toxicol. sci.-2000-kihara-392-9

Effects of Prenatal Aflatoxin B1 Exposure on Behaviorsof 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

The effects of prenatal aflatoxin B1 (AFB) exposure on eightbehavioral parameters in Jcl:Wistar rat offspring were assessed.Pregnant rats were injected subcutaneously with 0.3 mg/kg/day ofAFB dissolved in dimethylsulfoxide on days 1114 (Group A) or1518 (Group B) of gestation. Controls received the vehicle simi-larly on days 1118 of gestation. Before weaning, the offspringwere 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 usingthe rotarod test (5 weeks of age), the open field test (6 weeks), aconditioned avoidance learning test (14 weeks), an underwaterT-maze test (15 weeks), and a reproduction test (16 weeks). Thepreweaning offspring in the AFB-A group showed significantlylower success rates than controls in cliff avoidance responses. Inswimming development, the offspring in the AFB-A group hadsignificantly lower scores than controls for swimming direction. Inthe rotarod test, the AFB-A group remained on the rod for asignificantly shorter time than the controls at 15 rpm on both thefirst and second trial days. The avoidance performance of the ratsin AFB-A and AFB -B groups was significantly poorer than that ofcontrols. These results indicate that prenatal exposure to AFBproduced a delay of early response development, impaired loco-motor coordination, and impaired learning ability in the offspringof rats exposed to AFB during middle pregnancy, and the earlygestational exposure appears to produce more effects than latterexposure.

Key Words: aflatoxin B1; 2,3,6aa,9aa-tetrahydro-4-methoxycy-clopenta[c]furo[3*,2*,: 4,5]furo[2, 3-h][1] benzopyran-1, 11-dione;mycotoxin; behavioral teratology; prenatal exposure; developmen-tal toxicity; neurotoxicity.

Aflatoxin B1 (AFB), produced by several fungi such asAspergillus flavus and A. parasiticus (Wilson et al., 1968) (Fig.1), is one of the most important food borne mycotoxins and isone of the most potent hepatotoxins and carcinogens in manyanimal species (Eaton and Gallagher, 1994; Wogan and New-berne, 1967), and it has also been implicated in the etiology of

human liver cancer in a number of epidemiologic investiga-tions (IARC, 1993).

The developmental toxicity of AFB has been studied invarious animals (for review, see Hayes 1981; Hood, 1979;Hood and Szczech, 1983). AFB has been reported to be tera-togenic and/or embryotoxic in rats (Elegbe et al., 1974; Griceet al., 1973; LeBreton et al., 1964; Panda et al 1970; Tanaka,1975), in mice (Arora et al., 1981; DiPaolo et al., 1967; Roll etal., 1990; Tanimura et al., 1982), in hamsters (DiPaolo et al.,1967; Elis and DiPaolo, 1967), in chick embryos (Bassir andAdekunle,1970; Cilievici et al., 1980; Dietert et al., 1985), intadpoles (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), andselective immune depression in chick embryos (Dietert et al.,1985) have also been reported. A previous study in our labo-ratory demonstrated that AFB induced cleft palate, skeletalmalformations, and intrauterine growth retardation in mousefetuses of dams injected intraperitoneally with doses of 32mg/kg/day for 2 days of days 67, 89, 1011, 1213 ofgestation (Tanimura et al., 1982).

In the past 20 years, the importance of postnatal evalua-tion for behavioral teratology has received increasing rec-ognition, and the test battery system for assessment ofbehavioral teratogenic potential in reproductive and devel-opmental toxicity study has been used widely (Riley andVorhees, 1986; Tanimura, 1990, 1992; Ulbrich and Palmer,1996). However, there is little published data on the behav-ioral teratogenic effects of AFB in animals or in humans.There has been only one study to date on the functionaleffects caused by prenatal AFB exposure. Chentanez et al.(1986) showed that AFB 2.0 mg/kg administered intrave-nously to Fisher rats on days 8 10 or 1517 of gestationinduced a decrease in some types of behaviors and in motoractivity levels in 1-month-old offspring.

The present study, therefore, was conducted to determine thebehavioral teratogenic effects on the rat offspring of damsinjected subcutaneously with a subteratogenic dose of AFBduring mid or late organogenesis. The test battery system

1 To whom correspondence should be addressed at 1-3-4-303 Harayamadai,Sakai, Osaka 590-0132, Japan. Fax: 181-722-99-8437.

TOXICOLOGICAL SCIENCES 53, 392399 (2000)Copyright 2000 by the Society of Toxicology

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recently developed in our laboratory (Kihara, 1991; Kihara etal.,1995) was used for functional evaluations.

MATERIALS AND METHODS

Animals and Treatments

Nine-week-old male (230250 g) and female (170190 g) Jcl:Wistar ratswere purchased from Clea Japan, Osaka, Inc., and acclimated to the laboratoryfor 2 weeks prior to mating. Pellet diet OA-2 (Clea Japan, Osaka, Inc.) and tapwater were available . Animals were maintained in a room with a controlledtemperature of 23 6 20C, a relative humidity of 50 6 10%, and a 12-hlight:dark cycle (lights on at 07:00 A.M.). Eleven-week-old females were matedovernight for 16 h with one male each. Mating was confirmed if sperm wasfound 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 threegroups, each consisting of 10 female rats at conception. They were individuallyhoused in polypropylene cages with wooden shavings provided throughoutgestation and lactation, and left undisturbed except for treatment and weighinguntil 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 dimethylsulfoxide (Wako Pure Chemical Industries Ltd., Osaka) on days 1114 (GroupA) or 1518 (Group B) of gestation. Control animals received subcutaneouslythe vehicle only on days 1118 of gestation. The treatment volume was 1.0ml/kg body weight. Fresh solutions of AFB were prepared on the day of use.

The dams were allowed to deliver spontaneously and rear their offspringuntil weaning. At 4 days after birth, the litters were culled randomly to groupsof eight offspring with the same number of males and females as far aspossible. At 21 days, the offspring were weaned, separated by sex, and housedin hanging wire-mesh cages with four littermates of the same sex. All offspringwere identified by marking with dry ink before weaning and with picricacid-ethanol solution after weaning.

Body Weight and Physical Landmarks

The dams were weighed on days 0, 7, 14, and 21 of gestation, daily duringtreatment, and on days 0, 4, 7, 14, and 21 after delivery. After weaning theirlitters (21 days after delivery), all the dams were killed by ether overdose andexamined for numbers of implantation sites and any abnormalities of thereproductive organs.

At birth, all live and dead offspring were counted. Live offspring wereweighed, sexed, and examined for external malformations. The live offspringwere again counted and weighed on 4, 7, 14, and 21 days after birth. Afterweaning, all the offspring were counted and weighed weekly until 20 weeksof age.

The following physical landmarks were noted: bilateral pinna unfolding at 4days of age, abdominal hair emergence at 7 days, low incisor eruption andbilateral 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 thebehavioral testing procedures were conducted blind with regard to the treat-ment 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 andnose over the edge. The amount of time required to complete backing andturning away from the edge was recorded. Each offspring was tested in onetrial. (Altman and Sudarshan, 1975; Brunner et al., 1978; Kihara, 1991). Thenumber of rats with successful responses within 30 s was recorded.

Negative geotaxis. The time taken to complete a 180-degree turn whenplaced in a head-down position on a 25-degree inclined plywood surface wasmeasured. Each animal was given one trial. (Kihara, 1991; Vorhees et al.,1979a,b). The number of rats with successful responses within 30 s wasrecorded.

Swimming development. This procedure has been described elsewhere(Kihara, 1991; Schapiro et al., 1970; Vorhees et al.,1979a,b). Each rat wasindividually placed in a tank of water (280C) for 510 s and direction, angle inthe water (head position), and limb usage was observed. Direction scoresconsisted of sinking (0 points), floating (1), circling (2), and swimming straightor nearly straight (3). Angle scores consisted of head submerged (0), nose atthe surface (1), nose and top of head at or above the surface but ears stillbelow the surface (2), ears half way above the surface (3), and ears completelyabove the surface (4). Limb usage scores consisted of no paddling (0), paddlingwith all four limbs (1), and paddling with hind limbs only with forelimbsstationary (2).

Rotarod. The apparatus consisted of a rod 7 cm in diameter with a hardrubber surface, and linked to a variable-speed motor. The top of the rod was 34cm 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 rotatedat speeds of 5 or 15 rpm.. Rats were tested on two trials per day for 2consecutive days. The maximum trial duration was 180 s, and the intertrialinterval was about 30 min. The time each animal remained on the rod at eachrotation speed was recorded (Kihara, 1991; Kihara et al., 1995).

FIG. 1. Structural formula of aflatoxin B1.

TABLE 1Schedule of Behavioral Test Battery

Procedure Age of testing

PreweaningaCliff avoidance 5Negative geotaxis 7Swimming development 6, 8, 10

PostweaningbRotarod 5Open field 6Conditioned avoidance learning 14Underwater T-maze 15Reproduction 16

Note. Age of offspring for preweaning tests in days; age of offspring forpostweaning 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 onefemale from each litter were used.

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

Acquisition of conditioned avoidance. The apparatus was comprised of anoperant chamber (Ohara and Co. Ltd., Tokyo, Model GT-7710), and aprogramming 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 (ModelGT-7720). The conditioned avoidance schedule was as follows: a 20-s inter-trial 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. Stimuliwere immediately terminated by the first lever-press elicited during the CSperiod, and foot shock was avoided. Both lever-pressing at times other than theCS presentation period and lever-holding were ineffective. Each session con-sisted of 1 h of training per day; animals were tested every other day for 15sessions. The acquisition processes were considered to reflect discriminatedavoidance learning. The index of conditioned avoidance learning was rates ofavoidance.

The number of avoidance responses, stimuli presented, and shocks deliveredwere recorded, and the mean percent of avoidance and the mean response rateper day were calculated (Kihara, 1991; Kihara et al., 1995).

Underwater T-maze. The T-maze apparatus consisted of a grey polyvinylchloride cylinder 20 cm in diameter; each arm was 50 cm long, and all the exitswere curved upward. The maze was completely filled with water maintained ata temperature of 280C. Each animal was given 10 trials per day. A maximumswimming time of 60 s was allowed for each trial. An error was designated asentry into a blocked arm or re-entry into the stem. Swimming time and thenumber 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 selectedat random for the reproduction test; sibling matings did not occur. They weremated monogamously for 2 weeks (about three estrus cycles) and wereexamined every morning for the presence of a vaginal plug. If a vaginal plugwas observed (day 0 of gestation), the female was placed in an individual cagecontaining wooden chips for nesting. The dams were killed by ether overdoseand necropsied on day 21 of gestation. These uteri were examined and thenumber of implantation sites, resorptions, and dead and live fetuses wererecorded. The live fetuses were sexed, weighed, and examined for externalmalformations.

Brain Weight

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

Statistical Analysis

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

RESULTS

Maternal EffectsNeither death nor noticeable symptoms were observed in the

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

Growth and Physical Landmarks of the OffspringAt birth, the number of live offspring was significantly lower

in the AFB-B group than those of controls, and body weightsof male and female offspring in both the AFB-A and -B groupswere significantly lower than the controls. However, there wereno significant differences between the AFB groups and thecontrol group with regard to the number of implants, sex ratioof offspring, offspring with external malformations, live birth

TABLE 2Reproductive Performance of the Dams Exposed to Aflatoxin

B1, and Survival and Body Weight in the Offspring

GroupControl

Days 1118a

0.3 mg/kg

Days 1114(A)

Days 1518(B)

No. of dams 10 10 10No. of implantsb 16.2 6 0.4 15.8 6 0.7 15.2 6 0.9No. of live birthsb 15.2 6 0.5 14.4 6 0.4 12.9 6 0.9*No. of offspring

malformed at birth 0 0 0Live birth indexc 93.9 93.8 85.6Survival index at 4 days of

aged 98.8 96.0 95.5Weaning index at 21 days

of agee 93.8 97.5 98.8Survival index at 20 weeks

of age f 93.4 90.7 97.3Body weight of offspring

(g)bAt birth

M 5.7 6 0.1 5.1 6 0.1* 4.7 6 0.3*F 5.4 6 0.1 4.9 6 0.1* 4.7 6 0.2*

At weaningM 49.1 6 1.1 46.1 6 1.6 46.7 6 2.8F 46.6 6 1.4 42.7 6 2.0 46.0 6 2.3

At 20 weeksM 371 6 4.9 362 6 6.4 355 6 9.3F 234 6 4.3 223 6 4.3 220 6 7.0

Note. M, male; F, female.a Gestational days exposed.b Mean 6 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).

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index, survival rates at 4, 21 days of age (at weaning), or 20weeks of age, or body weights at 4 and 7 days of age (data notshown), 21 days, and 20 weeks of age (Table 2).

With regard to physical landmarks, there were no statisti-cally significant effects of AFB-exposure on pinna unfolding,hair emergence, incisor eruption, testis descent, or vaginalopening (data not shown). However, the mean percentage ofoffspring 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, respec-tively. The AFB-A group showed significantly delayed eyeopening 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 3and 4.

Reflex behavior. The results of reflex testing are presentedin Table 3. The AFB-A offspring were less successful in cliffavoidance than control offspring, and the AFB-A group alsohad significantly slower response times. Cliff avoidance wasnot affected in the AFB-B group. On the negative geotaxisreflex, there were no significant differences between the AFB-exposed groups and the control group.

Swimming development. The results are shown in Table 4.With regard to swimming direction, the AFB-A group scoredsignificantly lower than the control group at 6 days of age;however, there were no significant treatment effects at anyother age observed. There were no significant differences be-tween the AFB groups and the control group for swimmingangle and limb usage on 6, 8, and 10 days of age.

Rotarod performance. The rats in the AFB-A group re-mained on the rotarod for a significantly shorter time than thecontrols at 15 rpm on both the first and second test days.However, there were no statistically significant differencesbetween the AFB-B and control groups (Table 5).

Open field activity. There were no significant differencesbetween the AFB-exposed groups and the control group forambulation counts, rearing, and defecation frequencies, or thestart latency time on any of the test days. (The mean controlvalues [mean 6 SE] were the ambulation 1st day 29.8 6 7.2,2nd 22.0 6 4.7, 3rd 22.1 6 5.7; the rearing 1st 9.4 6 3.4, 2nd3.6 6 1.3, 3rd 2.5 6 0.8; the defecation frequencies 1st 1.7 60.5, 2nd 4.0 6 0.8, 3rd 4.5 6 0.6).

TABLE 3Reflex Development of Preweaning Rat Offspring of Dams

Exposed to Aflatoxin B1

GroupControl

Days 1118a

0.3 mg/kg

Days 1114(A)

Days 1518(B)

No. of offspring tested 80 80 77Cliff avoidance (5 days of

age)Success rateb 97.5 76.0* 91.2Response timec 6.4 6 0.7 16.7 6 2.3* 7.6 6 1.9

Negative geotaxis (7 daysof age)

Success rateb 95.8 98.7 97.5Response timec 17.7 6 2.6 13.9 6 1.1 20.2 6 2.6

a Gestational days exposed.b Percentage of offspring achieving criterion.c Mean positive response time (s) 6 SE.* Significantly different from the control (p , 0.05).

TABLE 4Swimming Development in Preweaning Rat Offspring of

Dams Exposed to Aflatoxin B1

GroupControl

Days 1118a

0.3 mg/kg

Days 1114 (A) Days 1518 (B)

No. of offspring 80 80 776 days of age

Direction 2.3 6 0.1 1.8 6 0.1* 2.2 6 0.2Angle 2.3 6 0.1 2.5 6 0.2 2.4 6 0.2Limb usage 1.0 6 0 1.0 6 0 1.1 6 0.1

8 days of ageDirection 2.1 6 0.1 2.0 6 0.1 2.2 6 0.2Angle 2.8 6 0.1 2.8 6 0.1 2.5 6 0.2Limb usage 1.0 6 0 1.0 6 0 1.0 6 0

10 days of ageDirection 2.3 6 0.1 2.2 6 0.1 2.2 6 0.1Angle 3.1 6 0.1 2.9 6 0.1 2.9 6 0.1Limb usage 1.0 6 0 1.0 6 0 1.0 6 0

Note. Values are expressed using rating scales; mean 6 SE.a Gestational days exposed.* Significantly different from the control (p , 0.05).

TABLE 5Rotarod Performance in Male Rat Offspring of Dams Exposed

to Aflatoxin B1

GroupControl

Days 1118a

0.3 mg/kg

Days 1114 (A) Days 1518 (B)

rpmb

1st day 5 136.0 6 22.6 84.5 6 26.6 70.5 6 29.615 30.4 6 10.5 6.9 6 2.0* 46.7 6 25.3

2nd day 5 152.7 6 18.4 110.8 6 20.9 176.6 6 3.415 91.6 6 25.6 16.5 6 4.9* 87.8 6 31.7

Note. Values are means of time (s) spent on the rod 6 SE. Maximum timewas 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).




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Conditioned avoidance. Acquisition rates of the condi-tioned avoidance test are presented in Figure 2. The meanavoidance rates in both AFB-exposed groups were lower thanthose in the control group during 15 training sessions. Both theAFB-A group (5th15th sessions) and the AFB-B group (12th,14th, and 15th sessions) showed significantly lower avoidancerates than the control group. Furthermore, rates of AFB-Agroup during 511 sessions were also lower than avoidancerates of the AFB-B group. There appears to be some increasein variance that may be treatment related. No significant dif-ferences in the mean lever-pressing responses were detectedamong the three groups throughout the 15 sessions of training(data not shown).

Underwater T-maze. There were no significant differencesbetween the AFB-treated groups and the control group forswimming time and number of errors on any of the trial days(The mean control values of swimming time (s 6 SE) were the1st trial day 6.2 6 0.5, 2nd 4.6 6 0.3, 3rd 4.3 6 0.3, 4th 8.1 60.7, 5th 5.1 6 0.6, and 6th 6.1 6 0.5; the number of errorsranged between 0.18 and 0.8).

Reproductive performance. No significant differenceswere seen between the AFB-exposed and control groups in anyreproductive parameters including copulation and pregnancyrates, litter size, sex ratio of offspring, fetal weight, or inci-dence of external malformations (data not shown).

Brain Weight

There were no statistically significant differences in brainweight between the AFB-exposed and the control groups. Thevalues (mean: g 6 SE) were 1.88 6 0.05, 1.83 6 0.04, and1.85 6 0.04 for the control group, the AFB-A and the AFB-Bgroups, respectively.

DISCUSSION

The present study is the first postnatal physical and behav-ioral evaluation of offspring development using a test batterysystem following prenatal exposure to AFB during mid or lateorganogenesis. The main effects observed were a smaller num-ber of live births, lower mean birth weights, a delayed physicaldevelopment, delayed behavioral developments in thepreweaning period, and a impaired locomotor coordination anddeficits in avoidance performance in the postweaning period.On the other hand, no effects on maternal mortality, maternalweight gains, gestation length, the offspring with malforma-tions, and the brain weight were observed in the prenatalexposure to AFB groups.

AFB treatment during late organogenesis (days 1518) re-sulted in decreased numbers of live pups; however, no signif-icant reduction in the live birth index was seen because themean number of implants in group AFB-B was smaller thanthat the control group (Table 2). AFB exposure during bothmid-organogenesis (days 1114) and late organogenesis (days1518) resulted in reduced birth weights in both male andfemale offspring, but body weight differences were completelyrecovered after 4 days of age. Thus, growth rates of offspringin the AFB groups were equivalent to the control group duringthe remainder of the study. There was also a delay in eyeopening in the prenatally exposed animals, but it was signifi-cant only when AFB was administered in the mid-organogen-esis period (days 1114).

The present study is the first to show that prenatal exposureto AFB affects behavioral performance in the preweaningoffspring and alters the rate of acquisition of a conditionedavoidance task, motor coordination, and body balancing in thepostweaning offspring. Treatment with AFB during mid-orga-nogenesis (days 1114) but not during late organogenesis (days1518) delayed the development of the cliff avoidance re-sponse. Swimming ontogeny is a measure of the developmentof neuromotor coordination and swimming ability (Kihara,1991; Schapiro et al., 1970; Vorhees et al., 1979a,b). In thepresent study, prenatal AFB exposure during mid-organogen-esis (days 1114) also results in a delay in the direction ofswimming but not during late organogenesis (days 1518). Onthe other hand, the negative geotaxis test results in the presentstudy did not distinguish exposed from control rats. Theseresult suggest that AFB has a significant toxic action on de-velopmental patterns of behavior in newborn rats, particularlyon development of motor coordination.

The rotarod test has been used during the postweaningperiod to determine forced coordinated motor and balancingabilities (Altman and Sudarshan, 1975; Kaplan and Murphy,1972; Kinnard and Carr, 1957). The results of the present studyshow a deficit in motor skills in the offspring of mothers treatedwith AFB during mid-organogenesis (days 1114) but notduring late organogenesis (days 1518). As rotarod perfor-mance has been related to cerebellar function (Pellegrino and

FIG. 2. Acquisition of conditioned avoidance responses in male rat off-spring of dams exposed to aflatoxin B1. Changes in the mean avoidance ratesare shown. AFB-A: period of exposure of aflatoxin B1 (0.3 mg/kg/day) on days1114 of gestation; AFB-B: days 1518; control (vehicle): days 1118. Tenmale offspring were tested per group. Vertical bars represent the mean standarderror. *Significantly different from control (p , 0.05).

