Effects of Genistein
Exposure on Maternal
Behavior and Immune
Function
Mary Kay Caniglia, Evan Ball,
Amy B. Wisniewski and
Dean Hoganson
Endocrine Disruptor Lab
Overview
What is Endocrine Disruption?
What is Genistein?
Why study Genistein?
Why Study Maternal Behavior?
Experimental Design
Preliminary Results: Maternal Behavior
and Immune Function
What is Endocrine Disruption?
Exogenous agents that have the ability to
interfere with endogenous hormone
physiology
Harmful to humans and non-human
animals
Early exposure is more harmful
What the heck?
The analogy of a lock and key is
often used to understand the
mechanism by which hormones
communicate information in our
bodies:
+ =
De-masculinized or
Lock=Hormone Receptor
feminized males
Key=Hormone
Unlocked door=biological response
+ = When receptors are
stimulated to initiate a
biological response
when they are not
supposed to, we have a
problem.
Endocrine Disruptor: Lock Pick
Hypospadias/10,000 births
19
0
5
10
15
20
25
30
35
40
45
70
19
73
19
76
19
79
19
82
19
85
Year of birth
19
88
Humans and Wildlife
19
91
What is Genistein?
Genistein is a phytoestrogen contained in
soy that binds estrogen receptors
Genistein is known to preferentially bind
ERβ
Why study Genistein?
We are all exposed to genistein
Long-Term effects of early exposure to genistein
are not fully understood, but have been shown
to be on the reproductive system of male infants
and various animals
The immune system is also responsive to sex
hormones, yet less attention has been given to
the effects of phytoestrogens on immune
development
Hypothesis:
The aim of the present study is to identify
critical time periods during perinatal
development that exposure to genistein
results in maximal atypical development of
endocrine related measures: e.g.
reproductive, immune.
We are assessing maternal behavior as a
potential confound.
Why Study Maternal Behavior
Rodent maternal behavior affects offspring
development
Direct or Indirect effects?
Maternal Behavior (cont.)
Endogenous estrogen stimulates maternal
behavior1,2
estradiol activates neuroendocrine substrates
during pregnancy to stimulate maternal
behavior
After parturition, estradiol levels diminish
Changes in estrogen levels via exogenous
sources may alter behavior of mother
Current Research Studies
Conflicting Results
Study 1: No differences1
oral genistein administration.
Study2: BPA treated animals show less time nursing
and more time removed2
BPA is a synthetic estrogen
oral oil administration
Study3: 17α-ethinylestradiol treated animals are less
removed and quicker to retrieve3
17α-ethinylestradiol is a synthetic estrogen
injection
Experimental Design
Lactation
Gestation
Genistein
Control
GD1 PND1 PND21
Experimental Design (cont.)
Observations done before feeding
Time 9 am to 11 am
30 second observations
10 minutes between 30 second sessions
3 total sessions per mother
60 total observations per mother
This begins on PND 2 and continues through
PND 21
Experimental Design (cont.)
Measures4
Nursing (no type distinction)
Grooming
Carrying
Nesting
Laying
Removed
Results
Maternal Behavior Round 1
60%
50%
40% Control
Genistein
30%
Gestation
20% Lactation
10%
0%
Nursing Grooming Carrying Nesting Laying Removed
Results (cont.)
% Rem oved
40%
35%
30%
25%
20%
15%
10%
9.75 9.5 12.5 7.5
5%
0%
Control Genistein Gestation Lactation
Results (cont.)
% Nursing
70%
60%
50%
40%
30%
20%
10%
0%
Control Genistein Gestation Lactation
Results (cont.)
% Grooming
20%
18%
16%
14%
12%
10%
8%
6%
4%
2%
0%
Control Genistein Gestation Lactation
Results (cont.)
% Laying
45%
40%
35%
30%
25%
20%
15%
10%
5%
0%
Control Genistein Gestation Lactation
Results (cont.)
% Carrying
3.00%
2.50%
2.00%
1.50%
1.00%
0.50%
0.00%
Control Genistein Gestation Lactation
Results (cont.)
