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THE IMPACT OF POLYCHLORINATED BIPHENYLS ON
MATERNAL CARE AND OFFSPRING SEXUAL BEHAVIOR
IN LABORATORY RATS - THE IMPORTANCE OF
EXAMINING ALL COMPONENTS OF A DYNAMIC SYSTEM

presented by
JENNIFER ANN CUMMINGS

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THE IMPACT OF POLYCHLORINATED BIPHENYLS ON MATERNAL CARE
AND OFFSPRING SEXUAL BEHAVIOR IN LABORATORY RATS - THE
IMPORTANCE OF EXAMINING ALL COMPONENTS OF A DYNAMIC SYSTEM

By

Jennifer Ann Cummings

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of -

DOCTOR OF PHILOSOPHY

Department of Zoology

2006

ABSTRACT

THE IMPACT OF POLYCHLORINATED BIPHENYLS ON MATERNAL CARE
AND OFFSPRING SEXUAL BEHAVIOR IN LABORATORY RATS - THE
IMPORTANCE OF EXAMINING ALL COMPONENTS OF A DYNAMIC SYSTEM

By

Jennifer Ann Cummings

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that remain
in the environment today even after their production was banned in the United States in
the 19708. While many studies have examined the effects of PCBs on a variety of
behaviors and developmental outcomes, the effects of the contaminants on maternal
behavior have been largely ignored. Those that have evaluated maternal behavior after
gestational exposure to PCB used the traditional paradigm in which a dam is treated
while she is pregnant and the resulting maternal behavior is evaluated. With this
approach, it is impossible to distinguish between effects on maternal behavior that are the
result of direct actions on the dams’ endocrine and/or nervous systems from those that
stem from indirect effects mediated by changes in the physical and behavioral proﬁles of
the exposed litter. In this dissertation, I examined the effects of different doses of the
dioxin-like PCB congener 3, 4, 3’, 4’-tetrachlorobipheny1 (PCB 77) on maternal behavior
in rats using a cross-fostering paradigm to gain insights about the contributions of direct
and indirect effects of PCB 77 exposure on maternal behavior. I also examined the

effects of the toxin on offspring partner preference and sexual behavior.

Various components of maternal behavior were affected when dams were treated with a

dose of 2 mg/kg b.w. from gestation day (GD) 6 — 18. Some of the effects on maternal

behavior can be ascribed to prenatal exposure of the litters to the PCB, while other effects
appear to be due to both direct effects of the PCB on the dams and effects mediated by

changes in the offspring.

A second experiment employed a dose of 3 mg/kg to determine how an intermediate dose
of PCB 77 affects maternal behavior and to expand the behavioral analysis. The results
from this experiment conﬁrm that exposing dams to PCB during gestation alters the care
they provide to their litters, and that many of the affected behaviors appear to be the
result of dams rearing prenatally PCB-exposed pups instead of being due to the treatment

the dams received while pregnant.

Finally, we examined the sexual behavior and partner preference of offspring that were
exposed to PCB pre- and/or postnatally. While PCB exposure did not affect male or
female sexual behavior, it did alter the display of partner preference behavior. These

effects were more salient in the females than the males.

This dissertation also addresses the traditional experimental paradigm that is often used,
in which treatment of the dam is simply considered an avenue through which toxins can
be delivered to the developing embryo without taking into consideration any possible
alterations in the dams’ behavior. Since such exposure is not speciﬁc to the pups the
effects of the contaminant on the dam must be evaluated in terms of its impact on the

offspring.

ACIGNI OWLEDGMEN TS

I would like to thank the people who made this undertaking possible, starting with my
committee: my advisor, Dr. Lynwood Clemens, for helping to guide me through this
incredible journey and for knowing when and how hard to push me; Dr. Antonio Nunez,
for the guidance and countless number of manuscript edits; Dr. Cynthia Jordan, for being
there to help me through both professional and personal situations; and Dr. Joseph

Lonstein, for his incredible knowledge of maternal behavior.

I would like to thank my family: my husband, for having faith in me even when I don’t
have faith in myself, and without whom I wouldn’t have made it through my ﬁrst
graduate course; my parents, for always believing in me and letting me know; and my
daughter, who helped me to get through graduate school by reminding me what’s really

important in life.

I am very thankful for those in my lab who helped me with a variety of tasks: Dr. Yu-
Wen Chung-Davidson, who taught me a number of laboratory techniques; Sawyer
Pouliot and Nancy Carlisle, for their help with maternal behavior video analysis; and
Sarah Paxton, for her assistance with daily laboratory activities. I would like to thank
Casey Henley for being my office mate and lab mate for 6 years, and all that that entails,

and for teaching me how to properly throw a softball; and Jamie J ohansen, for

everything.

iv

I would also like to thank everyone in the Zoology Department and Neuroscience
Program for their support; the Graduate School for my Food, Nutrition, and Chronic
Disease Dissertation Completion Fellowship; and the Biological Sciences Department for
employing me for many semesters. My research was supported by a NIEHS Superfund

grant (P42ESO4911) and a NSF grant (IBN9728883) to L. Clemens.

TABLE OF CONTENTS

LIST OF FIGURES ....................................................................................................... ix
LIST OF TABLES ......................................................................................................... xii
CHAPTER ONE: INTRODUCTION .......................................................................... 1
PCBs — The Nature of the Problem ..................................................................... 1
PCBs — The Intracellular Mechanism .................................................................. 3
PCBs and Human Exposure ................................................................................. 4
Using Animal Models to Evaluate the Effects of PCBs ...................................... 7
Effects of PCBs Using Animal Models ............................................................... 8
The Importance of Maternal Behavior in the Development of the Offspring ..... 11
Objectives ............................................................................................................ 12
CHAPTER TWO: EXPERIMENTATION WITH ORAL ADMINISTRATION OF
PCBs ............................................................................................................................... 13
Abstract ................................................................................................................ 13
Introduction .......................................................................................................... 14
Experiment One: PCBs, A Pilot Study ................................................................ 16
Experimental Design ................................................................................ 16
Results ...................................................................................................... 16
Discussion ........................................................................................... 17
Experiment Two: Subcutaneous Administration of PCB 77 to Pregnant Rats and
Consumption of an Untreated Cookie ................................................................. 19
Methods ............................................................................................................... 20
Animals and Housing ............................................................................... 20
Procedure ................................................................................................. 20
Results .................................................................................................................. 21
Discussion ............................................................................................................ 23
Experiment Three: Oral Administration of PCB 77 to Non-pregnant Rats ......... 24
Methods ............................................................................................................... 25
Animals and Housing ............................................................................... 25
Procedure ................................................................................................. 25
Results .................................................................................................................. 26
Discussion ............................................................................................................ 29
General Conclusions ............................................................................................ 31

CHAPTER THREE: HOW DOES PCB 77 (2 mg/kg b.w.) ON GESTATION DAYS
6 - 18 AFFECT THE MATERNAL CARE OF A RAT WHEN UTILIZING A

CROSS-FOSTERING DESIGN? ................................................................................. 32
Introduction .......................................................................................................... 32
Methods ............................................................................................................... 34

vi

Procedure ................................................................................................. 34

Analyses ................................................................................................... 36
Results .................................................................................................................. 37
Discussion ............................................................................................................ 48

Comparison of PCB Treatments with Controls ....................................... 48

Cross-fostering Analysis: Developmental Outcomes .............................. 49

Cross-fostering Analysis: Maternal Behavior .......................................... 50

CHAPTER FOUR: HOW DOES AN INTERMEDIATE DOSE OF PCB 77
AFFECT MATERNAL BEHAVIOR WHEN USING THE CROSS-FOSTERING

DESIGN? ........................................................................................................................ 56
Abstract ................................................................................................................ 56
Introduction .......................................................................................................... 57
Methods ............................................................................................................... 59

Animals and Housing ............................................................................... 59
Procedure ................................................................................................. 60
Analyses ................................................................................................... 61
Results .................................................................................................................. 62
Discussion ............................................................................................................ 73
Cross-fostering Analysis: Developmental Outcomes .............................. 73
Cross-fostering Analysis: Maternal Behavior .......................................... 74
Summary .................................................................................................. 81

CHAPTER FIVE: DOES THE SEXUAL BEHAVIOR OR PARTNER
PREFERENCE OF PRE- AND/OR POSTNATALLY-EXPOSED PCB PUPS
DIFFER FROM CONTROLS? IF SO, ARE THESE DIFFERENCES RELATED
TO THE ALTERED MATERNAL CARE EXHIBITED BY THE EFFECTD

DAMS? ............................................................................................................................ 83
Abstract ................................................................................................................ 83
Introduction .......................................................................................................... 84
Methods ............................................................................................................... 86

Animals and Housing ............................................................................... 86
Hormone Treatments ............................................................................... 87
Testing Procedure .................................................................................... 88
Partner Preference Tests .......................................................................... 88
Male-Paced Sexual Behavior Tests ......................................................... 89
Female-Paced Sexual Behavior Tests ...................................................... 90
General Analyses ..................................................................................... 91
Partner Preference Analyses .................................................................... 92
Sexual Behavior Analyses ....................................................................... 92
Results .................................................................................................................. 93
Male Partner Preference ........................................................................... 93
Female Partner Preference ....................................................................... 95
Male-Paced Sexual Behavior ................................................................... 100
F ernale-Paced Sexual Behavior ............................................................... 100
Discussion ............................................................................................................ 103

vii

Male Partner Preference ........................................................................... 103

Female Partner Preference ....................................................................... 104

Male-Paced Sexual Behavior ................................................................... 108

Female-Paced Sexual Behavior ............................................................... 110

Summary .................................................................................................. 112

CHAPTER SIX: GENERAL DISCUSSION ............................................................... 113
APPENDICES ................................................................................................................ 117
Appendix A .......................................................................................................... 118
Appendix B .......................................................................................................... 120
Appendix C .......................................................................................................... 121
Appendix D .......................................................................................................... 123
REFERENCES ............................................................................................................... 124

viii

LIST OF FIGURES

Figure l. The percent of ol-treated cookies eaten by pregnant rats on gestation day 6 —
18. The AN OVA did not detect any signiﬁcant main effects of subcutaneous PCB
treatment or interactions .................................................................................................. 22

Figure 2. The percent of oil- or PCB-treated cookies eaten by non-pregnant rats on
treatment days 1 — 13. The ANOVA detected a signiﬁcant interaction between PCB
treatments and days, and further analysis revealed a simple effect of treatment on days 3
— 13. ................................................................................................................................. 27

Figure 3. Top: A comparison of the oil-only group against the combined PCB groups
found a signiﬁcant difference (*) in the number of pups present on PND 6, with fewer
pups surviving in the combined PCB group. Bottom: A comparison of the four treatment
groups using a one-way ANOVA revealed a main effect of treatment on pup survival; the
groups sharing the same letter are not signiﬁcantly different from one another. For this
and all the other ﬁgures see the text for details of statistical analysis. ............................ 38

Figure 4. Top: A comparison of the oil-only group against the combined PCB group
found a signiﬁcant difference (*) in the average amount of weight that the pups gained
since PND 0, with less weight gained by the combined PCB group. Bottom: The
ANOVA of the data ﬁ'om the four treatment groups did not ﬁnd a signiﬁcant main effect
of treatment for average amount of weight gained by the pups ....................................... 39

Figure 5. Top: The difference between percent of time engaged in maternal
autogrooming between the oil-only group and the combined PCB group missed statistical
signiﬁcance (p = 0.06). Bottom: The ANOVA of the data ﬁom the four treatment groups
found a signiﬁcant main effect of treatment on maternal autogrooming; the groups
sharing the same letter are not signiﬁcantly different ﬁom each other. .......................... 41

Figure 6. Top: A comparison of the oil-only group against the combined PCB group
found a signiﬁcant difference (*) in the amount of licking and grooming directed to the
litter, with enhanced pup grooming seen in the combined PCB group. Bottom: The
ANOVA of the data from the four treatment groups did not ﬁnd a signiﬁcant main effect
of treatment (p = .08). ...................................................................................................... 43

Figure 7. Top: A comparison of the oil-only group against the combined PCB group
found a signiﬁcant difference (*) in the number of nursing bouts displayed by the dams,
with more bouts seen in the combined PCB group. Bottom: The ANOVA of the data
from the four treatment groups found a signiﬁcant main effect of treatment on the number
of nursing bouts; the groups sharing the same letter are not signiﬁcantly different ﬁom
each other. ........................................................................................................................ 44

Figure 8. Top: A comparison of the oil-only group against the combined PCB group
found a signiﬁcant difference (*) in the display of the high crouch nursing position, with
the PCB group showing less high crouch nursing. Bottom: The ANOVA of the data from
the four treatment groups found a signiﬁcant main effect of treatment on the display of
high-crouch nursing; the groups sharing the same letter are not signiﬁcantly different
from each other. ............................................................................................................... 45

Figure 9. Top: A comparison of the oil-only group against the combined PCB group
found a signiﬁcant difference (*) in the amount of time the darn spent on the nest, with
more time on the nest seen in the combined PCB group. Bottom: The ANOVA of the
data from the four treatment groups found a signiﬁcant main effect of treatment on time
spent on the nest; the groups sharing the same letter are not signiﬁcantly different from
each other. .................... 47

Figure 10. Top: A comparison of the oil-only group against the combined PCB group
found a signiﬁcant difference (*) in the number of pups present on PND 6, with fewer
pups surviving in the combined PCB group. Bottom: A comparison of the four treatment
groups using a one-way ANOVA revealed a main effect of treatment on pup survival; the
groups sharing the same letter are not signiﬁcantly different from each other ............... 63

Figure 11. Top: A comparison of the oil-only group against the combined PCB group
found a signiﬁcant difference (*) in the average amount of weight that the pups gained
from PND 0 to PND 6, with less weight gained by the combined PCB group. Bottom:
The ANOVA of the data from the four treatment groups revealed a signiﬁcant main
effect of treatment; groups with the same letter are not signiﬁcantly different from each
other. ................................................................................................................................ 64

Figure 12. Top: A comparison of the oil-only group against the combined PCB group
did not ﬁnd a signiﬁcant difference in the amount of time the dam spent on the nest (p =
0.55). Bottom: The ANOVA of the data from the four treatment groups found a
signiﬁcant main effect of treatment on time spent on the nest; the groups sharing the
same letter are not signiﬁcantly different from each other. ............................................. 66

Figure 13. Top: A comparison of the oil-only group against the combined PCB group
did not detect a signiﬁcant difference in the amount of pup-directed licking and grooming
(LG; p = 0.46). Bottom: The ANOVA of the data from the four treatment groups
revealed a signiﬁcant interaction between treatment and postnatal day for LG; groups
sharing the same letter are not signiﬁcantly different from each other. .......................... 67

Figure 14. A comparison of the oil-only group against the combined PCB group found a
signiﬁcant interaction between treatment and day for the amount of time the dam spent in
high crouch. Analysis of the simple main effects revealed a signiﬁcant difference (*) in
the display of high crouch nursing on PNDs 4 and 6, with the PCB group exhibiting less
high crouch nursing. Bottom: The ANOVA of the data from the four treatment groups
found a signiﬁcant main effect of treatment on the display of high-crouch nursing; the
groups sharing the same letter are not signiﬁcantly different from each other. .............. 69

Figure 15. Top: A comparison of the oil-only group against the combined PCB group
found a signiﬁcant difference (*) in the amount of time the dam engaged in ventral
maternal autogrooming, with an increase in ventral autogrooming seen in the combined
PCB group. Bottom: The ANOVA of the data ﬁ‘om the four treatment groups found a
signiﬁcant interaction between treatment and day for time spent autogrooming ventrally;
the groups sharing the same letter are not signiﬁcantly different from each other .......... 71

Figure 16. Top: The difference between percent of time spent nursing for the oil-only
group and the combined PCB group missed statistical signiﬁcance (p = 0.79). Bottom:
The ANOVA of the data from the four treatment groups found a signiﬁcant main effect
of treatment on time spent nursing; the groups sharing the same letter are not signiﬁcantly
different from each other. ................................................................................................ 72

Figure 17. A graph illustrating the differences found with the post hoc analysis that
followed the signiﬁcant interaction between treatment and week for percent of time males
spent in the female chamber. There were no signiﬁcant differences during the ﬁrst
partner preference test. During the second test, there was a signiﬁcant effect of
treatment; the groups sharing the same letter are not signiﬁcantly different from each
other. ................................................................................................................................ 94

Figure 18. A comparison of the combined PCB groups to the oil only group found a
signiﬁcant difference (*) in the amount of time females spent with the stimulus female,
with the PCB- exposed animals spending more time in the female chamber than the oil
animals. ............................................................................................................................ 96

Figure 19. A comparison of the combined PCB groups to the oil only group found a
signiﬁcant difference (*) in the amount of time females spent with the stimulus male,
with the PCB- exposed animals spending less time in the male chamber than the oil
animals. ............................................................................................................................ 97

Figure 20. The ANOVA of the data examining the four treatment groups found a
signiﬁcant main effect of treatment on percent of time females spent with the stimulus
female; the groups sharing the same letter are not signiﬁcantly different ﬁom each other.
......................................................................................................................................... 98

Figure 21. The ANOVA of the data examining the four treatment groups found a
signiﬁcant main effect of treatment on percent of time females spent with the stimulus

male; the groups sharing the same letter are not signiﬁcantly different from each other.
......................................................................................................................................... 99

xi

LIST OF TABLES

Table 1. Comparisons of treatment groups for the amount of PCB-laced cookie

consumption by day ......................................................................................................... 28

Table 2. Exposure to PCBs and male sexual behavior ................................................... 101
Table 3. Exposure to PCBs and female sexual behavior ............................................... 102
Table 4. Number of dams responding maternally .......................................................... 123

xii

Chapter 1. Introduction
PCBs — The Nature of the Problem

Polychlorinated biphenyls do not btun and are nearly indestructible — characteristics that
originally made them perfect for use in transformers and insulators, coolants and
hydraulic fuels. These same characteristics are why they are considered to be one of the
worst environmental villains of the twentieth century. Thirty years aﬁer their
manufacture was banned, polychlorinated biphenyls (PCBs) continue to move throughout
the world on winds, waves and rivers and threaten the destruction of entire populations of
humans as well as non-humans. The global pattern of distribution of PCBs is so dynamic
and unceasing that regions of the world far ﬁ'om where PCBs were used are suffering the

most damage -— damage that continues to worsen with every year.

The safe disposal of these chemicals is a persisting challenge. Thousands of old
transformers still exist today, harboring more than 100 million pounds of PCBs. All it
takes is for a ﬁre to erupt near one of these transformers, and poisonous chemicals spray
into the air and leak into the pavement. The molecules seep into our groundwater or
catch a ride on the closest gust of wind and are lifted up into the atmosphere where they
can travel hundreds of miles. When the air cools, the droplets condense and fall to the
ground where they can move easily with the currents. The cycle continues until the

contaminants reach a cold place where they can rest in liquid form. As it turns out, this

place happens to be one that is considered to be one of the most untouched, pristine

places on Earth — the Arctic.

An estimated 67 tons of PCBs arrive in the Arctic every year where about two-thirds
remains. PCBs are lipophilic (i.e. accumulate in adipose tissue) and work their way up
the food chain by magnifying in concentration as they get closer to the top. The people
of the Arctic are most vulnerable not only because where they reside is a virtual dumping
ground for the PCBs, but also because of their place at the top of the world’s dietary
hierarchy. They sustain themselves on sea otters and whale, animals that are themselves
near the top of the food chain and have a much higher concentration of PCBs compared
to land-raised animals such as beef and poultry. Because of this, Inuit women carry such
large loads of these chemicals that their bodies and breast milk could be classiﬁed as
hazardous waste, and children of the Arctic suffer extraordinarily high rates of infectious
diseases such as ear infections that recur so often that they cause permanent hearing loss

(Cone, 2005).

The detrimental effects of PCBs on these people are a clear concern and illustrate that no
one is safe from these toxins. According to Sheila Watt-Cloutier, chair of the Inuit
Circumpolar Conference, “the Arctic is the barometer of the health of the planet, and if

the Arctic is poisoned, so are we all” (Cone, 2005).

PCBs — The Intracellular Mechanism

There are 209 different PCB congeners that vary both in the number and location of
chlorine molecules. According to Safe (1990), the most toxic type of PCBs have a
coplanar, non-ortho-substituted structure, and are similar to 2, 3, 7, 8-tetrachlorodibenzo-
p-dioxin (TCDD; Safe, 1990). While PCB has recently been shown to directly activate
estrogen receptor (ER) alpha (Abdelrahim et al., 2006), most of the toxic and biological
effects of PCBs are mediated by the aryl hydrocarbon receptor (AhR) (Safe et al., 1998;
Mimura and Fujii-Kuriyama, 2003). The AhR is an intracellular cytosolic protein that is
ligand-activated and binds with high afﬁnity to TCDD or related compounds (i.e.
coplanar PCBs; Safe et al., 1998). After it is activated by the ligand, the AhR dissociates
from its chaperone complex and moves into the nucleus, where it binds to the AhR
nuclear translocator (ARNT) and then to the DNA recognition site where it can regulate
the transcription of a number of genes. Currently, there are no known endogenous

ligands for the AhR (Pravettoni et al., 2005).

