Male
and
Female
Pubertal
Protocols:

Identifying
Environmental
Compounds
That
Alter
The
Thyroid
axis
T.
Stoker
Endocrinology
Branch,
RTD
NHEERL,
ORD
U.
S.
EPA,
RTP,
NC
Applicability
Detects
compounds
that
display:

anti­
thyroid
Estrogenic
androgenic
or
anti­
androgenic
altered
puberty
via
changes
in
FSH,
LH,
PRL,

GH
or
hypothalamic
function.

anti­
estrogenic
[
estrogen
receptor
(
ER)]

steroid
enzyme
mediated
activity
Male
and
Female
Pubertal
Protocols
Questions

Are
the
male
and
female
pubertal
assays
useful
as
screens
for
thyrotoxicants
with
different
mechanisms
of
action?


Are
results
reliable?

 
What
is
Interlaboratory
variability?
Overview

Mechanisms
of
altered
thyroid
homeostasis.


Examples
of
chemicals
with
specific
mechanisms.


Contract
study
results
for
PB
and
PTU
 
What
was
observed
 
Published
studies
vs.
current
data.


New
studies
looking
at
other
thyrotoxicants
(
EPA,
ORD)
MECHANISMS
OF
ALTERED
THYROID
HOMEOSTASIS

Clearance
of
thyroid
hormones
(
ex.
liver
enzyme
induction
or
altered
TTR/
TBG)


Synthesis
of
thyroid
hormones
(
ex.
altered
thyroperoxidase
or
altered
uptake
of
iodine
into
the
thyroid
cell)


Tissue
deiodinase
(
peripheral
conversion
of
T4>
T3,
FD
&
C
Red
No.
3,
others?)


Receptor
Binding
(??
known)


Brain/
hypothalamic
control
of
TRF
(
not
known
??)
Liver
T4­
Gluc
Biliary
Excretion
Blood/
Plasma
Free­
TH
Bound­
TH
TTR/
TBG
Brain
Pituitary
Thyroid
TRH
TSH
+
+
T3,
T4
T3,
T4
Tissue
deiodinase
T4
>
T3
T4
UDPGTs
Iodine
Perchlorate
T4
T3
T4
Thyroperoxidase
I
+
tyrosine
T3
&
T4
PTU
Mancozeb
PBDE
Phenobarbitol
PCBs
Mechanisms
of
Thyroid
Disruption
FD&
C
Red
No.
3
Alachlor
DDT
Altered
Synthesis
Altered
Thyroid
Synthesis

By
altered
KI
symporter
 
Perchlorate
0
1
2
3
4
5
6
Control
62.5
mg/
kg
125
mg/
kg
250
mg/
kg
500
mg/
kg
Total
Serum
T4
(

ug/

dl)
Treatments
*

*

*
0
0.5
1
1.5
2
2.5
3
3.5
Control
62.5
mg/
kg
125
mg/
kg
250
mg/
kg
500
mg/
kg
Serum
TSH
(

ng/

ml)
Treatments
*

EPA,
RTP
Wistar
female
Altered
Synthesis
of
T4

By
inhibition
of
thyroperoxidase
 
PTU
Control
PTU240
0
1
2
3
4
5
6
7
8
T4
(

ug/

dl)
Control
PTU240
0
5
10
15
20
25
TSH
(

ng/

ml)
Control
PTU240
0
10
20
30
40
50
60
70
80
Thyroid
WT
(

g)

Marty
et
al.,

Tox.
Sci.,
2001)
Male
CD
Altered
Clearance
of
T4
Altered
Clearance
of
T4

Phenobarbital
in
Pubertal
Male
Marty
et
al.,

Tox.
Sci.,
2001
Male
CD
0
2
4
6
8
10
12
14
Control
50
PB
100PB
Liver
Wt.(

g)
*
0
10
20
30
40
50
60
70
Control
50PB
100PB
240PTU
Thyroid
Wt.