% Nesting
2.50%
2.00%
1.50%
1.00%
0.50%
0.00%
Control Genistein Gestation Lactation
Where we are now
Sample sizes are too small to determine statistical
significance
Some maternal behaviors may be altered
While a significant body of literature has assessed the
needs to develop a quality animal model without
additional confounding influences from a toxicological
perspective, less attention has been given to the need to
understand the effects of maternal behavior on
development in rats.
Immune Measures
Data so far:
Differential
WBC count
Spleen/Thymus: Relative Organ mass
Pending Data:
Total WBC count
Cell-mediated immunity (DTH)
Humoral Immune Response
Lymphocyte Proliferation Assay
Differential WBC Count
Control Genistein Gestation Lactation ANOVA
Agranulocytes
Lymphocytes 51.33 ± 3.29 50.00 ±0.00 60.00 ± 4.73 61.00 ± 7.00 F(3,8)=1
Monocytes 33.00 ± 3.79 43.00 ± 0.00 24.67 ± 4.63 27.00 ± 4.00 F(3,8)=1
Granulocytes
Neutrophils 8.67 ± 0.67 6.00 ± 0.00 6.33 ± 1.45 6.00 ± 1.00 F(3,8)=1
Eosinophils 4.33 ± 0.61 1.00 ± 0.00 4.00 ± 0.58 4.50 ± 0.50 F(3,8)=1
Basophils* 2.33 ± .42 6.00 ± 0.00 5.00 ± 1.15 1.50 ± 1.50 F(3,8)=4
Differential WBC Count Data Represent average number of cells ± SEM.
An asterik (*) indicates that experimental groups were significantly
different than controls based on one-way ANOVA, p<0.05
Male Thymus
0.0025
0.002
Relative Mass
N=5/3
0.0015
N=7/3
N=3/3
0.001
N=6/2
0.0005
0
Control Genistein Gestation Lactation
Group
Male Spleen
0.00285
0.00265
0.00245
N=7/3
0.00225
0.00205 N=5/3
N=3/3
0.00185 N=6/2
0.00165
Control Genistein Gestation Lactation
References
1. Flynn KM et al. Multigenerational Exposure to Dietary Genistein has No
Severe Effects on Nursing Behavior in Rats. NeuroToxicology 21(6):997-
1002 (2000).
2. Palanza P et al. Exposure to a Low Dose of Bisphenol A during Fetal Life
or Adulthood Alters Maternal Behavior in mice. Environmental Health
Perspectives 110(3):415-422 (2002).
3. Arabo A, Lefebvre M, Fermanel M, Caston J. Administration of 17α-
ethinylestradiol during pregnancy elicits modifications of maternal
behavior and emotional alteration of the offspring in the rat.
Developmental Brain Research 156:93-103 (2005).
4. Sanders BJ and Gray MJ. Early Environmental Influences Can Attenuate
the Blood Pressure Response to Acute Stress in Borderline Hypertensive
Rats. Physiology and Behavior 61(5): 749-754 (1997).
References
Paulozzi, L, Erickson, J, Jackson, R.: Hypospadias
trends in two US surveillance systems. Pediatrics,
100(5), 831, 1997
Klein, S, Wisniewski, AB, Marson, AL, Glass, G,
Gearhart, J.: Early exposure to genistein exerts long-
lasting effects on the endocrine and immune system in
rats. Molecular Medicine, 8(11), 742, 2002.
Yellayi, S, Naaz, A, Szewczykowski, MA, et al. The
phytoestrogen genistein induces thymic and immune
chagnes: a human health concern? PNAS, 99, 7616,
2002.
Images: Weston A. Price Foundation, Google Images
Acknowledgements
Liz and Erica
Previous Lab Members: Alicia Fisher (AS ’06), Kylie Lago (AS ’06),
Stacie Holmgren (AS ’06), Raime Robinson (AS ’06)
Current Lab Members: Stacy Knight, Jeremy DeFoe, Michelle
Gombas
Brian Sanders
Funding Support from:
Brady Urological Research Institute
Drake University
Iowa Academy of Sciences
Dr. Smith