TCDD and structurally-related compounds are considered to be endocrine disruptors.
They have the ability to modulate several endocrine response pathways by inducing AhR
gene expression and inhibiting estrogenic functions and ER-mediated signaling (Safe et
al., 1998). For example, AhR-induced gene expression has been shown to decrease ER
levels (Mortensen et al., 2006), inhibit cell proliferation and gene expression in breast,
endometrial and ovarian cancer cells (Safe et al., 2003), and induce germ cell apoptosis

and diminish ovarian follicular reserves (Franczak et al., 2006). The mechanisms by

which the AhR mediates changes in E-induced responses appear to be cell- and context-

speciﬁc.
PCBS and Human Exposure

PCBs cause a variety of neurological and behavioral deﬁcits in both naturally-exposed
humans and laboratory-exposed animal models. The earliest studies that examined the
effects of PCBs on neurological, behavioral and developmental measures in humans took
place in the 197 Os aﬁer two mass outbreaks of chlorobiphenyl poisoning. In 1968, the
ﬁrst mass outbreak occurred in Japan when rice oil used for cooking was contaminated
with Kanechlor 400, the main component of which is tetrachlorobiphenyl. Neurological
examination of some of the affected individuals revealed symptoms of sensory
neuropathy (e. g. numbness, pain, hypoesthesia, or areﬂexia of limbs) as well as reduced

sensory nerve conduction velocity (Murai and Kuroiwa, 1971).

The second outbreak occurred in Taiwan in 1979 when cooking oil was contaminated
with thermally degraded PCBs. Because these chemicals persist in human tissue for
years and offspring of exposed females continue to be born affected, Rogan et a1.
performed a ﬁeld survey of all living children who were born during or immediately after
the period of contamination (Rogan et al., 1988). In addition to physical abnormalities
such as reduced height and weight, hyperpigrnentation and an increased rate of
bronchitis, the exposed children demonstrated signiﬁcant delays in cognitive

development. For example, the exposed children were delayed compared to controls in

the age at which they performed tasks such as saying phrases, carrying out requests,
pointing to body parts or catching a ball. The exposed children also scored lower than
controls on developmental and cognitive tests (i.e. the Bayley Scale of Infant
Development, Stanford-Binet IQ test, and the WISC IQ test), and performed worse on the

behavioral assessment test (Rutter Scale).

Examinations of children exposed to PCBs in the United States via maternal consumption
of contaminated ﬁsh report similar detrimental ﬁndings. Gladen et al. (Gladen et al.,
1988) followed a cohort of children in North Carolina and found that infants 6 and 12
months of age exhibited lower psychomotor scores on the Bayley Scale after increased
transplacental PCB exposure. A similar study reports hypotonicity and hyporeﬂexia in
infants exposed to high PCB levels in breast milk (Rogan et al., 1986). Jacobson et al.
reported that Michigan children whose mothers consumed PCB-contaminated Great
Lakes Whiteﬁsh demonstrated various reﬂexive and motoric abnormalities similar to
those described above in Rogan et al., and that the children were also slower to
accommodate to novel visual stimuli at 7 months of age (Jacobson et al., 1984). When
assessed at 4 years of age, these children exhibited poorer short-terrn memory on verbal
and quantitative tests (Jacobson et al., 1990). More recently, attention deﬁcit
hyperactivity disorder (ADHD) has been indicated as one of the effects of developmental

exposure to PCB-like environmental contaminants (Schettler, 2001; Hardell et al., 2002).

PCB-altered male fertility has caused considerable global scientiﬁc and public concern.

Populations of men from Sweden (Richthoff et al., 2003) to Denmark (Andersen et al.,

2000) to the Midwestern (Swan et al., 2003) and Eastern (Hauser et al., 2002) United
States have experienced a decrease in sperm concentration and motility. In addition, a
Swedish report revealed a strong association between the level of PCB in their mothers’
blood and incidences of testicular cancer in men (Hardell et al., 2003). Not only are men
affected who have been directly exposed to the toxins, but a recent report has
demonstrated a transgenerational effect (i.e., germline transmission to multiple
generations) in males through the F4 generation after perinatal exposure of the males to

endocrine disruptors (Anway et al., 2005).

Additionally, while occupational exposure to PCBs has become less common, the
possibility for exposure still exists for certain individuals. For example, ﬁremen called to
a ﬁre in a Binghamton, New York, ofﬁce building were exposed to PCBs after a
transformer exploded and PCBs were sprayed into the air (Kilburn et al., 1989). The men
were exposed via contact with the skin and inhalation of the smoke and gases. All of the
men reported extreme fatigue, headache, muscle weakness and aching joints, while a
portion of the ﬁreﬁghters reported memory loss, irritability, insomnia, and an inability to

concentrate.

Studying neurological and behavioral deﬁcits in humans exposed to PCBs is challenging
for a variety of reasons. The number of occurrences, lack of data on affected subjects and
confounding variables are only a few of the problems associated with this type of

research. In light of these difﬁculties, many scientists have turned to utilizing animal

models to assess the effects of PCB exposure on a variety of developmental, neurological

and behavioral measures.

Using Animal Models to Evaluate the Eﬂects of PCBs

Experimentation with animal models allows for greater ﬂexibility than using humans to
evaluate the effects of PCBs. For example, examining the effects of PCBs on
developmental measures often requires that the animals be exposed to the toxins both
pre- and postnatally, due to the nature of the measures that are being examined. This is
accomplished by treating the female for varying lengths of time prior to and/or during
pregnancy and lactation, and examining the offspring. The duration of PCB exposure can
vary from a single dose of PCB during pregnancy (F aqi et al., 1998) to treatment that
begins prior to mating and stops at parturition (Hany et al., 1999a) or continues
throughout lactation (Rice and Hayward, 1999). Other experiments treat the dams for an
intermediate amount of time — three days (Gupta, 2000) or 13 days (Wang et al., 2002)
during pregnancy. Although the above mentioned paradigms differ in length of
treatment, it is important to note that because of the toxins’ ability to reach a developing
fetus in utero (Kodama and Ota, 1980) and their afﬁnity for accumulating in adipose
tissue (Heinrich-Hirsch et al., 1997), the offspring of exposed mothers are likely to be
directly affected by the PCB both during gestation and via lactation in all cases, albeit to

different degrees.

An additional beneﬁt of using animal models to learn about the effects of PCBs is the
freedom to manipulate the type of exposure that the animals receive (i.e. the speciﬁc PCB
congener(s) to which the animals are exposed). Generally, experiments can be separated
into those that evaluate the effects of PCB mixtures and those that examine a single
congener. One beneﬁt of utilizing mixtures is being able to assess the effects of
biologically relevant mixtures, such as those that are found in the environment (J onsson
et al., 1976; Seegal et al., 1991; Chung et al., 2001) and in human breast milk (Lilienthal
et al., 2000; Kaya et al., 2002). However, this method comes with its disadvantages. For
example, using mixtures does not allow for isolation of the effects of speciﬁc toxins, and
can lead to results in which the congeners that comprise the mixture act synergistically to
produce effects that cannot be achieved when using single congeners. In some cases, the
results may be synergistic and some compounds may amplify the effects of others. Thus,
experiments will examine either PCB mixtures or single congeners based on the speciﬁc

goals of the study that is being conducted.
Eﬂects of PCBs Using Animal Models

Regardless of the length or type of exposure, utilizing animal models to evaluate the
effects of PCBs has led to a wealth of literature revealing a variety of deﬁcits caused by
the contaminants. In rats, PCB exposure has been shown to reduce weight gain from
birth to weaning in exposed offspring (Rice and Hayward, 1999), increased ano-genital
distance in female (Wang et al., 2002) and male (Faqi et al., 1998) neonates, reduce

testes weights and serum testosterone levels and increase uterine weights (Hany et al.,

1999b) and reduce daily sperm production (Faqi et al., 1998). Neurological effects
resulting ﬁom PCB exposure include increased (Seegal etal., 1997; Roth-Harer et al.,
2001) and decreased (Seegal et al., 1997; Seegal et al., 1986; Seegal et al., 1994; Seegal
et al., 1998) dopamine concentrations in the brain depending on the type of PCB that is
used and brain areas that are examined, and decreased thyroid hormone levels (Seo et al.,

1995: Lilienthal et al., 1997; Rice and Hayward, 1999).

The behavioral effects of PCB exposure are equally numerous. Monkeys exposed to
PCB mixtures exhibited deﬁcits in spatial learning and memory (Schantz et al., 1991;
Rice and Hayward, 1997). Experimentation with rats has revealed a feminization in
sweet preference behavior in adult males (Hany et al., 1999a; Kaya etal., 2002), as well
as an alteration in male sexual behavior (Faqi et al., 1998) and female sexual behavior
and receptivity (Wang et al., 2002; Chung and Clemens, 1999; Chung et al., 2001 ).
PCB-exposed offspring have also showed impairments in passive avoidance behavior as
adults (W einand-Harer et al., 1997). Finally, and perhaps most important for this
dissertation, PCB exposure during gestation altered the display of various measures of
maternal behavior in rats (Simmons et al., 2005). Subcutaneous (sc) treatment with PCB
77 (3, 4, 3’, 4’-tetrachlorobiphenyl) on gestation days (GD) 6 — 18 decreased the amount
of time spent in high crouch nursing, altered the amount of time the dams spent on the

nest, and increased pup mortality.

In addition to the concern for organisms that have been exposed directly by consumption,

offspring of exposed individuals are at particular risk for being affected by maternal PCB

exposure both in utero and via lactation because of the toxin’s ability to enter the
placenta and its affinity for adipose tissue. Examining organisms that have been exposed
perinatally is particularly important considering the amount of neurological and

physiological development that takes place at this time.

However, exploring the effects of contaminants on individuals who have been exposed
via maternal treatment is confounded by the dynamic relationship between mother and
offspring. The traditional experimental paradigm used to evaluate the effects of a
contaminant involves treating a pregnant animal and examining her offspring for
subsequent effects. Rarely are the possibilities that the dam’s exposure to the
contaminant could have altered the care she provides to the offspring, or that her behavior
may change when caring for affected pups. It is not unusual for a dam to adjust her
behavior according to the behavior and physiology of her offspring (Stern and Keer,
2002; Stern and Johnson, 1990; Stern and Lonstein, 1996; Moore, 1982; Leigh and
Hofer, 1973). A change in the dam’s care can signiﬁcantly affect the developing pups
(Moore et al., 1992; Moore, 1992; Liu et al., 2000; Caldji et al., 1998; Francis et al.,
1999) — effects that may be mistakenly attributed to the direct effects of the toxin on the
offspring. It is for these reasons that maternal care must also be evaluated when

examining the effects of toxin on offspring via maternal exposure.

10

The Importance of Maternal Behavior in the Development of the Oﬁfspring

Maternal care has a major impact on the physiological and behavioral development of the
offspring. Since the 1960s scientists have noted the importance of infant stimulation and
its enduring effect on offspring (Denenberg, 1969; Levine and Thoman, 1969). One of
the most-studied behaviors, and arguably one of the most important behaviors, exhibited
by a newly parturient dam is pup-directed licking and grooming (LG). Natural variations
in LG exist among dams, with a percentage of the dams licking their offspring
signiﬁcantly more or less frequently than most dams. This difference is most prominent
within the ﬁrst postnatal week, especially on postnatal days 2 - 5 (Champagne et al. ,
2003). A large amount of pup-directed LG is directed toward the pups’ ano-genital
region. This type of LG is distributed unequally between a dam’s offspring with males
receiving more attention than their female littermates (Moore and Morelli, 1979), and is
mediated by testosterone (Moore, 1982). Pup-directed LG has been shown to be
involved in multiple aspects of the offsprings’ development such as “programming” their
hypothalamic-pituitary-adrenal response to stress (Liu et al., 1997), affecting the number
of motor neurons in a sexually dimorphic nucleus of the spinal cord (Moore et al., 1992),
altering male sexual behavior (Moore, 1984) and female reproductive success (Gomes et
al., 1999), and changing gene expression by altering DNA methylation and chromatin

structure in the offsprings’ epigenome (Weaver et al., 2004).

Taking into account the considerable role maternal care plays in the development of the

offspring, the ﬁnding of Simmons and colleagues that maternal behavior is altered after

11

exposure to polychlorinated biphenyls has enormous implications for offspring reared by
PCB-exposed dams. Further, as mentioned above, the offspring of exposed mothers are
likely to be directly affected by the PCB both during gestation and via lactation. This
may result in behavioral and/or physiological changes in the litter, leading to further

alterations in the dam’s behavior as she responds to affected pups.

Objectives

This dissertation will begin by addressing the feasibility of using an oral route of
administration to examine the effects of PCB 77 on maternal behavior in Long Evans

rats. Next, this paper will examine the effects of 2 mg/kg PCB 77 on various measures of
maternal care and early offspring development. Subsequently an intermediate dose of
PCB 77 will be examined to replicate ﬁndings and expand the behavioral analysis of the
previous experiment. Finally, this dissertation will evaluate the effects of perinatal PCB
exposure on offspring sexual behavior and partner preference. This paper will end with a
discussion of the implications of this research with regards to the direction of future
experiments and limitations of the original experimental paradigms used to evaluate the

effects of contaminants on exposed offspring.

12

Chapter 2. Experimentation with oral administration of PCB 77

ABSTRACT

Polychlorinated biphenyls (PCBs) are environmental toxins known to affect
neurobehavioral development in many laboratory studies using different animal models.
Because of their bioaccumulation and long half-life PCBs are a serious concern for our
own species. Recently, a study was conducted in our laboratory that examined the effects
of the dioxin-like PCB congener 3, 4, 3’, 4’- tetrachlorobiphenyl (PCB 77) on maternal
care in rats using a subcutaneous dose of PCB during the animals’ gestation period.
However, since human exposure typically occurs via food consumption, we decided to
examine the possibility of using an oral route of administration to investigate the effects
of PCB on maternal behavior. To this end, we conducted a series of experiments to test
the feasibility of treating pregnant rats orally with PCB 77. In the ﬁrst experiment,
pregnant rats were treated with PCB- or oil-laced vanilla wafers on gestation days (GD) 6
- 18. The majority of dams did not consume the wafer for the duration of the
experiment, so the second and third experiments were conducted to test if the animals
were developing a taste aversion to the wafers. In the second experiment, pregnant rats
were given an untreated vanilla wafer after being injected subcutaneously with either oil
or PCB 77 in the dose of 2 or 4 mg/kg. There was no signiﬁcant effect of treatment on
cookie consumption in any of the groups. Thus, subcutaneous administration of the PCB
did not support the development of a taste aversion to the wafer. In the third experiment

non-pregnant rats were given one-half of a vanilla wafer treated as in the ﬁrst experiment

13

with either oil or 2 or 4 mg/kg PCB 77 emulsiﬁed in oil. The animals in the oil group
consumed all or the majority of each cookie. The animals in the 2 mg/kg group began to
eat the cookies at the start of the experiment but stopped within a week, whereas the
females in the 4 mg/kg group never ate the cookies. Overall, the results indicate that
voluntary oral administration of PCB 77 is not an option for the study of effects of this
contaminant on maternal behavior, since as the animals develop an aversion to the

cookie, they discontinue or reduce their exposure to the PCB.

INTRODUCTION

Previously, the effects of PCB on maternal behavior were examined by our group after
subcutaneous (sc) injection of PCB 77 (Simmons et al., 2005). However, PCB exposure
in humans occurs during food consumption as a result of the toxin’s accumulation in the
food chain, and it is a concern whether the effects with so administration rrrirror those that
are experienced after oral ingestion. Therefore, it is important to explore the possibility
of studying the effects of PCB 77 on maternal behavior using an oral route of

administration.

Different methods have been employed to administer PCB orally to animals. A number
of experiments have used a gavage technique, in which the PCB is emulsiﬁed in oil and
force-fed to the animal through a tube inserted orally into the animal’s esophagus
(Crofton et al., 2000; Agrawal et al., 1981; Widholm et al., 2001; Faqi et al., 1998;

Schantz et al., 1996). However, this method may put the animal under a great deal of

14

stress, which could also signiﬁcantly affect maternal behavior (Moore and Power, 1986;
Smith et al., 2004) or modify the physiology and/or behavior of the pups that were
exposed to the stress in utero (Ward and Weisz, 1980; Vom Saal et al., 1990; Griffin et
al., 2003; Weinstock, 1997; Takahashi et al., 1992). Experiments have also presented the
toxin by lacing the animals’ daily food source with the PCB (Hany et al., 1999a; Kaya et
al., 2002) or by adding the oil-emulsiﬁed PCB to cookies or treats that were given to the
animals (Seegal et al., 1997; Rice and Hayward, 1999). Although both methods avoid
the stress of gavage, it is often difﬁth to be sure that each animal receives equivalent
doses of PCB when treating the animals’ food source with the toxin if all of the food is
not consumed. This becomes particularly problematic when investigating contaminants
that have estrogenic actions since estradiol is known to reduce food intake (see Wade and

Gray, 1979 for review).

In the interest of paralleling the route of exposure that occurs naturally in the human we
conducted a pilot study to determine the feasibility of using an oral route of
administration to evaluate the effects of PCB 77 on maternal care in rats. To this end we
followed procedures reported by Seegal et al. (1997) with slight modiﬁcations and

presented pregnant rats with cookies adulterated with PCB.

15

Experiment One: PCB Cookies, A Pilot Study

EXPERIMENTAL DESIGN

In order to compare the results of the current study with our earlier one that used sc PCB
administration (Simmons et al., 2005) the dams in this pilot study were treated on the
same gestation days (GDs) with the same doses of PCB. Thus, pregnant rats were fed
one-half of a vanilla wafer cookie treated with the corn oil vehicle, 2 mg/kg or 4 mg/kg
PCB 77 emulsiﬁed in corn oil on GDs 6 — 18. There were four dams in each treatment
group. Cookies were prepared the day before by dispensing the appropriate amount of
corn oil (with or without PCB) on to the wafers and placing them in the fume hood

overnight to allow for absorption of the oil/PCB mixture.

RESULTS

Early into the experiment, we noticed a few things. First, two of the twelve dams (both
from the 4 mg/kg group) never appeared to eat any of the cookies. The animals that did
eat the cookies (the remaining 10 animals) began doing so after one or two presentations.
Finally, regardless of dose, all the PCB-treated dams that began eating the cookies
stopped aﬁer a week. Thus, the only dams that consumed the cookies for the duration of

the experiment were those in the oil group.

16

DISCUSSION

It is unclear why our outcome differed from that of Seegal et al. (1997), the study from
which we derived our methods. However, there were some sigriﬁcant methodological
differences. Most notably is that the two studies used different strains of rat.

Seegal et al treated pregnant Sprague-Dawley rats with PCB while we used Long Evans.
These two strains exhibit a long history of signiﬁcant differences for a variety of
measures including likelihood of nicotine self—administration (Shoaib et al., 1997),
physiological responses to stress (Faraday et al., 2005) and length of time required prior
to pup sensitization (Stern, 1989), to name a few. In addition, while both strains of rat
are considered to be susceptible to the toxic effects of dioxin, the Long Evans strain is
known to be the most vulnerable (Long Evans, LD50 ~ 10 rig/kg; Sprague-Dawley, LD50
~ 50 ug/kg; Franc et al., 2001). Long Evans rats are often used when the purpose of an
experiment is to assess the effects of dioxin on a strain of rat that is highly susceptible to
TCDD (Unkila et al., 1994; Pohjanvirta et al., 1988; Pohjanvirta et al., 1993; Huuskonen
et al., 1994). Thus, since PCB 77 is a dioxin-like congener, it is not surprising that
pregnant rats from these two groups responded differently to ingestion of the PCB.
Additionally, Seegal et al. used a lower dose of PCB 77 (0.1 or 1 mg/kg) over a longer
period of time, and adjusted the dose of PCB that was applied to the cookie according to
weight twice weekly, as opposed to every day as was done in our experiment. This may
have decreased the actual amount of PCB the dams were consuming from the intended 1

mg/kg to a lower dose.

17

We were not surprised that many of the dams did not eat the cookie until the second or
third day as rats are extremely neophobic (Richter, 1953; Barnett, 1956) and will often
only sample a novel food item when it is ﬁrst presented (Rzoska, 1953) so that they can
“test” the safety of the novel food. What we did ﬁnd unusual, however, was that after the
dams appeared to overcome their neophobia and ate the cookies, many stopped after a
few days. These events suggested to us that the dams might have fallen ill as a result of
the ingested PCB and developed a taste aversion to the cookies. A learned taste aversion
occurs when an animal associates the ﬂavor of something it has eaten with an unpleasant
effect that occurs after consumption of the item, although the food may not have been
what directly caused the adverse effects. If consuming the PCB caused the dams to
become ill, they may have associated the sickness with the consumption of the cookie. A
taste aversion can often be learned in as little one trial (Garcia et al., 1966), but less toxic
stimuli may require up to four trials to be effective (Gomez, 2001). Thus, the dams could
have developed a taste aversion to the PCB-treated cookies if exposure to the toxin

caused them to become ill and they associated the sickness and the novel food.