(

mg)

0
5
10
15
20
25
Control
50PB
100PB
240PTU
TSH
(

ng/

ml)
0
1
2
3
4
5
6
7
8
Control
50PB
100PB
240PTU
T4
(

ug/

dl)
*

*

*
Pubertal
Assays
Detecting
Thyroid
Toxicants

Mechanism
Identified
by
Assay(
s)

 
Altered
KI
symporter
 
Altered
thyroperoxidase
activity
 
Altered
clearance
of
T4

Mechanisms
not
studied
to
date
 
Binding
proteins
(
organochlorines,
like
OH­
PCBs)

 
Deiodinase
(
FD&
C
Red
No.
3)

 
Hypothalamic
alterations
Results
from
Contract
Studies

Contract
I,
2000
(
PTU)

 
Thyroid
Hormones

Contract
II,
2003
(
PB)

 
Thyroid
Weights

Contract
III,
2003
(
PTU
&
PB)

 
Thyroid
Hormones,
Weights
and
Histology
16.5
16.9
16.8
17.7
18.1
20
17.2
18.5
16.9
Oil
EE0025
EE005
PB25
PB50
PB100
MET
12.5
MET
25
MET
50
15
16
17
18
19
20
21
22
THYROID
WEIGHT
**

Contract
Study
II
2003
Effect
of
chemicals
on
Thyroid
Weights
in
Female
Pubertal
21.6
21.2
20.4
20.4
21.9
23.2
19.3
21.9
21
Oil
F25
F50
PB25
PB50
PB100
V10
V30
V100
16
18
20
22
24
THYROID
WEIGHT
(
g)
NS
Contract
Study
II
2003
The
effect
of
chemicals
on
thyroid
weight
in
the
Male
Pubertal
19.3
16.2
16.1
15.4
15.1
14.9
16.8
14.3
12.6
13.4
14.7
14.4
38.5
62
Con
A
Atrazine
75
Atrazine
150
Fenarimol
50
Fenarimol
250
Methoxychlor
25
Methoxychlor
50
Con
B
BPA
400
BPA
600
Ketoconazole
50
Ketoconazole
100
PTU
2
PTU
25
0
10
20
30
40
50
60
70
THYROID
Wt
*
*
*
**
**

Contract
Study
III
2003
The
effect
of
chemicals
on
thyroid
weight
in
the
Female
Pubertal
20.5
18.6
88.6
24.2
18
.5
22
24.3
19.8
20.6
21
16.8
27.3
77
26.6
26.7
29.4
31.9
32.5
Con
a
ATR
75
ATR
150
p,

p'DDE
50
p,

p'DDE
100
Vin
30
Vin
100
Met
25
Met
50
Con
b
PTU
2
PTU
25
Keto
50
Keto
100
Linuron
50
Linuron
100
PB
50
PB
100
15
20
25
30
35
**
*

*

**

*

Contract
Study
III
2003
The
Effect
of
Chemicals
on
Thyroid
Weight
in
the
Male
Pubertal.
0
1
2
3
4
5
6
7
T4
ug/
dl
LE>
SD
BUT
NO
INTERACTION
WITH
TR
LE
SD
Oil
KETOMT
DBP
FL
PTU
PIM
6.54
5.88
6
3.45
5.97
0.6
5.58
4.87
5.16
4.15
3.86
4.14
0
4.98
0
5
10
15
20
25
30
TSH
ng/
ml
LE
SD
Oil
KETOMT
DBP
FL
PTU
PIM
3.13
2.86
2.72
2.11
4.16
26.2
2.1
3.06
2.98
2.67
2.43
2.36
27.7
2.68
*
**
**

Contract
Study
I
2000
The
effect
of
chemicals
on
thyroid
hormones
in
the
Male
Pubertal
0
1
2
3
4
5
6
T4
ug/
dl
Oil
KETO
MET
EE
TAM
PTU
PIM
4.12
3.71
4.01
4.3
5.51
0.03
3.34
1
10
100
TSH
ng/
ml
Oil
KETO
MET
EE
TAM
PTU
PIM
1.58
1.57
1.33
1.66
2
28.2
1.32
**
**
**

Contract
Study
I
2000
The
effect
of
chemicals
on
thyroid
hormones
in
the
Female
Pubertal
4.444.554.674.72
3.73
4.574.69
4.814.88
5.23
4.97
4.79
1.97
0.66
Con
A
Atrazine
75
Atrazine
150
Fenarimol
50
Fenarimol
250
Methoxychlor
25
Methoxychlor
50
Con
B
BPA
400
BPA
600
Ketoconazole
50
Ketoconazole
100
PTU
2
PTU
25
0
1
2
3
4
5
6
T4
9.629.9
8.52
19.7
12.5
8.349.83
7.88
6.977.518.92
8.24
42.4
99.4
Con
A
Atrazine
75
Atrazine
150
Fenarimol
50
Fenarimol
250
Methoxychlor
25
Methoxychlor
50
Con
B
BPA
400
BPA
600
Ketoconazole
50
Ketoconazole
100
PTU
2
PTU
25
0
20
40
60
80
100
120
TSH
**
**
**

