approach for Iodide Binding in Approach for Iodide Binding in ThyroidThyroid

127I intake (µg/day)0 50 100

150

200

250

300

350

400

Tota

l thy

roid

al 12

7 I (µg

)

0.1

1

10

Plas

ma

127 I (

µg/1

00m

L)

0

10

20

30

40

50

60

Plasma 127I

Thyroid 127I

Thyroid tissueBound Free

Thyroid Blood

PATRTNIS

THprod

QT*Ca_iQT*Cvt_i

dtdTHprod

dtdIB

dtidIB

iCIBKaibKVitV

iCfiKmiCfitV

dtdIB

aibbindb

b

b

−=

+=

+=

_

_*0_max

___*_max Binding equation

Binding inhibition equation

Rate of change in amount bound

Adapted from Pedraza et al., 2006

and Nagataki et al., 1966

Bound Bound 125125I in ThyroidI in Thyroid

Time (hr)0 2 4 6 8 10 12

Bou

nd T

hyro

idal

125 I (

ng/m

L)

103

104

105

Adult Male Rat33ug/kg i.v. dose

Time (hr)0 20 40 60 80

Bou

nd T

hyro

idal

125 I (

ng/m

L)

0

200

400

600

PND 14 Pup1 ug/kg oral gavage dose

Clewell et al., 2003 Yu et al., 2002

125125I PBPK Model PredictionsI PBPK Model Predictions

Time (hr)0 20 40 60 80 100

Free

Ser

um 12

5 I (ng

/mL)

0

20

40

60

80

Urin

e 12

5 I (µg

)

0

2

4

6

8

10

Urine 125I

Serum 125I

Time (hr)0 20 40 60 80

Free

Ser

um 12

5 I (ng

/mL)

0.0

0.2

0.4

0.6

0.8

1.0

Serum 125I

PND 14 Pup Adult Male Rat1 ug/kg oral gavage dose 33 ug/kg i.v. dose

Clewell et al., 2003 Yu et al., 2002

[[125125I]I]--T4 PBPK Model Structures and T4 PBPK Model Structures and PredictionsPredictions

Time (hr)0 2 4 6 8 10 12 14 16

125 I-T

4 (n

g/m

L)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Serum

Brain

Time (hr)0 5 10 15 20 25

125

I-T4

(ng/

mL)

0.00

0.02

0.04

0.06

0.08

0.10

Plasma

Adult Male Rat4.4 ng i.v. doseWong et al., 2005

PND 14 Pup9.6 ng i.v. doseSilva et al., 1984

Liver Blood

Liver Tissue

Volume of Distribution

Iv dose

Kel*AP_t4

QL*Ca_t4

QL*Ca_t4QL*Cvl_t4

T4 Elimination

RT4LU PAL

QL*Cvl_t4

M-M Type 1 5’DT4-G formationKglu*CL_t4

Brain Tissue

Brain Blood

Volume of Distribution

Iv dose

Kel*AP_t4

QB*Ca_t4

QL*Ca_t4QL*Cvl_t4

T4 Elimination

PAB

QB*Cv_t4

M-M Type 1 5’DT4-G formationKglu*CL_t4

Liver Blood

Liver Tissue

PALRT4LU

QBr*Ca_T4QBr*Cvb_T4

M-M Type 2 5’D

Thyroid (negative feedback loop)Thyroid (negative feedback loop)

( ) ( )( )

0,max40

TSH

0,TSH 4elim,TSH

T4

*[ ]*[ ]*[ 4] *[ ]*[ 4]

[ ]

**[ ]

[ 4]

d cat cat

Tpl

k TSHdTV k k UDPGT T k Deiod Tdt TSH k

k kdTSHV k TSHdt k T

⎛ ⎞⎛ ⎞ ′= + − −⎜ ⎟⎜ ⎟ +⎝ ⎠ ⎝ ⎠⎛ ⎞⎛ ⎞ = −⎜ ⎟⎜ ⎟ +⎝ ⎠ ⎝ ⎠

MOA (PCB)

Negative feedback, T4, TSH

TSH, T4 production

Wherek0= basal production rate of T4 (in absence of TSH)k0,max=maximal rate of T4 secretion to plasma (under TSH

stimulation)k0,TSH=rate of TSH production (T4 conc. approaches 0)

Steady state prediction Steady state prediction vsvs observation observation for serum T4 and TSH concentrationsfor serum T4 and TSH concentrations

in adult male ratin adult male rat

Time (hr)

0 20 40 60 80

T4 (u

g/dL

)

0

1

2

3

4

5

6

TSH

(ng/

mL)

0

20

40

60

80

100

120

140

T4

TSH

Experimental WorkExperimental WorkDuncan FergusonDuncan Ferguson

Blood / CSF

Free

T4

Nucleus Nucleus

T4

MCT8

RibosomesRibosomes

Neuronal Proteins

Neuronal Function, Growth,

Differentiation, Synaptogenesis

Pre-translational regulation

Post-translational

regulation

Nuclear Occupancy

Nuclear Occupancy

LTP, Synaptic Response, Learning, Memory

TRE Activation

TRE Activation

T3D2 T3

D3

T2

-

RC3 Neurogranin,

Synaptophysin

GFAPMBP

Glial Cell Function

Myelinogenesis

GlialProteins

OATP1C1

IncreasedIn hypothyroidism

Astrocyte/Tanycyte Neuron

Dosing ProtocolDosing Protocol• Dosing of timed pregnant Sprague-Dawley

dams started at GD2 and continued through PND21-PND30 depending upon sacrifice schedule

• Current dose levels are 0, 3, and 10 mg/L (ppm) PTU in the drinking water.