To further investigate this hypothesis a second experiment with pregnant rats receiving sc

injections of PCB 77 was conducted.

18

Experiment Two: Subcutaneous Administration of PCB 77 to Pregnant Rats and

Consumption of an Untreated Cookie

The dams in the previous study may have stopped eating the vanilla wafers because they
became ill as a result of ingesting the PCB. However, they may have become sick simply
as a result of the presence of the toxin in their bodies, regardless of the route of
administration. If the dams became sick as a result of the presence of PCB, then we
would expect that dams receiving PCB sc should also become ill. If these dams are given
oil-treated vanilla wafers at the time of PCB injection, they may associate negative
effects of PCB exposure (e. g. becoming sick) with the novel food item and stop eating it.
On the other hand, if oral delivery of the toxin is required for the dams to become ill, then
sc injection will not cause the dams to become sick and they should continue to eat the
oil-treated cookie. In order to determine if the presence of PCB in the body will cause
the dams to become sick and develop a taste aversion to a novel food item, we treated
pregnant rats with PCB sc, fed them an oil-treated vanilla wafer, and monitored their
cookie consumption. Thus, we used the development of a taste aversion as a way to
gauge whether the dams experienced negative side effects of PCB exposure, such as

becoming ill.

19

METHODS

Animals and Housing

Fourteen timed-pregnant Long-Evans rats from Charles River Laboratories (Raleigh,
North Carolina) arrived at our laboratory on GD 5 and were housed singly in plastic
cages (45 X 22 X 21 cm) with wood shavings as bedding in a laminar-ﬂow unit

(NU AIRE). Animals were maintained on a 14:10-h lightzdark cycle with lights off at
1100 h and a dim red light on at all times. The ambient temperature was 21 +/- 1°C. The
rats were allowed free access to water and Harlan Rodent Chow 8640 food pellets. Our
animal facilities are accredited by the Association for Assessment and Accreditation of
Laboratory Animal Care, and the Michigan State University Animal Care and Use

Committee approved experimental procedures.

Procedure

The dams were weighed daily and given subcutaneous injections of the oil vehicle or
PCB 77 emulsiﬁed in corn oil in the dose of 2 or 4 mg/kg from GD 6 to GD 18. At this
time, dams were also given a half of a vanilla wafer onto which corn oil had been
dispensed in accordance with their weight from the previous day. Cookies were weighed
just prior to being placed in the nest and again after the testing period if any piece(s)
remained. The female was allowed 10 min to consume the cookie, which had been

shown in a previous experiment to be plenty of time for the animal to eat the entire wafer

20

(Rice and Hayward, 1999). The number of dams in each group was oil = 4, 2 mg/kg = 6,
4 mg/kg = 4. Nest building materials in the form of thirty brown paper towel strips were
placed on the wire top of each cage on GD 20. Parturition took place during the mid- to

late-light phase of the animals’ light/dark cycle on GD 22 - 23.

RESULTS

The 3 X 13 ANOVA (treatments X days, with repeated measures on the second factor)

analyzing the effect of subcutaneous PCB treatment on cookie consumption did not

detect any signiﬁcant main effects or interactions (Figure 1).

21

Percent of
Cookie Eaten

 

4o-

20-

 

 

 

 

6789101112

Gestation Day

Figure l. The percent of oil-treated cookies eaten by pregnant rats on

l3

14

15

l6 l7

 

l8

~Elou
- I2mg/kg
’I4mg/kg

 

gestation days 6 — 18. The ANOVA did not detect any signiﬁcant main

effects of subcutaneous PCB treatment or interactions.

22

DISCUSSION

Feeding oil-treated cookies to pregnant rats at the time of so PCB administration did not
cause the dams to stop eating the wafers. Thus, either so administration of the toxin did
not cause the dams to become ill, or the length of time between the effects of sc injection
of the PCB and the ingestion of the cookies was too long for the rats to form a learned
taste aversion to the cookie which was consumed immediately following the injection.
Vagal sensory ﬁbers reside in the gastro-intestinal tract and send information to the
nucleus of the solitary tract (Torvik, 1956; Kalia and Mesulam, 1980) where it converges
with taste information (Travers et al., 1987) to form a learned taste aversion. When the
toxin is injected, then perhaps the timing of convergence of information after tasting the
cookie and experiencing any putative negative effects of the PCB treatment is not
conducive to the development of a taste aversion, as is the case when the cookie and the
PCB are ingested together. The animal may become ill as a result of the PCB injection,
but may not recognize the cookie as the source of the illness if the length of time between
ingesting the cookie and feeling sick is too great. Alternatively, although the eventual
fate of PCB in the body is probably similar (i.e. accumulation in adipose tissue), after
different routes of administration the ﬁnal concentration of each metabolite and what
chemical changes the toxin undergoes prior to being fully metabolized are likely to be
very different with oral and so administration. This differential metabolism may account
for the different results seen with the two routes of administration examined. These two

hypotheses are not mutually exclusive.

23

Experiment Three: Oral Administration of PCB 77 to non-pregnant rats

Pregnant rats that were given oil-treated vanilla wafers at the time of so PCB
administration did not develop a taste aversion, that is, the females did not stop eating the
cookies. As mentioned above, so PCB administration either does not cause dams to
become physically ill, or the length of time between tasting the cookie and experiencing
the effects of the toxin are too great for the animal to make the association. It is also
possible that the effect we saw in the pilot experiment (that the dams stopped eating the
PCB-treated vanilla wafers) was due in part to the pregnant state of the females.

Estrogen has been known to modulate learning (Leuner et al., 2004) including the rate of
extinction of conditioned taste aversions (Yuan and Chambers, 1999). Estrogen levels in
female rats are relatively low early in the pregnancy (30 pg/ml; Bridges, 1984) but can
reach 110 pg/ml during the proestrus phase of the estrous cycle (Schwartz, 1974; Nequin
et al., 1975) in a cycling female. This may cause the pregnant and non-pregnant rats to
respond differently to the PCB-treated cookie, resulting in the non-pregnant females
eating the PCB-laced cookies even though their pregnant counterparts did not. To this
end, we examined the effects of PCB consumption in non-pregnant females to see if these
animals would react in the same way to the ingested toxin and develop a similar taste
aversion to the PCB-treated wafers. Thus, our next experiment involved treating non-

pregnant females with PCB-laced cookies.

24

METHODS
Animals and Housing

Fourteen 60-day-old Long-Evans rats from Charles River Laboratories arrived at our
laboratory and were housed according to the Methods described above in the Pilot

Experiment.
Procedure

The day the females arrived at our laboratory was denoted day 0 with treatment
beginning on day 1. This was done in order to keep as close as possible to the treatment
received by the pregnant rats in Experiments One and Two, which arrived at our
laboratory on GD 5 and began treatment on GD 6. Females were randomly assigned to
one of three different groups and received one half of a vanilla wafer on days 1 — 13 that
was treated with the PCB or oil according to her weight the previous day, as described
above in the Pilot Experiment. One group received a cookie treated only with the corn
oil vehicle, one with 2 mg/kg PCB 77 and one with 4 mg/kg PCB 77 emulsiﬁed in corn

oil. The number of animals in each group was oil = 4, 2 mg/kg = 6, 4 mg/kg = 4.

Cookies were also weighed just prior to being placed in the cage, as described above in

Experiment One. Most animals that ate the cookie did so immediately. Animals that did

25

not consume the cookie usually buried it in the bedding in a comer or sniffed it and
walked away. Cages were searched after the 10 min were up and any remaining pieces of
the cookie were weighed. The percentage of cookie that was consumed was calculated

and recorded.

RESULTS

Using a 3 X 13 ANOVA to analyze cookie consumption (treatments X days, with
repeated measures on the second factor) we detected a signiﬁcant interaction between
PCB treatments and days [F (24, 132) = 8.37, p < 0.0001; Figure 2]. Further analysis did
not reveal a simple effect of treatment on days 1 and 2, but it did on days 3 — 13. For
days 3 — 13, the simple effect of treatment was evaluated using Bonferroni t-tests for

independent samples with the signiﬁcance level held at .05/3, or P < 0.017.

Every day on which the oil group was signiﬁcantly different from a PCB group, the oil
group ate more of the cookie. On days 3 and 4, the groups differed in a dose-dependant
fashion, with the oil group eating more than the other two groups, and the 2 mg/kg group
eating more than the 4 mg/kg group. On the rest of the treatment days, the oil group
differed from the 2 and 4 mg/kg groups, with the females in the oil group eating more of
the cookies than the other two groups (See Table 1 for a summary of the comparisons of

treatment means for each day).

26

Percent of

Cookie Eaten

120

 

100-
30.

60-

5H
P1
:3—0
m
raj-C

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1m , > .

678910111213

 

 

Treatment Day

Figure 2. The percent of oil- or PCB-treated cookies eaten by non-pregnant
rats on treatment days 1 — 13. The ANOVA detected a signiﬁcant
interaction between PCB treatments and days, and further analysis revealed
a simple effect of treatment on days 3 - 13.

27

TABLE 1

 

Comparisons of treatment groups for the amount of
PCB-laced cookie consumed by day

 

 

 

Day Group Differences in Cookie Consumption
1 Not signiﬁcant
2 Not signiﬁcant
3 oil > 4 mg (t = 5.68, df= 6,p = 0.001)
2 mg > 4 mg (t = 3.80, df= 8,p = 0.005)
4 oil>2mg(t=6.12,df= 8,p=0.001)
oil > 4 mg (t = 10.75, df= 6,p < 0.001)
2 mg > 4 mg (t = 4.68, df= 8,p < 0.002)
5 oil>2mg (t=3.36,df= 8,p =0.010)
oil > 4 mg (t = 36.24, df= 6,p < 0.001)
6 oil>2mg(t=6.51,df= 8,p<0.001)
oil > 4 mg (t = 20.91, df= 6,p < 0.001)
7 oil > 2 mg (t = 3.91, df= 8,p = 0.005)
oil > 4 mg (t = 5.16, df= 6,p = 0.002)
8 oil > 2 mg (t = 8.95, df= 8,p < 0.001)
oil > 4 mg (t = 14.46, df= 6,p < 0.001)
9 oil>2mg (t= 15.60, df= 8,p <0.001)
oil > 4 mg (t = 21.27, df= 6,p < 0.001)
10 oil>2mg(t= 10.25, df= 8,p <0.001)
oil >4mg (t= 11.50, df= 6,p < 0.001)
11 oil > 2 mg (t = 92.59, df= 8,p < 0.001)
oil > 4 mg (t = 73.20, df= 6,p < 0.001)
12 oil>2mg(t=19.14,df= 8,p <0.001)
oil > 4 mg (t = 18.05, df= 6,p < 0.001)
13 oil > 2 mg (t = 30.36, df= 8,p < 0.001)
oil > 4 mg (t = 24.00, df= 6, p < 0.001)

 

28

DISCUSSION

Ingestion of PCB cookies by non-pregnant rats

One of the purposes for conducting Experiment Three was to determine if the dams in the
ﬁrst experiment stopped eating the cookies because of an interaction between the
pregnancy and the ingested PCB. The data from this experiment indicate that this was
not the case. The non-pregnant females in Experiment Three that received cookies
adulterated with 2 mg/kg PCB also stopped eating them after a few days. Thus, the fact
that the pregnant dams in the ﬁrst experiment stopped eating the cookies does not appear
to be related to their state of pregnancy (i.e. decreased estrogen levels or elevated levels

of progesterone).

Again, it is unclear why the animals in our experiment stopped eating the PCB-treated
cookies and those in Seegal et al. (1997) did not. As mentioned in the Discussion section
of the Pilot Experiment, they used the same PCB congener that we do here, although at a
lower dose (1 mg/kg/day) but over a longer period (GD 6 through weaning), and the most
likely reason for our conﬂicting results is that we used a different strain of rat than Seegal

et al.

Interestingly, the 2 and 4 mg/kg doses of PCB evoked different reactions. Although
dams from each of the three treatment groups (oil, 2 mg/kg and 4 mg/kg) exhibited a

species-typical neophobia at the beginning of the experiment and ate very little of their

29

cookies, the oil and 2 mg/kg groups ate signiﬁcantly more of their cookies than the 4

mg/kg group on the third and fourth days. From the ﬁfth day onward, both PCB-treated
groups ate signiﬁcantly less of their cookies than the oil group. While the females. in the
lower PCB group continued to eat the cookie for a few days before reducing the amount
that they consumed and eventually stopping altogether, the 4 mg/kg group ate very little

from the start and even after repeated presentations of the cookies.

The data obtained for the 4 mg/kg group are consistent with two different hypotheses.
First, it is possible that the 4 mg/kg PCB that was dissolved in oil and applied to the
cookie was in a concentration too high to remain ﬁrlly emulsiﬁed in the oil, leaving an
unfavorable tasting residue on the cookie. Alternatively, there is a chance that the
amount of PCB ingested ﬁom the sampling of the cookie was enough to cause the
females to experience adverse physical side effects and develop a taste aversion. The 4
mg/kg dose has been shown to decrease the viability of offspring and interfere with
delivery of the litter when administered sc, indicating a high degree of toxicity (Simmons
et al., 2005). However, those effects were exhibited after the full treatment of PCB over
13 gestation days. Therefore, it seems unlikely that the small amount of PCB consumed
by the females in the 4 mg/kg group would result in such a strong negative reaction and
cause the development of a taste aversion, indicating that the females in this group likely
stopped eating the adulterated wafers because of an unpleasant taste of the PCB. But, our

data do not allow us to differentiate between these two competing hypotheses at this time.

30

General Conclusions

For Long Evans rats, the use of a laced cookie to administer PCB 77 does not seem to be
a viable approach at least for the doses of interest for the research of this dissertation.
The data indicate that there are complex interactions between the dose of PCB and the
route of administration used to deliver the toxin (e. g. compare the results of the Pilot
Experiment and Experiment 2), but the systematic investigation of those interactions is
outside the scope of this project. Since forced ingestion of the PCB using the gavage
technique is clearly too stressful to use with pregnant animals (see Introduction), the
remainder of the experiments in this dissertation will focus on evaluating the effects of
PCB on maternal behavior using subcutaneous administration of the toxin in order to
ensure that the animals are receiving an accurate dose of PCB and to minimize stress
experienced by the dam. Previous experiments have successfully used a subcutaneous
route of administration to examine the effects of PCB on a variety of measures (Hany et
al., 1999a; Hany et al., 1999b; Weinand-Harer et al., 1997; Lilienthal et al., 1997; Roth-
Harer et al., 2001). In addition, Hany et al. (1999b) have shown that PCB 77

accumulates in brain and adipose tissue of dams and offspring after sc maternal exposure.

31

Chapter 3. How does PCB 77 (2 mg/kg b.w.) on gestation days 6 - 18 affect the

maternal care of a rat when utilizing a cross-fostering design?

INTRODUCTION

Polychlorinated biphenyls (PCBs) are persistent contaminants that remain in the
enviromnent because of their stable chemical structure and long half-life. Although they
were banned from use in the 19703, PCBs can still be found today in sediment, run-off
and wildlife, and have also been detected in human umbilical cord plasma and breast
milk (V reugdenhil et al., 2002; Mes and Marchand, 1987; Noren et al., 1990; Noren and
Lunden, 1991; Duarte-Davidson et al., 1992; Gonzalez et al., 1995). These ﬁndings
indicate a risk not only to adult individuals that are exposed to the toxicants, but also to
the more vulnerable developing organism exposed in utero and via lactation. As
discussed in the Introduction to this dissertation, studies examining humans that have
come in contact with PCBs (Rogan et al., 1988; Gladen et al., 1988; Dewailly et al.,
1991) and laboratory studies with various animal models (Hany et al., 1999a; Hany et al.,
1999b; Faqi et al., 1998; Seegal et al., 1997; Schantz and Widholm, 2001) have
demonstrated that PCB exposure is associated with both neurological and behavioral

deﬁcits.
In the present experiment we focus on the dioxin-like PCB congener 3, 4, 3’, 4’-

tetrachlorobiphenyl (PCB 77) because it has high toxic potency and afﬁnity for the Ah

receptor, has a low degradation rate in vivo, and it is among the most abundant PCB

32

congeners in human tissues (Safe, 1994). Further, in vivo and in vitro studies have shown
that PCB 77 has both estrogenic and anti-estrogenic activity (Seegal et al., 2005; Garner
et al., 1999; Nesaretnam et al., 1996; Jansen et al., 1993; and see Hansen, 1998 for
review). PCB 77 is detected in the brains of the developing offspring as well as in the
brains of the dams when administered to rats subcutaneously during pregnancy (Hany et
al., 1999b) thus, it could affect the central nervous system and behavior of both the
developing litter and the pregnant adult. Developmental effects of PCB 77 on the
behavior of male and female offspring are well-documented (Faqi et al., 1998; Chung et
al., 2001; Wang et al., 2002), and recently we reported an effect of this contaminant on
the maternal behavior of rats (Simmons et al., 2005). In addition to its possible
estrogenic and anti-estrogenic actions, under some conditions PCB 77 exposure enhances
central dopaminergic activity (Seegal et al., 1997; Seegal et al., 2005), and both estrogen
and dopamine play signiﬁcant roles in several aspects of maternal behavior in rats
(Fahrbach et al., 1985; Stern and Taylor, 1991). Recently, Seegal et al. (1995) have
shown that PCB 77 can increase preﬁ'ontal cortex DA by acting estrogenically,

demonstrating a direct link between PCB 77, estrogen and DA.

The few studies that have reported effects of environmental contaminants on maternal
behavior of mammals (mice, Palanza et al., 2002a; Palanza et al., 2002b; rats, Simmons
et al., 2005) have used experimental protocols in which both the pregnant animal and the
developing litter are exposed to the toxic agent. With that approach it is impossible to
differentiate between effects on maternal behavior that are the result of direct actions on

the dams’ endocrine and/or nervous systems from those that stem from indirect effects

33

mediated by changes in the physical and behavioral proﬁles of the exposed litter. Here
we use a cross-fostering design in an attempt to gain insights about the contributions of
direct and indirect effects of PCB 77 exposure on maternal behavior in rats. We also
investigated if the maternal behavior of dams that were exposed to PCB, or who raised
pups exposed to PCB, was different from that of dams that were never exposed to the

contaminant and raised unexposed litters.

METHODS

Procedure

The dams were weighed daily and given subcutaneous injections of the oil vehicle or 3,
4, 3’, 4’-tetrachlorobiphenyl (PCB 77; Accustandard, New Haven, CT; >99% pure as
determined by gas chromatography performed by the vendor) emulsiﬁed in corn oil in the
dose of 2 mg/kg from GD 6 to GD 18. Selection of this dose and treatment period was
based on the ﬁndings that a dose of 1.5 mg/kg PCB 77 on GD 7-18 resulted in detectable
but relatively low concentration of the contaminant in the maternal nervous system (Hany
et al., 1999b), as well as on the observation that the higher dose used here affects the
maternal behavior of dams treated during this gestational period (Simmons et al., 2005).
Nest building materials in the form of thirty brown paper towel strips were placed on the
wire top of each cage on GD 20. Parturition took place during the mid- to late-light
phase of the animals’ light/dark cycle on GD 22 - 23. On the day of birth [day of birth =

postnatal day (PND) 0], the litters were culled to 8 animals (4 females and 4 males) and

34

cross-fostered or left with their own mothers to create the following groups: (1) oil
treated dams with their own oil pups, (2) oil treated dams with a new litter of oil pups, (3)
PCB treated dams with their own PCB pups, (4) PCB treated dams with a different litter
of PCB-exposed pups, (5) PCB treated dams with a litter of oil pups, and (6) oil treated

dams with a litter of PCB-exposed pups. Each group contained six dams.

Dams and litters were videotaped on PND 1, 2, 4 and 6 for the animals’ last hour of light
and ﬁrst hour of dark (2 hr/day/animal). Litters were weighed every day after taping.
Videos were analyzed using The Observer 3.0 (N oldus Information Technology), a
behavioral data acquisition computer program. During review of the videotapes, the
following behaviors were recorded: time on the nest, licking and grooming of the pups,
maternal autogrooming, and amount of time spent nursing. Nursing behaviors included
resting, or supine nursing, in which the dam was lying on the pups or on her side, and two
quiescent nursing postures: low and high crouch. The low-crouch nursing posture is one
in which the dam becomes immobile in response to stimulation ﬁom the pups as they
ventrally root in order to attach to a nipple prior to nursing. High-crouch nursing can be
distinguished by a distinct splaying rigidity of the limbs and increased dorsal arching of
the dam’s back, a position that is exhibited in response to persistent stimulation from the
pups, such as continued rooting and nipple attachment. Total time spent nursing is the
total of all time spent in one of the three nursing postures while on the nest. The number
of nursing bouts, deﬁned as visits to the nest that involved nursing of the litter, was also
recorded. Average pup weight gain and pup survival between PND 1 and PND 6 were

determined for all litters. All percentages are relative to the two-hour scoring period,

35

save the percentages for the high crouch nursing behavior, which was calculated from the

total time spent nursing.

Two investigators scored the maternal behaviors from the video recordings. Because of
procedural demands, it was impossible to keep one of these two investigators unaware of
the treatment received by each dam. A subset of the recordings was scored
independently by both investigators and a correlational analysis was used to evaluate the
inter-observer reliability. For all the maternal behaviors analyzed, the positive

correlation between the two data sets was greater than .90.

Analyses

A preliminary analysis revealed that the maternal care exhibited by dams rearing adopted
young of the same treatment did not differ ﬁ'om dams rearing their own young, regardless
of whether the dam was oil or PCB treated (t-tests, ps = 0.12 - 0.94 for all behaviors
examined). For this reason, data from the group of PCB mothers rearing their own PCB
pups (uncrossed) were combined with those ﬁ'om the group of PCB dams rearing a litter
of PCB pups that were not her own (crossed) resulting in a total of twelve dams and
litters in this group. The same was done for the crossed and uncrossed oil groups. The

remaining four treatment groups contained six dams each.

For each behavioral measure, a t-test was used to test the hypothesis that exposure to

PCB by the dam and/or the litter affects the dams’ behavior. This was evaluated by

36

comparing the oil group to a combined PCB group that included all groups receiving any
PCB exposure. In addition, 4 X 4 ANOVAs (treatment groups X days) with repeated
measures on the second factor were used to analyze the data. Signiﬁcant overall F ratios
were followed by post-hoc comparisons of the individual group means using the Fisher’s
PLSD test. The data on pup survival and weight gain of the litters were analyzed using t-
tests (oil vs. all PCB groups combined) and one-way ANOVAs, followed by appropriate
post hoc comparisons. The relationships between pup grooming and nursing bouts, as
well as number of pups and amount of maternal autogrooming, were examined using
correlational analyses (Pearson Product Moment). All differences considered statistically

signiﬁcant have p S 0.05.

RESULTS

A comparison of all PCB-exposed groups to the oil-only group showed that PCB
exposure decreased the number of pups present on PND 6 (t = -2.68, df = 29, p < 0.05;
Figure 3, Top). A one-way ANOVA of the four different treatment groups also detected
a signiﬁcant [F (3, 27) = 4.37, p < 0.05] treatment effect on pup survival (Figure 3,
Bottom). Post hoc tests demonstrated that the main effect of treatment was due to a
higher mortality for pups that had received PCB exposure in utero, regardless of their

postnatal condition. Figure 4 summarizes the data for average weight gained by the pups

37

 

 

Number of Pups
on PND 6

 

 

 

 

 

oil

 

Number of Pups
on PND 6

 

 

 

 

 

 

 

Prenatal pup PCB oil PCB oil
Treatment

(Treatment of biological dam)

 

Postnatal pup PCB PCB oil oil
Treatment

(Treatment of dam rearing litter)

FIGURE 3. Top: A comparison of the oil-only group against the
combined PCB group found a signiﬁcant difference (*) in the
number of pups present on PND 6, with fewer pups surviving in
the combined PCB group. Bottom: A comparison of the four
treatment groups using a one-way ANOVA revealed a main effect
of treatment on pup survival; the groups sharing the same letter are
not signiﬁcantly different ﬁom each other. For this and all the
other ﬁgures see the text for details of statistical analyses.

38

 

 

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Average Pup Weight
Gain on PND 6
(gram5)

 

 

 

 

 

 

Prenatal pup PCB oil PCB oil
Treatment
(Treatment of biological dam)

 

Postnatal pup PCB PCB oil oil
Treatment
(Treatment of dam rearing litter)

FIGURE 4. Top: A comparison of the oil-only group against
the combined PCB group found a signiﬁcant difference (*) in
the average amount of weight that the pups gained since PND
0, with less weight gained by the combined PCB group.
Bottom: The ANOVA of the data ﬁom the four treatment
groups did not ﬁnd a signiﬁcant main effect of treatment for
average amount of weight gained by the pups.

39

(weight gain of the litter divided by number of surviving pups) between PND l and PND
6. The t-test detected a signiﬁcant effect of treatment (1 = -2.06, df = 29, p < 0.05) when
comparing all PCB-treated groups to the oil only group, in that the combined PCB groups
gained less weight than the oil group (Figure 4, Top). Considering the four treatment
goups (Figure 4, Bottom), the ANOVA did not ﬁnd a signiﬁcant main effect of

treatment on this measure (p = 0.28).

For all the other dependent variables, the 4 X 4 ANOVAs found no signiﬁcant
interactions between treatments and days. A sigriﬁcant main effect of days (data not
shown) was found for percent of time spent on the nest [F (3, 139) = 41.02, p < 0.05],
percent of total time nursing [F (3, 139) = 38.77, p < 0.05], and in the high crouch
nursing posture [F (3, 139) = 3.35, p < 0.05], percent of time grooming the pups [F (3,
139) = 4.05, p < 0.05] and engaged in maternal autogooming [F (3, 139) = 8.29, p <
0.05]. Post hoc analyses showed that the amount of time spent on the nest, nursing, pup
grooming and autogooming was signiﬁcantly higher on PND 1 than on PND 2, 4 and 6.
The amount of time spent engaged in high crouch nursing was sigriﬁcantly higher on
PND 6 than on PND 1 and 2. A signiﬁcant effect of days was not found for the number

of nursing bouts.

The t-test failed to reach sigriﬁcance for the amount of time spent autogooming (p =
0.07; Figure 5, Top). The analysis of the four separate treatment goups identiﬁed a
signiﬁcant main effect of treatment on this measure [F (3, 140) = 3.28, p < 0.05]. Post

hoc comparisons showed that dams rearing pups that were prenatally exposed to PCB

40

 

 

 

 

 

 

 

 

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Maternal Autogrooming
(Percent)

 

 

 

 

 

 

 

 

Prenatal pup PCB oil

Treatment
(Treatment of biological dam)

 

Postnatal pup PCB PCB oil oil
Treatment

(Treatment of dam rearing litter)

FIGURE 5. Top: The difference between percent of time engaged
in maternal autogooming between the oil-only goup and the
combined PCB goup missed statistical signiﬁcance (p = 0.06).
Bottom: The ANOVA of the data from the four treatment goups
found a signiﬁcant main effect of treatrrrent on maternal
autogooming; the goups sharing the same letter are not
signiﬁcantly different ﬁom each other.

41

goomed themselves more than dams rearing prenatally oil-exposed pups (Figure 5,
Bottom). Correlational analysis revealed a sigriﬁcant negative correlation (r = -0.47, df
= 31, p < 0.05) between the number of pups present and the amount of autogooming

exhibited by the dam on PND 6.

Figure 6 shows the data on pup gooming. A sigriﬁcant increase in pup goonring due to
PCB exposure was detected by comparing the mean of all PCB-treated goups with that
of the oil-only goup (t = 2.52, df = 142, p < 0.05; Figure 6, Top). When the four
separate treatment goups were compared, the main effect of treatment missed the
signiﬁcance level (p = 0.08; Figure 6, Bottom). The number of nursing bouts was
sigriﬁcantly higher when the combined PCB goups were compared the oil-only goup (t
= 2.02, df = 142, p < 0.05; Figure 7, Top). Further, the ANOVA detected a sigriﬁcant
main effect of treatment on this measure [F (3, 140) = 3.84, p < 0.05], and post hoc
comparisons showed that the main effect was due primarily to an increase in number of
nursing bouts in the dams rearing pups that were exposed to PCB prenatally. There was
no sigriﬁcant correlation between number of nursing bouts and amount of time spent
licking and gooming the pups for the data from the PCB goups (r = 0.10, df = 96, p =

0.34) or for the oil-only goup (r = 0.12, df = 48, p = 0.41).

None of the tests detected sigriﬁcant effects of PCB exposure on the amount of time the
dams spent nursing (data not shown), but the PCB-exposed goups displayed the high
crouch nursing posture signiﬁcantly less than the oil-only goup (t = -2.06, df = 142, p <

0.05; Figure 8, Top). The ANOVA of the four treatment goups found a sigriﬁcant main

42

 

 

 

 

 

 

 

 

 

 

 

 

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Treatment

(Treatment of biological dam)

 

Postnatal pup PCB PCB oil oil
Treatment
(Treatment of dam rearing litter)

FIGURE 6. Top: A comparison of the oil-only goup against
the combined PCB goup found a signiﬁcant difference (*)
in the amount of licking and gooming directed to the litter,
with enhanced pup grooming seen in the combined PCB
group. Bottom: The ANOVA of the data ﬁom the four
treatment goups did not ﬁnd a signiﬁcant main effect of
treatment (p = .08).

43

 

 

Number of Nursing Bouts

 

 

 

 

 

oil

 

Number of Nursing Bouts

 

 

 

 

 

Prenatal pup PCB oil PCB oil
Treatment
(Treatment of biological dam)

 

Postnatal pup PCB PCB oil oil
Treatment
(Treatment of dam rearing litter)

FIGURE 7. Top: A comparison of the oil-only goup against the
combined PCB group found a signiﬁcant difference (*) in the
number of nursing bouts displayed by the dams, with more bouts
seen in the combined PCB goup. Bottom: The ANOVA of the
data from the four treatment groups found a signiﬁcant main
effect of treatment on the number of nursing bouts; the groups
sharing the same letter are not signiﬁcantly different from each
other.

 

 

 

 

 

 

 

 

 

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Trme in the High Crouch
Nursing Posture (Percent)

 

 

 

 

   

 

 

 

Prenatal pup PCB oil PCB oil
Treatment

(Treatment of biological dam)

 

Postnatal pup PCB PCB oil oil
Treatment
(Treatment of dam rearing litter)

FIGURE 8. Top: A comparison of the oil-only goup against the
combined PCB goup found a signiﬁcant difference (*) in the
display of the high crouch nursing position, with the PCB goup
showing less high crouch nursing. Bottom: The ANOVA of the
data ﬁom the four treatment goups found a sigriﬁcant main
effect of treatment on the display of high-crouch nursing; the
groups sharing the same letter are not signiﬁcantly different from
each other.

45

effect of treatment for the amount of time that the dams displayed the high crouch
nursing posture [F (3, 140) = 1.81, p < 0.05], and post hoc tests found a sigriﬁcant
difference between the oil-only goup and the goup with PCB-exposed dams rearing oil-

treated pups (Figure 8, Bottom).

The t-test showed that the PCB goups spent sigriﬁcantly more time on the nest when
compared to the oil-only goup (t = 1.99, df = 142, p < 0.05; Figure 9, Top). The
ANOVA of the four treatment goups found a signiﬁcant main effect of treatment for
time spent on the nest [F (3, 140) = 1.94, p < 0.05; Figure 9, Bottom]. The three goups
exposed to PCB tended to show more time on the nest, but post hoc analysis found a
signiﬁcant difference only between the oil-only goup and the goup with PCB-exposed

dams rearing pups prenatally exposed to PCB.

46

 

 

 

 

 

 

 

 

 

 

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Prenatal pup PCB oil PCB oil
Treatment

(Treatment of biological dam)

 

Postnatal pup PCB PCB oil oil
Treatment

(Treatment of dam rearing litter)

FIGURE 9. Top: A comparison of the oil-only goup against the
combined PCB group found a signiﬁcant difference (*) in the
amount of time the darn spent on the nest, with more time on the
nest seen in the combined PCB goup. Bottom: The ANOVA of
the data from the four treatment groups found a signiﬁcant main
effect of treatment on time spent on the nest; the goups sharing
the same letter are not signiﬁcantly different from each other.

47

DISCUSSION

Comparison of PCB Treatments with Controls

Various components of maternal behavior were affected when pregnant rats were
exposed to PCB 77 or when they raised pups that had been exposed to the contaminant
during fetal life. Comparing the combined PCB-exposed goups to the oil-only goup,

we found an increase in the amount of time the dams spent gooming the pups and an
increase in the number of nursing bouts as well as in the amount of time the dams spent at
the nest, but a reduction in the display of the high-crouch nursing posture. In a recent
study, using a conventional desi gr without cross-fostering, we also detected effects of
PCB 77 on the maternal behavior of rats, which also included an increase in time spent
on the nest, an increase in licking and gooming of the offspring, and a decrease in the
display of the high-crouch nursing posture (Simmons et al., 2005). In both studies there
were no effects of any type of PCB exposure on the amount of time the dams devoted to
nursing the pups. Using mice, experiments with estrogenic endocrine disrupters
(Bisphenol A and methoxychlor) administered during gestation without cross-fostering of
the litters have also documented changes in maternal behavior (Palanza et al., 2002a and
2002b). In contrast to our results with PCB 77 , these estrogenic agents produced a
decrease in time on the nest and in time nursing the litters. Despite differences between
the speciﬁc behavioral effects of particular contaminants in mice and rats, taken together
these studies serve to highlight the potential importance of maternal contributions in the

evaluation of developmental outcomes in toxicology studies involving gestational

48

exposure. Interestingly, ﬁeld studies of avian behavior have reported effects of PCB
exposure on nest-site presence and nest building in gulls (Bustnes et al., 2001) and in tree
swallows (McCarty and Secord, 1999a and 1999b). Therefore, effects of environmental
contaminants on parental behaviors are likely a general feature of contaminant exposure

in vertebrates.

Cross-fostering Analysis: Developmental Outcomes

Our results indicate that some developmental outcomes associated with PCB exposure
may be independent of any effects of the contaminant on maternal care, while others may
be sensitive to the condition of the lactating darn. Thus, the increase in pup mortality
appears to be due primarily to exposure of the litters to the contaminant in utero, with no
substantial protection seen when the PCB-treated pups are reared by oil-treated dams, and
with no added morbidity ascribable to being reared by PCB-treated dams. In contrast,
evidence for a contribution of postnatal conditions can be seen in the decreased amount
of weight gained by pups with gestational and/or lactational exposure to the PCB.
Deﬁcits in the display of high-crouch nursing shown by dams treated with PCB 77 or
raising PCB-exposed pups likely contributed to the slower gowth of the pups, since milk
. let-down is more likely to occur when the dam is engaged in the high crouch nursing
posture than any of the other nursing postures (Lincoln et al., 1980; Voloschin and

Tramezzani, 1979; Lonstein et al., 1998).

49

While some of the effects we see after PCB exposure (e. g., decreased weight gain of the
pups) have also been demonstrated after exposure to other teratogens (e. g., lead, mercury,
fetal alcohol exposure), it is not likely that these compounds are effecting their change in
the same way. For example, PCB-induced AhR gene expression can alter thyroxine
levels, which could help to explain the decrease in weight gain we saw after PCB
exposure. The potential mechanisms by which lead and ethanol reduce gowth in
exposed organisms could be much different, and are not likely to work via the AhR since

they are not potent ligands for the receptor.

Cross-fostering Analysis: Maternal Behavior

The use of a cross-fostering paradign allows us to begin to separate effects on maternal
behavior that are a result of the PCB actions on the darn from those that are produced in
response to a change in the litter brought about by PCB exposure. However, our results
show that the contributions of direct versus indirect effects of PCB on maternal behavior
are not always clear cut, and that for speciﬁc maternal behaviors, both types of effects are

likely to be involved.

For maternal autogooming, our results strongly suggest that the treatment received by
the pups in utero was responsible for the increase seen in this behavior. Thus, regardless
of the treatment that they received during gestation, dams rearing pups that were exposed
to PCB in utero showed increased autogooming. PCB exposure in utero leads to a

decrease in litter size and, therefore, a reduction in the amount of pup-related stimulation

50

that the darn receives. This reduction in stimulation (e. g. rooting and suckling) may be
responsible for the increase in maternal autogooming as supported by the negative
correlation seen here between this behavioral measure and litter size. In addition, direct
actions of the PCB on the offspring may have decreased the quality of stimulation
provided by the pups that were still present. In order for a dam to engage in the high
crouch nursing posture, she must receive an adequate amount of ventral stimulation
(Stern, 1996). It is possible that the dam is attempting to compensate for the reduced
stimulation provided by the pups by increasing the amount that she licks and gooms her
ventrum. In order for this to be the case the increase in autogooming would be speciﬁc
to the ventrum of the dams. However, we are not able to distinguish between ventral and

dorsal autogooming and cannot test this hypothesis at this time.

Palanza et al. (2002a and b) also observed an increase in autogooming when mice were
treated with estrogenic endocrine disrupters during pregrancy. However, they did not
report a reduction in the size of the litters, so PCB 77 and other estrogenic contaminants
may produce similar effects on maternal autogooming, but may act via different

mechanisms.

Using a conventional desigr with no cross-fostering, previous research in this laboratory
found an increase in pup gooming when a high dose of PCB 77 (4 mg/kg) was
administered during gestation (Simmons et al., 2005). In that study, a 2 mg/kg dose did

not affect this measure. Here we report a small facilitation of the behavior by the 2

51

mg/kg dose that was only detected when the combined data for all PCB goups were

compared to the data from the oil-only goup.

The PCB-induced alteration seen in pup-directed licking and gooming may be
attributable to some of the known biological actions of PCB 77. For example, PCB 77
has been shown to act as an estrogen (Seegal et al., 2005; Nesaretnam et al., 1996), and
as such, it may be acting via brain oxytocin systems (Bale and Dorsa, 1995; Pedersen and
Boccia, 2003) to increase pup-directed gooming (Champagre et al., 2001; Francis et al.,
2000; Champagre et al., 2003). Exposure to other estrogenic contaminants (Palanza et
al., 2002a and b) has not resulted in increases in pup-directed licking, but various dioxin-
like compounds have increased estradiol secretion in placental tissue in vitro and could,

as a consequence, activate OT secretion from the pituitary (Augustowska et al., 2003).

Enhanced licking may also reﬂect changes in brain dopamine (DA), since changes in
levels of brain DA have been shown to affect pup-directed licking and gooming
(Champagre et al., 2004; Keer and Stern, 1999; Stern and Taylor, 1991). Developmental
exposure to PCBs is known to affect brain DA in the adult (Seegal et al., 1997). If adult
exposure to PCBs has similar effects, then behavioral changes in the PCB-exposed dams
may stem from effects of the contaminant on brain DA systems. However, there are
conﬂicting data on whether or not PCB exposure to the adult animal causes a similar
outcome to perinatal PCB exposure (see Seegal et al., 2005 and Mariussen et al., 1999).
Alternatively, changes in the stimulus quality of pups exposed to PCB during gestation

and/or lactation may induce more pup-directed gooming from the dams.

52

The increase in licking of the pups may serve to buffer or compensate for some of the
toxic effects that the offspring may otherwise incur as a result of exposure to the
contaminant. Manipulations that increase licking and gooming have been shown to
protect the offspring from the effects of many early-life insults including fetal ethanol
exposure (Lee and Rabe, 1999) and cerebral hypoxia (Chou et al., 2001). It is possible
I that some developmental effects that would otherwise be seen in the pups as a result of
PCB exposure may be lessened because of the increase in licking and gooming that is

exhibited by the PCB-exposed goups.

The effect of PCB on the number of nursing bouts was examined in an attempt to
understand the effects of the contaminant on pup gooming. Dams engage in more
licking and gooming just prior to a nursing bout (Stern, 1989). Thus, an increase in the
frequency of nursing bouts induced by actions of PCB 77 could be responsible for the
increase in maternal licking. Although a comparison of all PCB 77-exposed goups to
the oil-only goup revealed an increase in the number of nursing bouts, there was no
signiﬁcant correlation between this measure and the amount of licking and gooming
displayed by the dams. Further, the pattern of results for nursing bouts was clearly
different from the one obtained for licking and gooming, and very similar to the one seen
for autogooming. Namely, dams rearing pups that had been exposed to PCB 77 during
gestation preferentially showed an increase in the display of the behavior. Reduced
stimulation by smaller litters may contribute to this behavioral effect as suggested for

autogooming.

53

Although nursing per se was not affected by PCB exposure, dams in the combined PCB
goups spent less time in the high crouch nursing posture than their oil-only counterparts.
A similar reduction in high crouch was seen previously with the dose given in this
experiment as well as with a higher one (4 mg/kg) (Simmons et al., 2005). As was
observed for the effects of PCB 77 on licking and gooming of the pups, changes in both
the dam and the litter appear to contribute to the reduction in high-crouch nursing. The
reduction in high crouch nursing may be due to effects of the contaminant on the
physiology of the dams. For example, hypothyroidism has been associated with a
reduced ability to lactate in multiple animal models (Ben-David et al., 1966; Swanson
and Miller, 1973; Tarnasy et al., 1984; Tucker, 1994), and contaminants similar to PCB
77 have been shown to interfere with thyroid functioning (Gorski et al., 1988; Gray et al. ,
1993; Morse et al., 1993; Seo et al., 1995). But changes in the pups may also contribute
to the reduction in high crouch nursing. The litters exposed to PCB either in utero or via
lactation were lighter in body weight than control litters. Therefore, the reduction in high
crouch nursing may result ﬁom a reduction in ventral stimulation of the dam by the
smaller pups of the PCB-exposed goups. Regardless of questions about mediating
mechanisms, the reduction in high crouch nursing induced by exposure to PCB 77 is
clearly not indicative of a general neglect for the litter by the dams. In fact, the dams
from the combined PCB-exposed goups spent more time on the nest than those from the

oil-only goup as was reported earlier (Simmons et al., 2005).

Finally, it is important to note that while the dose of PCB used here (2 mg/kg body

weight per day) is much larger than an average individual would experience, it is closer

54

to the amount of PCB exposure received by those individuals living in highly-
contaminated areas such as the Arctic. It is important to elucidate the effects of PCBs on
organisms so that we can better understand how we may be able to potentially ameliorate
the effects of the contaminant after exposure. In order to do this, a higher dose must be
used in order to better identify all of the consequences resulting from environmental

contaminants.

In summary, our results conﬁrm that exposure to PCB 77 during gestation and/ or
lactation can have sigriﬁcant effects on the maternal behavior of the dams. The patterns
of behavioral changes that are discerned using a cross-fostering paradign suggest that
changes in maternal behavior are likely to emerge from direct effects of PCB on the dams
as well as in response to effects of the PCB on the litter. The changes in maternal
behavior are not reﬂective of a global disruption of maternal care since some behaviors
that contribute to the survival of the litters are enhanced by the treatment. These and
other ﬁndings (Simmons et al., 2005; Palanza et al., 2002a and b) should guide and
inform future experiments involving gestational treatments that may alter maternal
behavior. This is especially important for those studies that examine the effects of
contaminants or other perinatal treatments on the behavior of the offspring, since changes
in offspring behavior may be rooted in alterations of maternal care brought about by the

perinatal treatment as well as any direct treatment effects.

55

Chapter 4. How does an intermediate dose of PCB 77 affect maternal behavior

when using a cross-fostering design?

ABSTRACT

Polychlorinated biphenyls, or PCBs, are environmental toxins that have known
behavioral and neurological effects. Research in our laboratory has shown an effect of 3,
4, 3’, 4’-tetrachlorobiphenyl (PCB 77) on the maternal care of rats when the PCB was
delivered during pregrancy. A dose of 4 mg/kg resulted in altered maternal behavior, but
also led to a sigriﬁcant decrease in the number of viable offspring. A subsequent
experiment using a cross-fostering design and a dose of 2 mg/kg demonstrated sigriﬁcant
effects on maternal behavior with some of these effects resulting primarily from effects
of the PCB on the dams and others driven by effects of the PCB on the pups. However,
not all of the changes demonstrated after treatment with 4 mg/kg PCB were observed
after exposure to 2 mg/kg. The current study, utilizing a dose of 3 mg/kg and a cross-
fostering desigr, was conducted in order to (1) use an intermediate dose of PCB in an
attempt to identify whether the changes in maternal care seen after treatment with 4
mg/kg (that were not seen after treatment with 2 mg/kg) were due to PCB-affected pups
or dams, and (2) expand the behavioral analysis of the effects of the PCB on the dams to
test the “maternal compensation hypothesis.” Pregnant rats were treated with
subcutaneous injections of either PCB 77 emulsiﬁed in corn oil (3 mg/kg b.w.) or vehicle
alone on gestation days 6 - l8. Litters were cross-fostered and maternal care was

examined on postnatal days 1, 2, 4 and 6. Our data indicate that exposure to PCB 77

56

altered the amount of time the dams spent on the nest, the time licking and gooming
their pups, the time engaged in high crouch nursing, ventral auto gooming, and nursing.
These results conﬁrm that exposing dams to PCBs during gestation or to pups that
received in utero PCB exposure affects maternal behavior in ways likely to contribute to

the development of the litter.

INTRODUCTION

Although the use of a cross-fostering desigr to examine the effects of PCB 77 on
maternal behavior answered many of the questions that arose after the Simmons et al.
study, new questions were also raised by the results. For example, behavioral measures
were altered in dams that did not receive PCB treatment directly but reared prenatally
PCB-exposed offspring. These dams goomed themselves more and exhibited less high
crouch nursing than dams rearing pups exposed to only oil prenatally. In the discussion
of those observations, I suggested that the stimulus properties of the PCB-treated pups
might have been altered, resulting in the observed change in the dams’ behavior. Dams
require a speciﬁc amount of ventral stimulation ﬁom pups in order to achieve the high
crouch nursing posture (Stern, 1996). This stimulation usually occurs while the pups are
ventrally rooting around for and attaching to a nipple prior to nursing. If the dams do not
receive the necessary stimulation, they will not engage in high crouch nursing (Stern and
Johnson, 1990; Stern and Lonstein, 1996). I hypothesized that the increase in
autogooming exhibited by dams rearing prenatally PCB-exposed pups was the result of

the dams’ attempt to compensate for the reduced stimulation provided by these pups.

57

However, since the original analysis did not separate ventral autogooming from that

directed toward the rest of the body, I was not able to test that hypothesis.

In addition, I was curious as to how maternal behavior was affected by a higher dose of
PCB 77 using a cross-fostering desigr. Even though treatment with the 4 mg/kg dose
affected maternal behavior (Simmons et al., 2005), the interpretation of the results was
complicated by the relatively high toxicity associated with that dose (as indicated by the
appearance of the pups, increase in mortality rate and decrease in the number of dams
that delivered litters). Furthermore, there were behaviors that were altered after treatment
with 4 mg/kg that were not affected with the 2 mg/kg dose (e. g., pup licking and
gooming), and since the experiment using 4 mg/kg did not utilize a cross-fostering
desigr, we were not able to identify if PCB-affected offspring lead to the increase in pup
gooming, or if this behavior was changed as a result of the PCB treatment the dam

received.

Finally, in a report that examined the effects of various teratogens on development, a 3
mg/kg dose of PCB 77 resulted in morphological effects on the offspring (Wardell et al.,
1982). Knowing that treatment with 3 mg/kg causes developmental effects in the
exposed offspring, it became important to explore how the same treatment would affect
maternal behavior, and how maternal care might be altered in dams responding to

prenatally PCB-exposed pups.

58

For the current experiment, I utilized a cross-fostering desi gr and a more detailed
behavioral analysis to examine the effects of 3 mg/kg of PCB 77 on maternal care inrats.‘
Similar to the earlier studies, pregrant females were treated on GD 6 — 18 and litters were
cross-fostered on the day of birth. Maternal behavior was analyzed on postnatal days
(PNDs) 1, 2, 4 and 6, and developmental outcomes of the offspring were studied on PND

6.
METHODS
Animals and Housing

Twenty-four timed-pregnant Long-Evans rats from Charles River Laboratories (Raleigh,
North Carolina) arrived at our laboratory on gestation day (GD) 5, and were maintained
on a 14:10-h light:dark cycle with lights off at 1100 h at an ambient temperature of 21°C
in a laminar-ﬂow unit (NUAIRE). A dim red light remained on at all times. The rats
were housed singly in plastic cages (45 X 22 X 21 cm) with wood shavings as bedding,
and were allowed ﬁ'ee access to water and Harlan Rodent Chow 8640 food pellets.
Animals were maintained in a facility accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care, and the Michigan State University Animal

Care and Use Committee approved experimental procedures.

59

Procedure

The dams were weighed daily and given subcutaneous injections of the oil vehicle or 3,
4, 3’, 4’-tetrachlorobiphenyl (PCB 77; Accustandard, New Haven, CT; >99% pure as
determined by gas chromatogaphy performed by the vendor) emulsiﬁed in corn oil in the
dose of 3 mg/kg from GD 6 to GD 18. Nest building materials in the form of thirty
brown paper towel strips were placed on the wire top of each cage on GD 20. Parturition
took place during the mid- to late-light phase of the animals’ light/dark cycle on GD 22 -
23. On the day of birth [day of birth = postnatal day (PND) 0], the litters were culled to 8
animals (4 females and 4 males) and cross-fostered or left with their own mothers to
create the following goups: (1) oil treated dams with their own oil pups, (2) PCB treated
dams with their own PCB pups, (3) oil treated dams with a litter of PCB-exposed pups,

and (4) PCB treated dams with a litter of oil pups. Each goup contained six dams.

Dams and litters were videotaped on PND 1, 2, 4 and 6 for the animals’ last hour of light
and ﬁrst hour of dark (2 hr/day/animal). Videos were analyzed using The Observer 4.1
(N oldus Information Technology), a behavioral data acquisition computer progam.
During review of the videotapes maternal behaviors were recorded as was described in
detail in Cummings et al., 2005. Brieﬂy, the behaviors that were analyzed include the
amount of time the darn spent on the nest, licking and gooming the pups, autogooming
ventrally and dorsally, and amount of time spent nursing. Nursing behaviors included
resting, low crouch and high crouch. Time spent nursing is the total of all time spent in

one of the three nursing postures while on the nest. The number of nursing bouts, deﬁned

60

as visits to the nest that involved nursing of the litter, was also recorded. Average pup
weight gain and pup survival between PND l and PND 6 were determined for all litters.
All percentages are relative to the two-hour scoring period, except for the percentages for

the nursing behaviors, which were calculated from the total time spent nursing.

Two investigators scored the maternal behaviors from the video recordings. Because of
procedural demands, it was impossible to keep the investigators unaware of the treatment
received by each darn. A subset of the recordings was scored independently by both
investigators and a correlational analysis was used to evaluate the inter-observer
reliability. For all the maternal behaviors analyzed, the positive correlation between the

two data sets was geater than .90.

Analyses

For each behavioral measure, a 2 X 4 ANOVA (treatment goups X days, repeated
measures on the second factor) was used to test the hypothesis that exposure to PCB by
the dam and/or the litter affects the dams’ behavior. This was evaluated by comparing
the oil goup to a combined PCB goup that included all goups receiving any PCB
exposure. The cross-fostering design included four treatment goups and 4 X 4 ANOVAs
(treatment goups X days) with repeated measures on the second factor were used to
analyze the data for sigriﬁcant main effects of treatment and day and the interactions.
Sigriﬁcant overall F ratios were followed by analysis of the simple effects and/or post

hoc comparisons of the individual goup means using the Fisher’s PLSD test. The data

61

on pup survival and weight gain of the litters were analyzed using t-tests (oil vs.
combined PCB goups) and one-way ANOVAs (comparing all four treatment goups),
followed by appropriate post hoc comparisons. All differences considered statistically

signiﬁcant have p S 0.05.

RESULTS

A comparison of all PCB-exposed goups to the oil-only goup showed that PCB
decreased the number of pups present on PND 6 (t = -2.84, df = 25, p = 0.009; Figure 10,
Top). The ANOVA analyzing the effect of the four treatment goups on pup survival
found a sigriﬁcant main effect of treatment [F (3, 23) = 6.97, p = 0.002], with post hoc
tests indicating that pups who received prenatal PCB exposure had a higher mortality rate

than pups who received prenatal oil exposure (Figure 10, Bottom).

The t-test comparing the combined PCB goups to the oil-only goup detected a
sigriﬁcant effect of PCB on the average amount of weight gained by the pups between
PNDs 1 and 6 (t = -3.27, df = 25, p = 0.003; Figure 11, Top). The analysis of the four
separate treatment goups identiﬁed a sigriﬁcant main effect of treatment on pup weight
gain [F (2, 23) = 3.36; p = 0.036], with post hoc analysis indicating that any exposure to
PCB decreased the average amount of weight gained by the pups over the ﬁrst six

postnatal days compared to the oil only goup (Figure 11, Bottom).

62

 

 

Number of Pups
on PND 6
0' a:

 

 

 

 

 

 

 

 

 

 

 

4 ..
3 ..
2 - ..
1 ~ -
0 —
PCB oil
8 A '1’
7 _
B
8. 6 B '
a \o
4... Q 5 ‘
° 2
I—
o 0.. 4 L
.o a (
E ° 3 .
Z l
2 .-
1 .-
0 . . . f
Prenatal pup PCB oil PCB oil
Treatment

(Treatment of biological dam)

 

Postnatal pup PCB PCB oil oil
Treatment

(Treatment of dam rearing litter)

Figure 10 Top: A comparison of the oil-only goup against the
combined PCB group found a signiﬁcant difference (*) in the
number of pups present on PND 6, with fewer pups surviving in
the combined PCB goup. Bottom: A comparison of the four
treatment goups using a one-way ANOVA revealed a main effect
of treatment on pup survival; the goups sharing the same letter
are not signiﬁcantly different from each other.

63

 

94 I

 

Average Pup
Weight Gain (g)

 

 

vlr‘lvrrIvrv

"l’

'I'I

 

 

 

 

 

 

 

 

 

 

 

oil
10 -
9 I -
a A -
:1. 3° 7 ‘
a .5 6 -
551:5 5 _
2 in 4 -
< g 3 .
2 L
1 I-
0 ' ' . : . ' -
Prenatal pup PCB oil PCB ’ oil
Treatment
(Treatment of biological dam)
Postnatal pup PCB PCB oil oil

Treatment

(Treatment of dam rearing litter)

Figure 11 Top: A comparison of the oil—only goup against the
combined PCB group found a signiﬁcant difference (*) in the

average amount of weight that the pups gained from PND 0 to PND
6, with less weight gained by the combined PCB group. Bottom:
The ANOVA of the data from the four treatment groups revealed a
signiﬁcant main effect of treatment; groups with the same letter are

not signiﬁcantly different from each other.

The two-way ANOVA comparing the combined PCB-exposed goups to the oil only
goup for percent of time the dams spent on the nest failed to reach sigriﬁcance for effect
of PCB (p = 0.548; Figure 12, Top), but did identify a sigriﬁcant effect of days [F (1, 25)
= 2.73, p = 0.050] with the dams spending more time on the nest on PND 1 than on PNDs
4 and 6 (data not shown). No sigriﬁcant interaction was detected for this measure (p =
0.586). The ANOVA of the four different treatment goups indicated that both days [F
(3, 96) = 5.17, p = 0.003] and treatment [F (3, 23) = 3.69, p = 0.026] had a sigriﬁcant
effect on percent of time on the nest, with no sigriﬁcant interaction (p = 0.401 ). For the
effect of days, post hoc analysis revealed that dams spent more time on the nest on PND
1 as compared to PNDs 4 and 6. For the effect of treatments (Figure 12, Bottom), post
hoc analysis showed that PCB-treated dams rearing prenatally PCB-exposed pups spent
more time on the nest than PCB-treated dams rearing oil pups. Also, oil-treated dams
rearing pups that had received PCB exposure prenatally spent more time on the nest than

dams rearing pups that had been exposed to oil prenatally (Figure 12, Bottom).

For percent of time spent engaged in pup-directed LG, the ANOVA for the combined
PCB goups and the oil-only goup failed to identify sigriﬁcant effects of PCB (p =
0.463; Figure 13, Top), of days (p = 0.923) or an interaction (p = 0.270). The analysis of
the four treatment goups detected a sigriﬁcant interaction between treatment and PND
[F (9, 69) = 2.09, p = 0.042]. There was a simple main effect of PND [F (3, 24) = 3.21, p
= 0.041] for oil dams rearing prenatally PCB-exposed pups, with dams spending more
time licking ”the offspring on PNDs 1 and 2 than PND 4 (data not shown). There was a

signiﬁcant simple main effect of treatment on LG on PND 1 [F (3, 23) = 2.97, p = 0.050]

65

45 ‘

 

 

401 -
35; L .
’8'? so: -
3% 2°: "
I" 15- -
1o: -
53 _
oi a

 

 

 

 

 

 

PCB oil

 

Time on Nest
(Percent)

 

 

 

 

Prenatal pup PCB oil PCB oil
Treatment

(Treatment of biological dam)

 

Postnatal pup PCB PCB oil oil
Treatment

(Treatment of dam rearing litter)

Figure 12 Top: A comparison of the oil-only goup against the
combined PCB goup did not ﬁnd a signiﬁcant difference in the
amount of time the darn spent on the nest (p = 0.55). Bottom:
The ANOVA of the data from the four treatment goups found a
signiﬁcant main effect of treatment on time spent on the nest; the
goups sharing the same letter are not signiﬁcantly different from
each other.

66

 

 

Time Spent Licking and
Grooming Pups (Percent)

 

 

 

 

 

 

oil

 

    

14 e
.57; 12- L
§§ -
Eng 10: - I PCBdamsandpups
ﬁg. 8‘ r PCBdamswith oil pups
gen 6- _ OildamswithPCBpups
ﬁg 4: L E] Oildamsandpups
Ea .
o 2_ _
1 l' P
o- l -

 

 

Postnatal Day 1 Postnatal Day 2

Figure 13 Top: A comparison of the oil-only group against the
combined PCB goup did not detect a signiﬁcant difference in the
amount of pup-directed licking and grooming (LG; p = 0.46).
Bottom: The ANOVA of the data ﬁ’om the four treatment goups
revealed a signiﬁcant interaction between treatment and postnatal
day for LG; goups sharing the same letter are not signiﬁcantly
different ﬁ'om each other.

67

and PND 2 [F (3, 23) = 4.36,p = 0.014], but not on PND 4 (p = 0.453) and PND 6 (p =
0.982) (data not shown). Dams rearing pups that had been prenatally exposed to PCB
spent more time gooming the pups compared to the oil only goup on PND 1 (Figure 13,
Bottom). On PND 2, dams rearing prenatally PCB-exposed pups spent more time

engaged in LG than PCB-treated dams with oil—treated pups (Figure 13, Bottom).

A sigiiﬁcant interaction was detected by the ANOVA of the combined PCB goups and
the oil only goup for percent of time spent in the high crouch nursing posture [F (3, 75)
= 3.22, p = 0.028]. T-tests for the simple main effects indicated that there was a
sigiiﬁcant effect of PCB on time in high crouch on PNDs 4 and 6, with the combined
PCB goups spending less time engaged in high crouch nursing than the oil only goup

(PND 4, t = -9.02, df= 5, p < 0.001; PND 6, t= -2.50, df= 5,p = 0.050; Figure 14, Top).

There was an effect of days for the oil only goup [F (3, 4) = 14.19, p = 0.013] with dams
‘ spending more time in the high crouch posture on PND 6 compared to PNDs 1, 2 and 4,
and more time on PND 4 than on PND 1. There was no effect of days for the combined
PCB goups. There was no effect of PCB exposure on high crouch nursing on PNDs 1 (p
= 0.403) and 2 (p = 0.350) (data not shown). The ANOVA of the four treatment goups
also detected a signiﬁcant effect of treatment on this measure [F (3, 23) = 3.82, p =
0.020], but no effect of PND (p = 0.284) and no signiﬁcant interaction (p = 0.381). Post
hoc analysis indicated that dams rearing pups that had been exposed to PCB prenatally
spent less time engaged in high crouch nursing than the oil-only control goup (Figure 14,

Bottom).

68

 

 

. . I Combined
15 . _ PCB Groups

’DOilonly

Time in High

Crouch (Percent)

 

 

 

 

 

 

 

 

PND4 PND 6

16 A A a a

 

14.

 

12-
10-
8d

 

Time in High

Crouch (Percent)

6‘
4..

v I v I T I v r v I v ' 1 I v

2,,

 

 

 

 

 

 

 

 

0 .1 . . . .th 1.4-2. ”NV-1.. v
Prenatal pup PCB oil PCB oil
Treatment

(Treatment of biological dam)

 

Postnatal pup PCB PCB oil oil
Treatment

(Treatment of dam rearing litter)

Figure 14 Top: A comparison of the oil-only goup against the
combined PCB goup found a signiﬁcant interaction between
treatment and day for the amount of time the darn spent in high
crouch. Analysis of the simple main effects revealed a
signiﬁcant difference (*) in the display of high crouch nursing
on PNDs 4 and 6, with the PCB goup exhibiting less high
crouch nursing. Bottom: The ANOVA of the data ﬁ'om the four
treatment goups found a signiﬁcant main effect of treatment on
the display of high-crouch nursing; the goups sharing the same
letter are not signiﬁcantly different ﬁ'om each other.

69

A sigiiﬁcant increase in maternal ventral autogooming as a result of PCB exposure was
detected when comparing the combined PCB goups to the oil only goup [F (l , 25) =
4.40, p = 0.046] (Figure 15, Top) with no effect of days (p = 0.067) and no interaction (p
= 0.395). When the four separate treatment goups were compared, the ANOVA detected
a sigiiﬁcant interaction between treatment and days [F (9, 69) = 4.34, p < 0.001].
Analysis of the simple main effects found a sigiiﬁcant effect of treatment on PND 1 [F
(2, 23) = 11.20,p < 0.001] and PND 2 [F (3, 22) = 4.59,p = 0.012; Figure 15, Bottom]
but not PND 4 (p = 0.968) and PND 6 (p = 0.772) (data not shown). Post hoc analysis
found that on PND 1, PCB-treated dams rearing prenatally PCB-exposed pups spent
more time gooming themselves ventrally than the other three treatment goups (Figure
15, Bottom). On PND 2, PCB-treated dams with prenatally PCB-exposed pups spent
more time engaged in ventral autogooming than PCB-treated dams rearing oil-treated
pups (Figure 15, Bottom). Also on PND 2, oil-treated dams rearing prenatally PCB-
exposed pups goomed themselves ventrally more than dams rearing prenatally oil-
treated pups (Figure 15, Bottom). There was a sigiiﬁcant simple main effect of days for
PCB dams rearing PCB pups [F (3, 20) = 12.77, p < 0.001] but not for the other three
goups. PCB dams rearing PCB pups spent more time engaged in ventral autogooming
on PND 1 than on the other three PNDs. The amount of time spent engaged in dorsal

autogooming was not affected by PCB exposure (p = 0.278; data not shown).

The one-way ANOVA did not detect a sigriﬁcant main effect of treatment or of days on

the percent of time spent nursing when comparing the combined PCB goups to the oil-

only control (Figure 16, Top). However, the ANOVA of the four treatment goups

70

Time Spent Autogooming

Time Spent Autogooming

Ventrally (Percent)

Ventrally (Percent)

 

1.8: l
1.6-
1.45
1.23

1‘:

 

.6-
.4-
.2-

 

 

 

 

 

oil

 

 

- - PCB dams and pups

: PCB dams with oil pups
- on dams with PCB pups
_ I] Oil dams and pups

    

m.

 

 

Postnatal Day 1 Postnatal Day 2

Figure 15 Top: A comparison of the oil-only goup against the
combined PCB goup found a signiﬁcant difference (*) in the
amount of time the darn engaged in ventral maternal autogooming,
with an increase in ventral autogrooming seen in the combined PCB
goup. Bottom: The ANOVA of the data ﬁom the four treatment
goups found a signiﬁcant interaction between treatment and day
for time spent autogooming ventrally; the groups sharing the same
letter are not signiﬁcantly different from each other.

71

 

 

Time Spent Nursing
(Percent)

 

 

 

 

 

 

 

Time Spent Nursing
(Percent)

 

 

 

 

Prenatal pup PCB oil PCB oil
Treatment

(Treatment of biological dam)

 

Postnatal pup PCB PCB oil oil
Treatment

(Treatment of dam rearing litter)

Figure 16 Top: The difference between percent of time spent nursing
for the oil-only goup and the combined PCB goup missed statistical
signiﬁcance (p = 0.79). Bottom: The ANOVA of the data ﬁom the
four treatment groups found a signiﬁcant main effect of treatment on
time spent nursing; the goups sharing the same letter are not
signiﬁcantly different from each other.

72

identiﬁed a sigiiﬁcant main effect of treatment [F (3, 23) = 2.92, p = 0.050] and of days
[F (3, 69) = 2.80, p = 0.046], with no signiﬁcant interaction between the two (p = 0.695).
Post hoc analysis indicated that oil-treated dams rearing prenatally PCB-exposed pups
spent more time nursing than PCB-treated dams rearing oil-exposed pups (Figure 16,
Bottom). Additionally, all dams spent more time nursing on PND 1 than on the other

three PNDs (data not shown).

DISCUSSION

Cross-fostering Analysis: Developmental Outcomes

The effects of 3 mg/kg PCB on the developmental outcomes of the offspring paralleled,
for the most part, those reported in previous experiments that used a lower dose (2 mg/kg;
Cummings et al., 2005) and a higher dose (4 mg/kg; Simmons et al., 2005) of PCB on the
same gestation days. Perinatal PCB exposure decreased the number of pups that
remained in the nest on postnatal day (PND) 6 when the pups received PCB exposure in
utero, similar to what was found with the 2 mg/kg dose when using the cross-fostering
desigr (Cummings et al., 2005). The 4 mg/kg dose also increased pup mortality using a
traditional treatment paradign (Simmons et al., 2005). The increase in mortality seen in
the prenatally PCB-exposed pups is most likely attributable to the toxic effects of the
PCB on the litter in utero. However, it cannot be ruled out that the dam’s behavior is
changed in response to an alteration in the physiology and/or behavior of the pups, which

in turn affects their viability.

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PCB exposure with the 3 mg/kg dose also decreased the average amount of weight gained
by all offspring that were exposed to PCB regardless of the timing of exposure. That is,
pups receiving pre- and/or postnatal PCB exposure gained less weight than offspring who
were not exposed to PCB. Treatment with 2 mg/kg in Cummings et al. (2005) also
decreased pup weight in the combined PCB goups compared to the oil only goup, and a
similar pattern was found when comparing the four treatment goups to one another.
Traditional treatment with 2 and 4 mg/kg (Simmons et al., 2005) also decreased average
pup weight gain in pups exposed pre- and postnatally. The reduction in weight gain seen
with the 3 mg/kg dose may be due, in part, to the ﬁnding that dams in the combined PCB
goup spent less time in the high crouch nursing posture than oil-treated dams on PNDs 4
and 6. Since the high crouch nursing posture is the one in which the majority of milk
letdown occurs (Lincoln et al., 1980; Voloschin and Tramezzani, 1979; Lonstein et al.,
1998), it is reasonable to suggest that pups who receive less high crouch nursing would
not gain as much weight as those pups whose mothers were engaged in the nursing

posture a higher percentage of the time.

Cross-fostering Analysis: Maternal Behavior

For the 3 mg/kg dose of PCB used here, the analysis comparing the four treatment goups
to one another revealed sigriﬁcant treatment effects on various aspects of maternal care.

Interestingly, the majority of the affected behaviors tended to be changed as a result of

dams rearing prenatally PCB-exposed pups.

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The amount of time dams spent licking and gooming their pups on PNDs 1 and 2 was
altered in dams rearing pups that were exposed to PCB prenatally compared to dams that
reared prenatally oil-exposed pups. Treatment with 4 mg/kg PCB using a conventional
desigr with no cross-fostering also increased LG (Simmons et al., 2005) and, while
treatment with the lower dose of PCB (2 mg/kg; Cummings et al., 2005) did not result in
sigiiﬁcant differences between the four goups for this measure, it did lead to an increase

in pup-directed LG when comparing the combined PCB goups to the oil-only goup.

After analyzing the effects of 2 mg/kg PCB on maternal behavior in the previous
experiment (Cummings et al., 2005), I hypothesized that the increase in pup-directed LG
may have been due, in part, to the PCB acting as an estrogen via brain oxytocin systems
to increase the amount of LG displayed by the dams. Additionally, I suggested that PCB-
induced alterations in brain dopamine (DA) could play a role in the increase seen in LG,
since developmental exposure to PCB has been shown to increase brain DA in adults and
alteration in brain DA have been shown to affect pup-directed LG. Support for these
hypotheses requires that the increase in LG be shown only by those dams that received
PCB treatment during pregrancy, but not by those dams that received oil treatment and
reared PCB-exposed offspring, as was the case here. Therefore, the increase in pup-
directed LG appears to be due not to the direct effects of the toxin on the dams, as
suggested previously, but to a change in the prenatally PCB-exposed pups that in turn

affects the behavior of the dams.

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One way in which pups could have been changed is through PCB-induced
masculinization. Moore and Morelli (1979) have shown that male pups receive more
pup-directed LG from the dam than their female siblings. Treatment of neonatal female
pups with testosterone or estrogen increased the amount of LG that the female pups
received so that they no longer differed from males (Moore, 1982). Since PCB can act as
an estrogen (Jansen et al., 1993; Nesaretnam et al., 1996) and pups that have been
exposed to PCB prenatally receive more LG than oil pups, it is possible that prenatal PCB
is acting to masculinize the female offspring, resulting in an increase in the amount of LG
provided by the dams. Thus, maternal behavior may have been altered in response to a

change in the stimulus properties of the pups, particularly in the female offspring.

In addition to masculinization, PCB may have affected how the pups interact with their
mother. For example, dams often goom their pups at the start of a nesting bout prior to
nursing (Stern, 1989). During this time, the dam receives ventral stimulation from the
pups while the pups are rooting around for and attaching to a nipple. After the dam
receives sufﬁcient stimulation she will become immobile and display the high crouch
nursing posture. If she does not receive the ventral stimulation required to assume the
posture, because of a change in the behavior of the pups or a decrease in the number of
pups present, the dam will not adopt the nursing posture (Stern and Johnson, 1990; Stern
and Lonstein, 1996). Leigh and Hofer (1973) have shown that when the number of pups
in the litter is reduced to one, the dam will continue to lick that pup longer than she
would have had she received enough stimulation from the offspring to stop licking and to

display the high crouch nursing posture. Pups that were exposed to PCB prenatally

76

weigh less and are present in fewer numbers on PND 6 than oil only controls, and the
dams that rear these pups spend more time licking their pups and less time engaged in
high crouch nursing than dams with prenatally oil-exposed pups. A litter that is smaller
in size and number may not provide enough ventral stimulation to stop the dam’s display
of pup-directed LG and elicit the high crouch nursing posture, leading to an overall

increase in LG and decrease in high crouch.

The interaction that occurred between treatments and days for high crouch nursing may
be related to the decrease in average amount of weight gained by PCB-exposed pups.
The display of high crouch is reduced on PNDs 4 and 6 in the combined PCB goups.
The average amount of weight gained by the offspring in these goups is also reduced
compared to the oil-only controls. Previous research has shown that high crouch nursing
is more prevalent in later postpartum compared to early postpartum (Stern and Johnson,
1989); the offspring are larger and can more easily elicit the high crouch behavior from
the dam. Since a dam will only engage in high crouch nursing if she receives adequate
ventral stimulation, pups that are smaller in size may be less able than larger pups to
stimulate the dam to display the high crouch posture. The difference in high crouch
nursing may not develop between treatment goups until PND 4 because the goups may

not exhibit a difference in weight until that time.
Since this experiment did not look at average pup weight gain on each PND, it is not

possible to know if the difference in weight gain emerged just after birth or later in the

ﬁrst postnatal week. However, a previous experiment using 2 and 4 mg/kg doses of PCB

77

did not ﬁnd a difference in pup weight gain until PND 2 (Simmons et al., 2005). This
ﬁnding may lend support to the suggestion that the interaction between treatment and
days is related to changes in the size of the offspring that are not present immediately
after birth. In addition, high crouch nursing is the posture from which the pups receive
the most nourishment. Thus, a reduction in high crouch may result in lower weight gain
by the young, causing in a circular pattern of cause and effect that would result in

decreased pup weight gain and decreased display of high crouch.

PCB exposure altered the pattern of effect of days for high crouch nursing, such that the
amount of time the dams in the combined PCB goup spent engaged in high crouch did
not increase on PNDs 4 and 6, as it did for the oil only goup. If the hypothesis presented
above is correct and PCB-exposed pups gain weight more slowly than oil-only pups, the
amount of high crouch nursing exhibited by the dams in the combined PCB goups may
increase at a later date when the size of PCB-exposed pups is sufficient to evoke the
behavior. On the other hand, high crouch nursing in the combined PCB goups may not
increase to the level of that exhibited by the oil-only females for the duration of lactation.
I am unable to address this question, as maternal behavior was not observed beyond PND

6.

Although the 2 and 4 mg/kg doses did not change the pattern of effects of days for any
measure, it is not unprecedented for an environmental contaminant to do so (Palanza et
al., 2002b). After treatment with the endocrine disrupter methoxychlor (MXC), Palanza

et al. report an alteration in the regulation of timing of maternal behavior in mice.

78

Control mice exhibit a natural decline in nest-related behaviors near the end of the second
postnatal week, an index of the start of the weaning process. Dams treated with MXC
began to decrease the amount of time they spent engaged in nest-related behaviors at the
end of the ﬁrst postnatal week during the dark period. Although the total amount of
maternal care that the pups received did not differ, altering the time weaning starts can
affect the neuroendocrine and/or behavioral proﬁle (Fleming et al., 1999). Therefore,
that PCB treatment altered the effect of days in this study may have sigiiﬁcant

implications for the offspring.

In an attempt to expand on the behavioral analysis of the previous cross-fostering report
(Cummings et al., 2005) the current study analyzed maternal ventral autogooming
separately ﬁ'om all other autogooming displayed by the dam. If PCB exposure does
change the stimulus properties of the pups in addition to reducing them in number, and
the dam is unable to achieve and sustain the high crouch, I hypothesized that the increase
in autogooming exhibited by dams rearing PCB-exposed pups may reﬂect an effort by
the dam to compensate for reduced stimulation from the pups through increased ventral
autogooming. The present data conﬁrm that on PND 2, dams rearing prenatally PCB-
exposed pups spent more time engaged in ventral autogooming than dams rearing oil-
exposed pups. This ﬁnding lends support to the hypothesis that the increase in maternal
autogooming displayed by dams rearing PCB pups may be the result of an attempt to

self-stimulate because of a decrease in pup-provided ventral stimulation.

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The data indicate that the amount of time the dam spent on the nest was also related to the
treatment that the pups received gestationally. That is, dams rearing pups that had been
exposed to PCB prenatally tended to spend more time on the nest than dams rearing
prenatally oil-exposed pups. This result is similar to what was reported after treatment
with 2 and 4 mg/kg. The increase in this behavior may be partly a result of the increase
in amount of time these dams spent gooming both themselves and their offspring on
PNDs l and 2. Oil-treated dams rearing prenatally PCB-exposed pups tended to spend
more time nursing than the other goups, which may also help to account for the

increased amount of time spent on the nest.

Finally, the amount of time spent nursing was altered after treatment with 3 mg/kg PCB.
This is the ﬁrst time PCB 77 has been shown to affect this measure, although time spent
nursing has been modiﬁed before with exposure to the environmental contaminants
methoxychlor (Palanza et al., 2002b) and Bisphenol A (Palanza et al., 2002a), which are i
also capable of estrogenic activity. Although PCB dams with oil pups and oil dams with
PCB pups differed from one another, they did not differ sigiiﬁcantly from the oil-only
goup or the goup of PCB-treated dams rearing prenatally PCB-exposed pups. This
pattern of behavior has not been exhibited in any other of the behaviors analyzed,
including the speciﬁc nursing postures that make up the total time nursing. Thus, while
oil dams rearing PCB pups did not spend sigiiﬁcantly more time in any speciﬁc nursing
posture (and even spent less time in high crouch than the oil only goup) the mean

amount of time spent in each position must have been elevated enough to put their total

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time nursing above that for PCB dams rearing oil offspring. The mechanism for this,

however, is unknown.

It is unclear as to why this dose (3 mg/kg PCB 77) resulted in interactions between
treatments and days and the 2 and 4 mg/kg doses did not. The lower dose may not have
had enough impact on the animal in order to affect the pattern of behavior over days,
whereas the main effect of the higher dose of PCB may have been so salient as to obscure
any potential interactions. It is possible that 3 mg/kg may be a threshold dose that is
strong enough to evoke interactions between treatment and postnatal days, but not so

strong as to cancel out the interactions because of its strong main effects.

Summary

Our results conﬁrm that treatment with 3 mg/kg PCB 77 alters maternal care and
offspring developmental measures. The patterns of behavioral change indicate that these
alterations in maternal care were most often due to the dams’ response to prenatally PCB-
exposed pups as opposed to being direct effects of PCB treatment on the darn. This
treatment yielded results that parallel those produced with a 2 mg/kg treatment. Both
doses caused a decrease in pup weight gain, number of pups on PND 6, and time spent
engaged in high crouch. A similar pattern resulted for percent of time PCB-exposed
animals spent on the nest and licking and gooming the pups, but only treatment with the
lower dose caused these behaviors to change sigriﬁcantly. The amount of time the dams

spent nursing was not sigiiﬁcantly different after either dose.

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It is clear that evaluation of the effects of environmental contaminants requires extensive
analysis to separate out those effects speciﬁc to the mother, those speciﬁc to the offspring

and those that arise as an interaction between a mother and her young.

82

Chapter 5. Does the sexual behavior or partner preference of pre- and/or
postnatally exposed PCB pups differ from controls? If so, are these differences

related to the altered maternal care exhibited by the effected dams?

ABSTRACT

Polychlorinated biphenyls (PCBs) are environmental contaminants that cause
developmental and behavioral problems in humans and animals. In rats, gestational
exposure to the PCB congener 3, 4, 3’, 4’- tetrachlorobiphenyl (PCB 77) affects the brain
and behavior of the offspring as well as the maternal behavior of the dams. In the present
study, a cross-fostering design was utilized to examine the effects of pre- and/or postnatal
exposure to PCB 77 on sexual behavior and partner preference in rats. Pregiant rats were
injected subcutaneously with either oil or PCB emulsiﬁed in oil (2 mg/kg b.w.) on
gestation days 6 — 18 and then given pups that had been exposed to either the oil vehicle
or PCB during gestation. The offspring were tested as adults in a ﬁve-week paradign
that included an initial partner preference test, three sexual behavior tests, and a ﬁnal
partner preference test. A 3-compartment chamber was used for partner preference tests
to examine the amount of time each experimental animal spent with the stimulus male
and female while allowing for sexual interaction to take place. Our results indicate that
PCB 77 affects male and female partner preference but not the display of sexual
behavior. Males exposed to PCB both pre- and postnatally showed a sigiiﬁcant increase
in their preference for the female following the three tests for sexual experience; this

effect of sexual experience was absent in the other goups. A comparison of the

83

combined PCB goups to the oil-only goup indicates that PCB increased the amount of
time females spent in the vicinity of the stimulus female and decreased the amount of
time spent with the stimulus male compared to controls. A comparison of the three
different goups of PCB exposure and the oil control showed that females that received
postnatal PCB exposure spent more time in the stimulus female chamber than females
that received no PCB exposure, and females receiving both pre- and postnatal PCB
exposure spent less time with the stimulus male than females receiving either no PCB
exposure or prenatal exposure only. The effects of PCB 77 on female partner preference
may stem from direct effects of the contaminant on the developing animal, but could be
also the result of the enhanced amount of maternal licking and gooming received by

these animals during the neonatal period.

INTRODUCTION

As previous experiments have shown (Simmons et al., 2005; this dissertation), rats that
receive PCB exposure during pregrancy and/or rats that rear prenatally PCB-exposed
offspring exhibit altered maternal behavior. The importance of maternal care in several
aspects of offspring deve10pment is well documented (see Dissertation Introduction for
review). For example, sigiiﬁcantly decreasing the amount of time a darn licks and
gooms her pups affects the timing of male sexual behavior (Moore, 1984), decreases the
number of motor neurons in the sexually dimorphic spinal nucleus of the
bulbocavemosus (Moore et al., 1992) and signiﬁcantly reduces total dendiitic arbor in the

SNB (Lenz and Sengelaub, 2005). On the other hand, increasing the amount of licking

84

and gooming (LG) the offspring receive decreases their level of fearfulness when
conﬁ'onted with a novel situation (Caldji et al., 1998) or in response to stress (Liu et al.,
97). Pup-directed LG has also been implicated in female reproductive success. Gomes et
al. (1999) demonstrated that females receiving more LG as a result of neonatal handling
showed an increase in the number of anovulatory cycles compared to non-handled
females. Thus, alterations in maternal care as a result of PCB exposure have the potential

to geatly affect the reproductive development of the offspring.

Previous work in this laboratory has indicated that PCB treatment alters female
receptivity and sexual behavior when administered neonatally (Chung et al., 2001) and
perinatally (Chung and Clemens, 1999). However, due to the desigrs of the experiments
that examined the effects of PCB on offspring behavior, it is not known whether the
effects seen in the offspring are attributable to the direct effects of pre- and or postnatal

PCB exposure, or to PCB-induced changes in maternal behavior.

Further, in vivo and in vitro studies have shown that PCB 77 can have both estrogenic
and anti-estrogenic activity (N esaretnam et al., 1996; Jansen et al., 1993). Estrogen (E)
has been shown to be necessary for the development of males’ preference for females
(Brand et al., 1991; Bakker et al., 1996; Houtsmuller et al., 1994), and the estrogenic
metabolite of testosterone is also required for the development of male sexual behavior
(see Baum, 2003 for review). Moreover, females given exogenous E early in

development demonstrate masculinized sexual behavior when tested as adults (Paup et

85

al., 1972; Paup et al., 1974). Consequently, PCB 77 may be acting estrogenically or anti-

estrogenically to alter male and female partner preference and sex behavior.

For the current experiment, I treated pregrant rats with PCB 77 during gestation and
tested their offspring as adults to evaluate the effects of prenatal exposure (PCB received
while the offspring were in utero) and/or postnatal exposure (PCB received via lactation)
on partner preference and sexual behavior. In the analysis, I also considered that the
effects seen in the offspring might be due, in part, to PCB-induced alterations in maternal

CEII'C.

METHODS

Animals and Housing

The animals used in this study were born to the Long-Evans dams from Chapter 2 that
received subcutaneous injections of 2 mg/kg PCB 77 on GD 6 - 18. Parturition took
place during the mid- to late-light phase of the animals’ light/dark cycle on GD 22 - 23.
On the day of birth [day of birth = postnatal day (PND) 0], the litters were culled to 8
animals (4 females and 4 males) and cross-fostered or left with their own mothers to
create the following goups: (1) oil treated dams with their own oil pups, (2) oil treated
dams with a new litter of oil pups, (3) PCB treated dams with their own PCB pups, (4)
PCB treated dams with a different litter of PCB-exposed pups, (5) PCB treated dams with

a litter of oil pups, and (6) oil treated dams with a litter of PCB-exposed pups. Each

86

goup contained six dams. Two males and two females were weaned from each dam on
PND 23 and housed in same-sex pairs for the duration of the study in plastic cages
similar to those previously described. Animals were maintained in a facility accredited
by the Association for Assessment and Accreditation of Laboratory Animal Care, and the
Michigan State University Animal Care and Use Committee approved all experimental

procedures.
Hormone Treatments

At 60 days of age, two female offspring from each litter were ovariectomized
(anesthetized with a ketamine/xylazine cocktail containing 44 mg/kg ketamine, 10 mg/kg
xylazine) and implanted subcutaneously with an estradiol benzoate-ﬁlled silastic capsule.
Capsules were made from silastic brand laboratory tubing (Dow Corning, cat. No. 508-
006) with an inner and outer diameter of 0.058 in. and 0.077 in., respectively. Estradiol
benzoate (EB, Signa) was dissolved in ethanol and allowed to sit in a Petri dish for 48 hr
or until all of the liquid evaporated ﬁ’om the dish. The crystalline EB was then packed in
the pre-cut silastic tubing to ﬁll a length of 5 mm. The ends of the capsules were sealed
with medical-gade silastic adhesive (Dow Coming), and implants were incubated in PBS
for 24 hr prior to implantation. After one week of recovery and four hours prior to the
start of the sexual behavior tests, females were injected with 0.5 mg/0.1 ml progesterone.
Stimulus females were at least 60 days of age at the start of testing. They were not
ovariectonrized but were treated with estrogen and progesterone in a manner similar to

experimental females.

87

Testing Procedure

Males and females began their 5-week testing paradign when they reached 90 and 60
days of age, respectively. The ﬁrst week consisted of a partner preference test during
which animals were sexually naive, followed by sexual behavior tests once a week for
three weeks, with behavioral data collected only on the third and ﬁnal test. The paradign
ended with a partner preference test during the ﬁfth week. All tests took place under dim

red illumination during the the dark phase of the light-dark cycle.

Partner Preference Tests

Animals were tested in a three-compartrnent apparatus made of Plexiglas. The middle
compartment was connected to the two outer compartments (30 X 59 X 39 cm each
chamber, 90 X 59 X 39 cm total) by openings in the back of the apparatus. One of the
outer compartments contained a sexually active male and the other a sexually receptive,
hormone-treated female. Both stimulus animals were tethered to the front of the
apparatus using a Velcro harness and wire. Stimulus animals were able to display sexual
behavior but were not able to leave their respective chambers. Experimental animals
were able to traverse the chambers freely through the openings in the back of the
apparatus. The experimental animal was placed in the center chamber and allowed to
acclimate for 5 min prior to the start of the test. After 5 min, the clear plastic divider that

closes the middle compartment off from the rest of the apparatus was removed. The test

88

lasted for 20 min, during which time the duration spent in each chamber was recorded.

No sexual behavior was recorded at this time.

Three investigators scored the female partner preference tests from the video recordings.
Because of procedural demands, it was impossible to keep one of these three
investigators unaware of the treatment received by each offspring. A subset of the
recordings was scored independently by all investigators and a correlational analysis was
used to evaluate the inter-observer reliability. The positive correlation between the three

data sets was geater than .80 for all behavioral measures.

Male-Paced Sexual Behavior Tests

Sexual behavior tests were conducted in a Plexiglas chamber, 56 X 44 X 49 cm, where
the experimental male had ﬁee access to a sexually receptive stimulus female who
received EB implants and progesterone injections as described above. Males were
allowed 5 min to acclimate to the chamber before the introduction of the female. The test
was terminated after the male achieved a post-ejaculatory intromission or 30 min had
elapsed since the female was introduced and no ejaculation was achieved. Behavioral
measures that were recorded include: number of mounts and intromissions, mount latency
(ML, the time until the male achieved his ﬁrst mount), intromission latency (IL, the time
until the male achieved his ﬁrst intromission), ejaculation latency (EL, the interval from
the ﬁrst intromission to ejaculation), copulatory efﬁciency (CE, number of intromissions

divided by the number of intromissions plus the number of mounts), inter-intromission

89

interval (111, the average amount of time between intromissions starting with the ﬁrst
intromission and ending with ejaculation), and post-ejaculatory interval (PEI, the interval

ﬁom ejaculation to the next intromission).

F emale-Paced Sexual Behavior Tests

Experimental females were tested with sexually active adult stimulus males in a two-
compartment Plexiglas cage. The testing chamber (56 X 44 X 49 cm) was divided into a
main chamber (34 X 44 X 49 cm) and an escape chamber (22 X 44 X 49 cm). During
these tests, the females could retreat to the escape chamber through one of four holes in
the Plexiglas divider that separated the main and escape chambers, and through which the
larger male was unable to pass. The stimulus male was introduced into the main chamber
5 min before the experimental female was placed in the escape chamber. The test ended
after the female received an ejaculation and returned to the main chamber after her
postejaculatory reﬁactory period. However, if the female did not leave the main chamber
within 60 8 following ejaculation, the test was stopped. Alternatively, if the female did
not receive an intromission within 15 min or an ejaculation within 20 min after the start

of the test, the test was terminated.

During this test, the following behavioral measures were recorded: approach latency (AL,
the amount of time it takes the female to cross into the main chamber after being
introduced in the escape chamber), mount latency (ML, the time between the female’s

entry into the main chamber until the ﬁrst mount), and intromission latency (IL, the time

90

between the female’s entry into the main chamber and the ﬁrst intromission). Mount
return latency (MRL), intromission return latency (IRL), and post-ej aculatory refractory
period (PER) refer to the length of time between the female leaving the main chamber
following a mount, intromission, and ejaculation, and returning. All latency measures are
expressed in seconds. The percentage of times that the female escaped ﬁ'om the male
following a mount or intromission was calculated by dividing the number of exits by the
number of each copulatory event. The percentage of time the female spent with the male
was also calculated by dividing the amount of time the female spent in the main chamber
by the total test time. In addition, the sexual receptivity of experimental females was
analyzed using the lordosis quotient (LQ). When the female is mounted, she shows a
reﬂexive posture that is characterized by a pronounced arching of the back called
lordosis. LQ is calculated by the following formula: (number of lordosis responses
during the ﬁrst 10 mounts/10) X 100, or (number of lordosis responses/number of

mounts) X 100 when the number of mounts was smaller than 10 during the pacing test.

General Analyses

Preliminary analyses revealed that the sexual behavior and partner preference exhibited
by male and female offspring were not different between those offspring reared by their
own mother or by a foster mother (t-tests, ps = 0.35 — 0.89 for all behaviors examined).

For this reason, data from the goup of PCB mothers rearing their own PCB pups

(uncrossed) were combined with those from the goup of PCB dams rearing a litter of

91

PCB pups that were not her own (crossed). The same was done for the crossed and

uncrossed oil goups.

Partner Preference Analyses

2 X 2 ANOVAs (treatment X ﬁrst or second test) with repeated measures on the second
factor were used to test the hypothesis that any exposure to PCB affected the offsprings’
partner preference. This was evaluated by comparing the offspring of the oil goup to a
combined PCB goup that included offspring of all goups receiving any PCB exposure.
In addition, to evaluate the effects of the three types of PCB exposure against the oil
goup, 4 X 2 ANOVAs (treatment X ﬁrst or second test) with repeated measures on the
second factor were used to analyze the data. Si giiﬁcant interactions and/or main effects

and simple effects were followed by Fisher’s PLSD. Differences were considered

statistically sigiiﬁcant if p S 0.05.

Sexual Behavior Analyses

If a male did not achieve ejaculation his score was left out of the analysis. For each
behavioral measure obtained ﬁom experimental animals of both sexes, a t-test was used
to test the hypothesis that any exposure of the litter to PCB affects the pups’ sexual
behavior. One-way ANOVAs were also used to evaluate the effects of the four different
PCB treatments on behavior and sigiiﬁcant F ratios were followed by post hoc tests. All

differences were considered statistically si giiﬁcant if p s 0.05.

92

RESULTS

Male Partner Preference

The 2 x 2 ANOVA comparing all PCB-exposed animals to the oil-only goup over the
two preference tests did not reveal sigiiﬁcant effects of treatment or sigiiﬁcant
interactions for the percent of time spent in each chamber (data not shown). However, a
sigriﬁcant effect of test was found for percent of time spent in the female chamber [F (l ,
33) = 5.33, p = 0.027] and percent oftime spent in the male chamber [F (1, 33) = 11.36, p
= 0.002], with males spending more time with the female and less time with the male

during the second test than during the ﬁrst test (data not shown).

The ANOVA of the four treatment goups detected a sigiiﬁcant interaction between
treatment and test for time spent in the stimulus female chamber [F (3, 31) = 3.14, p =
0.03 9]. For the goups that received both pre- and postnatal exposure to PCB, there was
a simple main effect of test (t = -2.41, df = 20, p = 0.026); these animals spent more time
with the female during the second partner preference test than the ﬁrst test (Figure 17).
There was no sigiiﬁcant simple main effect of treatment on percent of time spent with
the female during the ﬁrst test (Figure 17). However, there was a simple main effect of
treatment during the second partner preference test [F (3, 38) = 3.58, p = 0.023]; none of

the PCB-treated goups were different from the oil-only goup on this second test, but

93

Time in the Female

Chamber (Percent)

 

 

‘ I Pre- and Postnatal
PCB Exposure
5‘ Postnatal PCB Exposure
Prenatal PCB Exposure
E] No PCB Exposure

 

 

 

Figure 17. A gaph illustrating the differences found with the
post hoc analysis that followed the signiﬁcant interaction
between treatment and week for percent of time males spent
in the female chamber. There were no signiﬁcant
differences during the ﬁrst partner preference test. During
the second test, there was a signiﬁcant effect of treatment;
the goups sharing the same letter are not signiﬁcantly
different from each other.

94

males from both goups exposed to PCB prenatally spent more time in the female

chamber than those males who received only PCB exposure postnatally (Figure 17).
Female Partner Preference

For all dependent variables, the ANOVAs comparing the combined PCB goups to the
oil-only goup found no sigiiﬁcant effects of test and no sigiiﬁcant interactions between
treatments and test (data not shown). However, the PCB-exposed goup spent
signiﬁcantly more time with the stimulus female [F (1, 33) = 18.51, p < 0.001] and
sigiiﬁcantly less time with the stimulus male [F (l, 33) = 8.76, p = 0.006]. Those main
effects of PCB exposure are shown in Figures 18 and 19, respectively. There was no
effect of PCB exposure on percent of time spent in the middle chamber (p = 0.90; data

not shown).

The ANOVAs comparing the four treatment goups across the two tests found no
signiﬁcant effects of test and no signiﬁcant interactions. But there was a sigiiﬁcant main
effect of treatment on percent of time spent with the stimulus female [F (3, 31) = 7.60, p
< 0.001]. Post hoc analysis revealed that females receiving postnatal PCB exposure only
and those receiving both pre- and postnatal PCB exposure spent more time with the
stimulus female than the oil goup (Figure 20). For percent of time spent with the
stimulus male, the ANOVA detected a signiﬁcant main effect of treatment [F (3, 31) =

6.22, p = 0.002] and post hoc tests showed that females that received both me and post-

95

Time in the Female
Chamber (Percent)

35

30
25a

20-

10‘

15-

 

d

 

 

 

 

 

 

 

 

Combined PCB on Group
Groups

Figure 18. A comparison of the combined PCB groups
to the oil only goup found a signiﬁcant difference (*)
in the amount of time females spent with the stimulus
female, with the PCB- exposed animals spending more
time in the female chamber than the oil animals.

96

Time in the Male
Chamber (Percent)

 

so
45~
40.1
35-
304
251
20:
15:
1o:

5:

0:

 

 

 

 

 

 

 

f
F
*
I
L
r
L.
L
Combined PCB Oil Group
Groups

Figure 19. A comparison of the combined PCB goups

to the oil only goup found a signiﬁcant difference (*)
in the amount of time females spent with the stimulus
male, with the PCB- exposed animals spending less
time in the male chamber than the oil animals.

97

Time in the Female

Chamber (Percent)

 

 

    

 

 

 

 

‘ r
35 -‘ A A -
30 - ‘
25 - ‘
. B
20 - T ..
15 - ~
10 - '
5 -‘ .
Pre- and Postnatal Prenatal No Exposure
Postnatal Only Only
Type of PCB Exposure

Figure 20. The ANOVA of the data examining the
four treatment groups found a signiﬁcant main effect
of treatment on percent of time females spent with the
stimulus female; the goups sharing the same letter are
not signiﬁcantly different ﬁ'om each other.

98

Time in the Male

Chamber (Percent)

 

so
45.-
40.-
353
30:
25:
205
155
ro-Z

 

0-

 

 

 

 

 

Pre- and Postnatal Prenatal No Exposure
Postnatal Only Only

Type of PCB Exposure

Figure 21. The ANOVA of the data examining the
four treatment goups found a signiﬁcant main eﬁ‘ect
of treatment on percent of time females spent with the
stimulus male; the goups sharing the same letter are
not sigiiﬁcantly different from each other.

99

natal PCB exposure spent less time with the male than those females that received either
only prenatal or no PCB exposure (Figure 21). There was no effect of treatment on

percent of time spent in the middle chamber (p = 0.16; data not shown).

Male-Paced Sexual Behavior

A comparison of all PCB-exposed goups to the oil-only goup revealed no sigiiﬁcant
differences between the goups for any of the behavioral measures that were analyzed.
One-way ANOVAs of the four treatment goups also failed to reach sigiiﬁcance for all

behaviors examined (Table 2).

F emale-Paced Sexual Behavior

A comparison of all PCB-exposed goups to the oil-only goup revealed no sigriﬁcant

differences between the goups for any of the sexual behavior measures that were

investigated. One-way ANOVAs of the four treatment goups also failed to reach

sigiiﬁcance (Table 3).

100

Table 2
Exposure to PCBs and male sexual behavior

 

 

PCB/PCB PCB/oil oil/PCB oil only
(F8) ("=81 ("=8) ("=8)

#M 13.12% 1.2 12.0:24 15314.1 11.1:t 1.7
#1 14.4:t1.5 10.41 1.1 11.5:t3.5 11.5i3.9
ML (s) 40.6 :t 14.3 106.6 i 50.8 64.1 :t 19.8 140.6 :t 53.9
IL (3) 49.5 i 10.9 187.1 :t 75.4 74.6 :1: 32.9 219.4 :t 80.5
EL (5) 483.2 i 50.1 564.6 :t 59.2 535.1 i 76.5 376.5 :t 83.7
111 (s) 41.3 i 4.0 43.4 t 2.3 44.2 :t 5.3 45.0 :1: 8.4
PEI (s) 301.1 a; 33.7 260.8 :t 63.1 177.3 1 27.5 220.7 :1: 30.1
CE (%) 52.0 58.4 52.5 59.3

 

The data for the ﬁrst seven measures are presented as mean :t: S.E.M. No signiﬁcant differences between
goups were detected with p values ranging ﬁ'om 0.32 — 0.88. See text for statistical tests. PCB/PCB =
prenatally PCB-exposed pups reared by PCB-treated dams; PCB/oil = prenatally oil-exposed pups reared by
PCB-treated dams; oil/PCB = prenatally PCB-exposed pups reared by oil-treated dams; pups only exposed
to oil; # M = number of mounts; # I = number of intromissions; ML = mount latency; IL = intromission
latency; EL = ejaculation latency; 111 = inter-intromission interval; PEI = postejaculatory interval; CE =

copulatory efﬁciency; n = number of litters.

101

Table 3
Exposure to PCBs and female sexual behavior

 

 

PCB/PCB PCB/oil oil/PCB oil only
(I! = 8) (n = 8) ,.(n = 8) (n = 8)
AL(s) 4.63: 1.0 10.5:t4.3 6.1 $0.3 8.1 i 1.1
ML (5) 65.4 i 28.6 128.5 at 112.7 59.6 :t 20.9 86.7 :t 33.0
IL (s) 37.8 :t 27.0 140.0 a: 111.2 120.7 :1: 65.2 89.8 :t 38.9
MRL (s) 8.5 :1: 2.0 27.0 :1: 18.1 5.9 :t 2.3 15.8 :1: 7.5
IRL (s) 30.9 i 3.5 35.6 i 2.9 14.6 :t 5.7 39.0 :1: 13.7
PER (s) 67.7 :1: 11.8 107.6 :1: 26.7 65.1 :t 12.8 114.9 :t 25.7
LQ (%) 77.9 i 4.8 78.3 d: 4.9 75.0 :1: 7.2 71.9 :t 7.4

 

The data for the measures are presented as mean i S.E.M. No signiﬁcant differences between goups were
detected with p values ranging from 0.22 — 0.74. See text for statistical tests. PCB/PCB = prenatally PCB-
exposed pups reared by PCB-treated dams; PCB/oil = prenatally oil-exposed pups reared by PCB-treated
dams; oil/PCB = prenatally PCB-exposed pups reared by oil-treated dams; pups only exposed to oil; AL =
approach latency; ML = mount latency; IL = intromission latency; MRL = mount return latency, IRL =
intromission return latency; PER = postejaculatory refractory period; LQ = lordosis quotient; n = number of
litters.

102

DISCUSSION

It is important to note that although we refer to our testing paradign as a “partner
preference” situation, and even though sexual interaction was allowed between the
experimental and stimulus animals, we do not imply that we tested the animals for their
“mate choice.” We determined with whom the experimental animal would rather spend
time when given the choice between a sexually active male or a hormone-primed,
sexually receptive female when sexual interaction was allowed to take place. Prefening
to spend time with an animal of the same sex to one of the opposite sex does not imply '
that the experimental subject would prefer to mate with an animal of the 'same sex. It
means that given the choice, and under these conditions, the experimental animal would

rather spend time in the vicinity of one stimulus animal over another.

Male Partner Preference

The comparison of the combined PCB goups with the oil goup showed that regardless
of treatment, males spent more time with the stimulus female after receiving sexual
experience. However, analysis of the four separate treatment goups revealed that this
overall increase in female preference during the second partner preference test was
mainly due to the increase in preference exhibited by males that received double PCB
exposure. That is, no other goup’s preference changed signiﬁcantly after sexual
experience. This enhanced responsiveness to the effects of sexual experience on

preference for the female was the only feature that differentiated any PCB-exposed goup

103

from the oil controls. An increase in preference for the female induced by sexual
experience has been reported for control male rats (Brand and Slob, 1991; Bakker et al.,
1996), but the mechanisms responsible for this effect of experience are not known.
Consequently, it is difﬁcult to generate hypotheses to explain why we did not see this
increase in the control goup or how it may be augnented by perinatal exposure to PCB
77. In the present study, there were no differences across goups on any aspect of male
sexual behavior; therefore it is unlikely that differences in quantity or quality of sexual

experience were responsible for the goup differences.

Female Partner Preference

Females exposed to PCB perinatally spent more time with the stimulus female and less
time with the stimulus male than females that were not exposed to PCB. Comparison of
the combined PCB goups to the oil goup revealed that any exposure to PCB altered the
amount of time females spent with both stimulus animals compared to the control
females. With respect to time spent with the female stimulus, the more detailed analysis
revealed that females reared by dams that had been treated with PCB while pregrant
spent more time in the vicinity of a sexually receptive female than controls. In addition,
those females that received double exposure during development also showed a

sigiiﬁcant reduction in the time spent with the male when compared to controls.

The altered partner preference displayed by females who were reared by PCB-exposed

dams may be partially attributed to the changes in the care provided by their mothers.

104

Analysis of some aspects of the maternal care received by these animals (see Chapter 3)
revealed a pattern of licking and gooming behavior in the dams that was very similar to
the females’ partner preference proﬁle. PCB-exposed dams displayed an increased
amount of pup-directed LG, and the females from those dams exhibited an increased
preference for the stimulus female. In addition, while the differences in pup-directed LG
between the four treatment goups was not statistically sigiiﬁcant (Chapter 3), the three

PCB-exposed goups appeared to spend more time engaged in LG than the oil-only dams.

