Atrazine: Adverse Outcomes and MOARalph L. Cooper,Ph.D,Atrazine:AdverseOutcomesandMOAp p, ,Chief, Endocrine Toxicology BranchToxicity Assessment Division,National Health and Environmental EffectsResearch Laboratory,ORDU.S. EPA,RTP, NCJuly 2011History of MOA/and Hazard AssessmentResearchHow did we get to LH as a key event?•Initial ConcernsAt i d th.HowdidwegettoLHasakeyevent?•InitialConcerns:Atrazine exposure causedthepremature development of mammary gland tumors inthe female SD rat.•2000 SAP on Atrazine: Hazard and DoseResponse Assessment and Characterizationagreed that atrazine-induced alterations in the secretionof luteinizing hormone specifically the ovulatory surgeofluteinizinghormone,specificallytheovulatorysurgeof LH, was a key event in the development of themammary gland tumors.2The key events as described in the 2003IRED included:•Hypothalamic effects resulting in changes incatecholamine function and regulation of the pulsatilerelease of GnRH.•Attenuation of the LH surgeAttenuationoftheLHsurge.•Cessation of ovulation with the ensuing persistentrelease of estrogen.•Increased prolactin release by the pituitary as asecondary consequence resulting from the elevatedestrogen levelsestrogenlevels.•Prolactin and estrogen-induced proliferative processesin the mammaryland leadingto tumorigenesis.g g g3Relevanceofcancer/noncancereffects•Reproductive aging is driven by brain in rodents.e e a ce o ca ce / o ca ce e ects•Reproductive aging is driven by ovary in women.•Thus, the MoA for mammary gland tumordevelopment is not relevant to humans.•But, there are other LH-dependent adverseoutcomes that have been identified in the rat thatoutcomesthathavebeenidentifiedintheratthatmay be relevant.4Adverse Reproductive Effects in Rodents•Delayed puberty in male and female•LOAEL M l 12 5 F l 30 /k /kpLOAEL,Male =12.5;Female30mg/kg/kg•Ovarian cycle•LOAEL = 75 mg/kg/day 21 days•LOAEL <40 /k h tt 22 /k ltLOAEL<40mg/kg short-term; ~22mg/kglong-erm.•Early reproductive senescence 3.65 mg/kg 6 mo.•Pregnancy maintenance•FLR 50 /k GD 811FLR50mg/kgGD8-.•Prostatitis after lactational exposure.•LOAEL 25 mg/kg (12 5 mg/kg twice a day)LOAEL,25mg/kg(12.5mg/kgtwiceaday)•Altered mammary gland development.5HPG Axis Perturbations: Summary of Temporal andDose ConcordanceAxis Key Event Time Dose (NOEL/LOEL) ReferenceHypothalamus↓G RH l4 days N.A./25 mg/kg/day‡Cooperet al.2007↓GnRHreleaseygg yp4 days NA/50 mg/kg/day Foradori et al., 20091 day N.A./300 mg/kg/day Cooperet al. 20001 dayNA/100 mg/kg↑Goldman Internal report, 2011HPG AxisPituitaryAttenuation of LHsurge3 days N.A./50 mg/kg/day Cooperet al.20004 days N.A./6.25 mg/kg/day Cooperet al.20074 daysBMDL 2.56 Cooper et al., Internal Report,HPGAxis4daysmg/kg/day 20115days NA/30 mg/kg/day McMullin et al, 2004;26weeks 1.8/3.65 mg/kg/day Morsethet al.,1996cOvary1dayNA/300 mg/kg/dayCooper et al (2000)Estrous cyclicitydisruption1-dayN.A./300mg/kg/dayCooperetal.(2000)4 days25/75 mg/kgCooper et al., Internal report201121 days N.A./75 mg/kg/day Cooper et al. (1996)626 weeks 1.8/3.65 mg/kg/dayMorseth (1996c)2000 SAP on Atrazine•“The reproductive developmental effects in rats ( delayed puberty,prostatitis) are consistent with a primary action of atrazine on the HPGaxis. In the case of delayed puberty, a suppression of GnRH and LHwould obviously delay increases in estradiol secretion by the ovary thatcharacterize puberty in females This would lead to the observedcharacterizepubertyinfemales.Thiswouldleadtotheobserveddelays in vaginal opening. In the male, similar delays in increased LHsecretion would lead to delays in Televations that would result indelayed preputial separation etc. Therefore, the proposed mode ofti f d l t l ff t h id bl l ithactionfordevelopmentaleffects shares considerable overlap withthe proposed mode of action for carcinogenicity. Indeed, theneuroendocrine actions of atrazine are probably the dominantmechanisms bywhich atrazine exerts reproductive developmentalppeffects.The same endocrine perturbations that induce tumors alsoappear to play a role in at least some reproductive developmentaleffects.7Effect of Four Equimolar Doses of Atrazine orMetabolites on the Pre-Ovulatory Surge of LH inProestrous FemalesProestrousFemales.20 022.5LH: Vehicle vs. DIAV hi l12.515.017.520.0**