**
**

*

Contract
Study
III
2003
The
effect
of
chemicals
on
thyroid
hormones
in
the
Female
Pubertal.
6.35
6.28
0.37
3.4
6.46
5.9
5.32
4.87
4.28
6.11
6.66
5.78
0.53
5.58
5.65
4.6
5.66
5.73
Con
a
ATR
75
ATR
150
p,

p'DDE
50
p,

p'DDE
100
Vin
30
Vin
100
Met
25
Met
50
Con
b
PTU
2
PTU
25
Keto
50
Keto
100
Linuron
50
Linuron
100
PB
50
PB
100
0
1
2
3
4
5
6
7
T4
10.9
12.7
135
9.49
10.95
14.1
14.42
10.6
12.9
11
10.8
11.54
82.1
10
11.1
8.98
16.4
14.5
Con
a
ATR
75
ATR
150
p,

p'DDE
50
p,

p'DDE
100
Vin
30
Vin
100
Met
25
Met
50
Con
b
PTU
2
PTU
25
Keto
50
Keto
100
Linuron
50
Linuron
100
PB
50
PB
100
0
5
10
15
20
TSH
**
**

**
**
**
**
*

**
**

**

Effects
of
toxicants
on
Thyroid
Hormones
in
the
Male
Pubertal
Contract
Study
III
2003
Summary
of
Contract
Studies

All
studies
found
alterations
in
thyroid
parameters
that
were
consistent
with
the
chemical
action
 
Thyroid
Weights

Both
Contract
II
and
III
found
appropriate
differences
in
weights,
Contract
I
did
not
take
weights.

 
Thyroid
Hormones

PTU
 
Contract
I
and
III
identified
PTU
thyroid
hormone
effects
(
T4
and
TSH)

 
Contract
II
did
not
test
PTU

PB
 
Contract
III
identified
changes
in
TSH,
but
no
T4
decrease
 
Contract
II
did
not
measure
hormones
Identifying
other
chemicals
(
ORD
studies)


Perchlorate­
KI
symporter

DE­
71­
Liver
metabolism
of
T4
The
Effect
of
Perchlorate
on
Thyroid
Endpoints
in
the
Male
Pubertal
0
1
2
3
4
5
6
Control
62.5
mg/
kg
125
mg/
kg
250
mg/
kg
500
mg/
kg
Total
Serum
T4
(

ug/

dl)
Treatments
*
*
*
0
1
2
3
4
Control
62.5
mg/
kg
125
mg/
kg
250
mg/
kg
500
mg/
kg
Serum
TSH
(

ng/

ml)
Treatments
*
*
*

EPA
Special
Study
Wistar
Rat
The
Effect
of
Perchlorate
on
Thyroid
Endpoints
in
the
Female
Pubertal
Protocol
0
1
2
3
4
5
6
Control
62.5
mg/
kg
125
mg/
kg
250
mg/
kg
500
mg/
kg
Total
Serum
T4
(

ug/

dl)
Treatments
*

*

*
0
0.5
1
1.5
2
2.5
3
3.5
Control
62.5
mg/
kg
125
mg/
kg
250
mg/
kg
500
mg/
kg
Serum
TSH
(

ng/

ml)
Treatments
Females
*

EPA
Special
Study
Wistar
Rat
5
Day
31
Day
0
1
2
3
4
5
6
7
Control
3mg/
kg
30mg/
kg
60mg/
kg
Total
Serum
T4
(

ug/

dl)
*
*
*

*

*

Treatments
5
Day
31
Day
0
0.5
1
1.5
2
2.5
3
Control
3mg/
kg
30mg/
kg
60mg/
kg
Serum
TSH
(

ng/

ml)
*
*

Treatments
The
Effect
of
DE­
71
on
Thyroid
Endpoints
in
the
Male
Pubertal
Protocol
EPA
Special
Study
Wistar
Rat
5
Day
20
Day
0
1
2
3
4
5
Control
3
mg/
kg
30
mg/
kg
60
mg/
kg
Total
Serum
T4
(

ug/

dl)
Treatments
*
*
*
*
5
Day
20
Day
0
0.5
1
1.5
2
Control
3
mg/
kg
30
mg/
kg
60
mg/
kg
Serum
TSH
(

ng/

ml)
Treatments
The
Effect
of
DE­
71
on
thyroid
endpoints
in
the
Female
Pubertal
Protocol
EPA
Special
Study
Wistar
Rat
T3
in
DE­
71
Study
Comparison
of
Male
and
Female
5
Day
20
Day
0
50
100
150
200
Control
3
mg/
kg
30
mg/
kg
60
mg/
kg
Total
Serum
T3
(

ng/

dl)
Treatments
5
Day
31
Day
0
20
40
60
80
100
120
140
Control
3mg/
kg
30mg/
kg
60mg/
kg
*