A correlation was run to test the hypothesis that the amount of LG that the PCB-exposed
females received was correlated to the increased percent of time they spent with the
stimulus female and decreased amount of time spent with the stimulus male. The percent
of time that each PCB-exposed female spent with the stimulus male and stimulus female
was compared to the average amount of LG that her litter received over the four postnatal
days. Interestingly, the correlation did not reveal any signiﬁcant effects (rs = -0.01 —
0.29). It is possible that a correlation does exist but is obscured by other factors. For
example, the correlational analysis used the average amount of LG that the entire litter
received over the four postnatal days that were examined. Our procedural methods do
not allow us to examine the speciﬁc amount of LG that each female received on each
postnatal day. Thus, although the mean amount of LG that the litter received over all
four postnatal days does not correlate with the percent of time PCB-exposed females
spent with the stimulus animals, the amount of LG each individual female received may

correlate to the percent of time spent with the stimulus animals.

105

PCBs and other environmental contaminants have been detected in the milk of rats
(Crofton et al., 2000; You et al., 1999) and humans (Dewailly et al., 1991; Schulte and
Malisch, 1984; Jensen, 1987; Noren and Lunden, 1990) after exposure to the toxin,
indicating the possibility of lactational transfer to the offspring. Thus, in addition to
possible maternal inﬂuences, the offsprings’ change in partner preference may be due to
direct effects of the PCBs that they received from the dam’s milk. PCB exposure has
also been found to have geater developmental effects when the animal is exposed
postnatally rather than prenatally. For example, exposure to Aroclor 1254 caused
permanent low-ﬁ'equency hearing deﬁcits and hypothyroxinernia in rats who were
exposed postnatally, whereas offspring who received only prenatal exposure were not
sigiiﬁcantly affected (Crofton et al., 2000). Thus, it is not surprising that the females
who are reared by PCB-treated dams are different from females reared by oil-treated

dams.

It is possible that the preference PCB-exposed females exhibit for the stimulus females is
not be the result of increased sexual motivation but increased fearfulness associated with
the male. Conceivably, PCB-exposed females may be interpreting novel situations as
fearful, particularly those in which a sexually active male is tethered in one chamber and
where it is possible to escape to a compartment that contains a more familiar, less novel
animal (i.e. another female). The PCB-treated females may not be attracted to the
stimulus female as a result of sexual motivation, but because another female is a more
familiar or less novel stimulus. However, our ﬁndings that PCB-exposed offspring

received an increased amount of LG, and the work conducted by Meaney and colleagues

106

(Liu et al., 1997; Caldji et al., 1998), would indicate that this is not likely to be the case.
Females that receive an increased amount of LG from their mothers tend to be less fearful
and less reactive to novel situations. On the other hand, the increased amount of LG
demonstrated by PCB-treated dams did not reach the level of LG exhibited by the dams
in Meaney’s studies. Therefore, the offspring reared by PCB-exposed dams may not
have received the amount of LG that is required in order to decrease the females’ level of
fear. To test this hypothesis, one would need only to measure the stress response of these
females when they are placed in the vicinity of a stimulus male. If the stress response is
within the normal range, or similar to that of control females, then the PCB-exposed
females are not spending more time in the stimulus females’ chamber because of an

increased fear of the male.

It is also possible that the reason PCB-exposed females spent more time in the stimulus
females’ chamber is that the PCB-exposed females are more sexually attracted to the
stimulus females. One way to determine if PCB exposure is increasing the females’
sexual preference for other females is to measure the experimental animals’ proceptivity
level in either a partner preference or female-female pacing situation. If PCB exposure
increases the females’ sexual preference for females, then we would expect the affected
individuals to show an increase in proceptive behaviors such as car wiggling, hopping
and darting. An increase in these behaviors would lend evidence to the possibility that
PCB treatment has altered the females’ choice for a sexual partner. This hypothesis is
currently being tested in our laboratory by further analysis of the video recordings of the

partner preference tests. The possibility that PCB-exposed females are spending more

107

time with the stimulus females and less time with the males because of a decrease in
receptivity can be ruled out, since the lordosis quotients of the PCB- and oil-exposed

goups are not different.

While PCB affected the stimulus preference of both sexes, the effects were more salient
for the females. This difference may be due in part to the different procedures employed
for the two sexes. Experimental males remained intact for the tests, while experimental
females were gonadectomized and brought into receptivity using exogenous estrogen and
progesterone. It is possible that the PCBs are affecting the males and females similarly,
but the males are able to compensate for any changes caused by the PCBs because of

their functioning testes.

Male-Paced Sexual Behavior

In contrast to the changes seen in male partner preference, gestational and/or lactational
exposure to PCB 77 did not alter male sexual behavior in the pacing paradign.
Comparison of the combined PCB-exposed goups to the oil-only goup as well as
comparison of the four individual treatment goups did not reveal any signiﬁcant
differences. These ﬁndings parallel those of previous experiments in which a single oral
dose of PCB 77 on gestation day 15 (Faqi et al., 1998) and multiple intraperitoneal doses

on gestation days 7 — 18 (Wang et al., 2002) failed to alter male sexual behavior.

108

It is also important to note that the increased amount of pup-directed LG provided by
PCB-treated dams may serve to buffer or compensate for some of the toxic effects that
the offspring may otherwise incur as a result of exposure to the contaminant. As
mentioned in the Discussion section of Chapter Three, manipulations that increase licking
and gooming (e. g. neonatal handling) have been shown to protect the offspring from the
effects of early-life insults including fetal ethanol exposure (Lee and Rabe, 1999) and
cerebral hypoxia (Chou et al., 2001). Previous research has documented the importance
of maternal behavior, in particular licking and gooming, in the development of male
sexual behavior (Moore, 1984; Moore, 1995). Since PCB-exposed dams exhibited an
increased amount of pup-directed LG, it is possible that some developmental effects that
would otherwise be seen in the pups as a result of PCB exposure may be lessened

because of the increase in licking and gooming that is exhibited by the PCB-exposed

goups.

There are a few experiments that could be conducted in order to test whether the
increased LG male pups receive ﬁom PCB-exposed mothers reduces some of the effects
that PCBs might otherwise have of male sexual behavior. First, PCB-exposed male
offspring could be fostered to mothers that show either sigiiﬁcantly increased or
signiﬁcantly decreased LG. If males reared by a low LG dam demonstrate altered sexual
behavior compared to those males reared by a high LG darn, then it is likely that we did
not see effects of PCB on male sexual behavior in our study because the increased LG

exhibited by the dams attenuated the effects of the toxin on the offspring.

109

Another possible experiment to determine the role of LG in PCB-affected male sexual
behavior would be to administer zinc sulfate to PCB-treated dams in order to render them
anosmic. This procedure has been shown to sigiiﬁcantly decrease the amount of licking
that the pups receive (Moore and Power, 1992; Moore et al., 1992). However, there are
additional considerations that need to be taken into account if this method is employed.
For example, nearly eliminating the amount of stimulation provided by the darn as a
result of her being anosmic has been shown to affect the display of male sexual behavior
(Moore, 1992). Therefore, any changes seen in male sexual behavior exhibited by PCB-

exposed offspring reared by anosrrric dams cannot be conclusively attributed to the PCB.

Finally, an experiment that identiﬁes the exact amount of LG that each PCB-exposed pup
receives would help to elucidate the possible role of LG in attenuating the effects of PCB
on male sexual behavior. Our desigr did not allow for individual examination of the
amount of maternal behavior each offspring received. While we can identify if a dam
demonstrated a high or low amount of LG, the amount of individual stimulation each pup
receives often varies within the litter. If we could correlate the amount of LG that each
pup receives ﬁom PCB-exposed dams to his sexual behavior performance, we would be

able to identify if increased LG affected this behavior.

F emale-Paced Sexual Behavior

While PCB 77 affected female partner preference behavior, we did not see any sigiiﬁcant

effects of the toxin on the females’ copulatory behavior. No sigriﬁcant differences were

110

found in any behavioral measures when comparing the combined PCB-exposed goups to.
the oil-only goup, nor when comparing all four treatment goups to one another.
Previous research from our laboratory using commercial PCB mixtures demonstrated
sigiiﬁcant changes in female receptivity and sexual behavior after the offspring had been
exposed to the mixture in the early prenatal (Chung et al., 2001) and the perinatal period
(Chung and Clemens, 1999). However, it is difficult to compare our current ﬁndings to
those of earlier experiments because they used mixtures of PCBs that were comprised of
both coplanar (the class of PCBs of which PCB 77 is a part) and ortho-substituted PCBs,

whereas the current study employed a single PCB congener.

In a study in which PCB 77 was delivered during the same gestation days as used in the
current experiment but utilizing a different route of administration (IP), Wang et al.
(2002) describe reduced female sexual behavior and receptivity. The discrepancies
between their results and those reported here could be due to the differential metabolism
of the PCB as a result of different routes of administration. Hany et al. (1999a) examined
the relative amounts of PCB congeners that remained in various tissue types after
subcutaneous treatment of the dam on GD 7 — 18. The concentration of PCB 77 that
remained in tissue was extremely small, indicating that PCB 77 is metabolized quickly
when the toxin is administered subcutaneously. Although there are no data
demonstrating the how quickly the PCB would be metabolized after IP administration,
there is the possibility that the toxin would be degaded more slowly. Thus, the
differences between the female sexual data presented here and. those from Wang et al.

may be explained by the possibility that PCB 77 has a geater effect on female sexual

lll

behavior when it is delivered IP because it is not degaded as quickly as when the

contaminant is administered subcutaneously.

Summary

While perinatal PCB exposure did not affect sexual behavior, it did alter female and male
partner preference. There was an interaction between treatment and test for PCB-
exposed males with regards to their stimulus animal preference. Treatment with PCB
prenatally increased the effects of sexual experience on the males’ preference for a
stimulus female compared to postnatal PCB treatment. Any exposure to PCB altered the
females’ preference; pre- and/or postnatal exposure increased the amount of time females
spent with the stimulus female and decreased the amount of time spent with the stimulus
male. The changes seen in the females may be due in part to altered maternal care
provided to the offspring as a result of the PCB exposure their mothers received while
pregrant. Speciﬁcally, the pattern of licking and gooming exhibited by PCB-exposed
dams is similar to the preferences exhibited by the female offspring. While this does not
explain the pattern of behavior for the males, it is interesting that by increasing the
amount of licking and gooming the females receive (i.e., masculinizing the amount of

stimulation) the females’ preference for both stimulus males and females is masculinized.

112

Chapter 6. General Discussion

For decades the traditional toxicological paradign used to evaluate the effects of a toxin
on a developing organism involved treating pregnant rats with a speciﬁc contaminant and
examining the offspring. In light of the work presented in this dissertation as well as that
conducted by Simmons et al., it is clear that this traditional paradign needs to be revised
to include an analysis of maternal behavior. Taking into consideration the accumulating
evidence in recent years, it is essential that we control for the effects on the mother when
evaluating whether treatment effects on the offspring are a direct result of the treatment
on the developing organism or a reﬂection of alterations in maternal care, or some

combination thereof.

In the traditional paradign, treatment of the dam is simply considered an avenue through
which toxins can be delivered to the developing embryo (for example, see Loefﬂer and
Peterson, 1999; Simanainen et al., 2004). Clearly, such exposure is not speciﬁc to the
pups and effects of the contaminant on the dam must be factored into the eventual
outcome. Changes in maternal care are often accompanied by changes in the offsprings’
physiology and/or behavior. Without taking into account possible changes in the
behavior of the dam, we cannot state with certainty that the effects seen in the offspring
are due to the direct effect of the toxin on the pups as is ﬁequently reported (e. g. Loefﬂer

and Peterson, 1999).

113

As early as the 19603 the impact of neonatal stimulation on the development of the litter
was clearly established. At that time, the authors noted that their daily handling of the
litter changed various aspects of the pups’ behavior when they were tested as adults
(Levine and Thoman, 1969; Thoman and Levine, 1969). The authors also identiﬁed
changes in the dam that were due to handling of the pups (Levine and Thoman, 1969).

At that time, however, the mechanisms responsible for these changes were not known. It
was not until 30 years later that scientists linked handling of the pups to speciﬁc changes
in maternal care. Meaney and colleagues (Liu et al., 1997) identiﬁed an increase in the
amount of pup—directed licking and gooming provided by the dams of handled offspring,
and linked these changes in maternal care to a variety of physiological and behavioral

alterations seen in the offspring.

In this thesis, we have demonstrated not only that PCB treatment to rats during gestation
alters the care provided by the exposed dams, but that the behavior of the dams can also
be altered in response to rearing prenatally PCB-exposed offspring (Chapters 3 and 4).
Although the dynamic interactions that take place between a mother and her pups are not
new, this is an area which has been virtually igrored in the ﬁeld of toxicology.
Additional analyses must be conducted to further address how early treatments can
modify adult behavior via direct or indirect action of the treatment on both the offspring

and their mother.

We have also demonstrated effects of PCB exposure on partner preference behavior in

female rats (Chapter 5). New hypotheses arose out of our analysis of loffspring partner

ll4

preference that warrant further consideration. For example, the sexual motivation of the
PCB-exposed females may have been altered in the direction of preferring stimulus
females rather than males. The additional analyses will include correlating offspring
behavior to the amount of pup-directed licking and gooming that the offspring received
as neonates to evaluate potentially confounding effect of PCB-induced alterations in

maternal care on the offspring.

Environmental contamination with man-made chemicals is unfortunately a fact of
modern life and is a global problem. Even though PCBs are no longer produced they
continue to enter and exist in the ecosystem and move throughout the world, threatening
the existence of both human and non-human populations. Saving these populations from
extinction and protecting ourselves ﬁom the effects of exposure requires an
understanding both of the global dynamics of PCBs as well as how these contaminants
affect the organisms with which they come in contact. Since it is clear that we can not'
fully protect ourselves against PCB exposure, we must ﬁnd ways to attenuate the effects.
While there is no miracle “cure,” there are some measures that may be taken in order to
help ameliorate the effects of PCB exposure. For instance, environmental enrichment,
deﬁned as “a combination of complex inanimate objects and social stimulation” (Guilarte
et al., 2003), has been shown to attenuate deﬁcits produced by developmental lead
exposure (Guilarte et al., 2003). There also have been several reports demonstrating the
efﬁcacy of early intensive behavioral therapy, sensory stimulation and environmental
enrichment in reversing (or overcoming) some of the core autistic symptoms (Schneider

et al., 2006; Harris et al., 1991; Scheinkopf and Siegel, 1998; Ozonoff and Cathcart,

115

1998), that surprisingly seem also to be triggered by early PCB exposure. Thus, it is
possible that employing similar techniques to children exposed to PCBs developmentally
may help to reduce the observed deﬁcits. Irnportantly, these interventions have the
geatest effect when the disorders are detected early (Filipek et al., 2000; Sandler et al.,
2001), stressing the importance of identifying possible effects and symptoms of PCB

exposure in order to better diagiose and treat the deﬁcits early on.

This thesis is a small step when considering the enormity of the problem. However, it
does establish that the earlier paradigns used to analyze the effects of toxins on
developing organisms needs to be expanded to include consideration of the complex

interaction between the contaminant and the dynamic mother-infant relationship.

116

APPENDICES

117

Appendix A: The effect of developmental PCB exposure on open ﬁeld behavior.

This experiment was conducted in order to test the hypothesis that developmental PCB
exposure affected the animals’ display of fear in an open, well-lit arena. The animals that
were tested in this study were born to the dams ﬁ'om Chapter Two, Experiment Two in
which the pregrant rats were treated with a subcutaneous dose of PCB 77 during
gestation and given an untreated cookie immediately after the injection. The offspring

were weaned ﬁ'om the dam on postnatal day (PND) 23 and housed in same-sex pairs.

Testing began at 60 and 90 days of age for females and males, respectively. All animals
were left intact. The open ﬁeld apparatus was made of opaque Perspex and was 120 X
120 cm. Lines were drawn on the ﬂoor of the apparatus using a large permanent marker
in order to determine the mobility of the animal. The experimental animal was placed in
a small, black, Perspex box and put in the corner of the apparatus before the test began.
The box was completely enclosed save for one of the walls that the experimenter could
remove at the start of the test to let the animal out. Tests lasted 10 mins from the time at
which the wall of the black box was removed. The following parameters were recorded:
latency to emerge from the black box, amount of time spent in the ﬁeld, percent of time
spent in the perimeter compared to the center of the ﬁeld, number of lines crossed,

number of boluses in the black box, and number of boluses in the ﬁeld.

None of the males exposed to 4 mg/kg PCB were still alive when testing began on PND

90. Thus our analysis included females from the 2 and 4 mg/kg goups and males from

118

the 2 mg/kg goup. Our analysis did not reveal any sigiiﬁcant main effects of treatment
on any of the parameters measured. Thus, it appears that developmental exposure to 2 or
4 mg/kg does not affect the animals’ performance in the open ﬁeld. However, this does
not mean that the animals’ stress or fear responses are not altered by exposure to PCB,
but that the behaviors measured with our experiment were not affected. Examining the
animals’ responses in a different testing situation designed to analyze different behaviors
would help to address this question of whether or not the animals’ fear responses are
affected by PCB exposure (e.g., measuring the animal’s stress response in a restraint

situation).

119

Appendix B: The effect of developmental estrogen (E) on maternal behavior.

Since PCB has been shown to act as an estrogen, we desigied an experiment to see if
PCB 77 was altering maternal behavior by acting estrogenically. Pregnant dams were to
be treated with varying amounts of E during gestation, after which their maternal
behavior would be recorded on the same PNDs as those examined in the PCB studies
(PND 1, 2, 4 and 6) and compared to the maternal behavior exhibited by dams exposed to

PCB 77.

We used a total of six different doses over the duration of our experiment, yielding an
increase in serum levels of E to approximately 110 pg/ml, 55 pg/ml, 35 pg/ml, 25 pg/ml,
20 pg/ml and 10 pg/ml (exposure via silastic implants, approximations from Bridges,
1984). However, no dams carried their fetuses to full term; the pregnancies of all dams
were aborted or the embryos resorbed. It is likely that our doses of E were too high for
the dam to maintain the pregnancy. Throughout the pregrancy, E remains low and rises
only at the end before parturition (for example see Siegel and Rosenblatt, 1975). The
increased level of E produced by our implants may have signaled to the dam’s body that

her current hormonal conditions were not optimal and aborted the pregrancy.

120

Appendix C: The effect of PCB-exposed dams on offspring nipple attachment.

In the experiment examining the effects of 2 mg/kg PCB on maternal care, we found a
decrease in the amount of time the dams spent in the high crouch nursing posture after
PCB exposure. Since the darn cannot engage in high crouch nursing unless she receives
the appropriate amount of ventral stimulation (including rooting around for and attaching
to a nipple), we wondered if the offspring being reared by PCB-exposed dams found

them aversive and were not motivated to attach to their nipples.

In order to test this, we treated dams with 3 mg/kg PCB 77 during gestation. On the day
of birth, dams and litters were cross-fostered to make the following four goups: PCB-
treated dams rearing PCB-exposed offspring, PCB-treated dams rearing oil-exposed
offspring, oil-treated dams rearing PCB-exposed offspring, and oil-treated dams rearing
oil-exposed offspring. On PND 7, litters were removed and placed in an incubator for 2
hours to induce hunger in the pups. At the end of the 2 hr period, dams were anesthetized
and placed on her side. Pups were placed 2 cm from the darn, one pup close to the
anterior and one close to the posterior ends of her ventrum. Tests were stopped either
when both pups attached to the nipples or after 3 min had elapsed. Three trials were run
per dam, one trial with each goup of pups. Dams were not tested with the litters they

reared, and the order of presentation of pups was randomly varied from dam to dam.

Very few pups attached to the dam within the time limits of the test, so we were not able

to run any analyses to test the hypothesis that PCB-exposed dams spent less time engaged

121

in high crouch nursing because the offspring found the dams to be less attractive, or

aversive, and spent less time suckling.

122

Appendix D: The effect of PCB-treatment on expression of c-fos in maternal brain

areas.

In order to see if PCB-treatment to the dams or the pups altered the expression of c-fos in
maternal brain areas, dams were treated with 3 mg/kg PCB 77 dining gestation. On the
day of birth, dams and litters were cross-fostered to make the four exposure goups. On
PND 4, all pups were removed from the dams and given back during the light cycle 48 hr
later. This was done in order to induce the expression of maternal behavior in the hopes
of also inducing expression of c-fos. The behavior of the dams was recorded for 2 hr
after the pups were replaced for conﬁrmation of behavioral expression and to perhaps

compare the amount of behavior displayed with the expression of c-fos.

A total of 5 of the 14 dams tested responded maternally after replacement of the pups
(Table 4). The reasons for this remain unclear; the paradign we followed has often been
used to successfully induce maternal behavior. However, since we were not able to
induce maternal behavior, we did not examine the brains for PCB-induced alterations in

fos expression.

 

 

Table 4.
Number of Dams Responding Matemally.
Treatment of Treatment of Number of Dams Responding
Darn Pups Matemally
PCB PCB 2 out of 3
PCB oil 1 out of 3
oil PCB 1 out of 4
oil oil 1 out of 4

 

 

 

123

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