g /ml2025Vehicle10 mg/kg DIA60.2 mg/kg DIA+

L)Vehicle12.5 mg/kg ATR75 mg/kg ATR2 55.07.510.0

LH n g51015++***

LH (ng/m81600180020000.02.5Hours1600180020000Hours8MoA and ToxicityPathwayLH is a key event leading to the disruption of hypothalamic gonadal functionand as a consequence altered reproductive performance and cancer( t ) i th ty y(mammarytumors)inthe rat.OrganSystemMolecular Cellular Tissue Organ IndividualT i it P thOrganSystemLH alterationsToxicityPathwayCh i lReceptor/LigandGeneactivationAlteredToxicantCellularResponsesOrganResponsesOrganismResponsesMacroMolecularInteractionsChemicalPropertiesp gInteractionDNA BindingProtein OxidationProteinproductionAlteredsignalingphysiologyDisruptedhomeostasisAltered tissuedevelopment/ImpairedDevelopmentImpairedReproduction9development/functionMammaryGlandDevelopment•Effect of gestational Atrazine exposure on mammary glandtumor development was reviewed at the September 2010 SAP.a a y G a d e e op e t•Atrazine stunted mammary gland growth through PND67. Rayner et al. (2005); Enochet al.(2007).•Currently Rayner and Enoch findings can not be replicated•Hovey et al. (2011) observed no differences in mammarygland morphology in Long Evans rats exposed toatrazine from GD 13-19.•In response to the September 2010 SAP’s suggestions,ORD used both subjective and objective measures in aneffort to compare and contrast the two approaches. SDrats exposed GD 14-21 on PND 45 (dose range 1-100mg/kg/day) Davis et al. (2011).10Mammary Whole Mount Morphometric(Quantitative)Evaluations2520

x 3 mmb.i.d.(Blocks 2 & 4)(Quantitative)Evaluations151050

ranches / 3 x015201001110 910 6

o f TEBs B0152010060504030

u ct

Number o01520 100302010011 89 9615105

a nce (mm) d e - Most Distal D u50ATR Treatment (mg/kg/d)

Dist a

Lymph No d01520 10011 91010 611Mammary Whole Mounts Subjective(Qualitative)Assessments4Treatment GD 14-21Assessment PND 45()33.5AtrazineAssessmentPND4501001 522.5

Scoremg/kg/d1000 511.500.5s.i.d.b.i.d.Blocks 1 & 3Blocks 2 & 4Dosing RegimenBlocks1&3Blocks2&412Summary•The reproductive/developmental effects in rats (delayedpuberty ovarian cycling FLR) are consistent with a primarypuberty,ovariancycling,FLR)areconsistentwithaprimaryaction of atrazine on the HPG axis, namely LH secretion.•Alt ti i LH id th l t LOEL dAlterationsin serumLHprovidesthelowestLOELs andNOELs available and these serum hormone measures serve asa sentinel for two of the three adverse outcomes identified(puberty and ovarian function)(pubertyandovarianfunction).•Although the proposed MoA for prostatitis is different, theLOEL d NOEL f LH lt ti t ti fLOELs andNOELsforLHalterations are protectiveforprostatitis as well.13One vs four-days of dosing on LHDuring the September 2010 SAP, the Panel raised the issue of singlevsmultiple atrazine exposure effects on the LH surge. In their report to theA th SAP t dAgencytheSAPcommented:“Data are clear in identifying thata greater-than-one pulse of exposure toatrazine is necessary for attenuation of the LH surge For example single highatrazineisnecessaryforattenuationoftheLHsurge.Forexample,singlehighdoses (over 100 mg/kg) administered on the morning of proestrus did not altercharacteristics of the LH surge occurring later the same day. Additional dataclearly demonstrate a once daily dose for 4 days and beginning on estrus caninduce significant inhibition of the LH surge peak. In this instance, a doseresponse is observed. However, what is not clear is if less than 4, but greaterthan 1 days’ exposure is sufficient to alter the LH surge. Further complicatingthe matter it is not clear if a 4-day exposure beginning on a different day of thethematter,itisnotclearifa4dayexposure,beginningonadifferentdayofthecycle, will result in changes in the LH surge similar to those when dosingbegins on the morning of proestrus.Understanding of the relationshipbetween duration of exposure and phase of the cycle will be key in14translating rodent data to humans for risk assessment purposes.”Role of estrogen and progesterone in theregulation of the LH surgeg•Estrogen alone will induce small LH surge 4days later (E)4 0O V X - E strad io l Im p lan t / s c P ro g estero n e•Estrogen for 3-4 days followed byprogesterone on day 4 will significantlyincrease the amplitude of the LH surge(E+P).3 02 0E + PP E•The opposite order, P followed by E, willeliminate any E-dependent LH surge (P+E)•Hypothesis:One dose of atrazineadministered on vaginal proestrus (AM) or0- 6-4 -2 P eak +2 +4 +6Ho ursfrom Pe ak1 0E o n lyP+Eon the morning the expected surge in theOVXed, E-primed female will increase theamount of LH as a result of the atrazineinduced increase in progesterone (Laws etou s o eaal., 2009; Fraites et al. 2009).•Atrazine dosing for 4 days will inhibit theamplitude because of the daily release ofprogesterone.15•But we thought one dose had no effect!Effect of Atrazine on Ovariectomized,Estrogen-primed LH SurgeOne-Day Exposure Three-Day Exposure•Long-Evans Rats•A th d ti f d i i th ff ti16Astheduration ofdosingincreases,the effectivedose decreases.Cooper et al., Tox Sci. 2000One Dose of Atrazine increases peak height andArea under the Curve.AreaundertheCurve.40*10/k*Sampling30*7890100(11)(10)mg/kg

ml) m l)1400-1600-1800-2000hAUC20181%456( )*

m LH (ng/ LH (ng/ m10234

Seru m Serum00 100-6 -4 -2 Peak +2 +401Ti R l ti t P kAt i ( /k )17TimeRelativetoPeakAtrazine(mg/kg)Two Doses of Atrazine decreases peak heightand Area under the Curve10mg/kgSampling 1400-1600-1800-2000h40AUCandAreaundertheCurve.789

n g/ml)0100(9)(9)g/ g g /ml)3035456

e rum LH ( n( )152025

r um LH (n g1234

S e51015

Se r77%-4 -2 Peak +2 +4 +601Time Relative to Peak05Atrazine (mg/kg)0 10018TimeRelativetoPeakAtrazine(mg/kg)Four Doses of Atrazine decreases peakheight and Area under the CurveheightandAreaundertheCurve.10mg/kg40Sampling 1400-1600-1800-2000hAUC7890100(10)(10)mg/kg3035

