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Endocrine Disruptor Screening Program:
Ralph L. Cooper
Endocrinology Branch
Reproductive Toxicology Division
NHEERL, U.S. EPA
Male and Female Pubertal Assays
PubertyPuberty is a period of dramatic neuroendocrine development that culminates in reproductive maturation.
Requires extensive interplay between a variety of hormones, organs and tissues.
Period of increased sensitivity to environmental agents.
Pubertal Assays for Endocrine Disrupting Chemicals
Many endpoints have been standardized
Many endpoints are currently being used in EPA testing
Large Toxicology database
Female Protocol is recommended
Male Pubertal is alternate
Objectives
EDSP Pubertal Protocols
Review Protocols for Male and Female Rat
Discuss data from a variety of sources indicating the ability of these protocols to detect EDCs
Discuss advantages and potential problems
Assessment of Pubertal Development and Thyroid
Function in Juvenile Female Rats
Assessment of Pubertal Development and Thyroid Function in Juvenile
Female Rats
Purpose
The purpose of this protocol is to quantify the effects of environmental compounds on pubertal development and thyroid function in the intact juvenile female rat.
Assessment of Pubertal Development and Thyroid Function in Juvenile
Female Rats
Applicability This assay detects agents that display
anti-thyroid, estrogenic, anti-estrogenic [estrogen receptor (ER)] or steroid enzyme mediated activity, or alter luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, growth hormone (GH) secretion or hypothalamic function.
Female Pubertal Protocol
pups(8-10)
21 25 30 35 42
Wean
4
BW
Postnatal Day
DoseCull
Necropsy
Assign toTreatmentWeight Rank
VOVO
Daily exam for VO, then cyclicity
Serum
Required Endpoints(Female pubertal assay)
Growth (body weight)
Age and weight at vaginal opening
Serum thyroxin (T4) and thyroid stimulating hormone (TSH)
Liver, kidney, pituitary and adrenal weight
Thyroid histology
Uterine and ovarian weights and histology
Vaginal cytology
Optional Measures (Female pubertal assay)
Serum estradiol, Prolactin and tri-iodothyronine (T3)Thyroid weightLiver, kidney, pituitary, adrenal & vaginal histologyEx-vivo ovary and pituitary hormone productionHypothalamic neurotransmitter concentrationEstrous cycle in adulthood
20
25
30
35
40
Con
trol
Eth
ynyl
-E
Tam
oxife
nP
TU
Ket
ocon
Pim
ozid
e
Met
hoxy
* *
* * *
*
Vaginal OpeningA
ge
(day
s)
Summary of Significant Effects on Major Endpoints inFemale Sprague-Dawley and Long-Evans rats
Treatment Mode of Action Age at
VO
Age at
First E
Histo-pathology
TSH T4
Ethynyl Estradiol
0.005 mg/kdER agonist = =
Tamoxifen
10 mg/kg
ER antagonist Partial agonist SD
Propylthiouricil
240 mg/kg
Inhibits T4 Synthesis SD SD
Ketoconozole
100 mg/kg
Inhibits Steroidogenesis SD =
Pimozide
30 mg/kg
DA receptor blocker = LE
Methoxy-chlor
100 mg/kg
ER agonist LE =
Daily oral (mg/kg/d) Methoxychlor or Octylphenol treatment induces pseudoprecocious puberty in the 21 day old female rat. Data are expressed as the number of days that VO was accelerated (Gray and Ostby, 1998).
0 50 100 150 2000
2
4
6
8
Octylphenol Methoxychlor
Days that VO was accelerated
Subchronic DBP oral treatment fails to accelerate pseudo precocious puberty (vaginal opening) in female rats. Treatments were initiated at 22 days of age. In contrast the xenoestrogen Methoxychlor markedly accelerates this estrogen-dependent process.
0 250 500 1000Dose of DBP (mg/kg/d)
20
25
30
35
Age at Vaginal Opening (Days)
0 3 6 12 25 50 100 200
Dose of Methoxychlor (mg/kg/d)
20
25
30
35
Age at Vaginal Opening (Days)
Gray et al., 1999
The xenoestrogen Methoxychlor induces early VO and markedly increases the occurrence of Estrous and Proestrous smears. Similar effects on estrous cyclicity have been reported for dietary estradiol treatment. In contrast, DBP did not alter the age of VO or vaginal cycling following VO
39 41 42 42
0 250 500 1000Dose of DBP (mg/kg/d)
0
20
40
60
80
100
Percentage of Smears with Cornified Cells
44 47
61 62
95
0 25 50 100 200Dose of Methoxychlor (mg/kg/d)
0
20
40
60
80
100
Percentage of Smears with Cornified Cells
* **
Gray et al., 1999
Effect of Chlorotriazines on Puberty
Atrazine Alters Neuroendocrine Control of gonadal Function
GnRH pulses, LH and prolactin surges decreased Hypothalamic catecholamines decreased, GABA
neurotransmission altered
Hypothesis: Atrazine will alter onset of puberty in male and female
Dose response using pubertal protocol Evaluated Wistar strain
Cooper et al., 2000
Effect of Atrazine on Puberty in Female Wistar Rats
0
25
30
35
40
0 12.5 1005025
Atrazine (mg/kg)
200
**
*
0
80
90
100
110
120
130
BW
T (
g)
0 12.5 5025 200100
BWT at Vaginal Opening
* * *
Atrazine (mg/kg)
Ag
e (D
ays
)
Age at Vaginal Opening
Laws et al., 2000
Effect of Atrazine on Puberty in Female Wistar Rats
0 200 Pair-fed0
20
40
60
80
(gra
ms)
0 200 Pair-fed0
50
100
150
(gra
ms)
0 200 Pair-fed0
10
20
30
40
50
(day
s)
0 200 Pair-fed0
50
100
150(g
ram
s)
BWT (PND22) BWT (PND 41)
Age at VO BWT at VO* *
* ** *
* *
Laws et al., 2000
Assessment of Pubertal Development and Thyroid Function in Immature Male
Rats
Assessment of Pubertal Development and Thyroid Function in Immature Male
Rats
Purpose
The purpose of this protocol is to quantify the effect of environmental compounds on pubertal development and thyroid function in the intact juvenile/peripubertal male rat
Assessment of Pubertal Development and Thyroid Function in Immature Male
RatsApplicability This assay detects compounds that
display anti-thyroid, estrogenic, androgenic, anti-androgenic [androgen receptor (AR)] or steroid enzyme mediated activity or alters puberty via changes in follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin, growth hormone (GH) or hypothalamic function.
