EndocrineEndocrine EffectsEffects ofof SelectiveSelective SerotoninSerotonin ReuptakeReuptake InhibitorsInhibitors ((SSRIsSSRIs)) onon AquaticAquatic OrganismsOrganisms

Marsha C. Black & Emily D. Rogers University of Georgia Athens, Georgia, USA

Theodore B. Henry University of Tennessee Knoxville, TN OutlineOutline

¾¾ SSRIsSSRIs -- MOAMOA andand clinicalclinical significancesignificance ¾¾ PresencePresence inin thethe environmentenvironment ¾¾ StudyStudy objectivesobjectives ¾¾ ResultsResults andand DiscussionDiscussion

z AcuteAcute toxicitytoxicity ((macroinvertebratemacroinvertebrate,, fish)fish)

z ChronicChronic effectseffects ((macroinvertebratemacroinvertebrate,, fish,fish, frog)frog) ¾¾ SummarySummary andand conclusionsconclusions ¾¾ FutureFuture researchresearch directionsdirections SelectiveSelective SerotoninSerotonin ReuptakeReuptake InhibitorsInhibitors ((SSRIsSSRIs))

¾¾ TreatTreat clinicalclinical depression,depression, obsessiveobsessive-- compulsivecompulsive andand panicpanic disorders,disorders, PMS,PMS, etc.etc. ¾¾ ClinicalClinical MOA:MOA: blockblock serotoninserotonin reuptakereuptake ¾¾ Examples:Examples:

z FluoxetineFluoxetine (Prozac(Prozac®® andand SarafemSarafem®®))

z SertralineSertraline (Zoloft(Zoloft®®))

z CitalopramCitalopram ((CelexaCelexa®® andand LexaproLexapro®®))

z FluvoxamineFluvoxamine ((LuvoxLuvox®®))

z ParoxetineParoxetine ((PaxilPaxil®®)) SSRI Structures

O F3C OCHCH2CH2NHCH3 FNH O O ® Fluoxetine (Prozac®) CN Paroxetine (Paxil )

F O

CH2CH2CH2N(CH3)2 Citalopram ® (Celexa ) Cl

CH2CH2CH2CH2OCH3 Cl NHCH3 F3C

N CH2CH2NH2 O

Fluvoxamine (Luvox®) Sertraline (Zoloft®) SourcesSources ofof SurfaceSurface WaterWater ContaminationContamination byby HumanHuman PharmaceuticalsPharmaceuticals

Pharmaceuticals

Disposal Metabolism, Excretion

Landfill WWTP

Groundwater Surface Waters SSRIsSSRIs:: DetectionDetection inin thethe EnvironmentEnvironment

z FluoxetineFluoxetine detecteddetected inin surfacesurface waterswaters • 0.012 ppb detected in USGS reconnaissance study (Kolpin et al. 2002) • 0.030-0.099 ppb in Canada (Metcalfe et al. 2003) • 0.031-0.076 ppb in Mississippi (Wook-Kwon and Armbrust, unpublished) z FluoxetineFluoxetine,, sertralinesertraline andand metabolitesmetabolites detecteddetected inin fishfish tissuestissues (Brooks et al., 2005) PhysicochemicalPhysicochemical PropertiesProperties ofof SSRIsSSRIs (data from Wook-Kwon and Armbrust)

a b c Compound Log KOW Log KOC Photolysis t½ (d)

Citalopram 1.39 5.63 39

Fluoxetine 1.22 4.65 122

Fluvoxamine 1.21 3.82 0.57; 29

Paroxetine 1.37 4.47 0.67

Sertraline 1.37 4.17 23

aMeasured on salt form bAverage calculated from experiments with 5 different soils and sediments c Average calculated from experiments with 2 different lake water samples WhyWhy Worry?Worry?

¾ PharmaceuticalsPharmaceuticals areare designeddesigned toto havehave aa therapeutictherapeutic (=biological)(=biological) effecteffect

z Effects on non-target organisms are mostly unknown ¾ AquaticAquatic organismsorganisms areare exposedexposed throughoutthroughout theirtheir lifetimelifetime ¾ PotentialPotential forfor multigenerationalmultigenerational exposureexposure ¾ LittleLittle isis knownknown aboutabout persistence,persistence, fatefate ofof drugsdrugs inin thethe environmentenvironment ¾ SSRIsSSRIs knownknown toto promotepromote spawningspawning inin mollusksmollusks OverallOverall ResearchResearch PlanPlan……

¾ DetermineDetermine environmentalenvironmental fatefate ofof SSRIsSSRIs

z Techniques used for pesticide registration

z Measure hydrolysis, photolysis, metabolism, etc. ¾ MeasureMeasure parentparent andand majormajor degradationdegradation productsproducts

z Wastewater effluent

z Downstream receiving water ¾ DetermineDetermine acute,acute, chronicchronic impactsimpacts toto aquaticaquatic organismsorganisms

z Ceriodaphnia dubia (macroinvertebrate)

z Gambusia affinis (Western mosquito fish)

z Xenopus laevis (frog) ToxicityToxicity TestsTests

¾ TestTest organism:organism: CeriodaphniaCeriodaphnia dubiadubia

¾ AcuteAcute toxicitytoxicity (48(48 h)h) z Single compound exposures z Binary, quaternary mixture exposures z Mortality (LC50) as endpoint