• Water intake recorded and animals weighed q48h

• Gender of offspring determined in the third week after birth, and female pups culled midway through that week

TimelineTimeline• Pups were sacrificed from PND21-PND31.• Dams were sacrificed on PND31, when the pups were

weaned.• Adults were sacrificed starting 2 months after weaning

(average PND100)• Female pups were culled on approximately PND 24; 2

males per litter studied at each timepoint• 14 litters

– 0 ppm (n= 5)– 3 ppm (n=5)– 10 ppm (n=4)

• Additional analyses for D2 activity were performed on Hooded Long-Evans rats (1 dam and 1 PND21 pup) dosed at 0 (n=12),1 (n=13),2 (n=13) and 3 (n=12) ppm PTU from GD6 to PND21.

Serum Thyroid Hormone and TSH Serum Thyroid Hormone and TSH Concentrations: DamsConcentrations: Dams

*

**

*

*

*

Serum Thyroid Hormone Serum Thyroid Hormone Concentrations:Concentrations:

PND25PND25

**

*

Hepatic D1 ActivityHepatic D1 Activity

*

* * *

PND25 Cortical T3 and D2 Activity:PND25 Cortical T3 and D2 Activity:PND25 vs. DamPND25 vs. Dam

*

**

*

*

*

*

*

Serum T4 vs. Cortical T3Serum T4 vs. Cortical T3PND25

Comparison of D2 response to Fall in Total Comparison of D2 response to Fall in Total T4: T4:

PND21PND21--30 vs. Dams30 vs. Dams

Comparison of Thyroid mRNA response:Comparison of Thyroid mRNA response:PND21PND21--30 vs. Dams30 vs. Dams

PND100 Serum HormonesPND100 Serum Hormones

PND100 Cortical T3, PND100 Cortical T3, and D1 and D2 Activitiesand D1 and D2 Activities

HippocampalHippocampal Electrophysiology:Electrophysiology:Stimulus/Response Curves: PND21Stimulus/Response Curves: PND21--3030

fEPSP S/R curve

0

0.5

1

1.5

2

2.5

3

0 20 40 60 80 100 120 140stimulus intensity (µA)

slop

e (m

V/m

sec)

control3ppm10ppm

LongtermLongterm PotentiationPotentiation::PND100PND100

0.5

1.0

1.5

2.0

2.5

3.0

15 35 55 75 95 115 135 155 175 195 215Time (minutes)

norm

aliz

ed fE

PS

P s

lope

control3 ppmbaseline1.35

1.66

1.44

1.89

1.32 1.18

1.551.35

(1 x 100Hz)

(3 x 100Hz)

LTP timecourse

Serum T4 vs. Synaptic Response:Serum T4 vs. Synaptic Response:PND21PND21--3030

Cortical T3 vs. Synaptic Response:Cortical T3 vs. Synaptic Response:PND21PND21--3030

Correlation of D2 vs. Maximum Correlation of D2 vs. Maximum Synaptic Response at PND21Synaptic Response at PND21--3030

Conclusions: Thyroid Conclusions: Thyroid ParametersParameters

• Thyroid hormone depletion by gestational/neonatal PTU exposure is ameliorated within the cerebral cortex by D2 induction, whereas hepatic D1 activity is maximally inhibited by 10 ppm PTU.

• Cortical T3 concentrations in PND21-30 pups were maintained in the euthyroid range until a fall of about 75% of serum T4.

• A highly significant negative exponential relationship was observed between serum T4 concentration and D2 activity, with a doubling in D2 with every 1.3 ug/dl fall in T4 in both dams and pups. The relative D2 maximal response was ~8-fold higher in the pups.

• Both cortical D2 and thyroid NIS mRNA induction, likely tissue biomarkers of T4 deficiency/TSH elevation, demonstrate greater sensitivity of the offspring to thyroid hormone deficiency.

• All serum and tissue thyroid parameters returned to normal following 2 months of PTU withdrawal.

Conclusions: Conclusions: ElectrophysiologyElectrophysiology

• Baseline synaptic transmission was significantly reduced in the CA1 region of hippocampal slices obtained from PND21-30 rats under the ongoing influence of PTU exposure.

• Slices obtained from littermates allowed to mature in the absence of PTU until PND90-100 did not exhibit any persisting change in baseline synaptic transmission, however a significant reduction in the magnitude of LTP was observed.

• The decreased ability of the synapses to undergo synaptic plasticity even after the animal has recovered to euthyroid status suggests that although some of the acute impact of hypothyroidism can be restored, the potential remains for significant persisting impairments on the processing of information through neuronal networks.

• D2 enzymatic activity is tightly and positively correlated with synaptic potential at PND25, and may serve as a useful biomarkerof thyroid hormone sufficiency in the brain.

Ongoing and Future WorkOngoing and Future Work• Tissue thyroid markers

– Cortical D3 activity– Tissue T4 concentrations – In situ hybridization: D2,D3, RC3, GFAP, MCT8,

OATP1C1• Thyroid markers

– Histomorphometry– NIS and Tg immunohistochemistry

• Anatomical – Brain histopathology– Immunohistochemistry for BDNF, synaptophysin

• Refined dose studies: 0.3,1 and 3 ppm• Behavioral studies: locomotor and cognitive function

as adults