*

Total
Serum
T3
(

ng/

dl)
Treatments
EPA
Study
Wistar
Rat
Thyroid
Histopathology
(
hypo)


Decreased
Colloid
Area

Increased
Follicular
proliferation
and
cell
heights.
Hypothyroidism
EPA
DE­
71
Study
Control
Male
Thyroid
60
mg/
kg
DE­
71
Male
Thyroid
Also
observed
in
female
60
mg/
kg
DE­
71.
Conclusions

Male
and
Female
Pubertals.

 
No
failures
so
far
 
3
mechanisms
examined
 
Do
we
need
to
do
other
mechanisms,
before
moving
on?
5'
Deiodinase
inhibitor,
TTR,
hypothalamus??

 
Some
disappointments
(
maybe)


One
contract
lab
missed
PB
effect
on
T4
 
Still
thyroid
effects
observed!
(
TSH,
liver,
thyroid
wt.)
0
2
4
6
8
10
12
14
Control
50
PB
100PB
Liver
Wt.(

g)
*
*
0
10
20
30
40
50
60
70
Control
50PB
100PB
240PTU
Thyroid
Wt.

(

mg)

0
5
10
15
20
25
Control
50PB
100PB
240PTU
TSH
(

ng/

ml)
*

*
0
1
2
3
4
5
6
7
8
Control
50PB
100PB
240PTU
T4
(

ug/

dl)
*

*

*

Marty
et
al.,

Tox.
Sci.,
2001)
Control
PB5
PB25
PB50
PB100
0
1
2
3
4
5
6
SE
Control
*
*
*

Liver
(%

BW)
Control
PTU.
025
PTU.
25
PTU1
PTU10
0
1
2
3
4
5
SE
Mean
Liver
(%

BW)

O'Connor,
1999
15
Day
SD
Male
The
Effect
of
PB
and
PTU
on
Liver
in
the
Intact
15
Day
Male
Assay
Control
PB5
PB25
PB50
PB100
0
1
2
3
4
5
SE
Mean
*
*
*

T4
(

ug/

dl)
Control
PB5
PB25
PB50
PB100
0
5
10
15
20
25
30
35
SE
Mean
TSH
(

ng/

ml)
*
*

O'Connor,
1999
The
Effect
of
PB
on
Thyroid
Hormones
in
the
15
Day
Intact
Male
SD
Control
PTU.
025
PTU.
25
PTU1
PTU10
0
10
20
30
40
50
60
SE
Mean
*
*
*

O'Connor
15
day
male
SD
TSH
(

ng/

ml)

Control
PTU.
025
PTU.
25
PTU1
PTU10
0
0.5
1
1.5
2
2.5
3
3.5
SE
Mean
*
*
*

T4
(

ug/

dl)
O'Connor
Studies

Contract
studies
 
Therimmune
2000

PTU
(
240)
,
male
and
female
in
SD
and
LE
 
Therimmune
2003

PB
(
25,
50,
100
mg/
kg)
male
and
female
SD
 
RTI

PTU
(
2
and
25
mg/
kg)
male
and
female
SD

PB
(
50
and
100
mg/
kg)
male
and
female
SD

EPA
studies
 
PBDE
and
perchlorate.


Industry
studies
 
Marty
et
al.,
PB
50
and
100
mg/
kg
and
PTU
240
mg/
kg
in
male
SD
 
O'Connor
et
al.,
PB
5,
25,
and
50
mg/
kg
or
PTU
.025,
.25,
1
and
10
mg/
kg
in
SD
males
(
15
day
intact)

 
Yamada,
PB
at
125
mg/
kg
and
PTU
at
2.5
mg/
kg
(
10
day
HERSH)
0
5
10
15
20
25
Control
PB50
PB100
PTU2
PTU25
Males
Liver
wt.

(

g)
*
*
*
*

0
2
4
6
8
10
12
Control
PTU2
PTU25
Female
Liver
wt
(

g)
*

Contract
Study
III
2003
The
effect
of
chemicals
on
liver
weight
in
the
Male
and
Female
Pubertal
Effect
of
chemicals
on
liver
weight
in
Male
and
Female
Pubertals
Control
PB25
PB50
PB100
0
5
10
15
Liver
wt
(

g)

Contract
Study
II
2003
Control
PB25
PB50
PB100
0
5
10
15
20
25
Female
Male
*

*

*

*
