A Proposed MOA for Atrazineand Atrazine MetabolitesPresentation to Science Advisory PanelRalph L. Cooper, Ph.D.Chief, Endocrinology Branch,Toxicity Assessment Division, National Health andEnvironmental Effects Research Laboratory,U.S. EPA,RTP, NCApril, 20102OverviewzAlteration of the hypothalamic-pituitary-gonadalaxis as the Mode of Action (MOA) for the adversereproductive effects of atrazine.zReview of rational and studies that indicate thatatrazine affects hypothalamic-pituitary-adrenalhormone secretion.zSynthesis of neuroendocrine effects of atrazine inthe rat33DIESTRUS I DIESTRUS II PROESTRUS ESTRUS

serum estradiol serum LHM M

Vaginal cytologyMdiestrus 1 diestrus 2 Proestrus EstrusCooper et al.,, 1986, JAGS, 34:735-751Hormonal Control of Rat Ovarian Cyclez4-day estrous cycle is ahighly coordinatedneuroendocrine processessential for successfulreproduction.zAmenable to studyasvaginal smear indicateovarian status (e.g.,regular or altered cycles).zConsiderablehomologybetween rodent andhuman in the process ofovulation34Brain-Pituitary Gonadal AxisHypothalamusGnRHLH, FSH& PrlOvaryEstrogen andProgesteroneAtrazinePituitaryHypothesis:Atrazine alters CNS (hypothalamic)control of pituitary hormone secretionTarget Tissue5Luteinizing Hormone1600180020000510152025****Hours

ng/mlcontrol6.2 mg/kg12.52575Effect of Atrazine on the Proestrous LH SurgeCooper et al., 2007Effect of Atrazine on Ovariectomized,Estrogen-primed LH SurgezLong-Evans RatszAs the duration of dosing increases, the effectivedose decreases.One-Day ExposureThree-Day Exposure1600180020000.02.55.07.510.0Control50100200300Time (h)

ng/ml14001600180020000123456Control50100200300Time (h)** *

ng/mlLHLHCooper et al., Tox Sci. 2000614001600180020000.00.51.01.52.02.53.03.54.04.55.05.5Control50100200300Time (h)

ng/mlCon75150300Con751503000510Dose (mg/kg)

ng/mlLELHzSprague Dawley vs. Long EvanszAs the duration of dosing increases, the effectivedose decreases.Three-Day Exposure21-Day Exposure: 1800hLHEffect of Atrazine on Ovariectomized,Estrogen-primed LH SurgeSD*****7Cooper et al., Tox Sci. 20008Atrazine alters CNS control of PituitaryPulsatile Release of GnRHTreatment (ip) X 3 days02040 6080 100120 1400123456789Time (min)

Serum LH (ng/ml)VehicleATR 50ATR 200Tyrey et al., 19970246810Time (min)ATR300ATR300+GnRH-10 05 15 30 45 60

Serum LH (ng/ml)Cooper et al., Tox Sci. 2000GnRH Restores Surge123456100200300400500LHHours0GnRH / TRH GnRH / TRHKCl0μM (n=10) 100μM (n=10)

pg LH / min mg pitEx vivo LH release (no change)Cooper et al., Tox Sci. 2000Median Eminence GnRH

GnRH, pg/mg Tissue*1200h1400h012345Con2575Cooper et al., 20079DI I DI II PRO ESTRUSM MMMiddle-ageThreshold(ovulatory quota) serum estradiolDI I DI II PRO ESTRUSM MMOldPersistent/ConstantEstrusWith advancing age, the amplitude of theLH surge is lower and the onset delayedDI I DI II PRO ESTRUSM MMYoung

serum LHzWith aging, cycles become irregular and cease because of loss ofCNS regulation.zThe non-cycling female’sendocrine milieuconducive todevelopment of mammary gland tumors (high E and prolactin).10SummaryzAtrazine disrupts the hypothalamic control ofgonadal function, contributes to earlyreproductive senescence and produces theendocrine milieu conducive to the developmentof mammary tumors.Reproductive aging is driven by brain in rat.Reproductive aging is driven by ovary inhuman.zThus, MOA for mammary gland tumordevelopment is not relevant to humans,but thereare other relevant reproductive effects have beenidentified.11•Delayed pubertyin male and female rat•LOAEL, Male =12.5; Female 30 mg/kg/kg•Prostatitisafter lactational exposure•LOAEL, 25 mg/kg (12.5 mg/kg twice a day)•Ovarian cycles•LOAEL <40 mg/kg short-erm; ~22 mg/kg long-term.•Early reproductive senesecence.•Impairedtestostosteronesynthesis•LOEL, 50 mg/kgEffect of Atrazine on Reproduction12z2003 SAP agreed that atrazinecaused aCNS-mediateddecrease inLH was the relevant MOA.zAtrazine-induced decrease of LHleads toadversereproductiveeffects.Atrazine MOAIncorporating recent findings into theMOA for atrazinezIncreased serum estrogens in pubertal males (Stoker et al., 2000).zOther studies reported that atrazine increases aromatase activity.zWhat is the impact of atrazine on aromatase and steroidogenesis?13Atrazine (mg/kg)0 200 0 200020406080100120

(pg/ml)0 200 0 200020406080100120140

(pg/ml)PrepubertalExposurePost-pubertalExposure****EstradiolEstradiolEstroneEstroneLaws et al., 200514Aromatase:zAromatase (CYP19):Converts androgensto estrogensPresent in multiple target tissues(Brain, gonads, adipose, placenta).Regulation of gene expression differs amongtarget tissues and between speciesAltered aromatase activity or gene expressiondisrupts estrogen availability impactingreproductive development, function andenhancing total lifetime exposure to estrogens.TestosteroneAndrostenedioneEstradiolEstroneCYP 19CYP 1917β-HSD15•Is theactivityor synthesis of aromatase, the enzymethat converts androgens to estrogens,alteredbychlorotriazine herbicides?•What is the evidence for altered aromatase activity?–In vitro–In vivo•What is the evidence for a broader effect of atrazineon steroidogenesis?Aromatase and Steroidogenesis16H295R Human Adrenocarcinoma Cell Line:Stimulation of Aromatase ActivityzAtrazineand metabolites,DIA and DEA,increased aromataseactivity, CYP19 mRNA, and estrone/estradiol concentrationszDACThad no effect on these endpointszReplicated in JEG 3 cellsSanderson et al., EHP, 2001, 109: 1027-3017In vitroEffects of Atrazine: SteroidogenesisReferenceObservationLOEL/NOEL(μM)ConclusionsHeneweer 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 cells10.0/0.310.0/3.00.1/0.011.0/0.1↑estrogen (E) w/ ATR exposure≥24 hrs;↑E likely involves↑aromatase gene transcription.ATR alters estrogen synthesisFan et al., 2007a, bATR 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 binds and activates SF-1 to induceArPII dependent↑CYP19 expression.Suzawa & Ingraham,2008No direct binding to SF-1, JEG3 cells,Stimulates signaling pathways that activateNR5A receptors (SF-1, LRH-1,zff1d),↑gene expression ofStAR, P450scc,11βHSD1/0.1:ATR activates broad range of intracellulareffects through 3 signaling pathways knownto activate NR5A receptors.ATR can affect the synthesis ofmultiple steroidsAlbanito et al., 2008No activation of ERα(10 μM), ovarian cancercellsNo effect on aromatase activity (1 μM)↑ERK phosphorylation resulting in↑expression c-fos and other estrogen targetgenes--1ATR may bind to GPR30, a membranereceptor, and modify signaling pathwaysassociated w/ c-AMP, ERK, and P13kactivation.Additional evidence of effects onsignaling pathways18zDose-dependent increase in serum estradiol andestrone (LOEL 50 mg/kg, x3 days). However,nochangeobserved in:aromataseactivity in testes.CYP19 mRNAexpression in brain, testes or adipose.Does Atrazine alter Aromatasein vivo?OVXED Females(4 days)0255075100125150175**SerumEstrone**SerumEstradiol0 50 2000 50 200

pg/mlMaleW. Modic, 200419Summary:zAtrazineincreases serum estrogensin male andfemale rats (NOEL ~25 mg/kg, but short term).zLittle or noevidence to support a direct atrazineinduced change in aromatase mRNA (severaltissues) or activityin vivo.zSuggest that atrazine increasesadrenalsteroidogenesis, which, in turn, could drive theincrease in serum estrogens.zWhat is the evidence that atrazine stimulates adrenalsteroidogenesis? Direct? Indirect (throughhypothalamic-pituitary activation)?20Brain-Pituitary adrenal AxisHypothalamusCRF/AVPACTHAdrenalCorticosterone,Progesterone,Target TissueAtrazine?21*******051530601800100200300400500Control5 mg/kg50 mg/kg100 mg/kg200 mg/kg*ACTH

(pg/ml)**********051530601800100200300400Time (minutes)

(ng/ml)CorticosteroneSerum Hormones After a Single Dose of Atrazinein the Male Rat051530601800246810TestosteroneTime (min.)

(ng/ml)*******051530601800246********

(ng/ml)8Progesterone**Laws et al., Tox Sci., 2009, 112: 78-87Serum Hormones After a Single DoseDACT vs. ATRACTH051530600100200300400500Control3.37 mg/kg33.7 mg/kg67.5 mg/kg135.0 mg/kg*****

(pg/ml)Corticosterone010020030040050051530 60*****0

ng/mlTime (Minutes)DACTDACT051530601800100200300400500*******

(pg/ml)ACTHControl5 mg/kg50 mg/kg100 mg/kg200 mg/kg**********051530601800100200300400

(ng/ml)CorticosteroneATRATRLaws et al., Tox Sci., 2009, 112: 78-87Time (Minutes)2223*****************051530601800100200300400500Control5 mg/kg50 mg/kg100 mg/kg200 mg/kg*ACTHTime (minutes)

(pg/ml)051530601800100200300400Time (minutes)

(ng/ml)CorticosteronezControl/DACT notaffectedzDose responsezNot gastrointestinaldistress (vagotomy/measurement ofghrelin)zActivation withintravenousadministrationAtrazine Induces a Chemical SpecificActivation of HPA24240100200300400500ControlDIA 4 mg/kgDIA 10 mg/kgDIA 40 mg/kgDIA 80 mg/kgATR 100 mg/kg1530Post-Dosing Time (minutes)DACT 67.5 mg/kg*******

CORT (ng/ml)***4 or 21-daily Doses of Atrazine04210100200300ControlAtr 50 mg/kgAtr 200 mg/kgTime (Days)

CORT (ng/ml)Serum Corticosterone After a Single vs. Multiple Doses•ATR and DIA, but notDACT,stimulate adrenal steroidswithin 15 minutes after asingle dose in the male rat.•Corticosterone release inresponse to ATRcontinuesfollowing multiple exposures(4 or 21 days).24Comparison after 1 or 4-Days Exposure toAtrazine and metabolitesFour Doses

Vehicl e ATR 1 2 . 5 ATR 7 5 DIA 10 DIA 60.2 DAC T 8.4 DAC T 50.602004006008001000bbb

ACTH (pg/mL) Vehicl e ATR 1 2 . 5 ATR 7 5 DIA 10 DIA 60.2 DAC T 8.4 DAC T 50.60300600bbaa

Corticosterone ( ng/mL)One DoseVehicleATR 75DIA 60.2DACT 50.602004006008001000ab

ACTH (pg/mL)VehicleATR 75DIA 60.2DACT 50.60200400600bb

Corticosterone ( ng/mL )Fraites, et al., Tox Sci., 2009, 112: 88-992526261600180020000.02.55.07.510.012.515.017.520.022.5**Hours

LH ng/mlLH: Vehicle vs. DIA1600180020000510152025Vehicle10 mg/kg DIA60.2 mg/kg DIA++***Hours

LH (ng/mL)LH: Vehicle vs. DACT1600180020000510152025Vehicle8.4 mg/kg DACT50.6 mg/kg DACTHours

LH (ng/m L)Effect of Four EquimolarDoses of Atrazine orMetabolites on the PreOvulatory Surge of LH inProestrous Females.Vehicle12.5 mg/kg ATR75 mg/kg ATR27Summary of Adrenal Effects ofAtrazine and MetabolitesIn the male, ACTH and adrenal steroidsincrease within15 minutesfollowing a singledose of ATR, DIA, or DEA.Dose-responsive (ATR LOEL for1xCort > 5mg/kg; LOEL for DIA1xCort = 10 mg/kg,).Compared to ATR and DIA, the increase inACTH and adrenal steroid release inresponse to DACT is markedly less.zCorticosterone release is sustained for upto three weeks (♂) and 4 days (♀), whileACTH response appears to acclimate (↓4days) suggesting some direct effect onadrenal.LOEL for Cort., (12.5 mg/kg in female)zImportantly, atrazine induces a release ofadrenal progesterone in the intact femalethat was significant after both the 1 and 4day exposure to ATR and DIA.Summary of Adrenal Effects ofAtrazine and Metabolites28Comparison of HPA w/HPG effectszThe LH surge is not decreased by a single 200mg/kg dose of atrazine in two different rat strains(OVX + E2 model).zThe LH surge is significantly suppressedfollowing three days exposure in LE females witha LOEL of 50 mg/kg (OVX + E2 model).zThe LOEL for suppression of LH in Cooper et al.,(2007) was 6.25 mg/kg (NOEL of 3.25) after 4-dayexposure in the intact rat.zThese observations indicate that changes in theHPA axis do precede and may contribute to thedisruption of the LH surge and subsequentchanges in reproductive function.2930GnRHHypothalamusLH, FSH& PrlGonad♂Testosterone♀Estrogen andProgesteronePituitaryTarget TissueHypothalamusCRF/AVPACTHAdrenalCorticosterone,Progesterone,PituitaryTarget Tissue↓Steroidogenesis inLeydig Cell



# ↓ L H

v ia

B

r a in / p i t u i t a r yCRF decreases GnRH pulses31SummaryzAtrazine has a clear, acute effect on the HPA increasingboth ACTH and adrenal steroid hormone secretion.zThe temporal aspects of the atrazine-induced changes inthe HPA and HPG, along withthe similarities in the doseresponse following extended dosing (e.g., 4 days or more)suggest that the changes inthe adrenal axis contribute toor the disruption of LH secretion and impairments inreproductive function identified with this compound.zThe current studies broadens our understanding of theMOA for atrazineIncorporates observed changes in HPA that likely contribute tothe disruption of the the LH surge in the rat.75 mg/kg ATR 60.2 mg/kg DIA 50.6 mg/kg DACTATR14±2.1(0.065 uM)DEA101.7±14.6(0.54uM)DIA475.0±62.2(2.7 uM)3886.0±356.0(22uM)DACT4138.0±1780.0(28uM)13136.0±2401.0(90uM)12687.7±1660.6(87uM)12.5 mg/kg ATR 10 mg/kg DIA 8.4 mg/kg DACTATR8.8±1.2(0.041 uM)DEA63.8±13.7(0.34uM)DIA252.2±51.7(1.5uM)1877.7±253.9(11uM)DACT747.5±55.5(5.1uM)2088.3±435.5(14uM)5977.0±2119.1(41uM)Plasma Metabolitesng/mL +SEMppb(μM)Four Equimolar Doses3⁄4Dose-dependent increases in plasma ATR & metabolite3⁄4Plasma metabolites 10-100x greater than ATR75 mg/kg ATR 60.2 mg/kg DIA 50.6 mg/kg DACTATR14±2.1(0.065 uM)DEA101.7±14.6(0.54uM)DIA475.0±62.2(2.7 uM)3886.0±356.0(22uM)DACT4138.0±1780.0(28uM)13136.0±2401.0(90uM)12687.7±1660.6(87uM)12.5 mg/kg ATR 10 mg/kg DIA 8.4 mg/kg DACTATR8.8±1.2(0.041 uM)DEA63.8±13.7(0.34uM)DIA252.2±51.7(1.5uM)1877.7±253.9(11uM)DACT747.5±55.5(5.1uM)2088.3±435.5(14uM)5977.0±2119.1(41uM)Plasma Metabolitesng/mL +SEMppb(μM)Four Equimolar Doses3⁄4Equimolar doses of ATR or metabolites do notyield comparable plasma levelsOne DoseFour Doses

Veh i cle A TR 12 . 5 A T R 75 DI A 1 0

DI A 6 0. 2 DACT 8.4

D ACT 50.602004006008001000bbb

ACTH (pg/mL) Vehic l e ATR 12.5 ATR 75 DI A 10 DI A 60.2 DA CT 8 . 4

DA CT 5 0.60300600bbaa

Corticosterone (ng/mL)VehicleATR 75DIA 60.2DACT 50.602004006008001000ab

ACTH (pg/mL)VehicleATR 75DIA 60.2DACT 50.60200400600bb

Corticosterone (ng/mL)VehicleATR 75DIA 60.2DACT 50.60102030bb

Progesterone (ng/mL) Vehicle ATR 12.5 ATR 75 D I A 1 0 D IA 60.2 D A CT 8.4 DACT 5 0.60102030bbba

Progesterone (ng/mL)37Serum Androstenedione012Intact0Castrated50 200050 200Atrazine (mg/kg/d)

(ng/ml)Serum Estradiol0102030405060IntactCastrated***050 200050 200Atrazine (mg/kg/d)

(pg/ml)Serum Testosterone01234567IntactCastrated*050 200050 200Atrazine (mg/kg/d)

(ng/ml)Serum Estrone050100150IntactCastrated*050 200050 200Atrazine (mg/kg/d)**

(pg/ml)17-B-HSD17-B-HSDP450 AromP450 AromzStudies using castrated rats show that serum estradiol isstill increased following atrazine exposure.zThusadrenal androstenedioneis a likely substrate for theatrazine-induced increase in serum estradiol..zThis also raises thepossibility that theincrease in serumestrogens may besubstrate driven.zAdrenal androgensare substrate foradiposearomataseto produce estrone.Does Atrazine alter Aromatasein vivo?