Atrazine ImmunotoxicityAtrazine ImmunotoxicityPresentation to the Science Advisory PanelApril 26-29, 2010Robert Luebke, Ph.D.Cardiopulmonary and Immunotoxicology BranchEnvironmental Public Health DivisionNHEERL, ORD2ImmunotoxicologyzUnintended modulationof immune functionSuppression/stimulationAllergy/hypersensitivityAutoimmune diseasezMode of actionAltered supply of cellsAltered maturation or function of cellsFailure to control autoreactivityzProximal causeDirectIndirect3ImmunotoxicologyzHost factors influence the outcome of exposureAge of the immune system• Development→maturation→maintenance→senescenceGender/genotype• Relative abundance of hormones• Wide range of “normal”Life4ImmunotoxicologyzHazard identificationClues from general toxicity studies• Observational snapshotsKnown or suspected MoAMandated or planned testing of immune function• Functional assays5ImmunotoxicologyzWhy evaluate atrazine forimmunotoxicity?Endocrine-active compounds also tend tobe immunomodulatory• Pesticides (synthetic pyrethroids, propanil)• Industrial chemicals (PBDEs, organotins,hexachlorobenzene, plasticizers)6 

# Evaluation of ATR Developmental Immunotoxicity Evaluation of ATR Developmental ImmunotoxicityzSprague Dawley rats exposed GD10-PND22ATR (35 mg/kg/d, p.o.)• NOEL (25 mg/kg; Laws et al., 2000) for delayed onset of puberty infemale pups and reduced prolactinNOEL (12.5 mg/kg; Stoker et al.,1999)Bromocryptine (0.2 mg/kg, 2X/d)• Induces hypoprolactinemia• LOAEL for prostatitis (Stoker et al.)Propylthiouracil (2 mg/kg/d)• Suppress TH synthesis to induce hypothyroidismRooney et al., ToxSci 76, 366–375, 20037zPND2: Pups counted, weighed, pooled by treatment and genderand distributed to dams (5 males and 5 females) withintreatment groupzPND14: One pup/gender/litter weighed, bled for TH levels,spleen and thymus weighedzPND49 or later: Immune function(antibody and cell mediatedimmune responses, phagocytosis and NK cell activity) assessed;humoral and cellular responses were reassessed in olderoffspring to evaluate persistenceRooney et al., ToxSci 76, 366–375, 2003 

# Evaluation of ATR Developmental Immunotoxicity Evaluation of ATR Developmental Immunotoxicity8PND 2-14ControlAtrazineBromocryptinePropylthiouracilAverage %mortality/litter2.3±1.2 %13.2±2.8 %**7.4±2.1 %0.8±0.6 %Litters with 1 ormore pup death419**10*2n31 litters29 litters23 litters24 littersTABLE 1Early Offspring Survival by Litter



# Note.Data are the mean values from 2 separate experiments ± SEM.* difference from control litter ( 

# p< 0.05).** difference from control litters ( 

# p< 0.001).Rooney et al., ToxSci 76, 366–375, 2003 

# Evaluation of ATR Developmental Immunotoxicity Evaluation of ATR Developmental Immunotoxicity9ControlAtrazine Bromocryptine PropylthiouracilMale Female Male Female Male Female Male FemalePND 28.0±0.11n=807.6±0.09†n=808.0±0.09n=1007.6±0.09†n=1007.4±0.11**n=577.1±0.12**†n=608.2±0.08n=597.7±0.08‡n=60PND 720.1±0.21n=15518.9±0.18‡n=15419.0±0.30**n=13918.3±0.34n=13714.0±0.21**n=11313.7±0.21**n=12018.2±0.17**n=11917.6±0.16*†n=120Body Weight (g)TABLE 2Early Body Weight of OffspringNote. PND 2 values from a single experiment; PND 7 values are the mean of 2 separate experiments ± SEM. No effects on maternalbody weight gain* difference from same-sex control (p < 0.05)** difference from same-sex control (p < 0.001)† difference from treatment-matched males (p < 0.05)‡ difference from treatment-matched males (p < 0.001)Rooney et al., ToxSci 76, 366–375, 2003 

