Atrazine Immuno

Atrazine Immuno

Presentation to the Science Advis

April 26-29, 2010

Robert Luebke, Ph.D.

Cardiopulmonary and Immunotoxico

Environmental Public Health D

NHEERL, ORD
Immunotoxicology

z Unintended modulation of immune function

 Suppression/stimulation Allergy/hypersensitivity Autoimmune disease

z Mode of action

 Altered supply of cells Altered maturation or function of cells Failure to control autoreactivity

z Proximal cause

 Direct Indirect 
Immunotoxicology

z Host factors influence the outcome of exposure 

 Age of the immune system

• Development 

→ maturation 

→ maintenance 

→ sen

 Gender/genotype

• Relative abundance of hormones• Wide range of “normal”

 Life
Immunotoxicology

z Hazard identification 

 Clues from general toxicity studies 

• Observational snapshots

 Known or suspected MoA Mandated or planned testing of immune function

• Functional assays
Immunotoxicology

z Why evaluate atrazine for 

immunotoxicity?

 Endocrine-active compounds als

be immunomodulatory

• Pesticides (synthetic pyrethroids, • Industrial chemicals (PBDEs, orga

hexachlorobenzene, plasticizers)
Evaluation of ATR Developmental Immuno

Evaluation of ATR Developmental Immuno

z Sprague Dawley rats exposed GD10-PND

 ATR (35 mg/kg/d, p.o.)

• NOEL (25 mg/kg; Laws et al., 2000) for delaye

female pups and reduced prolactin NOEL (12.5 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 hypothyroidis

Rooney et al., ToxSci 76, 366–375, 2003
z PND2: Pups counted, weighed, pooled by treatm

and distributed to dams (5 males and 5 femalestreatment group

z PND14: One pup/gender/litter weighed, bled for 

spleen and thymus weighed

z PND49 or later: Immune function (antibody and 

immune responses, phagocytosis and NK cell achumoral and cellular responses were reassessed offspring to evaluate persistence

Rooney et al., ToxSci 76, 366–375, 2003

Evaluation of ATR Developmental Immuno

Evaluation of ATR Developmental Immuno
PND 2-14 Control Atrazine Bromocry

Average %

mortality/litter 2.3 ± 1.2 % 13.2 ± 2.8 %** 7.4 ± 2.1 

Litters with 1 or 

more pup death 4 19** 10* 

n 31 litters 29 litters 23 litte

TABLE 1

Early 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 Immuno

Evaluation of ATR Developmental Immuno
Control Atrazine Bromocry

Male Female Male Female Male 

PND 2 8.0±0.11

n=80

7.6±0.09†

n=80

8.0±0.09

n=100

7.6±0.09†

n=100

7.4±0.11**

n=57

7

PND 7 20.1±0.21

n=155

18.9±0.18‡

n=154

19.0±0.30**

n=139

18.3±0.34

n=137

14.0±0.21**

n=113

1

Body Weight (g)

TABLE 2

Early Body Weight of Offspring

Note. PND 2 values from a single experiment; PND 7 values are the mean of 2 separate experiments ± SEM. No effec* 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 Immuno

Evaluation of ATR Developmental Immuno
PND 14

PND 62

Rooney et al., ToxSci 76, 366–375, 2003

Evaluation of ATR Developmental Immuno

Evaluation of ATR Developmental Immuno
Humoral immunity: Humoral immunity: 

response to immunization response to immunization 

at 8 wks of age at 8 wks of age

MalesFemales

0

1000020000300004000050000

IgM (ng/ml)

0

1000020000300004000050000600007000080000

Control ATR 

Treat

IgG (ng/ml)

*

A

B

Rooney et al., ToxSci 76, 366–375, 2003

Evaluation of ATR Developmental Immuno

Evaluation of ATR Developmental Immuno
Cellular immunity: Cellular immunity:

delayed type delayed type 

hypersensitivity hypersensitivity

MalesFemales

05

10152025

Control ATR BCR 

05

10152025

Control 

Treatment

A

B

Mean Difference in Footpad Thickness 

(Change as % Control Foot Thickness) * 

Rooney et al., ToxSci 76, 366–375, 2003

Evaluation of ATR Developmental Immuno

Evaluation of ATR Developmental Immuno
z Negative observations

 No effect on maternal weight gain d No effect on phagocytic activity of m No effect on NK cell activity No effect on relative or absolute sple

weights

 Pharmacologic induction of hypoprol

hypothyroidism did not replicate ATR 

Rooney et al., ToxSci 76, 366–375, 2003

Evaluation Evaluation of ATR Developmental Immun

of ATR Developmental Immun
z Hypothesis: Suppression of immune function in m

exposed to ATR is the result of dysregulated GnRGnRHR expression.

