A Proposed MOA for 

and Atrazine Metabo

Presentation to Science Adviso

Ralph L. Cooper, Ph.D.

Chief, Endocrinology Branch

Toxicity Assessment Division, National 

Environmental Effects Research Lab

U.S. EPA, 

RTP, NC

April, 2010 
Overview

z Alteration of the hypothalamic-pituit

axis as the Mode of Action (MOA) foreproductive effects of atrazine.

z Review of rational and studies that i

atrazine affects hypothalamic-pituitahormone secretion.

z Synthesis of neuroendocrine effects 

the rat 
serum estradiol DIESTRUS I DVaginal cytology

M

diestrus 1 die

 Cooper et al.,, 1986, JAGS, 34

Hormonal Control of Rat Ovar

z 4-day estrous cycle is a 

highly coordinated neuroendocrine processessential for successful reproduction.

z Amenable to study as 

vaginal smear indicate ovarian status (e.g., regular or altered cycles).

z Considerable homology 

between rodent and human in the process of ovulation
Brain-Pituitary Gonadal 

Hypothalamus

GnRH

LH, FSH & Prl

Ovary

Estrogen and Progesterone

Atrazine

Pituitary

Hypothesis: Atrazine alters CNS (

control of pituitary hormone secretio

Target Tissue
Luteinizing Hormone

1600 1800 2

05

10152025

*

*

*

*

Hours

ng/ml

control6.2 mg/kg12.525 75

Effect of Atrazine on the Proestrou
Effect of Atrazine on Ovariecto

Estrogen-primed LH Sur

z Long-Evans Ratsz As the duration of dosing increases

dose decreases.

One-Day Exposure Three-Day Exp

1600 1800 2000

0.02.55.07.5

10.0

Control50100200300

Time (h)

ng/ml

1400 1600 

0123456

Time (h

*

ng/ml 

LH LH
1400 1600 1800 2000

0.00.51.01.52.02.53.03.54.04.55.05.5

Control50100200300

Time (h)

ng/ml

Con 75 15

05

10

ng/ml

LE

LH

z Sprague Dawley vs. Long Evansz As the duration of dosing increases

dose decreases.

Three-Day Exposure 21-Day E

LH

Effect of Atrazine on Ovariecto

Estrogen-primed LH Sur

*

*
Atrazine alters CNS control of 

Pulsatile Rel

Treatment (i

0 20 40 60 

0123456789

Tim

Serum LH (ng/ml)

Tyrey et al., 1997

02468

10

Tim

-10 0 5 

Serum LH (ng/ml)

Cooper et al., Tox Sci. 2000

GnRH Res

123456

100200300400500

LH

Hours

0

GnRH / TRH GnRH / TRH KCl

0 μM (n=10) 100 μM (n=10)

pg LH / min mg pit

Ex vivo LH release (no change)

Cooper et al., Tox Sci. 2000

Median Eminence GnRH

GnRH, pg/mg Tissue

*

1200h 1400h

012345

Con

2575

Cooper et al., 2007
DI I DI II PRO ESTRUS

M M M

Middle-age

Threshold

(ovulatory quota)

DI I 

With advancing age, the ampli

LH surge is lower and the ons

DI I DI II PRO ESTRUS

M M M

Young

serum LH 

z With aging, cycles become irregular and cease b

CNS regulation.

z The non-cycling female’s endocrine milieu cond

development of mammary gland tumors (high E 
Summary

z Atrazine disrupts the hypothalamic c

gonadal function, contributes to earreproductive senescence and produendocrine milieu conducive to the dof mammary tumors.

 Reproductive aging is driven by b Reproductive aging is driven by o

human.

z Thus, MOA for mammary gland tumo

development is not relevant to humaare other relevant reproductive effecidentified. 
• Delayed puberty in male and femal

• LOAEL, Male =12.5; Female 30 mg/kg/

• Prostatitis after lactational exposu

• LOAEL, 25 mg/kg (12.5 mg/kg twice a 

• Ovarian cycles

• LOAEL <40 mg/kg short-erm; ~22 mg/• Early reproductive senesecence. 

• Impaired testostosterone synthesi

• LOEL, 50 mg/kg

Effect of Atrazine on Repro
z 2003 SAP agreed that atrazi

caused a CNS-mediated dec

LH was the relevant MOA. 

z Atrazine-induced decrease o

leads to adverse reproductiveffects.

Atrazine MOA
Incorporating recent finding

MOA for atrazine

z Increased serum estrogens in pubertal males (Stoz Other studies reported that atrazine increases aroz What is the impact of atrazine on aromatase and s

Atrazine (mg/kg)

0 200 0 200

0

20406080

100120

(pg/ml)

0 200 

0

20406080

100120140

(pg/ml)

PrepubertalExposure

Post-pubertalExposure

*

*

*

Estradiol

Estradiol

Estrone

Estro

Laws et al
Aromatase: 

z Aromatase (CYP19):

 Converts androgens

to estrogens

 Present in multiple target tissues

(Brain, gonads, adipose, placenta).

 Regulation of gene expression differs 

target tissues and between species 

 Altered aromatase activity or gene ex

disrupts estrogen availability impactireproductive development, function aenhancing total lifetime exposure to e

Androstenedion

Estrone 

CYP 19 1
• Is the activity or synthesis of aromata

that converts androgens to estrogens

chlorotriazine herbicides?

• What is the evidence for altered arom

– In vitro– In vivo

• What is the evidence for a broader ef

on steroidogenesis?

Aromatase and Steroidogenesi
H295R Human Adrenocarcinoma 

Stimulation of Aromatase Ac

z Atrazine and metabolites, DIA and DEA, inc

activity, CYP19 mRNA, and estrone/estradi

z DACT had no effect on these endpointsz Replicated in JEG 3 cells

Sa
In vitro Effects of Atrazine: Stero

Reference Observation

LOEL/NOEL

(μM)

Heneweer et al., 2004Laville et al., 2006Holloway et al., 2008Higley et al. 2010

↑ aromatase activity (AA), H295R cells↑AA, JEG3 cells

↑AA, human granulosa-lutein primary cells↑AA, testosterone, estradiol, H295R cells

10.0/0.310.0/3.00.1/0.01

1.0/0.1

↑ est↑ E l

nscrip

ATR 

Fan et al., 2007a, b

ATR binds to SF-1SF-1 interaction w/ co-activator,TIF2 

↑SF-1 binding to aromatase promotor (ArPII)

(H295R and KGN cells used in studies)

0.1

: ATR ArPII 

Suzawa & Ingraham, 2008

No direct binding to SF-1, JEG3 cells,Stimulates signaling pathways that activate NR5A receptors (SF-1, LRH-1,zff1d),↑gene expression of StAR, P450scc, 11βHSD 

1/0.1

: ATR 

effectto act

 AT

multi

Albanito et al., 2008

No activation of ERα (10 μM), ovarian cancer cellsNo effect on aromatase activity (1 μM)↑ERK phosphorylation resulting in ↑expression c-fos and other estrogen target 

genes

--

1

ATR recepassoc

activa

 Ad

signa
z Dose-dependent increase in serum est

estrone (LOEL 50 mg/kg, x3 days). Howchange observed in:

 aromatase activity in testes.

 CYP19 mRNA expression in brain, testes o

Does Atrazine alter Aromatas

0

255075

100125150175

**

SerumEstrone

* *

SerumEstradiol

 0 50 200 0 50 200

pg/ml

Male

W. Modic, 2004
Summary:z Atrazine increases serum estrogens in 

female rats (NOEL ~25 mg/kg, but sho

z Little or no evidence to support a direc

induced change in aromatase mRNA (tissues) or activity in vivo.

z Suggest that atrazine increases adren

steroidogenesis, which, in turn, could increase in serum estrogens.

z What is the evidence that atrazine stim

steroidogenesis? Direct? Indirect (thrhypothalamic-pituitary activation)?
Brain-Pituitary adrenal Axis

Hypothalamus

CRF/AVP

ACTH

Adrenal

Corticosterone, Progesterone, 

Target Tissue

Atraz
*

*

*

*

*

*

*

0 5 15 30 60 180

0

100200300400500 Control

5 mg/kg50 mg/kg100 mg/kg200 mg/kg

*

ACTH

(pg/ml)

*

*

*

**

*

*

*

*

*

0 5 15 30 60 180

0

100200300400

Time (minutes)

(ng/ml)