ng/ml) g /ml)456(10)152025*p= 0.06

e rum LH ( r um LH (n g23451015**54%

S e

Se r-4 -2 Peak +2 +40105010054%Time Relative to PeakAtrazine (mg/kg)19TimeRelativetoPeakAtrazine(mg/kg)Summary•Responding to the September 2010 SAP’s comments regarding singlevs. multiple exposures, ORD scientists examined what changes in LHl if ld i tt fd fSummaryrelease,ifany, wouldoccurin responsetoone,two, orfourdoses ofATR•One doseof ATR significantlyincreased LH(in OVXed + E rats)demonstrating a clear effect on LH regulation in a manner consistentwith an effect of the increase in progesterone observed followingt i d i i t tiT df ATR l dh iatrazine administration.woosesofATRrevealedno cangenLHfrom control.Four dosesled to the previously describeddecreasein LH. Again, all of these changes are consistent with a response tothe atrazine induced changes in adrenal progesteronetheatrazineinducedchangesinadrenalprogesterone.•These findings speak onlyto the duration of dosing and changes inLH Th d t i l d ff t fi d LH i thLH.Theydo notimply an adverse effectforincreasedLHintheOVXed E-primed rat.20MoA and ToxicityPathwayLH is a sensitive key event leading to the disruption of hypothalamic gonadalfunction and as a consequence altered reproductive performance and cancer( t ) i th ty y(mammarytumors)inthe rat.OrganSystemMolecular Cellular Tissue Organ IndividualT i it P thOrganSystemLH alterationsToxicityPathwayCh i lReceptor/LigandGeneactivationAlteredToxicantCellularResponsesOrganResponsesOrganismResponsesMacroMolecularInteractionsChemicalPropertiesp gInteractionDNA BindingProtein OxidationProteinproductionAlteredsignalingphysiologyDisruptedhomeostasisAltered tissuedevelopment/ImpairedDevelopmentImpairedReproduction21development/functionThank YouORD NHEERL TAD NTBOCSPP OPP HEDThankYouORD,NHEERL,TAD,NTB•Jerome Goldman, Ph. D•Susan Laws, Ph.DOCSPP,OPP,HED•Elizabeth Mendez, Ph. D.•Vicki Dellarco, Ph. D.•Tammy Stoker, Ph. D.•Michael Narotsky, Ph. D•Melanie Fraites, Ph.D.•Jack Fowle, Ph.DMelanieFraites,Ph.D.•Lori Davis, Ph.D.•Angela Buckalew,Deborah Best•DeborahBest•Ashley Murr•Michelle Hotchkiss2223200300***

m L)Female Studies.Eff t fAt iDIA100200 

ACTH (pg/ mEffectofAtrazine,DIAorDACT for 4 days onpituitary adrenal activity.

V e hi c l e ATR 12.5 ATR 75 D IA 1 0 DIA 60. 2

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TH (pg/mL)EffectofAtrazine,DIAorDACT for 1 day onpituitary adrenal activity.VehicleATR 75DIA highDACT high0200