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Altman, 1979), prenatal AFB exposure may produce alter-ations in the morphology and physiology of this area. Withregard to the avoidance learning task, it is of considerableinterest to note that rat offspring exposed to AFB during bothmid and late organogenesis showed impaired learning ability,and that the impaired avoidance learning in the prenatal AFBexposure during mid-organogenesis was more severe than thatduring late organogenesis. Therefore, mid-organogenesis (rat,days 1114 of gestation) appears to be a more vulnerableperiod for AFB disruption of the acquisition of the conditionedavoidance learning than is late organogenesis (days 1518).Generally, learning ability is a very important parameter forassessment of developmental neurotoxicity (Riley and Vo-rhees, 1986, Tanimura, 1992). In this respect, the most impor-tant finding in the present study is that prenatal exposure of ratsto AFB impaired performance in the conditioned avoidancetest.

The open field test is a measure of emotional reactivity andexploratory behavior in the rat (Hall, 1934), and has commonlybeen used in developmental toxicology studies. It may also beviewed as a measure of activity level (Adams, 1986; Walsh andCummins, 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 sometypes of behavior (sniffing, grooming, exploring, rearing lick-ing, and gnawing) and motor activity in the open field box in1-month-old offspring of dams treated intraperitoneally withAFB, at 2.0 mg/kg/day, during both days 79 or days 1416 ofgestation in Fisher rats but not in 2- and 3-month-old offspring.Thus, the results of the present study differ from those ofChentanez et al. (1986) with regard to the effect of the AFB onthe open field activity. This may be due to dose differences, ratstrain differences, treatment route differences, or observer dif-ferences.

The most important feature of the present study is thatprenatal exposure of rats to AFB, at a dose lower than thatwhich causes other gross malformations or growth retardation,altered neurobehavioral performance in offspring in thepreweaning and postweaning periods. Therefore, the behav-ioral changes observed here were the result of persistent effectsof AFB on the fetal nervous system.

Furthermore, the behavioral teratogenic effect of AFB isgreater when administered during mid-organogenesis than dur-ing late organogenesis. It was widely believed that late orga-nogenesis or the early postnatal period might be the mostsensitive stage for disrupting behavioral development, as brainmaturation actively takes place within those periods (Hutch-ings, 1983; Leonard, 1982). More recently, several studies inaddition to this one have demonstrated that mid-organogenesisin rats is a more sensitive period for some behavioral terato-gens (Kihara, 1991; Rodier et al., 1979).Vorhees (1983, 1987)also suggested that mid-organogenesis is the most vulnerableperiod. Mid-organogenesis, approximately days 1115 in therat, is an extremely active phase of neurogenesis for the visual

areas, cerebral cortices, basal ganglia and forebrain, and forthalamic, hypothalamic, and limbic regions (Vorhees, 1987).

The period of administration for the control group was twiceas long as that of the AFB treatment groups. This additionalhandling stress of the control dams could influence somebehavioral parameters, especially basic values in the vehiclecontrol.

The mechanisms of AFB-induced behavioral teratogenesisare unknown at present. However, AFB produces central ner-vous system malformations (Geissler and Faustman, 1988;Tanaka, 1975), inhibition of DNA replication and binding toDNA (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 (Go-erttler et al., 1980; Grice et al., 1973; Tanaka, 1975). It issurmised that some of the actions of AFB mentioned aboveare developmental toxic actions, including behavioral terato-genicity.

However, further studies are required to characterize anybehavioral effects of developmental AFB exposure in post-weaning female rats, behavioral effects in progeny of damstreated during the lactation period, neurobiochemical effects ofAFB, and the mechanisms of its effects on developmentalneurotoxicology.

ACKNOWLEDGMENTS

This work was supported by the Special Research Project on EnvironmentalScience, Grant-in-Aid for Scientific Research Ministry of Education, Cultureand Science, Japan, and in part by grants from the Environmental ScienceResearch Institute, Kinki University, and Grant-in-Aid for Science Researchfrom Japan Private School Promotion Foundation.

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