Corticosterone

Serum Hormones After a Single Dos

in the Male Rat

0 5 1

02468

10

Testosterone

T

(ng/ml)

*

0 5 1

0246

(ng/ml)

8 Progesterone

Laws et al., T
Serum Hormones After a Sing

DACT vs. ATR

ACTH

0

5 15 30 60

0

100200300400500 Control

3.37 mg/kg

33.7 mg/kg

67.5 mg/kg

135.0 mg/kg

*

* ***

(pg/ml)

Corticosterone

0

100200300400500

5 15 30 60

*

*

*

*

*

0

ng/ml

Time (Minutes)

DACTDACT

0

5 

0

100200300400500

(pg/ml)

ACTH 

0

5 

0

100200300400

(ng/ml)

Corticoste

ATR

ATR

Laws et al., Tox Sci., 2009, 112: 78-87
0 5 

0

100200300400500 

ACTH

T

(pg/ml)

0 5 1

0

100200300400

Tim

(ng/ml)

Corticoster

z Control/DACT not 

affected

z Dose responsez Not gastrointestinal 

distress (vagotomy/ measurement of ghrelin)

z Activation with 

intravenous administration

Atrazine Induces a Chemical SpeActivation of HPA
0

100200300400500

Control

DIA 4 mg/kgDIA 10 mg/kgDIA 40 mg/kgDIA 80 mg/kg

ATR 100 mg/kg

15 30

Post-Dosing Time (minutes)

DACT 67.5 mg/kg

* * *

*

*

*

*

CORT (ng/ml)

4 or 21-

0 

0

100200300 

CORT (ng/ml)

Serum Corticosterone After a Single vs. 

• ATR and DIA, but not DACT,

stimulate adrenal steroids within 15 minutes after a single dose in the male rat.

• Corticosterone release in 

response to ATR continues following multiple exposures (4 or 21 days).
Comparison after 1 or 4-Days E

Atrazine and metabolites

F

Vehicle

ATR 12.5

ATR 

0

200400600800

1000

ACTH (pg/mL)

Vehicle

ATR 12.5

ATR 

0

300600

a

Corticosterone (ng/mL)

One Dose

Vehicle ATR 75 DIA 60.2 DACT 50.6

0

200400600800

1000

a

b

ACTH (pg/mL)

Vehicle ATR 75 DIA 60.2 DACT 50.6

0

200400600

b

b

Corticosterone (ng/mL)

Fraites, et al., Tox Sci., 2009, 112: 88-99
1600 1800 2000

0.02.55.07.5

10.012.515.017.520.022.5

*

*

Hours

LH ng/ml

LH: Vehi

1600 1

05

10152025

H

LH (ng/mL)

LH: Vehicle vs. DACT

1600 1800 2000

05

10152025

Vehicle8.4 mg/kg DACT50.6 mg/kg DACT

Hours

LH (ng/m L)

Effect of 

Doses 

Metabo

Ovulator

Proest

Vehicle

12.5 mg/kg ATR

75 mg/kg ATR
Summary of Adrenal Eff

Atrazine and Metabo

 In the male, ACTH and adrenal st

increase within 15 minutes followdose of ATR, DIA, or DEA.

 Dose-responsive (ATR LOEL for 1x 

mg/kg; LOEL for DIA 1x Cort = 10 m

 Compared to ATR and DIA, the in

ACTH and adrenal steroid releasresponse to DACT is markedly le
z Corticosterone release is sustai

to three weeks (♂) and 4 days (♀

ACTH response appears to accl

days) suggesting some direct efadrenal.

 LOEL for Cort., (12.5 mg/kg in fem

z Importantly, atrazine induces a 

adrenal progesterone in the intathat was significant after both thday exposure to ATR and DIA. 