# Evaluation of ATR Developmental Immunotoxicity Evaluation of ATR Developmental Immunotoxicity10PND 14PND 62Rooney et al., ToxSci 76, 366–375, 2003 

# Evaluation of ATR Developmental Immunotoxicity Evaluation of ATR Developmental Immunotoxicity11Humoral immunity:Humoral immunity:response to immunizationresponse to immunizationat 8 wks of ageat 8 wks of ageMalesFemales01000020000300004000050000

IgM (ng/ml)01000020000300004000050000600007000080000Control ATRBCRPTUTreatment

IgG (ng/ml)**ABRooney et al., ToxSci 76, 366–375, 2003 

# Evaluation of ATR Developmental Immunotoxicity Evaluation of ATR Developmental Immunotoxicity12Cellular immunity:Cellular immunity:delayed typedelayed typehypersensitivityhypersensitivityMalesFemales9 wks12 wks0510152025Control ATRBCRPTU0510152025ControlATRTreatmentAB

Mean Difference in Footpad Thickness (Change as % Control Foot Thickness)**Rooney et al., ToxSci 76, 366–375, 2003 

# Evaluation of ATR Developmental Immunotoxicity Evaluation of ATR Developmental Immunotoxicity13zNegative observationsNo effect on maternal weight gain during dosingNo effect on phagocytic activity of macrophagesNo effect on NK cell activityNo effect on relative or absolute spleen or thymusweightsPharmacologic induction of hypoprolactinemia orhypothyroidism did not replicate ATR effectsRooney et al., ToxSci 76, 366–375, 2003



# Evaluation Evaluation of ATR Developmental Immunotoxicity of ATR Developmental Immunotoxicity14zHypothesis: Suppression of immunefunction in male offspringexposed to ATR is the result ofdysregulated GnRH production andGnRHR expression.GnRH and GnRHR expression is set early in development and is sexuallydimorphicNeonatal exposure to GnRH antagonists causes long term suppressionof cell-mediated and humoral immunity in male primatesGnRH agonists have little effect on males, but suppress lymphocytepopulations of B and T cells in juvenile female ratsLymphocytes express R for GnRH and GnRH upregulates cytokine andantibody production 

# Evaluation of ATR Developmental Immunotoxicity Evaluation of ATR Developmental Immunotoxicity15zCaveatIndependent replicate studies produced similar resultsAttempt to generate a dose response curve unsuccessful aftermoving to a new facility• Confidence in both data sets• Unknown environmental factor(s) responsible?? 

# Evaluation of ATR Developmental Immunotoxicity Evaluation of ATR Developmental Immunotoxicity16 

# DIT of ATR in Mice DIT of ATR in Mice Rowe et al., TAAP 214, 69 Rowe et al., TAAP 214, 69

– 77, 2006 77, 2006zExposure of Balb/c mice began GD10-12zWhy mice?zControlled-release pellet containing ATR or placeboPellets released 700μg ATR/24 h X 21 d: ATR (≅35→ ≅23mg/kg/d)Dose based on Rooney et al. (> NOEL (25 mg/kg) for delayof sexual maturation (Laws et al.) and prolactin effects(Stoker et al.)Weaned at 28 days; assayed at 10-12 wks old17 

# DIT of ATR in Mice DIT of ATR in Mice Rowe et al., TAAP 214, 69 Rowe et al., TAAP 214, 69

– 77, 2006 77, 2006Ab titers were similar in all offspringAb titers were similar in all offspringHumoral immunity: response to pneumococcal vaccinationHumoral immunity: response to pneumococcal vaccinationStandardized to B cellsStandardized to B cellsStandardized to spleen cellsStandardized to spleen cellsATRControlControlATRControlControl18 

# DIT of ATR in Mice DIT of ATR in Mice Rowe et al., TAAP 214, 69 Rowe et al., TAAP 214, 69

– 77, 2006 77, 2006Mixed lymphocyte responseCytotoxic T cell activityCellular immunity: proliferation and target cell lysisCellular immunity: proliferation and target cell lysis19 

# DIT of ATR in Mice DIT of ATR in Mice Rowe et al., TAAP 214, 69 Rowe et al., TAAP 214, 69

– 77, 2006 77, 2006zPotential implications of unexpected “enhanced”functionAutoimmune diseaseHypersensitivity/allergy20zNegative observationsNo effects on maternal weight gain, pregnancy rate or littersizeNo effect on offspring weight gainNo effect on and spleen weights or lymphocyte subsetdistribution 

# DIT of ATR in Mice DIT of ATR in Mice Rowe et al., TAAP 214, 69 Rowe et al., TAAP 214, 69

– 77, 2006 77, 200621



# Summary DIT Summary DITzBasis for different outcomes in rats and miceDosing schemes?• Daily gavage vs. constant subcutaneousFunctional assessment endpoints?Species differences?Mouse strain?22



# Summary DIT Summary DITzAre the immunotoxicity results in conflict?Gender: effects constantSupply of cells: constantGenotype: not constantImmunomodulation rather than suppression orenhancement23 

# Late Development Exposure Study Late Development Exposure Study

Filipov Filipov et al. ToxSci 86, 324 et al. ToxSci 86, 324

– 332 (2005) 332 (2005)zExposure of 4 week old male C57Bl/6 mice to5, 25, 125, and 250 mg ATR/kg/d for 14 dzHigh dose effects that were generally shortlived at the lower doses; most resolved 7 dafter exposure ended.zData are observational; may have beenhelpful to understand changes in function,but no functional tests were done.24 

# Adult Exposure Studies Adult Exposure Studies25 

# Adult Exposure Studies Adult Exposure Studies

Karrow et al., Toxicology 209, 15 et al., Toxicology 209, 15

- 28, 2005 28, 2005 (published version of NTP study) (published version of NTP study)zGavage exposure of female B6C3F1 mice to 0, 25, 250or 500 mg ATR/kg/d X 14 dzFew effects at 25 mg/kg, except 35% increase inantibody production not observed at higher doseszReduced resistance to tumor cell challenge at≥250mg/kg, but activity of primary effector cells was notzObservational endpoints were mixed and do not suggesta consistent pattern of immunotoxicity26 

# Adult Exposure Studies Adult Exposure Studies Fournier et al., Tox. Lett. 60, 263 Fournier et al., Tox. Lett. 60, 263

- 274, 1992 274, 1992zC57Bl/6J mice exposed to a single oral dose of 27.3,109.4, 4337.5 or 845 mg ATR/kg (2% - 50% of theLD50)No effect on body weightsMixed bag of inconsistent changes in organ weightsEssentially no effect on spleniccellularity or distribution ofCD4+ and CD8+ cellsNo effect on phagocytic activity27 

# Adult Exposure Studies Adult Exposure Studies Pruett et al., ToxSci75, 343 Pruett et al., ToxSci75, 343

– 354 (2003) 354 (2003)zB6C3F1 female mice exposed to 100,200 or 300 mg ATR/kg, single i.p.injectionzGoal was to evaluate corticosteroneAUC as predictor of immune effects, notas an immunotoxicity hazard ID study.Results presented as linear regressionof corticosterone AUC vs. endpoints28



# Adult

vs Developmental Exposure Developmental ExposurezThe developing immune system is moresensitive to atrazine exposure than theadult immune systemzThe developing immune system doesnot appear to be more sensitive thanthe neuroendocrine system29 

# In vitro Exposure Studies In vitro Exposure Studies30 

# In vitro Exposure Studies In vitro Exposure StudiesWhalen et al., Chem. Bio. Interact. 145 311-319, 2003Enriched human NK cellsEnriched human NK cellsExposure concentration 10Exposure concentration 10μM24 h exposureRowe et al., TAAP 221, 179–188, 2007Enriched human NK cellsEnriched human NK cellsBut, from NTP study:But, from NTP study: