Low dose effect of in utero exposure to bisphenol A anddiethylstilbestrol on female mouse reproduction

Shizuka Honmaa, Atsuko Suzukib,c, David L. Buchananb,c, Yoshinao Katsub,c,Hajime Watanabeb,c, Taisen Iguchib,c,d,*

aGraduate School of Integrated Science, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, JapanbCenter for Integrative Bioscience, Okazaki National Research Institutes, 38 Nishigonaka, Myodaiji, Okazaki 444-8585, Japan

cCREST, Japan Science and Technology Corporation, Kawaguchi, JapandDepartment of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan

Received 13 September 2001; received in revised form 20 November 2001; accepted 2 December 2001

Abstract

In utero exposure to bisphenol-A (BPA) at doses relevant to human consumption has been reported to accelerate weight gain and pubertyin female mice, but the effect of low dose BPA on female reproduction has not been described. In this study, we investigated low dose effectsof BPA on sexual maturation and reproduction in female ICR/Jcl mice. Pregnant ICR mice (F0) were injected (s.c.) with BPA (2 and 20�g/kg), diethylstilbestrol (DES; 0.02, 0.2, and 2�g/kg) or oil vehicle once per day from gestational days 11–17. For both female and maleoffspring (F1), body weights were measured on postnatal day (PND) 0 (the day of birth), 11, 22, and 60, and anogenital distance (AGD)was measured on PNDs 22 and 60. Pups were weaned at PND 22 and males were caged separately from females. Vaginal smears were takendaily beginning the day of vaginal opening for 30 days. The age at vaginal opening was significantly earlier in all exposed females exceptfor 2 �g/kg BPA females compared to oil controls. Body weight at vaginal opening was lower than controls in all exposed females. Thefirst vaginal estrus was earlier in all exposed females except for the 2�g/kg BPA group females compared to controls. From PND 90 to120, gestationally exposed F1 female mice were mated with unexposed males. Total numbers of pups and sex ratio in F1 mice exposed toBPA or DES, and those of their offspring (F2) were not different from controls in any treatment group. The present results indicate thatprenatal exposure to low doses of BPA and DES induces early vaginal opening, but does not affect reproductive functioning at the firstbreeding. © 2002 Elsevier Science Inc. All rights reserved.

Keywords: Low dose effect; Prenatal exposure; Bisphenol-A; Diethylstilbestrol; Vaginal opening; Estrous cycle; Anogenital distance; Female mousereproduction

1. Introduction

A large number of chemicals that have been released intothe environment may mimic the action of estrogen andpossibly disrupt endocrine functioning in wildlife and hu-mans [1,2]. Exposure to estrogenic agents in early life ishypothesized to be associated with reproductive and devel-opmental abnormalities [3,4].

Diethylstilbestrol (DES) effects on reproductive tractshave been widely studied in humans and experimental an-imals. Perinatal exposure to estrogenic agents includingDES induced reproductive abnormalities such as cryp-

torchidism, reduction in sperm count, and testicular hyper-plasia in men and male mice [5–7], and vaginal adenocar-cinoma and uterine abnormalities in women and femalemice [8–17]. Bisphenol A (BPA), a raw material of poly-carbonate and epoxy resins, is a widely used chemicalproposed to have estrogenic qualities. Human exposure toBPA can occur due to leaching from autoclaved polycar-bonate plastics [18] and inner plastic coatings of food cans[19]. In female mice, prenatal exposure to BPA as well asDES induced reproductive abnormalities such as polyovularfollicles and ovary-independent vaginal epithelial cornifica-tion [20,21]. Recently, several studies have examined theeffects of estrogenic chemicals, such as BPA, at dosesrelevant to those consumed by humans. In male mice, pre-natal exposure to estrogenic chemicals such as DES, BPAand methoxychlor induced increases in prostate weight and

* Corresponding author. Tel.:�81-564-55-7525; fax:�81-564-55-7527.

E-mail address: taisen@nibb.ac.jp (T. Iguchi).

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0890-6238/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.PII: S0890-6238(02)00006-0

size [22–24]. Prenatal exposure to low dose BPA (2.4 �g/kgbody weight) accelerated weight gain and puberty in femaleCF-1 mice [25]. Negative results regarding low dose effectsof estrogenic chemicals in mice have also been reported[26,27]. In order to clarify the effect of low dose BPAexposure on prenatal development, we examined femalesexual maturation and reproductive capacity in mice thatwere gestationally exposed to BPA and DES.

2. Materials and methods

2.1. Animals and treatments

ICR/Jcl mice kept under 12 h light/12 h dark at 23–25°Cwere given a commercial diet (CE-2, CLEA, Tokyo, Japan)

and tap water ad libitum. Female mice (F0 generation) weretime-mated for 12 h. The presence of a vaginal plug wasconsidered day 0 of gestation. Bisphenol A (BPA, TokyoKasei, Tokyo, Japan) and diethylstilbestrol (DES, SigmaChemical Co., St. Louis, MO) were dissolved in sesame oil.Pregnant mice were given 2 or 20 �g/kg body weight ofBPA, 0.02, 0.2 or 2 �g/kg body weight of DES, or the oilvehicle alone by subcutaneous (s.c.) injection on days 11through 17 of gestation. Following spontaneous parturition,the number of offspring (F1) and their body weights wererecorded. Sex of the offspring was determined by externalgenitalia on day 0 and confirmed by autopsy at the end of theexperiment. Litter size was adjusted to eight and offspringwere weaned at 22 days of age, at which time males andfemales were caged separately.

Table 1Offspring number and sex ratio in the F1 generation (n � 10 mated mice per dose group)

Treatments(�g/kg)

No. of pups/litter Sex ratio (% male)

Total Females Males

Control 14.2 � 0.6 6.1 � 0.6 8.1 � 0.5 57.4 � 3.5DES 0.02 12.4 � 0.9 5.4 � 0.7 7.0 � 0.7 56.7 � 4.6DES 0.2 12.6 � 0.8 6.5 � 0.5 6.1 � 0.9 46.2 � 5.7DES 2 11.6 � 1.4 5.5 � 0.7 6.1 � 1.0 46.7 � 7.1BPA 2 13.9 � 0.5 6.1 � 0.6 7.8 � 0.5 56.5 � 3.7BPA 20 13.1 � 0.6 6.8 � 0.5 6.3 � 0.7 47.4 � 3.8

Data are expressed as mean � sem. Sex was determined by external genitalia at the day of birth and confirmed by autopsy at the end of experiment.

Table 2Body weight and anogenital distance of F1 females

Treatments(�g/kg)

No. of litters(no. of females)

Body weight (g) Anogenital distance(mm/100g BW)

Day 0 Day 11 Day 22 Day 60 Day 22 Day 60

Control 10 (49) 1.65 � 0.04 8.3 � 0.3 17.3 � 0.5 33.7 � 0.9 32.6 � 1.1 19.2 � 0.6DES 0.02 10 (41) 1.82 � 0.11 7.3 � 0.3* 15.5 � 0.6* 32.9 � 0.7 33.7 � 1.2 19.6 � 0.6DES 0.2 10 (43) 1.77 � 0.04 7.1 � 0.3* 15.6 � 0.5* 32.7 � 1.1* 34.0 � 1.4* 20.6 � 0.9*DES 2 10 (48) 1.95 � 0.13* 7.8 � 0.2 16.5 � 0.7 33.4 � 1.2 32.8 � 1.5 19.8 � 1.0BPA 2 10 (48) 1.75 � 0.03 7.5 � 0.3 15.6 � 0.8* 33.2 � 1.0 34.4 � 1.4* 19.6 � 0.8BPA 20 10 (51) 1.72 � 0.02 7.8 � 0.3 16.1 � 0.8* 32.3 � 1.1* 33.8 � 1.6 20.0 � 0.9

Data are expressed as mean � sem. * Significantly different from controls at P � 0.05 (Student’s t-test).

Table 3Body weight and anogenital distance of F1 males

Treatments(�g/kg)

No. of litters(no. of males)

Body weight (g) Anogenital distance(mm/100g BW)

Day 0 Day 11 Day 22 Day 60 Day 22 Day 60

Control 10 (28) 1.91 � 0.01 8.3 � 0.2 17.7 � 0.7 43.3 � 0.6 58.4 � 1.2 42.1 � 0.4DES 0.02 10 (35) 1.91 � 0.05 7.6 � 0.2* 16.9 � 0.6 41.5 � 0.6* 60.1 � 1.3 45.7 � 0.7*DES 0.2 10 (28) 1.85 � 0.04 7.4 � 0.3* 16.7 � 0.5 41.1 � 0.5* 61.8 � 1.1 45.5 � 0.6*DES 2 10 (33) 1.94 � 0.09 7.8 � 0.2 16.7 � 0.6 42.3 � 0.3 60.5 � 1.6 44.7 � 0.7*BPA 2 10 (35) 1.83 � 0.05 7.8 � 0.3 16.0 � 1.0 41.8 � 0.7 60.5 � 0.8 44.6 � 0.9*BPA 20 10 (24) 1.81 � 0.03* 8.0 � 0.3 17.7 � 0.8 40.9 � 0.9* 57.5 � 1.1 45.6 � 0.9*

Data are expressed as mean � sem. * Significantly different from controls at P � 0.05 (Student’s t-test).

118 S. Honma et al. / Reproductive Toxicology 16 (2002) 117–122

2.2. Data collection

F1 males and females were weighed on postnatal days 0(day of birth), 11, 22, and 60 and their anogenital distance(AGD) was measured on days 22 and 60. AGD was ex-pressed as mm/100 g BW. After weaning, female mice werechecked for vaginal opening every day, and body weightwas recorded at the day of vaginal opening. Vaginal smearswere observed daily for 30 days from vaginal opening andthe day that only cornified cells were detected was consid-ered as the first estrus. To examine reproductive ability, F1female mice from 90 to 120 days of age were placed withuntreated males. After parturition in the F1 females, thenumber of offspring and sex ratio in the F2 generation wererecorded.

2.3. Statistical analysis

Statistical analysis was performed using litter-based val-ues by one-way analysis of variance with Student’s t-test orWelch’s t-test. Means were considered significantly differ-ent when P � 0.05.

3. Results

3.1. Reproduction of F0 female mice

All pregnant mice underwent parturition normally. Ges-tation length was about 19 days in all groups (Control,19.2 � 0.1; 2 �g/kg BPA, 19.1 � 0.1; 20 �g/kg BPA,19.3 � 0.30; 0.02 �g/kg DES, 19.2 � 0.1; 0.2 �g/kg DES,19.0 � 0; 2 �g/kg DES, 19.4 � 0.3). The number offemales and males, and sex ratio were not different betweencontrols and any BPA- or DES-treated group (Table 1).

3.2. Body weight and anogenital distance (AGD) in F1female mice

At day 0, sex was determined by external genitalia andconfirmed by autopsy at the end of experiment. The bodyweight of pups in the 2 �g/kg DES group was greater than

Table 4Estrous cycle length

Treatments(�g/kg)

No. of litters(no. of females)

Estrous cycle length (days) Days with cornified cells Days with lymphocytes

Control 10 (49) 4.5 � 0.4 16.1 � 0.6 10.7 � 0.5DES 0.02 10 (41) 6.1 � 0.4* 19.6 � 0.7* 8.1 � 0.5*DES 0.2 10 (43) 7.4 � 0.5* 20.1 � 0.6* 7.2 � 0.5*DES 2 10 (48) 7.7 � 0.7* 20.5 � 0.7* 7.8 � 0.7*BPA 2 10 (48) 5.8 � 0.4* 19.2 � 0.6* 8.5 � 0.5*BPA 20 10 (51) 5.5 � 0.4* 17.6 � 0.7 9.9 � 0.6

Data are expressed as mean � sem. * Significantly different from controls at P � 0.05 (Student’s t-test).Vaginal smears were examined daily for 30 days from the day of vaginal opening.

