53, 392399 (2000)

Copyright 2000 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Effects of Prenatal Aflatoxin B 1 Exposure on Behaviors

of Rat Offspring
Takahide Kihara,* ,1 Takuya Matsuo,* Michiko Sakamoto,* Yoshiko Yasuda,* Yoshitame Yamamoto,
and Takashi Tanimura
*First Department of Anatomy, Kinki University School of Medicine and Life Science Research Laboratory, Kinki University, Osakasayama, Osaka,
589-8511, Japan; and Medical Information Department, Santen Pharmaceutical Co. Ltd., Higashiyodogawa-ku, Osaka, 533-0021, Japan
Received March 4, 1999; accepted June 22, 1999

Aflatoxin B 1 (AFB), produced by several fungi such as

Aspergillus flavus and A. parasiticus (Wilson et al., 1968) (Fig.
1), is one of the most important food borne mycotoxins and is
one of the most potent hepatotoxins and carcinogens in many
animal species (Eaton and Gallagher, 1994; Wogan and Newberne, 1967), and it has also been implicated in the etiology of
1

To whom correspondence should be addressed at 1-3-4-303 Harayamadai,

Sakai, Osaka 590-0132, Japan. Fax: 81-722-99-8437.

human liver cancer in a number of epidemiologic investigations (IARC, 1993).

The developmental toxicity of AFB has been studied in
various animals (for review, see Hayes 1981; Hood, 1979;
Hood and Szczech, 1983). AFB has been reported to be teratogenic and/or embryotoxic in rats (Elegbe et al., 1974; Grice
et al., 1973; LeBreton et al., 1964; Panda et al 1970; Tanaka,
1975), in mice (Arora et al., 1981; DiPaolo et al., 1967; Roll et
al., 1990; Tanimura et al., 1982), in hamsters (DiPaolo et al.,
1967; Elis and DiPaolo, 1967), in chick embryos (Bassir and
Adekunle,1970; Cilievici et al., 1980; Dietert et al., 1985), in
tadpoles (Gabor et al., 1973), and Japanese medaka eggs
(Llewellyn et al., 1977). Tranplacental carcinogenesis in rats
(Goerttler et al., 1980; Grice et al., 1973; Tanaka, 1975), and
selective immune depression in chick embryos (Dietert et al.,
1985) have also been reported. A previous study in our laboratory demonstrated that AFB induced cleft palate, skeletal
malformations, and intrauterine growth retardation in mouse
fetuses of dams injected intraperitoneally with doses of 32
mg/kg/day for 2 days of days 6 7, 8 9, 10 11, 1213 of
gestation (Tanimura et al., 1982).
In the past 20 years, the importance of postnatal evaluation for behavioral teratology has received increasing recognition, and the test battery system for assessment of
behavioral teratogenic potential in reproductive and developmental toxicity study has been used widely (Riley and
Vorhees, 1986; Tanimura, 1990, 1992; Ulbrich and Palmer,
1996). However, there is little published data on the behavioral teratogenic effects of AFB in animals or in humans.
There has been only one study to date on the functional
effects caused by prenatal AFB exposure. Chentanez et al.
(1986) showed that AFB 2.0 mg/kg administered intravenously to Fisher rats on days 8 10 or 1517 of gestation
induced a decrease in some types of behaviors and in motor
activity levels in 1-month-old offspring.
The present study, therefore, was conducted to determine the
behavioral teratogenic effects on the rat offspring of dams
injected subcutaneously with a subteratogenic dose of AFB
during mid or late organogenesis. The test battery system

392

Downloaded from http://toxsci.oxfordjournals.org/ by guest on April 14, 2015

The effects of prenatal aflatoxin B 1 (AFB) exposure on eight

behavioral parameters in Jcl:Wistar rat offspring were assessed.
Pregnant rats were injected subcutaneously with 0.3 mg/kg/day of
AFB dissolved in dimethylsulfoxide on days 1114 (Group A) or
1518 (Group B) of gestation. Controls received the vehicle similarly on days 1118 of gestation. Before weaning, the offspring
were examined using the cliff avoidance response (5 days of age),
the negative geotaxis reflex (7 days), and swimming development
(6, 8, and 10 days). After weaning, animals were examined using
the rotarod test (5 weeks of age), the open field test (6 weeks), a
conditioned avoidance learning test (14 weeks), an underwater
T-maze test (15 weeks), and a reproduction test (16 weeks). The
preweaning offspring in the AFB-A group showed significantly
lower success rates than controls in cliff avoidance responses. In
swimming development, the offspring in the AFB-A group had
significantly lower scores than controls for swimming direction. In
the rotarod test, the AFB-A group remained on the rod for a
significantly shorter time than the controls at 15 rpm on both the
first and second trial days. The avoidance performance of the rats
in AFB-A and AFB -B groups was significantly poorer than that of
controls. These results indicate that prenatal exposure to AFB
produced a delay of early response development, impaired locomotor coordination, and impaired learning ability in the offspring
of rats exposed to AFB during middle pregnancy, and the early
gestational exposure appears to produce more effects than latter
exposure.
Key Words: aflatoxin B 1; 2,3,6a,9a-tetrahydro-4-methoxycyclopenta[c]furo[3,2,: 4,5]furo[2, 3-h][1] benzopyran-1, 11-dione;
mycotoxin; behavioral teratology; prenatal exposure; developmental toxicity; neurotoxicity.

393

PRENATAL AFLATOXIN B 1 AND BEHAVIOR

The following physical landmarks were noted: bilateral pinna unfolding at 4

days of age, abdominal hair emergence at 7 days, low incisor eruption and
bilateral eye opening at 14 days, descent of both testes in each male at 28 days,
and vaginal opening at 42 days in females.
Behavioral Test Battery
The behavioral tests and ages at testing are listed in Table 1. All the
behavioral testing procedures were conducted blind with regard to the treatment groups, and tests were performed between 09:00 A.M. and 05:00 P.M.
Cliff avoidance. A rat was placed on a table edge with the forepaws and
nose over the edge. The amount of time required to complete backing and
turning away from the edge was recorded. Each offspring was tested in one
trial. (Altman and Sudarshan, 1975; Brunner et al., 1978; Kihara, 1991). The
number of rats with successful responses within 30 s was recorded.
FIG. 1.

Structural formula of aflatoxin B 1.

MATERIALS AND METHODS

Animals and Treatments
Nine-week-old male (230 250 g) and female (170 190 g) Jcl:Wistar rats
were purchased from Clea Japan, Osaka, Inc., and acclimated to the laboratory
for 2 weeks prior to mating. Pellet diet OA-2 (Clea Japan, Osaka, Inc.) and tap
water were available . Animals were maintained in a room with a controlled
temperature of 23 2 0C, a relative humidity of 50 10%, and a 12-h
light:dark cycle (lights on at 07:00 A.M.). Eleven-week-old females were mated
overnight for 16 h with one male each. Mating was confirmed if sperm was
found in a vaginal smear or if a plug was observed. The following morning
(day 0 of gestation), the pregnant rats were randomly assigned to one of three
groups, each consisting of 10 female rats at conception. They were individually
housed in polypropylene cages with wooden shavings provided throughout
gestation and lactation, and left undisturbed except for treatment and weighing
until parturition.
Pregnant rats were injected subcutaneously with 0.3 mg/kg/day of AFB
(Makor Chemical Ltd., Jerusalem, Israel) dissolved in 0.9 mg/kg of dimethyl
sulfoxide (Wako Pure Chemical Industries Ltd., Osaka) on days 1114 (Group
A) or 1518 (Group B) of gestation. Control animals received subcutaneously
the vehicle only on days 1118 of gestation. The treatment volume was 1.0
ml/kg body weight. Fresh solutions of AFB were prepared on the day of use.
The dams were allowed to deliver spontaneously and rear their offspring
until weaning. At 4 days after birth, the litters were culled randomly to groups
of eight offspring with the same number of males and females as far as
possible. At 21 days, the offspring were weaned, separated by sex, and housed
in hanging wire-mesh cages with four littermates of the same sex. All offspring
were identified by marking with dry ink before weaning and with picric
acid-ethanol solution after weaning.
Body Weight and Physical Landmarks
The dams were weighed on days 0, 7, 14, and 21 of gestation, daily during
treatment, and on days 0, 4, 7, 14, and 21 after delivery. After weaning their
litters (21 days after delivery), all the dams were killed by ether overdose and
examined for numbers of implantation sites and any abnormalities of the
reproductive organs.
At birth, all live and dead offspring were counted. Live offspring were
weighed, sexed, and examined for external malformations. The live offspring
were again counted and weighed on 4, 7, 14, and 21 days after birth. After
weaning, all the offspring were counted and weighed weekly until 20 weeks
of age.

Swimming development. This procedure has been described elsewhere

(Kihara, 1991; Schapiro et al., 1970; Vorhees et al.,1979a,b). Each rat was
individually placed in a tank of water (28 0C) for 510 s and direction, angle in
the water (head position), and limb usage was observed. Direction scores
consisted of sinking (0 points), floating (1), circling (2), and swimming straight
or nearly straight (3). Angle scores consisted of head submerged (0), nose at
the surface (1), nose and top of head at or above the surface but ears still
below the surface (2), ears half way above the surface (3), and ears completely
above the surface (4). Limb usage scores consisted of no paddling (0), paddling
with all four limbs (1), and paddling with hind limbs only with forelimbs
stationary (2).
Rotarod. The apparatus consisted of a rod 7 cm in diameter with a hard
rubber surface, and linked to a variable-speed motor. The top of the rod was 34
cm above the base of the apparatus (Shinano Seisakusho Ltd., Tokyo, SN-498).
Animals were placed individually on the rod for at least 5 s, and it was rotated
at speeds of 5 or 15 rpm.. Rats were tested on two trials per day for 2
consecutive days. The maximum trial duration was 180 s, and the intertrial
interval was about 30 min. The time each animal remained on the rod at each
rotation speed was recorded (Kihara, 1991; Kihara et al., 1995).

TABLE 1
Schedule of Behavioral Test Battery
Procedure
Preweaning a
Cliff avoidance
Negative geotaxis
Swimming development
Postweaning b
Rotarod
Open field
Conditioned avoidance learning
Underwater T-maze
Reproduction

Age of testing

5
7
6, 8, 10
5
6
14
15
16

Note. Age of offspring for preweaning tests in days; age of offspring for
postweaning tests in weeks.
a
All offspring in each litter were tested.
b
One male from each litter was randomly assigned to each of the four
postweaning tests, except the reproductive test, for which one male and one
female from each litter were used.

Downloaded from http://toxsci.oxfordjournals.org/ by guest on April 14, 2015

recently developed in our laboratory (Kihara, 1991; Kihara et

al.,1995) was used for functional evaluations.

Negative geotaxis. The time taken to complete a 180-degree turn when

placed in a head-down position on a 25-degree inclined plywood surface was
measured. Each animal was given one trial. (Kihara, 1991; Vorhees et al.,
1979a,b). The number of rats with successful responses within 30 s was
recorded.

394

KIHARA ET AL.

Open field. Rats were tested in a circular open field (100-cm diameter
circular black polyvinyl chloride) on 3 consecutive days for 180 s per day. The
time to leave the start area (latency), number of sections entered with all the
four legs (ambulation), number of rearings, and number of fecal boluses were
recorded (Kihara, 1991; Kihara, et al., 1995).

Underwater T-maze. The T-maze apparatus consisted of a grey polyvinyl

chloride cylinder 20 cm in diameter; each arm was 50 cm long, and all the exits
were curved upward. The maze was completely filled with water maintained at
a temperature of 28 0C. Each animal was given 10 trials per day. A maximum
swimming time of 60 s was allowed for each trial. An error was designated as
entry into a blocked arm or re-entry into the stem. Swimming time and the
number of errors in the maze were recorded for each trial (Kihara, 1991;
Kihara et al., 1995).
Reproduction test. One male and one female from each litter were selected
at random for the reproduction test; sibling matings did not occur. They were
mated monogamously for 2 weeks (about three estrus cycles) and were
examined every morning for the presence of a vaginal plug. If a vaginal plug
was observed (day 0 of gestation), the female was placed in an individual cage
containing wooden chips for nesting. The dams were killed by ether overdose
and necropsied on day 21 of gestation. These uteri were examined and the
number of implantation sites, resorptions, and dead and live fetuses were
recorded. The live fetuses were sexed, weighed, and examined for external
malformations.

0.3 mg/kg

Group
No.
No.
No.
No.

of
of
of
of

dams
implants b
live births b
offspring
malformed at birth
Live birth index c
Survival index at 4 days of
age d
Weaning index at 21 days
of age e
Survival index at 20 weeks
of age f
Body weight of offspring
(g) b
At birth
M
F
At weaning
M
F
At 20 weeks
M
F

Control
Days 1118 a

Days 1114
(A)

Days 1518
(B)

10
16.2 0.4
15.2 0.5

10
15.8 0.7
14.4 0.4

10
15.2 0.9
12.9 0.9*

0
93.9

0
93.8

0
85.6

98.8

96.0

95.5

93.8

97.5

98.8

93.4

90.7

97.3

5.7 0.1
5.4 0.1

5.1 0.1*
4.9 0.1*

4.7 0.3*
4.7 0.2*

49.1 1.1
46.6 1.4

46.1 1.6
42.7 2.0

46.7 2.8
46.0 2.3

371 4.9
234 4.3

362 6.4
223 4.3

355 9.3
220 7.0

Note. M, male; F, female.

a
Gestational days exposed.
b
Mean SE.
c
Percentage of implants.
d
Percentage of offspring at birth.
e
Percentage of offspring at 4 days of age.
f
Percentage of offspring at weaning.
* Significantly different from the control (p 0.05).

RESULTS
Brain Weight

Maternal Effects

One male offspring from each litter was killed at 20 weeks of age by ether
overdose and autopsied. The brain was removed, weighed, and stored in 10%
neutral formalin solution.

Neither death nor noticeable symptoms were observed in the

dams of any group throughout gestation and lactation. AFB
had no significant effect on either the length of gestation or
maternal weight during the gestation and lactation periods
(data not shown).

Statistical Analysis
The number of implantation sites and live fetuses and offspring as well as
the body weight of dams and offspring were analyzed by one-way analysis of
variance (ANOVA), followed by Dunnetts test if differences were found. The
survival index data were analyzed by the chi-square test. The behavioral
measures and physical developmental observations were evaluated by using
the Mann-Whitney U-test for nonparametric comparison of group means
(Siegel, 1956; Sokal and Rohlf, 1973). Before weaning, the data from individual subjects were averaged, and the litter was used as the unit of analysis.
After weaning, the analysis was performed on the basis of individual animals
from each litter. The accepted level of significance was p 0.05.

Growth and Physical Landmarks of the Offspring

At birth, the number of live offspring was significantly lower
in the AFB-B group than those of controls, and body weights
of male and female offspring in both the AFB-A and -B groups
were significantly lower than the controls. However, there were
no significant differences between the AFB groups and the
control group with regard to the number of implants, sex ratio
of offspring, offspring with external malformations, live birth

Downloaded from http://toxsci.oxfordjournals.org/ by guest on April 14, 2015

Acquisition of conditioned avoidance. The apparatus was comprised of an

operant chamber (Ohara and Co. Ltd., Tokyo, Model GT-7710), and a
programming unit (Model GT-7715). As a visual and auditory warning device,
a pilot lamp (24W) and a loud speaker (400 Hz) were provided over the lever.
The chamber was placed inside a wooden sound-attenuating box (Model
GT-7720). The conditioned avoidance schedule was as follows: a 20-s intertrial interval (ITI), a 5-s warning duration (conditioned stimuli; CS), and shock
(approximately 8590V, 0.5 mA, 60 Hz AC) for a maximum of 5 s. Stimuli
were immediately terminated by the first lever-press elicited during the CS
period, and foot shock was avoided. Both lever-pressing at times other than the
CS presentation period and lever-holding were ineffective. Each session consisted of 1 h of training per day; animals were tested every other day for 15
sessions. The acquisition processes were considered to reflect discriminated
avoidance learning. The index of conditioned avoidance learning was rates of
avoidance.
The number of avoidance responses, stimuli presented, and shocks delivered
were recorded, and the mean percent of avoidance and the mean response rate
per day were calculated (Kihara, 1991; Kihara et al., 1995).

TABLE 2
Reproductive Performance of the Dams Exposed to Aflatoxin
B 1, and Survival and Body Weight in the Offspring

395

PRENATAL AFLATOXIN B 1 AND BEHAVIOR

TABLE 3
Reflex Development of Preweaning Rat Offspring of Dams
Exposed to Aflatoxin B 1

TABLE 4
Swimming Development in Preweaning Rat Offspring of
Dams Exposed to Aflatoxin B 1

0.3 mg/kg

Group
No. of offspring tested
Cliff avoidance (5 days of
age)
Success rate b
Response time c
Negative geotaxis (7 days
of age)
Success rate b
Response time c

0.3 mg/kg

Control
Days 1118 a

Days 1114
(A)

Days 1518
(B)

80

80

77

97.5
6.4 0.7

76.0*
16.7 2.3*

91.2
7.6 1.9

95.8
17.7 2.6

98.7
13.9 1.1

97.5
20.2 2.6

index, survival rates at 4, 21 days of age (at weaning), or 20

weeks of age, or body weights at 4 and 7 days of age (data not
shown), 21 days, and 20 weeks of age (Table 2).
With regard to physical landmarks, there were no statistically significant effects of AFB-exposure on pinna unfolding,
hair emergence, incisor eruption, testis descent, or vaginal
opening (data not shown). However, the mean percentage of
offspring with eyes opening at 14 days of age were 57.9, 17.9,
and 30.0 for the control, AFB-A, and AFB-B groups, respectively. The AFB-A group showed significantly delayed eye
opening compared to the controls.
Behavioral Testing
There were no sex differences on any of preweaning tests;
therefore, males and females were combined for data analysis.
The values presented the mean of the litter means in Table 3
and 4.
Reflex behavior. The results of reflex testing are presented
in Table 3. The AFB-A offspring were less successful in cliff
avoidance than control offspring, and the AFB-A group also
had significantly slower response times. Cliff avoidance was
not affected in the AFB-B group. On the negative geotaxis
reflex, there were no significant differences between the AFBexposed groups and the control group.
Swimming development. The results are shown in Table 4.
With regard to swimming direction, the AFB-A group scored
significantly lower than the control group at 6 days of age;
however, there were no significant treatment effects at any
other age observed. There were no significant differences between the AFB groups and the control group for swimming
angle and limb usage on 6, 8, and 10 days of age.

No. of offspring
6 days of age
Direction
Angle
Limb usage
8 days of age
Direction
Angle
Limb usage
10 days of age
Direction
Angle
Limb usage

Days 1114 (A)

Days 1518 (B)

80

80

77

2.3 0.1
2.3 0.1
1.0 0

1.8 0.1*
2.5 0.2
1.0 0

2.2 0.2
2.4 0.2
1.1 0.1

2.1 0.1
2.8 0.1
1.0 0

2.0 0.1
2.8 0.1
1.0 0

2.2 0.2
2.5 0.2
1.0 0

2.3 0.1
3.1 0.1
1.0 0

2.2 0.1
2.9 0.1
1.0 0

2.2 0.1
2.9 0.1
1.0 0

Note. Values are expressed using rating scales; mean SE.

a
Gestational days exposed.
* Significantly different from the control (p 0.05).

Rotarod performance. The rats in the AFB-A group remained on the rotarod for a significantly shorter time than the
controls at 15 rpm on both the first and second test days.
However, there were no statistically significant differences
between the AFB-B and control groups (Table 5).
Open field activity. There were no significant differences
between the AFB-exposed groups and the control group for
ambulation counts, rearing, and defecation frequencies, or the
start latency time on any of the test days. (The mean control
values [mean SE] were the ambulation 1st day 29.8 7.2,
2nd 22.0 4.7, 3rd 22.1 5.7; the rearing 1st 9.4 3.4, 2nd
3.6 1.3, 3rd 2.5 0.8; the defecation frequencies 1st 1.7
0.5, 2nd 4.0 0.8, 3rd 4.5 0.6).
TABLE 5
Rotarod Performance in Male Rat Offspring of Dams Exposed
to Aflatoxin B 1
0.3 mg/kg
Group

1st day
2nd day

rpm b
5
15
5
15

Control
Days 1118 a

Days 1114 (A)

Days 1518 (B)

136.0 22.6
30.4 10.5
152.7 18.4
91.6 25.6

84.5 26.6
6.9 2.0*
110.8 20.9
16.5 4.9*

70.5 29.6
46.7 25.3
176.6 3.4
87.8 31.7

Note. Values are means of time (s) spent on the rod SE. Maximum time
was 180 s per trial. Ten male offspring were tested per group.
a
Gestational days exposed.
b
Revolutions per min.
* Significantly different from the control (p 0.05).

Downloaded from http://toxsci.oxfordjournals.org/ by guest on April 14, 2015

Gestational days exposed.

b
Percentage of offspring achieving criterion.
c
Mean positive response time (s) SE.
* Significantly different from the control (p 0.05).

Group

Control
Days 1118 a

396

KIHARA ET AL.

DISCUSSION

Conditioned avoidance. Acquisition rates of the conditioned avoidance test are presented in Figure 2. The mean
avoidance rates in both AFB-exposed groups were lower than
those in the control group during 15 training sessions. Both the
AFB-A group (5th15th sessions) and the AFB-B group (12th,
14th, and 15th sessions) showed significantly lower avoidance
rates than the control group. Furthermore, rates of AFB-A
group during 511 sessions were also lower than avoidance
rates of the AFB-B group. There appears to be some increase
in variance that may be treatment related. No significant differences in the mean lever-pressing responses were detected
among the three groups throughout the 15 sessions of training
(data not shown).
Underwater T-maze. There were no significant differences
between the AFB-treated groups and the control group for
swimming time and number of errors on any of the trial days
(The mean control values of swimming time (s SE) were the
1st trial day 6.2 0.5, 2nd 4.6 0.3, 3rd 4.3 0.3, 4th 8.1
0.7, 5th 5.1 0.6, and 6th 6.1 0.5; the number of errors
ranged between 0.18 and 0.8).
Reproductive performance. No significant differences
were seen between the AFB-exposed and control groups in any
reproductive parameters including copulation and pregnancy
rates, litter size, sex ratio of offspring, fetal weight, or incidence of external malformations (data not shown).
Brain Weight
There were no statistically significant differences in brain
weight between the AFB-exposed and the control groups. The
values (mean: g SE) were 1.88 0.05, 1.83 0.04, and
1.85 0.04 for the control group, the AFB-A and the AFB-B
groups, respectively.

Downloaded from http://toxsci.oxfordjournals.org/ by guest on April 14, 2015

FIG. 2. Acquisition of conditioned avoidance responses in male rat offspring of dams exposed to aflatoxin B 1. Changes in the mean avoidance rates
are shown. AFB-A: period of exposure of aflatoxin B 1 (0.3 mg/kg/day) on days
1114 of gestation; AFB-B: days 1518; control (vehicle): days 1118. Ten
male offspring were tested per group. Vertical bars represent the mean standard
error. *Significantly different from control (p 0.05).

The present study is the first postnatal physical and behavioral evaluation of offspring development using a test battery
system following prenatal exposure to AFB during mid or late
organogenesis. The main effects observed were a smaller number of live births, lower mean birth weights, a delayed physical
development, delayed behavioral developments in the
preweaning period, and a impaired locomotor coordination and
deficits in avoidance performance in the postweaning period.
On the other hand, no effects on maternal mortality, maternal
weight gains, gestation length, the offspring with malformations, and the brain weight were observed in the prenatal
exposure to AFB groups.
AFB treatment during late organogenesis (days 1518) resulted in decreased numbers of live pups; however, no significant reduction in the live birth index was seen because the
mean number of implants in group AFB-B was smaller than
that the control group (Table 2). AFB exposure during both
mid-organogenesis (days 1114) and late organogenesis (days
1518) resulted in reduced birth weights in both male and
female offspring, but body weight differences were completely
recovered after 4 days of age. Thus, growth rates of offspring
in the AFB groups were equivalent to the control group during
the remainder of the study. There was also a delay in eye
opening in the prenatally exposed animals, but it was significant only when AFB was administered in the mid-organogenesis period (days 1114).
The present study is the first to show that prenatal exposure
to AFB affects behavioral performance in the preweaning
offspring and alters the rate of acquisition of a conditioned
avoidance task, motor coordination, and body balancing in the
postweaning offspring. Treatment with AFB during mid-organogenesis (days 1114) but not during late organogenesis (days
1518) delayed the development of the cliff avoidance response. Swimming ontogeny is a measure of the development
of neuromotor coordination and swimming ability (Kihara,
1991; Schapiro et al., 1970; Vorhees et al., 1979a,b). In the
present study, prenatal AFB exposure during mid-organogenesis (days 1114) also results in a delay in the direction of
swimming but not during late organogenesis (days 1518). On
the other hand, the negative geotaxis test results in the present
study did not distinguish exposed from control rats. These
result suggest that AFB has a significant toxic action on developmental patterns of behavior in newborn rats, particularly
on development of motor coordination.
The rotarod test has been used during the postweaning
period to determine forced coordinated motor and balancing
abilities (Altman and Sudarshan, 1975; Kaplan and Murphy,
1972; Kinnard and Carr, 1957). The results of the present study
show a deficit in motor skills in the offspring of mothers treated
with AFB during mid-organogenesis (days 1114) but not
during late organogenesis (days 1518). As rotarod performance has been related to cerebellar function (Pellegrino and

397

PRENATAL AFLATOXIN B 1 AND BEHAVIOR

areas, cerebral cortices, basal ganglia and forebrain, and for

thalamic, hypothalamic, and limbic regions (Vorhees, 1987).
The period of administration for the control group was twice
as long as that of the AFB treatment groups. This additional
handling stress of the control dams could influence some
behavioral parameters, especially basic values in the vehicle
control.
The mechanisms of AFB-induced behavioral teratogenesis
are unknown at present. However, AFB produces central nervous system malformations (Geissler and Faustman, 1988;
Tanaka, 1975), inhibition of DNA replication and binding to
DNA (Benasutti et al 1988; Jacobson et al., 1987; Sporn et al.,
1966), mutagenicity (Ong, 1975; Wong and Hsieh, 1976;
Yourtee and Kirk-Yourtee, 1987), chromosome aberrations
(Adgigitov et al.,1984), and transplacental carcinogenesis (Goerttler et al., 1980; Grice et al., 1973; Tanaka, 1975). It is
surmised that some of the actions of AFB mentioned above
are developmental toxic actions, including behavioral teratogenicity.
However, further studies are required to characterize any
behavioral effects of developmental AFB exposure in postweaning female rats, behavioral effects in progeny of dams
treated during the lactation period, neurobiochemical effects of
AFB, and the mechanisms of its effects on developmental
neurotoxicology.
ACKNOWLEDGMENTS
This work was supported by the Special Research Project on Environmental
Science, Grant-in-Aid for Scientific Research Ministry of Education, Culture
and Science, Japan, and in part by grants from the Environmental Science
Research Institute, Kinki University, and Grant-in-Aid for Science Research
from Japan Private School Promotion Foundation.

REFERENCES
Adams, J. (1986). Methods in behavioral teratology. In Handbook of Behavioral Teratology (E. P. Riley, and C. V. Vorhees, Eds.), pp. 6797. Plenum
Press, New York, London.
Adgigitov, F. I., Kosichenko, L. P., Popandopulo, P. G., Gubeladze, D, A., and
Djemilev, Z. A. (1984). Frequency of chromosome aberrations in bone
marrow of monkeys and their F 1 after aflatoxin B 1 injection. Exp. Pathol. 26,
163169.
Altman, J., and Sudarshan, K. (1975). Postnatal development of locomotion in
the laboratory rat. Anim. Behav. 23, 896 920.
Arora, R. G., Frolen, H., and Nilsson, A. (1981). Interference of mycotoxins
with prenatal developmentof the mouse. I. Influence of aflatoxin B 1 ochratoxin A and Zearalenone. Acta Vet. Scand. 22, 524 534.
Bassir, O., and Adekunle, A. (1970). Teratogenic action of aflatoxin B 1,
palmotoxin B 0 and palmotoxin G 0 on the chick embryo. J. Pathol. 102,
49 51.
Benasutti, M., Ejadi, S., Whitlow, M. D., and Loechler, E. L. (1988). Mapping
the binding site of aflatoxin B 1 in DNA: Systematic analysis of the reactivity
of aflatoxin B 1 with guanines in different DNA sequences. Biochemistry 27,
472 481.
Brunner, R. L., McLean, M., Vorhees, C. V., and Butcher, R. E. (1978). A

Downloaded from http://toxsci.oxfordjournals.org/ by guest on April 14, 2015

Altman, 1979), prenatal AFB exposure may produce alterations in the morphology and physiology of this area. With
regard to the avoidance learning task, it is of considerable
interest to note that rat offspring exposed to AFB during both
mid and late organogenesis showed impaired learning ability,
and that the impaired avoidance learning in the prenatal AFB
exposure during mid-organogenesis was more severe than that
during late organogenesis. Therefore, mid-organogenesis (rat,
days 1114 of gestation) appears to be a more vulnerable
period for AFB disruption of the acquisition of the conditioned
avoidance learning than is late organogenesis (days 1518).
Generally, learning ability is a very important parameter for
assessment of developmental neurotoxicity (Riley and Vorhees, 1986, Tanimura, 1992). In this respect, the most important finding in the present study is that prenatal exposure of rats
to AFB impaired performance in the conditioned avoidance
test.
The open field test is a measure of emotional reactivity and
exploratory behavior in the rat (Hall, 1934), and has commonly
been used in developmental toxicology studies. It may also be
viewed as a measure of activity level (Adams, 1986; Walsh and
Cummins, 1976). In the present study, no effects of 0.3 mg/
kg/day AFB were observed in the open field test in Wistar rats.
However, Chentanez et al. (1986) found a decrease in some
types of behavior (sniffing, grooming, exploring, rearing licking, and gnawing) and motor activity in the open field box in
1-month-old offspring of dams treated intraperitoneally with
AFB, at 2.0 mg/kg/day, during both days 79 or days 14 16 of
gestation in Fisher rats but not in 2- and 3-month-old offspring.
Thus, the results of the present study differ from those of
Chentanez et al. (1986) with regard to the effect of the AFB on
the open field activity. This may be due to dose differences, rat
strain differences, treatment route differences, or observer differences.
The most important feature of the present study is that
prenatal exposure of rats to AFB, at a dose lower than that
which causes other gross malformations or growth retardation,
altered neurobehavioral performance in offspring in the
preweaning and postweaning periods. Therefore, the behavioral changes observed here were the result of persistent effects
of AFB on the fetal nervous system.
Furthermore, the behavioral teratogenic effect of AFB is
greater when administered during mid-organogenesis than during late organogenesis. It was widely believed that late organogenesis or the early postnatal period might be the most
sensitive stage for disrupting behavioral development, as brain
maturation actively takes place within those periods (Hutchings, 1983; Leonard, 1982). More recently, several studies in
addition to this one have demonstrated that mid-organogenesis
in rats is a more sensitive period for some behavioral teratogens (Kihara, 1991; Rodier et al., 1979).Vorhees (1983, 1987)
also suggested that mid-organogenesis is the most vulnerable
period. Mid-organogenesis, approximately days 1115 in the
rat, is an extremely active phase of neurogenesis for the visual

398

KIHARA ET AL.

comparison of behavioral and anatomical measures of hydroxyurea induced

abnormalities. Teratology 18, 379 384.

Kihara, T. (1991). Effects of the prenatal ochratoxin A exposure on behaviors

of rat offspring. Acta Med. Kinki Univ. 16, 122.

Chentanez, T., Glinsukon, T., Patchimasiri, V., Klongprakit, C., and Chentanez, V. (1986). The effects of aflatoxin B 1 given to pregnant rats on the
liver, brain and the behaviour of their offspring. Nutr. Rep. Int. 34,
379 386.

Kihara, T., Matsuo, T., Sakamoto, M., Yasuda, Y., and Tanimura, T. (1995).
Effects of the neonatal vitamin A exposure on behaviors of adult rats. J.
Toxicol. Sci. 20, 93101.

Cilievici, O., Gordos, I., Ghidus, E., and Moldovan, A. (1980). The toxic and
teratogenic effect of aflatoxin B 1 on the chick embryo development. Morphol. Embryol. (Bucur). 4, 309 314.
Dietert, R. R., Qureshi, M. A., Nanna, U. C, and Bloom, S. E. (1985).
Embryonic exposure to aflatoxin B 1: Mutagenicity and influence on development and immunity. Environ. Mutagen. 7, 715725.
DiPaolo, J. A., Elis, J., and Erwin, H. (1967). Teratogenic response by
hamsters, rats and mice to aflatoxin B 1. Nature 215, 638 639.
Eaton, D.L. and Gallagher, E. P. (1994) Mechanisms of aflatoxin carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 34, 135172.
Elegbe, R. A., Amure, B. O., and Adekunle, A. A. (1974). Induction of
implantation by aflatoxin B 1 in the rat. Acta Embryol. Exp. 2, 199 201.

Kinnard, Jr., W. J., and Carr, C. J. (1957). A preliminary procedure for the
evaluation of central nervous system depressants. J. Pharmacol. Exp. Ther.
120, 354 361.
LeBreton, E., Frayssinet, C., Lafarge, C., and deRecondo, A. M. (1964).
Aflatoxine-Mecanisme de laction. Food. Cosmet. Toxicol. 2, 675 677.
Leonard, B. E. (1983). Behavioral teratology and toxicology. In Psychopharmacology 1, Part 1 Preclinical Psychopharmacology (D. G. GrahameSmioth, Ed., pp. 248 299. Excerpta Medica, Amsterdam, Oxford, Princeton.
Llewellyn, G. C., Stephenson, G. A., and Hofman, J. W. (1977). Aflatoxin B 1
induced toxicity and teratogenicity in Japanese Medaka eggs (Oryzias
Latipes). Toxicon. 15, 582587.
Ong, T. (1975). Aflatoxin mutagenesis. Mutat. Res. 32, 3553.
Panda, P. C., Sreenivasamurthy, V., and Parpia, H. A. B. (1970). Effect of
aflatoxin on reproduction in rats. J. Food. Sci. Technol. 7, 20 22.

Gabor, M., Puscariu, F., and Deac, C. (1973). Action teratogene des aflatoxins
sur les tetards. Arch. Roum. Pathol. Exp. Microbiol. 32, 269 275.

Pellegrino, L. J., and Altman, J. (1979). Effects of differential interference with

postnatal cerebellar neurogenesis on motor performance, activity level, and
maze learning of rats: A developmental study. J. Comp. Physiol. Psychol.
93, 133.

Geissler, F., and Faustman, E. M. (1988). Developmental toxicity of aflatoxin

B 1 in the rodent embryo in vitro: Contribution of exogenous biotransformation systems to toxicity. Teratology 37, 101111.
Goerttler, K., Lohrke, H., Schweizer, H. and Hesse, B. (1980). Effects of
aflatoxin B 1 on pregnant inbred Sprague-Dawley rats and their F 1 generation. A contribution to transplacental carcinogenesis. J. Natl. Cancer Inst.
64, 1349 1354.
Grice, H. C., Moodie, C. A., and Smith, D. C. (1973). The carcinogenic
potential of aflatoxin or its metabolites in rats from dams fed aflatoxin preand postpartum. Cancer Res. 33, 262268.
Hall, C. S. (1934). Emotional behavior in the rat. I. Defecation and urination
as measures of individual differences in emotionality. J. Comp. Psychol. 18,
385 403.
Hayes, A. W. (1981). Aflatoxins. In Mycotoxin Teratogenicity and Mutagenicity. (A.W. Hayes Ed.), pp. 44 47. CRC Press, Boca Raton.
Hood, R. D. (1979). Effects of mycotoxins on development. Teratological
testing. In Advances in the Study of Birth Defects (T. V. N. Persaud, Ed.),
Vol.2, pp. 191- 210. MTP press, Lancaster.
Hood, R. D., and Szczech, G. M. (1983). Teratogenicity of fungal toxins and
fungal-produced antimicrobial agents. In Handbook of Natural Toxins. Plant
and Fungal Toxins (R. F. Keeler and A. T. Tu, Eds.), Vol. 1, pp. 201235.
Marcel Dekker, Inc., New York.
Hutchings, D. E. (1983). Behavioral teratology: A new frontier in neurobehavioral research. In Teratogenesis and Reproductive Toxicology (E. M.
Johnson and D. M. Kochhar, Eds.), pp. 207235. Springer-Verlag, Berlin,
Heidelberg, New York.
IARC (1983) Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. In IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Vol. 56.
International Agency for Research on Cancer, Lyon, France.
Jacobsen, J. S., Refolo, L. M., Conley, M. P., Sambamurti, K., and Humayun,
M. Z. (1987). DNA replication- blocking properties of adducts formed by
aflatoxin B 12, 3- dichloride and aflatoxin B 12, 3- oxide. Mutat. Res. 179,
89 101.
Kaplan, M. L., and Murphy, S. D. (1972). Effect of acrylamiode on rotarod
performance and sciatic nerve -glucuronidase activity of rats. Toxicol.
Appl. Pharmacol. 22, 259 268.

Riley, E. P., and Vorhees, C. V. (1986). Handbook of Behavioral Teratology.

Plenum Press, New York, London.
Rodier, P. M., Reynolds, S. S., and Roberts, W. N. (1979). Behavioral
consequences of interference with CNS development in the early fetal
period. Teratology 19, 327336.
Roll, R., Matthiaschk, G., and Korte, A. (1990). Embryotoxicity and mutagenicity of mycotoxins. J. Environ. Pathol. Toxicol. Oncol. 10, 17.
Schapiro, S., Salas, M., and Vukovich, K. (1970). Hormonal effects on
ontogeny of swimming ability in the rat: Assessment of central nervous
system development. Science 168, 147151.
Siegel, S. (1956). Nonparametric Statistics: For the Behavioral Sciences.
McGraw-Hill Book Co., Inc., New York.
Sokal, R. R., and Rohlf, F. J. (1973). Introduction to Biostatics. W. H. Freeman
and Company, San Francisco.
Sporn, M. B., Dingman, C. W., Phelps, H. L., and Wogan, G. N. (1966).
Aflatoxin B 1 : Binding to DNA in vitro and alteration of RNA metabolism
in vivo. Science 151, 1539 1541.
Tanaka, T. (1975). Direct and delayed effects of aflatoxin B 1 on rat fetus. Proc.
Jpn. Assoc. Mycotoxicol. 1, 9 12.
Tanimura, T. (1990). Japanese perspectives on the reproductive and developmental toxicity evaluation of pharmaceuticals. J. Am. Coll. Toxicol. 9,
2737.
Tanimura, T. (1992). Update on the activities of the Japanese Behavioral
Teratology Meeting. Congenital Anom. 32(suppl), S7S20.
Tanimura, T., Kihara, T., and Yamamoto, Y. (1982). Teratogenicity of aflatoxin B 1 in the mouse. Rep. Environ. Sci. Res. Instit. Kinki Univ. No. 14,
247256.
Ulbrich, B. and Palmer, A. K. (1996). Neurobehavioral aspects of developmental toxicity testing. Environ. Health Perspect. 104(suppl 2), 407 412.
Vorhees, C. V. (1983). Fetal anticonvulsant syndrome in rats: Dose- and
period-response relationships of prenatal diphenylhydantoin, trimethadione
and phenobarbital exposure on the structural and functional development of
the offspring. J. Pharmacol. Exp. Ther. 227, 274 287.
Vorhees, C. V. (1987). Dependence on the stage of gestation: Prenatal drugs
and offspring behavior as influenced by different periods of exposure in rats.

Downloaded from http://toxsci.oxfordjournals.org/ by guest on April 14, 2015

Elis, J., and DiPaolo, J. A. (1967). Aflatoxin B 1, induction of malformations.

Arch. Pathol. 83, 5357.

PRENATAL AFLATOXIN B 1 AND BEHAVIOR

In Functional Teratogenesis (T. Fujii, and P. M. Adams, Eds.), pp. 39 51.
Teikyo University Press, Tokyo.
Vorhees, C. V., Brunner, R. L., and Butcher, R. E. (1979a). Psychotropic drugs
as behavioral teratogens. Science 205, 1220 1225.
Vorhees, C. V., Butcher, R. E., Brunner, R. L., and Sobotka, T. J. (1979b). A
developmental test battery for neurobehavioral toxicity in rats: A preliminary analysis using monosodium glutamate calcium carrageenan, and hydroxyurea. Toxicol Appl. Pharmacol. 50, 267282.
Walsh, R. N., and Cummins, R. A. (1976). The open-field test: A critical
review. Psychol. Bull. 83, 482504.
Wilson, B. J., Campbell, T. C., Hayes, A, W., and Hanlin, R. T. (1968).

399

Investigation of reported aflatoxin production by fungi outside the Asperigillus flavus group. Appl. Microbiol. 16, 819 821.
Wogan, G. N., and Newberne, P. M. (1967). Dose-response characteristics of
aflatoxin B 1 carcinogenesis in the rat. Cancer Res. 27, 2370 2376.
Wong, J. J., and Hsieh, D. P. H. (1976). Mutagenicity of aflatoxins related to
thier metabolism and carcinogenic potental. Proc. Natl. Acad. Sci. USA. 73,
22412244.
Yourtee, D. M., Kirk-Yourtee, C. L., and Searles, S. (1987). The direct
mutagenicity of aflatoxin B 1 and metabolites to Salmonella typhimurium:
Structure mutagenicity relationships and mechanisms of action. Res. Comm.
Chem. Pathol. Pharmacol. 57, 5576.

Downloaded from http://toxsci.oxfordjournals.org/ by guest on April 14, 2015