In higher vertebrates, estrogens exert an
organizational effect on the central nervous
system (CNS) during the perinatal phase of
development, and estrogen is the main hor-
mone responsible for sex differences in
behavior (1,2). There is great concern that
estrogenic pollutants, at low concentrations,
may mimic the action of estrogens on the
CNS at an early age and produce long-last-
ing or irreversible effects on behavior (3–5).
Bisphenol A (BPA) is a compound widely
used in the food industry for polycarbonate
bottles and linings of cans, as well as in den-
tistry as a hardener for sealants and for tooth
lacquering. It is known to have an estrogenic
action (6–8). Low doses of BPA adminis-
tered perinatally can modify explorative
behavior and anxiety in rats (9) and can
advance puberty in female mice (10),
although some effects are controversial (11).
These observations indicate the importance
of studying the effects of this compound on
complex behaviors expressed during puberty.
In mammals, play is considered crucial
for the maturation of adult behavior
(12,13). Many of the behaviors expressed in
a play situation are sexually dimorphic and
under the control of sex steroids. For exam-
ple, rough-and-tumble play is controlled by
androgens (13,14). Immature sexual ele-
ments of play represent a developmental
process leading to adult sexual behavior;
this process is under the perinatal organiza-
tional control of estrogens. Therefore,
because of its multiplicity in behavior, play
is a useful tool for the study of behavioral
alterations (15).
To study the potential effects of BPA on
behavior, we administered it to dams in two
treatments: long periods of exposure to a
low-dosage regimen, from conception to
weaning of their pups, and short periods of
exposure to a high-dosage regimen, from
gestation day (GD) 14, when the differentia-
tion of many sexual characters begins (16),
to postnatal day (PND) 6. The two dosages
are in the range of human environmental
exposure to BPA and are below concentra-
tions generally considered in toxicological
studies. The lower concentration for a longer
period of time can be encountered in human
food; the higher concentration for a shorter
period of time in human saliva after dental
interventions (17,18). We then studied the
behavior of the offspring of the treated
mothers in a play situation and considered
all behaviors expressed.
Materials and Methods
Subjects
We used 84 immature Sprague-Dawley rats
(42 females and 42 males) that were born
and bred in the Department of Animal
Biology, University of Florence (Florence,
Italy), and were the offspring of mothers
treated during gestation and lactation, as
described below. The subjects were separated
into three groups on the basis of their expo-
sure to BPA during the perinatal period: a)
high-dose treatment, 15 males and 15
females offspring of mothers treated with
short periods of exposure to a high dosage of
BPA; b) low-dose treatment, 12 male and
12 female offspring of mothers treated with
long periods of exposure to a low dosage of
BPA; c) control, 15 female and 15 male off-
spring of vehicle-treated mothers. The litters
were weaned on PND 21. For each treat-
ment group, the animals were randomily
chosen from different litters and housed in
groups of three males and three females in
polysulfone cages, 42 × 26 × 15 cm
(Tecniplast, Italy), under a natural light–
dark cycle. No cage contained siblings. Food
and water were available ad libitum.
Treatment Procedure
Bisphenol A (Fluka Ltd., Buchs,
Switzerland) dissolved in arachis oil was
administered daily at two dosages (40 and
400 µg/kg body weight) to two groups of
mothers during pregnancy and lactation.
Thirty-one female rats of reproductive
age were randomly allocated to three groups
and subjected to the following treatments: a)
low dose (n = 11), receiving 40 µg/kg/day
BPA, administered from day 10 before mat-
ing with a mature male until weaning of the
pups (PND 21); b) high dose (n = 11),
receiving arachis oil from day 10 before mat-
ing until GD 13, followed by 400 µg/kg
BPA from GD 14 (± 1 day) to PND 6, and
then arachis oil again until weaning; c) con-
trol (n = 9), receiving arachis oil from day 10
before mating until weaning. BPA was dis-
solved in arachis oil at 5.32 and 53.2 µg/mL,
for the low-dose and high-dose modalities.
Controls received a comparable amount of
oil, according to body weight. BPA was
administered orally with a pipette. Because
the animals enjoyed receiving the oil, the
procedure was not stressful. For all females,
mating took place 10 days after the begin-
ning of treatment: they were housed three
Environmental Health Perspectives • VOLUME 110 | SUPPLEMENT 3 | JUNE 2002 403
Effects of Perinatal Exposure to Bisphenol A on Play Behavior of Female
and Male Juvenile Rats
Francesco Dessì-Fulgheri,1 Stefania Porrini,2 and Francesca Farabollini2
1Department of Animal Biology and Genetics, University of Firenze, Firenze, Italy; 2Institute of Human Physiology, University of Siena,
Siena, Italy
This article is part of the monograph Impact of
Endocrine Disruptors on Brain Development and
Behavior.
Address correspondence to F. Dessì-Fulgheri,
Dept. of Animal Biology and Genetics, University
of Firenze, Via Romana 17, Firenze, Italy I-50125.
Telephone: 39 55 2288228. Fax: 39 55 222565.
E-mail: dessi@unifi.it
We are grateful to P. Palanza for her valuable
suggestions, A.F. Pantani and A. Cianfanelli for
technical support, and P. Christie for linguistic
revision. This research received financial support
from the University of Firenze (60% to F.D.-F.),
University of Siena (60% to F.F.), and MURST
(Cofin to F.D.-F. and F.F.).
Received 8 January 2002; accepted 27 March
2002.
Endocrine Disruptors
In higher vertebrates, estrogen can exert an organizational effect on sexually dimorphic areas of
the central nervous system (CNS) during the perinatal phase of development. The possibility that
estrogenic pollutants may mimic estrogen action on the CNS during development and produce
long-lasting or irreversible effects is an issue of great concern. Bisphenol A (BPA), a compound
widely used in the food industry and in dentistry, has proven estrogenic actions. To study its
potential developmental effects on behavior, we gave female Sprague-Dawley rats 40 µg/kg/day
BPA from conception to weaning postnatal day 21 and 400 µg/kg/day BPA from gestation day
14 to postnatal day 6. After exposure, we studied social behavior in a play situation in juvenile
male and female offspring. The attempt to use play behavior to study the effects of BPA yielded
some interesting results. We observed an early action of BPA on several behavioral categories in
both males and females. In particular we observed a masculinization of female behavior in two
behavioral categories (play with females and sociosexual exploration), an effect probably mediated
by the estrogenic activity of BPA in the CNS. These long-lasting effects of BPA could have
important consequences at individual and population levels. Key words: bisphenol A, environmen-
tal estrogens, play behavior, rat, sex differences, social behavior. Environ Health Perspect
110(suppl 3):403–407 (2002).
http://ehpnet1.niehs.nih.gov/docs/2002/suppl-3/403-407dessi-fulgheri/abstract.html

per cage for 48 hr with a sexually mature
male and then transferred to single cages.
The litters were culled to eight at birth and
weaned at PND 21. For each treatment
group, the pups were randomized and
housed in groups of three males and three
females such that no cage contained siblings.
Behavioral Testing
Behavioral observations were conducted at
PNDs 35, 45, and 55 between 1500 and
1900 under artificial dim white light. Rats
belonging to the same cage were tested
together for each age, and subjects were
individually marked with cosmetic dye.
Behavioral observations took place in a
neutral arena (36 × 61 × 34 cm) with the
floor covered with clean sawdust. The six
cagemates were transferred directly to the
arena from the home cage and video-
recorded with a Sony Hi8 video recorder for
6 min, starting after 1 min of familiariza-
tion. The video recordings were then ana-
lyzed by one observer blind to treatment,
using Noldus Observer in combination with
Noldus Video Tape Analysis System
(Version 3.0; Noldus Information
Technology, The Netherlands). All behav-
iors were recorded and the actor and
receiver identified for social interactions.
For the purposes of the present research,
only the frequency of behaviors displayed
during min 2 and min 3 of each session
were considered.
Play, defined as “any activity involving
exaggerated movements and inhibited
attacks; it appears to achieve no obvious
goal,” was identified according to the
description of Poole and Fish (19), while
those behaviors similar to adult behavior
were identified according to the description
of Grant and Mackintosh (20). Table 1 lists
the behaviors we considered.
Statistical Analysis
Principal component analysis (PCA) (21),
with varimax rotation and Kaiser normaliza-
tion (SPSS software), was performed on fre-
quencies of behaviors of all experimental
subjects. Individual factor scores of each
principal factor were subsequently used as
independent variables in a three-way analysis
of variance (ANOVA) considering treat-
ment, sex, and age.
When appropriate, post hoc analysis
(Fisher least significant difference test) sepa-
rately in males and females for comparison
of the treatment groups.
Animal Welfare
Experimental procedures followed the regu-
lations of the European Communities
Council Directive 86/609/EEC (22).
Endocrine Disruptors • Dessí-Fulgheri et al.
404 VOLUME 110 | SUPPLEMENT 3 | JUNE 2002 • Environmental Health Perspectives
Table 1. List of social and nonsocial behaviors
considered.
Nonsocial behaviors
Air-smelling
Exploration of the environment
Rearing (animal stands up and explores the
environment)
Ground exploration
Digging
Self-grooming
Crouching
Social behaviors
Approaching (moving toward another)
Crawl-over (moving over another)
Crawl-under (moving under another)
Social investigation (sniffing another’s body except
anogenital area)
Anogenital sniffing
Allogrooming (gentle grooming of another’s fur)
Aggressive grooming (vigorous grooming of another)
Pouncing (bouncing over another)
Charging (rushing toward another with vigorous
bouncing gait)
Chasing
Riding (forepaws over the back of a moving partner)
Sideways posture (the animal orientates itself
broadside on to another)
Aggressive posture (the animal orientates itself at
right angle to and over another)
Submissive posture (lying on the back with belly
exposed to another)
Biting
Withdrawing (all movements away from another)
Upright posture (with erect posture the rat exposes
its belly to another)
Boxing (both rats stand up facing each other and
boxing with forepaws)
Jumping (animal leaps vigorously into the air)
Table 2. Results of PCA applied to behaviors of immature rats: rotated component matrix (varimax) (total
variance explained: 69.3%).
Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Factor 6 Factor 7 Factor 8
(12.9%) (11.0%) (9.0%) (8.7%) (7.4%) (7.3%) (7.0%) (6.0%)
Nape–m 0.80 — — — — — — —
Pounce–m 0.79 — — — — — — —
Crawl over–m 0.73 — — — — — — —
Riding–m 0.73 — — — — — — —
Pounce–f — 0.81 — — — — — —
Chase–f — 0.74 — — — — — —
Nape–f — 0.71 — — — — — —
Withdraw–f — 0.44 — — — — — —
Air smelling — — 0.88 — — — — —
Rearing — — 0.84 — — — — —
Sideways posture–m — — — 0.80 — — — —
Withdraw–m — — — 0.79 — — — —
Crawl under–f — — — — 0.73 — — —
Crawl under–m — — — — 0.57 — — —
Anogenital sniffing–f — — — — — 0.70 — —
Social investigation–f — — — — — 0.67 — —
Self–grooming — — — — — 0.49 — —
Digging — — — — — — 0.77 —
Ground exploration — — — — — — 0.63 —
Approach–m — — — — — — — 0.82
Approach–f — — — — — — — 0.55
Abbreviations: f, female directed; m, male directed.
Only largest correlation coefficients of each behavior are reported. Percentage of total variance accounted for by each
factor is given in parentheses.
Table 3. Three-way ANOVA applied to factor scores.
Treatment Treatment Treatment
Treatment Sex Age × sex × age Sex × age × sex × age
(df = 2, 234) (df = 1, 234) (df = 2, 234) (df = 2, 234) (df = 4, 234) (df = 2, 234) (df = 4, 234)
F p F p F p F p F p F p F p
Factor 1 1.13 NS 0.57 NS 9.28 <0.000 1.15 NS 1.26 NS 2.63 <0.1 0.27 NS
Factor 2 3.46 <0.03 14.80 <0.000 5.88 <0.003 0.17 NS 0.70 NS 0.86 NS 0.64 NS
Factor 3 2.13 NS 0.07 NS 9.23 <0.000 0.99 NS 1.60 NS 0.82 NS 0.48 NS
Factor 4 2.09 NS 1.09 NS 9.02 <0.000 1.88 NS 1.72 NS 0.19 NS 0.88 NS
Factor 5 5.15 <0.01 4.69 <0.05 5.00 <0.007 0.07 NS 2.89 <0.02 0.58 NS 1.56 NS
Factor 6 7.21 <0.001 34.31 <0.000 8.43 <0.000 2.60 <0.1 1.27 NS 4.93 <0.01 0.76 NS
Factor 7 0.74 NS 14.76 <0.000 0.28 NS 1.96 NS 0.47 NS 2.09 NS 0.26 NS
Factor 8 14.69 <0.000 3.24 <0.1 11.30 <0.000 0.53 NS 2.46 <0.05 2.57 <0.1 0.89 NS
Abbreviations: df; degrees of freedom; NS, not significant, p > 0.1.

sexual exploration). Moreover, in one
category (play with females), we observed an
intensification of male behavior in males due
to BPA. Both BPA doses were below con-
centrations generally considered in toxico-
logic studies. Our results appear to agree
with the hypothesis of an early action of
BPA mediated by its estrogenic activity at
the CNS.
Conclusions
We have attempted to use a complex behav-
ior model—play behavior—to study the
effects of potential endocrine disruptors on
behaviors controlled by sex steroids during
development. Using this approach, we
observed many significant effects of the envi-
ronmental estrogen BPA. The action of BPA
was long-lasting. Our research suggests that
very important mechanisms underlying cer-
tain behaviors are involved, and thus, in the
long run, even small changes can have conse-
quences on individual fitness and on popula-
tion structure. The mechanisms underlying
the observed effects need to be clarified by
further research.
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Environmental Health Perspectives • VOLUME 110 | SUPPLEMENT 3 | JUNE 2002 407