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n1600180020000Hours1600180020000.0Hours26Plasma Chlorortriazine Concentrations(15 minutes post gavage)1 Dose75 mg/kg ATR 60.2 mg/kg DIA 50.6 mg/kg DACTATR+26.4 ± 4.0(9)DEA+153.1 ± 17.8(9)(15minutespostgavage)DIA+600.9 ± 50.5(9)4611.1 ± 359.0(9)DACT+78.0 ± 10.5(9)271.9 ± 53.6(9)980.4 ± 179.9(7)4 Doses12.5 mg/kg ATR 10 mg/kg DIA 8.4 mg/kg DACTATR* 8.8 ± 1.2(6)DEA* 63.8 ± 13.7(6)4 d75 /k ATR60 2 /k DIA50 6 /k DACTDIA* 252.2 ± 51.7(6)1877.7 ± 253.9(7)DACT** 747.5 ± 55.5(2)2088.3 ± 435.5(3)5977.0 ± 2119.1(3)4doses75mg/kgATR60.2mg/kgDIA50.6mg/kgDACTATR* 14 ± 2.1(6)DEA* 101.7 ± 14.6(6)27DIA* 475.0 ± 62.2(6)3886.0 ± 356.0(9)DACT** 4138.0 ± 1780.0(3)13136.0 ± 2401.0(2)12687.7 ± 1660.6(3)Serum Hormones After a Single Dose ofAtrazinein the Male Rat*500Control5 mg/kg50 /k*ACTH8Progesterone**AtrazineintheMaleRat******20030040050mg/kg100 mg/kg200 mg/kg

(pg/ml)**246******

(ng/ml)*0515306018001000515306018002********300400

/ ml)Corticosterone6810Testosterone

/ ml)*********051530601800100200

(ng /024 (ng /2805153060180Time (minutes)0 5 15 30 60 1800Time (min.)Serum Hormones After a Single Dose :DACT vs. ATRACTH400500Control3.37 mg/kg33.7 mg/kg67.5 mg/kg*DACT400500*ACTHControl5 mg/kg50 mg/kg100 mg/kgATR10020030067.5mg/kg135.0 mg/kg****

(pg/ml)DACT100200300******

(pg/ml)g g200 mg/kgATR051530600100 051530601800100Corticosterone300400500

m lDACT** ****300400

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ng/ m******100200

(ng/ml )051530 600Time (Minutes)051530601800Time (Minutes)29Atrazine Induces a Chemical SpecificActivation of HPA*500Control*ACTHActivationofHPA*****2003004005 mg/kg50 mg/kg100 mg/kg200 mg/kg

(pg/ml)•Control/DACT notaffected,•Dose response*051530601800100Time (minutes)Doseresponse•Not gastrointestinaldistress (vagotomy/measurement of*******200300400

(ng/ml)Corticosteronemeasurementofgherlin)•Activation withi t***051530601800100Time (minutes)intravenousadministration.30Time(minutes)Serum Corticosterone After a Singlevs. Multiple Doses•ATR DEAandDIAstimulateCorticosterone - 15 min aftersingle dose400***•ATR,DEAandDIAstimulaterelease of adrenal steroids within15 min. after a single dose in themale rat100200300

CORT ng/mlmaleratControlATRDIADEADACT0100Dose Equimolar to 200 mg/kg ATR400500ControlDIA 4 mg/kgDIA 10 mg/kg*****

g /ml)100200300DIA10mg/kgDIA 40 mg/kgDIA 80 mg/kgATR 100 mg/kgDACT 67 5 mg/kg**

CORT (n g3101530Post-Dosing Time (minutes)DACT67.5mg/kgComparison of after 1 or 4-DaysExposure toAtrazineand metabolites1000One Dose Four Doses1000bExposuretoAtrazineandmetabolites400600800b

TH (pg/mL)400600800a

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Cort i3220.022.5Eff t f FEi lVehicle12.5 mg/kg ATR75 /k ATR12.515.017.520.0**

n g/mlEffectofFourEquimolarDoses of Atrazine orMetabolites on the Pre75mg/kgATR2.55.07.510.0

LH nOvulatory Surge of LH inProestrous Females.1600180020000.05HoursLH: Vehicle vs. DIA25LH: Vehicle vs. DACT251520Vehicle10 mg/kg DIA60.2 mg/kg DIA+

g /mL)152025Vehicle8.4 mg/kg DACT50.6 mg/kg DACT

g /m L)510+***

LH (n g510 LH (n g33331600180020000Hours1600180020000Hours