31-Day Pubertal Male Assay
pups(8-10)
2322 33 38 53
Wean
4
3or
BW
BloodCollection
Postnatal Day
Dose
Cull
NecropsyAssign to Trt. (gavage)
(weight-rank)
28 43 48
PPS
examine for PPS, daily BWCheck BW and PPS Daily
Required Endpoints(Male pubertal assay)
Growth (body weight)Age and weight at preputial separationSerum thyroxin (T4) and thyroid stimulating hormone (TSH)Thyroid histologySeminal vesicle plus coagulating gland (with and without fluid) Liver, kidney, adrenal, pituitary and ventral Prostate weightLevator ani plus bulbocavernosus weightEpididymal and testis weight & histology
Optional Measures (Male pubertal assay)
Serum testosterone, estradiol, LH, prolactin and tri-iodothyronine (T3)Liver, kidney, adrenal, pituitary histologyEx-vivo testis and pituitary hormone productionHypothalamic neurotransmitter concentrations
30
35
40
45
50
55
60
Con
trol
Flut
amid
e
M-T
esto
s
PTU
Ket
ocon
Pim
ozid
e
DB
P
*
*
*
**
Preputial SeparationA
ge
(day
s)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0
0.1
0.2
0.3
0.4
Seminal Vesicles
Ventral Prostate
Treatment Mode of Action
Age at Preputial
Separation
Histo-
pathology
TSH T4
Fluatamide
(50 mg/kg/d)
AR
Antagonist = =
Methyl
Testosterone
80 mg/kg/d
AR
Agonist = =
Propylthiouracil
240 mg/kg/d
Inhibits
T4 synthesis
Ketoconozole
100 mg/kg/d
Inhibits
Steroido- genesis
= = =
Pimozide
30 mg/kg/d
Dopamine
Receptor
Blocker
LE = =
Dibutylphthalate
1000 mg/kg/d
Anti-androgenic LE LE
=
LE
Summary of Significant Effects on Major Endpoints inMale Sprague-Dawley and Long-Evans rats
0 10 30 100Dose of Vinclozolin (mg/kg/d)
38
40
42
44
46
48
Ag
e in
Da
ys
p < .0001
Effect of DBP and vinclozolin on preputial separation in LE rats.
39.5
42.643.4
44.4
0 250 500 1000Dose DBP (mg/kg/d)
36
38
40
42
44
46
Monosson et al., 1999Gray et al., 1999
Monosson et al., 1999
0 10 30 100Vinclozolin (mg/kg/d)
160
180
200
220
240
Ventral Prostate
0 10 30 100Vinclozolin (mg/kg/d)
500
600
700
800
900
Seminal Vesicle
p <0.0005
p <0.002
p<0.04
p<0.0003
0 10 30 1002
2.5
3
3.5
Testes
0 10 30 100440
460
480
500
520
540
Epididymis
Organ weights following vinclozolin exposure
Monosson et al., 1999
Treatments(mg/kg/d)
40
41
42
43
44
45
46
47
48P
ND
Effect of Atrazine on Preputial Separation
Pubertal Male Protocol (PND 23-53)
aa
CON 12.5 25 50 100 150 200 PF
a aaaa
a
a,b
Stoker et al., 2000
0
50
100
150
200
250
300
PND 53B
od
y W
eig
ht
(g)
aa
Effect of Atrazine on Male Pubertal Development
0
50
100
150
200
250
300
a
Ven
tral P
rosta
te (
mg
)
a
a aa
Treatment (mg/kg/d)
CON 12.5 25 50 100 150 200 PF
Stoker et al., Tox Sci., 2000
0
1
2
3
4
Effect of Atrazine on Serum Testosteronen
g/m
l
SummaryPubertal protocols detect a wide variety of EDCs
Advantages Tests are apical Dose response information, metabolism, dose
individual rats Provide information for MOA and mechanistic
studies Appear to be robust across strains Protocols involve relatively simply procedures
Summary
Pubertal protocols detect a wide variety of EDCs
Difficulties/drawbacks Precise measures of hormones Body weight issues Dosing not done during organogenesis (i.e., not a
transplacental assay) Length of assay Cost
Single Dose Study
Discrepancy between the ages of preputial separation identified in the two strains of rats.
Large degree of variation associated with the means of the fluid-filled and small tissue weights
Single Dose Study
Improve descriptive text in protocols such that every key step is clear.Establish performance criteria for inclusion into the protocolsEvaluate lower limits of detection of protocols by examining dose response for weaker EDCsShould strain be recommended?
Collaborators
Endocrinology Branch, Reproductive Toxicology Division, NHEERL
L. Earl Gray Jr., Ph.D.
Susan C. Laws, Ph.D.
Tammy Stoker, Ph.D.
Jerome M. Goldman, Ph.D.
Robert J. Kavlock, Ph.D.
Office of Science Coordination and Policy
OPPTS
Jim Kariya
Gary Timm