 GnRH and GnRHR expression is set early in develo

dimorphic

 Neonatal exposure to GnRH antagonists causes lon

of cell-mediated and humoral immunity in male pri

 GnRH agonists have little effect on males, but supp

populations of B and T cells in juvenile female rats

 Lymphocytes express R for GnRH and GnRH upreg

antibody production

Evaluation of ATR Developmental Immuno

Evaluation of ATR Developmental Immuno
z Caveat

 Independent replicate studies produced simila Attempt to generate a dose response curve u

moving to a new facility

• Confidence in both data sets• Unknown environmental factor(s) responsible?

Evaluation of ATR Developmental Immuno

Evaluation of ATR Developmental Immuno
DIT of ATR in Mice DIT of ATR in Mice

Rowe et al., TAAP 214, 69 Rowe et al., TAAP 214, 69–77, 200

77, 200

z Exposure of Balb/c mice began GD10-12z Why mice?z Controlled-release pellet containing ATR 

 Pellets released 700 

μg ATR/24 h X 21 d: AT

mg/kg/d)

 Dose based on Rooney et al. (> NOEL (25 m

of sexual maturation (Laws et al.) and prola(Stoker et al.)

 Weaned at 28 days; assayed at 10-12 wks o
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, 2006

Ab titers were similar in all offsp

Ab titers were similar in all offsp

Humoral immunity: response to pneumoco

Humoral immunity: response to pneumoco

Standardized to B cells Standardized to B cells Standa

Standa

ATR

Control Control
DIT of ATR in Mice DIT of ATR in Mice

Rowe et al., TAAP 214, 69 Rowe et al., TAAP 214, 69–77, 200

77, 200

Mixed lymphocyte response Cytot

Cellular immunity: proliferation and targ

Cellular immunity: proliferation and targ
DIT of ATR in Mice DIT of ATR in Mice

Rowe et al., TAAP 214, 69 Rowe et al., TAAP 214, 69–77, 200

77, 200

z Potential implications of unexpected “en

function

 Autoimmune disease Hypersensitivity/allergy
z Negative observations

 No effects on maternal weight gain, pregnan

size 

 No effect on offspring weight gain No effect on and spleen weights or lymphoc

distribution

DIT of ATR in Mice DIT of ATR in Mice

Rowe et al., TAAP 214, 69 Rowe et al., TAAP 214, 69–77, 200

77, 200
Summary DIT Summary DIT

z Basis for different outcomes in rat

 Dosing schemes?

• Daily gavage vs. constant subcutaneous

 Functional assessment endpoints?  Species differences? Mouse strain?
Summary DIT Summary DIT

z Are the immunotoxicity results in c

 Gender: effects constant Supply of cells: constant Genotype: not constant Immunomodulation rather than supp

enhancement
Late Development Exposure S

Late Development Exposure S

Filipov Filipov et al. ToxSci 86, 324 et al. ToxSci 86, 324–332 (20

332 (20

z Exposure of 4 week old male C57B

5, 25, 125, and 250 mg ATR/kg/d 

z High dose effects that were gener

lived at the lower doses; most resafter exposure ended. 

z Data are observational; may have 

helpful to understand changes in fbut no functional tests were done.
Adult Exposure Stud

Adult Exposure Stud
Adult Exposure Studies Adult Exposure Studies

Karrow et al., Toxicology 209, 15 et al., Toxicology 209, 15-28, 2

28, 2

(published version of NTP study) (published version of NTP study)

z Gavage exposure of female B6C3F1 mice 

or 500 mg ATR/kg/d X 14 d

z Few effects at 25 mg/kg, except 35% inc

antibody production not observed at high

z Reduced resistance to tumor cell challeng

mg/kg, but activity of primary effector ce

z Observational endpoints were mixed and 

a consistent pattern of immunotoxicity
Adult Exposure Studies Adult Exposure Studies

Fournier et al., Tox. Lett. 60, 263 Fournier et al., Tox. Lett. 60, 263-274, 

274, 

z C57Bl/6J mice exposed to a single oral d

109.4, 4337.5 or 845 mg ATR/kg (2% - LD50)

 No effect on body weights Mixed bag of inconsistent changes in organ  Essentially no effect on splenic cellularity or 

CD4+ and CD8+ cells

 No effect on phagocytic activity
Adult Exposure Studie

Adult Exposure Studie

Pruett et al., ToxSci75, 343 Pruett et al., ToxSci75, 343–354 (200

354 (200

z B6C3F1 female mice exposed 

200 or 300 mg ATR/kg, single injection

z Goal was to evaluate corticost

AUC as predictor of immune eas an immunotoxicity hazard IResults presented as linear reof corticosterone AUC vs. end
Adult vs Developmental Exp

Developmental Exp

z The developing immune syste

sensitive to atrazine exposure adult immune system

z The developing immune syste

not appear to be more sensitivthe neuroendocrine system
In vitro Exposure Stu

In vitro Exposure Stu
In vitro Exposure Studie

In vitro Exposure Studi

Whalen et al., Chem. Bio. In

Enriched hum

Enriched hum

24 h exposure Exposure conce

Exposure conce

Rowe et al., TAAP 221, 179–188, 2007

Enriched human NK cells Enriched human NK cells

But, from NTP study: But, from NTP study: