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journal homepage: www.intl.elsevierhealth.com/journals/demaDistribution of 14 C-bisphenol A in pregnant andnewborn mice
Mitsuro Tanaka a,∗ , Tadafumi Kawamoto b , Hiroki Matsumoto aa Department of Pediatric Dentistry, Iwate Medical University, 19-1 Uchimaru, Morioka 020-8505, Japanb Radioisotope Research Institute, Tsurumi University, 2-1-3 Tsurumi, Yokohama 230-8501, Japana r t i c l e

i n f oa b s t r a c t

Article history:

Objectives. The purpose of the present investigation was to trace the fate of bisphenol A

Received 20 February 2009

injected into pregnant mice, focusing on its potential accumulation in the fetus and theReceived in revised form

brain, critical targets of hormonal chemicals, using whole-body autoradiography.

14 June 2009

Methods. Pregnant mice were injected intraperitoneally with 0.46 MBq of 14 C-BPA andAccepted 9 October 2009

then killed at 1 h or 1, 3, or 5 days after injection. Sections for autoradiography wereprepared in a cryomicrotome and the exposed imaging plate was processed using a ﬂuorescent/radioisotope image analyzer.

Keywords:

Results. Intraperitoneally injected 14 C-BPA was distributed throughout the body, including

Autoradiography

the fetus and the brain, within 1 h. Radioactivity faded gradually from the whole body by

Bisphenol A

the ﬁfth day, and no accumulation in any speciﬁc organ was found. However, although 14 C

Brain

was detected in the fetuses immediately after injection, the transfer of BPA from mother to

Dental materials

newborn was not observed.

Fetus

Signiﬁcance. The routes of rapid BPA discharge were conﬁrmed, and BPA neither accumulated
in the body nor was it transferred to newborn mice. No evidence was observed to suggest
the existence of a blood–placenta or blood–brain barrier for BPA. This information should betaken into consideration when assessing the risks of using dental materials that contain BPA.
© 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

The problem of endocrine-disrupting chemicals was ﬁrst recognized in the ﬁeld of dentistry when bisphenol A (BPA) was
found to be leaking into the saliva from sealant applied topatients [1]. Because sealants are commonly applied in children, who are generally more sensitive to chemical exposure,
this observation created great concern among dentists. Particular concern was expressed regarding the possibility that
BPA may disrupt the human hormonal system. Although
there is evidence to suggest that these initial estimates of
BPA leakage were rather high [2], establishing the safety of∗

BPA is difﬁcult. Glass ionomer cement represents an alternative sealing material for pits and ﬁssures, but its retention
is unsatisfactory and therefore its beneﬁt for patients islimited.
Despite extensive discussion of the safety of BPA [3–9]
no decisive conclusion has been reached because extremely
small amounts of BPA, as low as 10 pM, can have hormonal
effects [10]. Previous studies indicated that BPA modulates
cellular function at concentrations between 1 pM and 1 nM
[11]. When high-performance liquid chromatography (HPLC)
is used to detect low concentrations of BPA, the lower limit ofdetection is generally in the nanomolar range [12,13]; therefore, precise detection of BPA is difﬁcult.

Corresponding author. Tel.: +81 19 651 5111; fax: +81 19 653 6538.
E-mail address: mtanaka@iwate-med.ac.jp (M. Tanaka).
0109-5641/$ – see front matter © 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.dental.2009.10.006

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More thorough investigation is required to assess the safetyof BPA, especially with regard to its effect on the fetus and thebrain. The fate of BPA is an important concern in the use of
dental materials containing BPA. The purpose of this study
was to investigate whether BPA injected into pregnant mice
accumulates in a speciﬁc part of the body and whether it istransmitted to fetuses through the placenta or to the brain
through the blood–brain barrier. BPA distribution was monitored via autoradiography and transfer to pups was examinedboth in utero and after delivery.

2.

Materials and methods

This study used eight mice in the 13th day of pregnancy(Sankyo Labo-Service Inc., Tokyo, Japan). Pregnant mice weighing 46–48 g were injected intraperitoneally with 0.46 MBq of
14 C-BPA (Moravek Biochemicals, Inc., Brea, CA) and then killed
at 1 h or 1, 3, or 5 days after injection. Sections for autoradiography were prepared following the methods outlined by
Kawamoto and Shimizu [14] and Kawamoto [15]. Brieﬂy, theentire mouse was frozen in hexane cooled to −94 ◦ C immediately after sacriﬁce, and then immersed in 5% carboxymethyl
cellulose (CMC) gel in a stainless steel container. The wholesample was immersed again in the cooled hexane and completely frozen. The embedded frozen CMC block was placed ina cryomicrotome (CM 3500; Leica Instruments, Germany) andwhole-body sections of approximately 20 ␮m were cut along
the sagittal plane. By the ﬁfth day after injection, all mice haddelivered. Four pups were selected and processed as describedfor adult mice.

The frozen CMC block was placed in the cryomicrotome
and trimmed to the area of interest. The exposed surface wascovered with an adhesive ﬁlm (Cryoﬁlm Type 2C, FINETEC Co.
Ltd., Tokyo, Japan). For histological staining and autoradiography, 7- and 20-␮m-thick sections, respectively, were cut
along the sagittal plane and lined with adhesive ﬁlm. The
sections for autoradiography were completely freeze-driedin the cryochamber and stored in a desiccator containing
silica gel inside the cryochamber. Sections covered with a2-␮m-thick plastic ﬁlm were placed on the imaging plate(BAS-MS2040, Fuji-ﬁlm Inc., Tokyo, Japan) and exposed for 4
days. The exposed imaging plate was processed using a ﬂuorescent/radioisotope image analyzer (FLA-3000, Fuji-ﬁlm Inc.,Tokyo, Japan) and autoradiograms were obtained and analyzed. Freeze-dried sections were quickly thawed, ﬁxed with
4% paraformaldehyde, stained with hematoxylin and eosin (HE), and mounted on a slide glass under an adhesive ﬁlm using
30% glycerol as an adhesive. To quantitatively assess the dataobtained from the ﬂuorescent/radioisotope image analyzer,
several areas were cut out from sections and analyzed using aliquid scintillation counter. Data from the imaging plate wereconverted to radioactivity and expressed in Bq per mm3 .
All experimental procedures followed the guidelines foranimal studies established by Tsurumi University and Iwate
Medical University.

3.

Results

Fig. 1 shows an H-E section and the corresponding 14 C-BPA
distribution of a whole mouse at 1 h after intraperitoneal

Fig. 1 – Whole-body section of a pregnant mouse stained with hematoxylin and eosin and its corresponding autoradiogram
1 h after 14 C-bisphenol A injection. The small arrows indicate fetuses. The red color in the autoradiogram indicates thehighest region of radioactivity.

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Fig. 2 – Whole-body section of a pregnant mouse and its corresponding autoradiogram 1 day after 14 C-bisphenol A
injection. The small arrows indicate fetuses.

Fig. 3 – Whole-body section of a pregnant mouse and its corresponding autoradiogram 3 days after 14 C-bisphenol A
injection. The small arrows indicate fetuses.

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Fig. 4 – Whole-body section of a mouse that had delivered within 1 day and its corresponding autoradiogram 5 days after
14 C-bisphenol A injection.

injection. 14 C-BPA was distributed throughout the body,
including the fetus and brain. High amounts of 14 C weredetected in the kidney, liver, lower part of the stomach, andfetal liver, and to a lesser extent in the brain. The highly
radioactive area in the stomach area appears to be derived
from the liver rather than the duodenum. The white arrows
in the autoradiogram indicate the same position in the H-E
section. Another mouse showed strong radioactivity in theupper intestine and the area of the submandibular gland 1 hafter injection. In the kidney, a high concentration of 14 C
was detected and a difference in distribution was observed
between the cortex and the medulla. Blood did not exhibit
strong radioactivity, as evidenced by the relative lack of radiation in the heart. The fetal liver displayed higher levels of 14 C
than other parts of the fetus.
The results obtained from mice at 1, 3, and 5 days after
injection, are shown in Figs. 2–4, respectively. Fig. 5 shows thechange in the average radioactivity of each area over time. As
there was considerable variation between animals even within
the same areas, several typical points were selected and theirradioactivity was measured, and the resulting average values were plotted. At 1 day, high levels of 14 C were detectedin the intestine and, to a lesser extent, in the stomach, liver,and kidney, but radioactivity was very low in the brain andfetus. These trends were similar at 3 days, and the injected
BPA appeared to be excreted to the intestine through the liver,and to the urine through the kidneys. The thick contents of
the intestine appeared to dilute the 14 C label per mm3 in the
3-day animals. By the ﬁfth day, all mice had delivered andwhole-body radioactivity was lower. Activity was limited toFig. 5 – Change in average radioactivity in each area overtime.

the intestine and, to a lesser extent, the liver, stomach, andkidney. Almost no radioactivity was detected in the brain. 14 C
activity was not detectable in the pups, which were born on
the fourth day after injection (Fig. 6).

4.

Discussion

This experiment provides insight into the fate of intraperitoneally injected BPA in mice. The routes of rapid BPA
discharge were conﬁrmed, and BPA neither accumulated in
the body nor was it transferred to newborn mice. No evidence
was observed to suggest the existence of a blood–placenta or

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Fig. 6 – Whole-body sections of newborn mice and their corresponding radiograms. No radioactivity was detected innewborn mice.

blood–brain barrier for BPA. This information should be taken
into consideration when assessing the risks of using dental
materials that contain BPA.
A review article concerning the effect of BPA from dental
materials [16] concluded that the short-term risk of estrogenic
effects from dental treatments using BPA-based resins was
insigniﬁcant because of the small dosages used, although BPA
can induce changes in estrogen-sensitive organs or cells inanimal or cell-culture experiments. On the other hand, among
115 published papers concerning the low-dose effects of BPA,
94 reported signiﬁcant estrogenic effects [10] and disruptionof cellular function was shown even at doses as low as 1 pM
or 0.23 ppt [17]. The deﬁnition of “small dosage” is therefore
problematic, and decisive judgment seems almost impossible. The ﬁeld of dentistry should remain aware that BPA may
lead to hormonal disruption in human beings.

4.1.

Effect of BPA on fetuses

Moors et al. studied the disposition and transplacental transfer of intravenously administered BPA in pregnant rats using
gas chromatography–mass spectrometry [18]. The authors
noted that BPA was distributed rapidly, and peak concentrations for total BPA were attained 20–30 min after intravenous
administration, with high mean values in the maternal liver,kidneys, and uterus, and lower mean values in the placenta
and fetal liver. In all tissues, BPA levels declined in parallelwith those in maternal blood. The authors concluded that BPA
is readily transferred across the placenta of rats to the fetus,
based on the similar concentrations in placenta and fetal liver.
The present study demonstrates that intraperitoneallyinjected BPA spreads throughout the body, including fetuses,

within 1 h. No evidence for the existence of a placental barrierfor BPA was observed, suggesting that BPA exerts direct effects
on the fetuses when introduced into the pregnant body. However, although 14 C was detected in fetuses immediately after
injection, the transfer of BPA from mother to newborn pup
was not observed, suggesting that BPA may have been excreted
before the time of delivery. Although the endocrine-disrupting
effects of BPA are relatively weak in adults, fetuses are generally much more sensitive to endocrine-disrupting chemicals.
Because BPA may cause long term adverse effects if exposure
occurs during critical periods of differentiation [19], caution
should be practiced when using BPA-containing sealants orcomposite resins in pregnant women.

4.2.

BPA passes though the blood–brain barrier

In a previous study, Sun et al. [20] used a HPLC-ﬂuorescence
detecting system to monitor the concentrations of BPA in rat
brain and plasma for 8 h after a single i.v. or oral dose, andfound that BPA was capable of penetrating the blood–brain
barrier. The monitored maximum concentration of BPA in theplasma and brain after oral administration (200 mg/kg) was
1624 ppb and 20 ppb, respectively. Kim et al. [21] also showedthat BPA in the rat brain increased in a dose-dependent
manner after oral administration, and that the blood–brain
barrier system did not limit the access of BPA to the brain.
These results are consistent with the autoradiographic data
presented here, in which female mice showed body-wide distribution at 1 h after intraperitoneal BPA injection.
Subcutaneous exposure to BPA in mice was reported tocause alterations in brain sexual differentiation when dams
were exposed to 25 ng BPA/kg body weight per day from day 8

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of pregnancy through day 16 [22]. Supposing that the rat body
is 60% water [23], the 25 ng BPA/kg body weight is calculated
to be 40 ppt. According to a model experiment by Imai and
Komabayashi [2], the amount of BPA that leaches out fromcommercial Bis-GMA-based polymerized resins in the longterm is negligible. However, the BPA content of unpolymerizedcomposite resins reportedly ranges from 1.5 ␮g/g to 10.2 ␮g/g[24]. Although the simulation of BPA concentrations is impossible, it is reasonable that some dentists will think it wiser toavoid the use of Bis-GMA-based resins for pregnant women
due to possible estrogenic effects in the brain.

4.3.

Route of BPA discharge

The injected radioisotope activity faded gradually from thewhole body by the ﬁfth day, and no accumulation in any speciﬁc organ was found, indicating that a rapid route of BPA
discharge must exist in mice. In an experiment using 14 Clabeled BPA, 28% and 56% of orally administered BPA was
excreted into the urine and feces, respectively [25]. BPA wasalso excreted into the urine and feces in the present study.
High concentrations of 14 C-BPA were detected in the kidney,
liver, small intestine, and colon. Previous studies showed that
BPA is excreted into urine through the kidney and into thefeces through the intestine and colon after conjugation withglucuronic acid in rat liver microsomes [26].
Although the activity of one glucuronidation enzyme isreportedly reduced in the pregnant rat and is absent in the
rat fetus [27,11], BPA levels in the liver declined fairly rapidly
in this experiment by the ﬁfth day after injection. Although thehalf-life of BPA is difﬁcult to determine precisely, radioactivity
in the liver declined to one-tenth within 5 days in the present
study. The existence of a discharge route for BPA is important,because it indicates that BPA will be eliminated after a certain
period of time.

4.4.

The safety of BPA in dental materials

The amount of BPA leaking from dental materials such as
sealants and composite resins is very small [3,8,2,6] and BPA
is rapidly excreted from the body [25]. These factors support the safety of dental materials that contain BPA. However,exposure to very low concentrations does not exclude thepossibility of endocrine disruption, because this possibility
cannot be judged based on analytical data obtained using
conventional methods. As the information concerning thissubject is very limited, no decisive conclusion can be drawn
at present. Although we should not underestimate the beneﬁtthat patients derive from Bis-GMA-based materials, we must
continue to collect precise information and avoid unnecessary exposure in sensitive individuals. For the present, every
possible effort should be made to reduce BPA levels in dental materials. Although polymerized resin is stable and theamount of BPA leaking from it is very limited, unpolymerizedresin in the surface layer of sealant may mix with saliva andbe absorbed after swallowing [6]. For this reason, care should
be taken to remove or reduce unpolymerized resin. According to data from the authors’ laboratory, brushing the sealantsurface for 10–20 s was effective in reducing undesirable substances that could leach out from the cured surface of thesealant (Matsumoto et al., unpublished observations). In addition to routine practices such as this, the ﬁeld of dentistry
should promote the development and use of alternative dental
materials.

Acknowledgments
This study was supported by Research Grants from the Ministry of Education and Science of Japan (No. 14571964), byGrants for High Performance Biomedical Materials Researchfrom the Ministry of Education and Science of Japan, and also
by the research fund of Iwate Medical University.

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