Endocrine Disruptor Screening Program: Ralph L. Cooper Endocrinology Branch Reproductive Toxicology Division NHEERL, U.S. EPA Male and Female Pubertal.

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