Summary of Adrenal Eff

Atrazine and Metabo
Comparison of HPA w/HPG effe

z The LH surge is not decreased by a s

mg/kg dose of atrazine in two differen(OVX + E2 model). 

z The LH surge is significantly suppres

following three days exposure in LE fa LOEL of 50 mg/kg (OVX + E2 model

z The LOEL for suppression of LH in C

(2007) was 6.25 mg/kg (NOEL of 3.25) exposure in the intact rat.

z These observations indicate that cha

HPA axis do precede and may contribdisruption of the LH surge and subsechanges in reproductive function. 
GnRH

Hypothalamus

LH, FSH & Prl

Gonad

♂ Testosterone♀ Estrogen and Progesterone

Pituitary

Target Tissue

Hypothal

ACTH

Adrenal

CortProg

Pituitary

↓ Steroidogenesis in 

Leydig Cell

↓LH via Brain/pituitary

CRF decreases GnRH pulses
Summary

z Atrazine has a clear, acute effect on the HPA 

both ACTH and adrenal steroid hormone se

z The temporal aspects of the atrazine-induce

the HPA and HPG, along with the similarities response following extended dosing (e.g., 4 suggest that the changes in the adrenal axis or the disruption of LH secretion and impairreproductive function identified with this co

z The current studies broadens our understan

MOA for atrazine

 Incorporates observed changes in HPA that l

the disruption of the the LH surge in the rat. 

75 mg/kg ATR 60.2 mg/kg DIA 

ATR 14 

± 2.1 (0.065 uM)

DEA

101.7 

± 14.6 (0.54 

uM)

DIA 475.0 

± 62.2 (2.7 uM)

3886.0 

± 356.0 (22 

uM)

DACT

4138.0 

± 1780.0 (28 

uM)

13136.0 

± 2401.0 (90 

uM)

12.5 mg/kg ATR 10 mg/kg DIA 

ATR 8.8 

± 1.2 (0.041 uM)

DEA

63.8 

± 13.7 (0.34 

uM)

DIA

252.2 

± 51.7 (1.5 

uM) 

1877.7 

± 253.9 (11 

uM)

DACT

747.5 

± 55.5 (5.1 

uM)

2088.3 

± 435.5 (14 

uM)

Plasma Metabolites

ng/mL + SEM 

ppb

(μM)

Four Equimolar D

3⁄4 Dose-dependent increases in plasma A

3⁄4 Plasma metabolites 10-100x grea
75 mg/kg ATR 60.2 mg/kg DIA 

ATR 14 

± 2.1 (0.065 uM)

DEA

101.7 

± 14.6 (0.54 

uM)

DIA 475.0 

± 62.2 (2.7 uM)

3886.0 

± 356.0 (22 

uM)

DACT

4138.0 

± 1780.0 (28 

uM)

13136.0 

± 2401.0 (90 

uM)

12.5 mg/kg ATR 10 mg/kg DIA 

ATR 8.8 

± 1.2 (0.041 uM)

DEA

63.8 

± 13.7 (0.34 

uM)

DIA

252.2 

± 51.7 (1.5 

uM)

1877.7 

± 253.9 (11 

uM)

DACT

747.5 

± 55.5 (5.1 

uM)

2088.3 

± 435.5 (14 

uM)

Plasma Metabolites

ng/mL + SEM 

ppb

(μM)

Four Equimolar 

3⁄4 Equimolar doses of ATR or meta

yield comparable plasma 
One Dose F

Vehicle

ATR 12.5

ATR 

0

200400600800

1000

ACTH (pg/mL)

Vehicle

ATR 12.5

ATR 

0

300600

a

Corticosterone (ng/mL)

Vehicle ATR 75 DIA 60.2 DACT 50.6

0

200400600800

1000

a

b

ACTH (pg/mL)

Vehicle ATR 75 DIA 60.2 DACT 50.6

0

200400600

b

b

Corticosterone (ng/mL)
Vehicle ATR 75 DIA 60.2 DACT 50.6

0

102030

b

b

Progesterone (ng/mL)

Vehicle

ATR 12.5

ATR 75

0

102030

b

b

Progesterone (ng/mL)
Serum Androstenedione

012

Intact

0

Castrated

50 200 0 50 200

Atrazine (mg/kg/d)

(ng/ml)

Serum Testosterone

01234567

Intact

* Castrated

0 50 200 0 50 200

Atrazine (mg/kg/d)

(ng/ml)

17-B-HSD 

P450 Arom

P450 Arom

z Studies using castrated rats show that seru

still increased following atrazine exposure. 

z Thus adrenal androstenedione is a likely su

atrazine-induced increase in serum estradio

z This also raises the 

possibility that the increase in serum estrogens may besubstrate driven.

z Adrenal androgens 

are substrate foradipose aromataseto produce estrone.

Does Atrazine alter Aromatas