Abstract
Objectives
The purpose of the present investigation was to trace the fate of bisphenol A injected into pregnant mice, focusing on its potential accumulation in the fetus and the brain, critical targets of hormonal chemicals, using whole-body autoradiography.
Methods
Pregnant mice were injected intraperitoneally with 0.46 MBq of 14 C-BPA and then killed at 1 h or 1, 3, or 5 days after injection. Sections for autoradiography were prepared in a cryomicrotome and the exposed imaging plate was processed using a fluorescent/radioisotope image analyzer.
Results
Intraperitoneally injected 14 C-BPA was distributed throughout the body, including the fetus and the brain, within 1 h. Radioactivity faded gradually from the whole body by the fifth day, and no accumulation in any specific organ was found. However, although 14 C was detected in the fetuses immediately after injection, the transfer of BPA from mother to newborn was not observed.
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 be taken into consideration when assessing the risks of using dental materials that contain BPA.
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 to patients . 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 , 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 is limited.
Despite extensive discussion of the safety of BPA no decisive conclusion has been reached because extremely small amounts of BPA, as low as 10 pM, can have hormonal effects . Previous studies indicated that BPA modulates cellular function at concentrations between 1 pM and 1 nM . When high-performance liquid chromatography (HPLC) is used to detect low concentrations of BPA, the lower limit of detection is generally in the nanomolar range ; therefore, precise detection of BPA is difficult.
More thorough investigation is required to assess the safety of BPA, especially with regard to its effect on the fetus and the brain. 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 is transmitted 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 examined both 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 and Kawamoto . Briefly, the entire 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 whole sample was immersed again in the cooled hexane and completely frozen. The embedded frozen CMC block was placed in a cryomicrotome (CM 3500; Leica Instruments, Germany) and whole-body sections of approximately 20 μm were cut along the sagittal plane. By the fifth day after injection, all mice had delivered. Four pups were selected and processed as described for adult mice.
The frozen CMC block was placed in the cryomicrotome and trimmed to the area of interest. The exposed surface was covered with an adhesive film (Cryofilm Type 2C, FINETEC Co. Ltd., Tokyo, Japan). For histological staining and autoradiography, 7- and 20-μm-thick sections, respectively, were cut along the sagittal plane and lined with adhesive film. The sections for autoradiography were completely freeze-dried in the cryochamber and stored in a desiccator containing silica gel inside the cryochamber. Sections covered with a 2-μm-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 (H-E), and mounted on a slide glass under an adhesive film using 30% glycerol as an adhesive. To quantitatively assess the data obtained from the fluorescent/radioisotope image analyzer, several areas were cut out from sections and analyzed using a liquid scintillation counter. Data from the imaging plate were converted to radioactivity and expressed in Bq per mm 3 .
All experimental procedures followed the guidelines for animal studies established by Tsurumi University and Iwate Medical University.
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 and Kawamoto . Briefly, the entire 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 whole sample was immersed again in the cooled hexane and completely frozen. The embedded frozen CMC block was placed in a cryomicrotome (CM 3500; Leica Instruments, Germany) and whole-body sections of approximately 20 μm were cut along the sagittal plane. By the fifth day after injection, all mice had delivered. Four pups were selected and processed as described for adult mice.
The frozen CMC block was placed in the cryomicrotome and trimmed to the area of interest. The exposed surface was covered with an adhesive film (Cryofilm Type 2C, FINETEC Co. Ltd., Tokyo, Japan). For histological staining and autoradiography, 7- and 20-μm-thick sections, respectively, were cut along the sagittal plane and lined with adhesive film. The sections for autoradiography were completely freeze-dried in the cryochamber and stored in a desiccator containing silica gel inside the cryochamber. Sections covered with a 2-μm-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 (H-E), and mounted on a slide glass under an adhesive film using 30% glycerol as an adhesive. To quantitatively assess the data obtained from the fluorescent/radioisotope image analyzer, several areas were cut out from sections and analyzed using a liquid scintillation counter. Data from the imaging plate were converted to radioactivity and expressed in Bq per mm 3 .
All experimental procedures followed the guidelines for animal 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 injection. 14 C-BPA was distributed throughout the body, including the fetus and brain. High amounts of 14 C were detected in the kidney, liver, lower part of the stomach, and fetal 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 the upper intestine and the area of the submandibular gland 1 h after 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 the change 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 their radioactivity was measured, and the resulting average values were plotted. At 1 day, high levels of 14 C were detected in the intestine and, to a lesser extent, in the stomach, liver, and kidney, but radioactivity was very low in the brain and fetus. 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 mm 3 in the 3-day animals. By the fifth day, all mice had delivered and whole-body radioactivity was lower. Activity was limited to the intestine and, to a lesser extent, the liver, stomach, and kidney. 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 ).