¾ ChronicChronic toxicitytoxicity z 7 day mini-chronic test z Brood size, # broods as endpoints

¾ AllAll teststests followedfollowed USUS EPAEPA protocolsprotocols AcuteAcute ToxicityToxicity (LC50)(LC50) ofof SSRIsSSRIs

SSRI LC50 ppba Citalopram (Celexa®) 3180 (220) Fluvoxamine (Luvox®) 1260 (830) Paroxetine (Paxil®) 470 (60) Fluoxetine (Prozac®) 590 (130) Sertraline (Zoloft®) 140 (20) aMean (± SD) of 3 tests

Henry et al. 2004, Environ Toxicol Chem 23:2229-2233 ChronicChronic ToxicityToxicity ofof SSRIsSSRIs

SSRI NOECa LOECa (ppb) (ppb) Citalopram (Celexa®) 800 4000 Fluvoxamine (Luvox®) 366 1466b Paroxetine (Paxil®) 220 440b Fluoxetine (Prozac®) 89 447b Sertraline (Zoloft®) 9 45 aTotal number of neonates produced over 7-8 d bNumber of broods also significantly reduced

(Henry et al. 2004, Environ Toxicol Chem 23:2229-2233) MixtureMixture ToxicityToxicity (In(In preparation,preparation, HenryHenry andand Black)Black)

Equitoxic Mixture Equal Concentration Mixture

100 100

80 80

60 60

Observed 40 40 % Mortality Model Observed

20 Model CA 20

Model IA

0 0 0 5 10 15 20 25 012345 µM SSRI (quaternary mixture) AcuteAcute ToxicityToxicity ofof FluoxetineFluoxetine toto WesternWestern MosquitofishMosquitofish

¾ 77--dd acuteacute teststests ¾ Endpoints:Endpoints:

z Mortality (LC50)

z Fish behavior

Western mosquitofish Gambusia affinis AcuteAcute ToxicityToxicity ofof FluoxetineFluoxetine toto WesternWestern MosquitofishMosquitofish

¾¾ MortalityMortality

z 77--dayday LC50LC50 == 614614 ppbppb

¾¾ BehavioralBehavioral effectseffects (0.6(0.6 andand 66 ppb)ppb)

z UncoordinatedUncoordinated swimmingswimming

z Lethargy,Lethargy, lacklack ofof responseresponse toto stimulistimuli

z LessLess aggression,aggression, interactioninteraction betweenbetween individualsindividuals ChronicChronic ExposuresExposures inin OutdoorOutdoor MesocosmsMesocosms

•110-L plastic tanks •50 fish/tank •85-d exposure •Water change 1x/wk ChronicChronic TestsTests (140(140 d)d) withwith MosquitofishMosquitofish

z TimeTime toto reproductivereproductive maturitymaturity • Fully developed gonopodium (males) • Formation of black spot (females)

z HistologicalHistological effectseffects onon gonads?gonads?

Male Female

Gonopodium Black Spot EffectEffect ofof FluoxetineFluoxetine onon MaleMale SexualSexual DevelopmentDevelopment

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d 40 60 ppb

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0 020406080100 Days of Exposure*

*Fish were 39-d old at t=0 EffectEffect ofof FluoxetineFluoxetine onon FemaleFemale SexualSexual DevelopmentDevelopment

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0 0 1020304050 Days of Exposure* *Fish were 39-d old at t=0 ResearchResearch withwith thethe AfricanAfrican ClawedClawed FrogFrog ((XenopusXenopus laevislaevis))

¾ EasyEasy toto breedbreed inin thethe lablab

z Inject with HCG ¾ TadpoleTadpole toto frogfrog inin 6060--7070 dd ¾ ManyMany measurablemeasurable endpointsendpoints

z Mortality

z Developmental malformations

z Time to metamorphosis WhyWhy StudyStudy Frogs?Frogs?

¾ ThyroidThyroid hormoneshormones (T(T3,T,T4)) cuecue metamorphosismetamorphosis ¾ TadpolesTadpoles withwith nono thyroidthyroid –– metamorphosismetamorphosis inhibitedinhibited

¾ ExposureExposure toto chemicalschemicals thatthat reducereduce circulatingcirculating TT3 willwill delaydelay oror inhibitinhibit metamorphosismetamorphosis RegulationRegulation ofof ThyroidThyroid AxisAxis inin MammalsMammals

Serotonin X X

X Fluoxetine

www.dpcweb.com/images/medicalconditions/thyroid/thyroid%20illustration.jpg

¾ Serotonin inhibits the release of TRH from the hypothalamus in rats

z Mitsuma et al. 1983; Mitsuma et al. 1996 ¾ Fluoxetine reduces circulating T3 and T4; increases TSH

z Golstein et al., 1983 DoesDoes FluoxetineFluoxetine InhibitInhibit FrogFrog Metamorphosis?Metamorphosis?