Fig. 1. Days of age at vaginal opening (A), body weight at vaginal opening(B) and age at first estrus (C). Values are mean � sem. *, **, and ***indicate significantly different from the control at P � 0.05, 0.01, and0.001 (Student’s t-test), n � 10 for each dose group.

119S. Honma et al. / Reproductive Toxicology 16 (2002) 117–122

controls (P � 0.05) at day 0. At day 11, 0.02 and 0.2 �gDES-exposed mice were lighter than controls (P � 0.05).At day 22, the body weights of all BPA- and DES-exposedfemale offspring except for those in the 2 �g/kg DES groupwere significantly less than controls. However, at day 60, 20�g/kg BPA- and 0.2 �g/kg DES-exposed females weresignificantly lighter than controls.

AGD at day 22 was significantly longer in the 2 �g/kgBPA- and 0.2 �g/kg DES-exposed female offspring than inthe controls, while at day 60, there was no difference inAGD between controls and any BPA- and DES-exposedfemale offspring except for those in the 0.2 �g/kg DESgroup (Table 2).

3.3. Body weight and AGD in F1 male mice

At day 0, male offspring body weight was not differentbetween BPA- or DES-exposed mice and controls exceptfor the 20 �g BPA group, which was lighter than controls.At day 11, body weight of only the 0.02 and 0.2 �g/kgDES-exposed males was less than controls (P � 0.05). Atday 60, 20 �g/kg BPA-, and 0.02 and 0.2 �g/kg DES-exposed males were significantly lighter than controls. Onday 60, all BPA- and DES-exposed males had significantlylonger AGD than did controls (Table 3).

3.4. Vaginal opening and estrous cycle

The age at vaginal opening was significantly earlier thancontrols in the 20 �g/kg BPA- and all DES-exposed groups.Female body weight at vaginal opening was significantlylower in all BPA and DES groups. Age at first estrus wasalso significantly earlier in all F1 females compared tocontrols except for the 2 �g/kg BPA group (Fig. 1). Totaldays showing cornified cells (totally and partially) in thevaginal smear were significantly longer in the 2 �g/kg BPAgroup and all DES-treated groups than in the controls (Table4).

3.5. Reproductive capacity of F1 female mice

To examine the reproductive ability of female mice afterprenatal exposure to BPA or DES, F1 females were mated

with untreated male mice. Total number of pups per motherand sex ratio were not different between controls and anyBPA- or DES-exposed group (Table 5).

4. Discussion

Slight alterations of steroid hormone concentration dur-ing the prenatal period can accelerate reproductive tractdevelopment and growth [28,29]. These phenomena suggestthat very low dose estrogenic chemicals relevant to humanexposure levels may also influence reproductive organs.However, the low dose effect is a matter of debate [26,27,30,31]. We investigated whether low doses of estrogenicchemicals during the prenatal period affect female repro-duction and sexual maturation.

BPA can easily cross the placenta and has been found infetal tissues including brain, liver, and reproductive organsas early as 30 min after injection in mice and 1 h afterinjection in Japanese monkeys [32]. BPA has also beenfound in fetal human umbilical cords [33–36].

Howdeshell et al. [25] reported that prenatally 2.4 �g/kgBPA-exposed CF-1 mice showed accelerated weight gainand early onset of vaginal opening. In the present study,however, body weight in 2 �g/kg BPA-exposed ICR fe-males at weaning was lower and the first vaginal estrus wasearlier in prenatally 20 �g/kg BPA- and 0.02–2 �g/kgDES-exposed mice compared to controls. Both studies arein disagreement in showing accelerated body weight gain byin utero BPA exposure. The conflicting results betweenthese studies may be due to differences in experimentaldesign and mouse strains. Hawdeshell et al. [25] exposedpregnant dams orally, delivered offspring by cesarean sec-tion, and foster-nursed the pups with untreated females.Since we did not foster-nurse pups with untreated females,offspring from treated mice possibly continued to receivechemicals through milk. Both studies, however, are inagreement in showing early puberty. Physical parameterssuch as body weight and estrus cycle also depend on inter-nal and external environmental factors such as feed, roomtemperature, and humidity. Spearow et al. [37] reportedstrain differences in sensitivity to estrogenic agents.

Table 5Reproduction of F1 females

Treatment group(�g/kg)

No. of micemated

No. of F2 pups/litter F2 sexratio (%male)Total Females Males

Control 10 14.9 � 0.8 7.7 � 0.7 7.2 � 0.6 47.9 � 3.4DES 0.02 9 14.4 � 0.8 7.8 � 0.8 6.7 � 0.6 46.8 � 4.4DES 0.2 10 13.7 � 0.7 6.7 � 0.9 7.0 � 0.7 51.9 � 5.0DES 2 10 13.5 � 1.2 6.0 � 0.9 7.5 � 1.0 55.1 � 4.7BPA 2 10 16.5 � 0.4 7.6 � 1.0 8.9 � 0.8 54.4 � 5.2BPA 20 10 15.9 � 1.1 8.6 � 0.8 7.3 � 0.7 45.9 � 2.9

Data are expressed as mean � sem. Sex was determined by external genitalia at the day of birth and confirmed by autopsy at the end of experiment. F1females were mated with non-treated intact males. The data represent only the first breeding.

120 S. Honma et al. / Reproductive Toxicology 16 (2002) 117–122

Takai et al. [38] demonstrated nonlinear effect of BPAon mouse embryo development in vitro. Development ofcultured mouse 2-cell embryos was accelerated by 1–3 nMBPA, however, it was retarded by 100 nM BPA-containing.Since the BPA effect was preventeded by the addition oftamoxifen, estrogen receptor mediation was suggested.These investigators also transferred embryos cultured inBPA-containing medium to untreated mouse uteri andfound that BPA-exposed embryos showed accelerated bodyweight gain at puberty, similar to the results of Howdeshellet al. [25], but Takai et al. [38] did not see any differencesbetween implantation and offspring number.

In the present study, the total number of pups per litterand the sex ratio were not different between controls and allBPA- or DES-exposed groups. McLachlan et al. [39], how-ever, described the reduced fertility of mice exposed inutero to DES at doses as low as 0.01 �g/kg. Their studyshowed no difference in reproductive performance for lowdose exposures at the start of the breeding experiment,similar to the present study. However, as McLachlan’s re-petitive breeding study progressed, mice exposed to lowdoses of DES also showed a decrease in fertility, suggestingearly reproductive senescence. The present study did notinclude repetitive breeding; therefore, the possibility oflongintudinal changes in fertility induced by in utero BPAand DES exposure could not be evaluated.

In conclusion, prenatal exposure to low doses of BPA orDES induced early vaginal opening. However, female re-productive function was not affected by low dose BPA orDES under the conditions of the present study.

Acknowledgments

The authors thank Emeritus Professor Noboru Takasugi,Yokohama City University, for his advice and critical read-ing of this manuscript. This work was partly supported by aGrant-in-Aid for Scientific Research on Priority Areas (A)“The Environmental Risk of Endocrine Disruptor” from theMinistry of Education, Culture, Sports, Science and Tech-nology of Japan, Special Coordination Funds of Science andTechnology Agency of Japanese Government, and theHealth Sciences Research Grant from the Ministry ofHealth, Labour and Welfare, Japan (No. H10-seikatsu-016).

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