¾ ExposeExpose tadpolestadpoles fromfrom hatchhatch untiluntil metamorphosismetamorphosis

z FluoxetineFluoxetine (FL):(FL): 0.059,0.059, 0.295,0.295, 2.95,2.95, 29.529.5 ppbppb (measured)(measured)

z AmmoniumAmmonium perchlorateperchlorate (AP):(AP): 1010 ppbppb

z ControlControl (clean(clean exposureexposure water)water) ¾ ObserveObserve dailydaily forfor limblimb developmentdevelopment untiluntil metamorphosismetamorphosis isis completecomplete EffectsEffects ofof ChronicChronic ExposureExposure toto FluoxetineFluoxetine (Xenopus)(Xenopus)

Tadpoles at 57 d* ¾ DevelopmentalDevelopmental delaysdelays

z Forelimb formation

z Tail resorbtion ¾ IncreasedIncreased timetime toto metamorphosismetamorphosis ¾ MortalityMortality

Control 38 ppb FL 9.5 ppb AP

*Data from range-finder experiment. Similar effects at 29.5 ppb in 2nd experiment. EffectEffect ofof ChronicChronic ExposureExposure toto FluoxetineFluoxetine onon TimeTime toto MetamorphosisMetamorphosis

s i * s o 35

h

p r 30

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a t 25

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me 15

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n 0 I Control 0.059 0.295 2.95 29.5

% Fluoxetine Concentration (ppb) EffectEffect ofof ChronicChronic ExposureExposure toto FluoxetineFluoxetine onon MassMass atat MetamorphosisMetamorphosis

45 * * * 40 * 35 Mass 30 25 20 15

% Reduction in % Reduction 10 5 0 Control 0.059 0.295 2.95 29.5 Fluoxetine Concentration (ppb) EffectsEffects ofof ChronicChronic ExposureExposure toto FluoxetineFluoxetine (Exp.(Exp. 2)2)

¾ LimbLimb malformationsmalformations

z Primary rotation of hindlimbs

z Micromelia of forelimbs

z Dorsal flexure of the tail TimeTime toto OnsetOnset ofof MalformationsMalformations ConclusionsConclusions (so(so farfar……))

¾¾ SSRIsSSRIs areare acutelyacutely toxictoxic toto CeriodaphniaCeriodaphnia andand mosquitofishmosquitofish ¾¾ FluoxetineFluoxetine affectsaffects fishfish behaviorbehavior ¾¾ FluoxetineFluoxetine delaysdelays sexualsexual developmentdevelopment inin fishfish ¾¾ FluoxetineFluoxetine delaysdelays developmentdevelopment andand metamorphosismetamorphosis inin frogsfrogs

None of these effects observed at environmentally-relevant concentrations. ConclusionsConclusions (cont(cont’’d)d)

¾ ReducedReduced massmass andand limblimb malformationsmalformations observedobserved withwith chronicchronic exposureexposure toto FLFL

z Both effects occured at environmentally relevant concentrations

z Mass reductions confirmed in 2 experiments

z Malformation data not yet confirmed • Lower temperature in experiment 2 (19ºC) • Increased exposure duration, TTM • Increased susceptibility of Exp. 2 frogs to developmental disorders? ImplicationsImplications ofof thethe ResearchResearch

¾ DelayedDelayed developmentdevelopment (fish,(fish, frogs)frogs)

z ↑ Predation, dessication (frogs),(frogs), populationpopulation decline?decline? ¾ ReducedReduced massmass andand limblimb malformationsmalformations (frogs)(frogs)

z ↑ Predation, ↓ reproductive success, population decline? Future Research Questions Generated by Research

¾ Conduct additional FL exposure with Xenopus ¾ Validate apparent impact of FL on the thyroid axis by measuring TH, TSH during frog development (with/without FL) ¾ Do other SSRIs have similar effects on frog development and growth? ¾ What is the toxicity of mixtures of SSRIs in the amphibian model? ¾ What are environmentally-relevant SSRI concentrations? AcknowledgementsAcknowledgements

¾ Project Personnel (University of Georgia) z Ted Henry (now at the University of Tennessee) z Emily Rogers (MS Tox 2004; PhD student) z Ben Hale (BS EH, 2004) z Nicole Campbell (BS EH 2003) z Tricia Smith (retired)

¾ Analytical Support (Mississippi State Chemical Lab) z Kevin Armbrust (Project Co-PI) z Jeong-Wook Kwon

¾ Outside Expertise z Kay Millar (US EPA Region IV Lab, Athens, GA) z James Rayburn (Jacksonville State University, AL) AcknowledgementsAcknowledgements

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