Archives of Toxicology

, Volume 79, Issue 5, pp 243–252 | Cite as

Disposition of low doses of 14C-bisphenol A in male, female, pregnant, fetal, and neonatal rats

  • Hideo Kurebayashi
  • Shin-Ichiro Nagatsuka
  • Hiroyuki Nemoto
  • Hideyo Noguchi
  • Yasuo Ohno
Toxicokinetics and Metabolism

Abstract

Bisphenol A (BPA) is a weak xenestrogen (ADI=50 μg kg−1, US EPA) which is mass-produced, with potential for human exposure. To study absorption, distribution, excretion, and metabolism of BPA, BPA labeled with carbon-14 was administered p.o. to male and female Fischer (F344) rats at relatively low doses (20, 100, and 500 μg kg−1), and i.v. injected at 100 and 500 μg kg−1. 14C-BPA (500 μg kg−1) was also administered orally to pregnant and lactating rats to examine the transfer of radioactivity to fetuses, neonatal rats, and milk. Radioluminographic determination using phosphor imaging plates was employed to achieve highly sensitive determination of radioactivity. Absorption ratios of radioactivity after three oral doses were high (35–82%); parent 14C-BPA in the circulating blood was quite low, however, suggesting considerable first-pass effect. After an oral dose of 100 μg kg−1 14C-BPA, the radioactivity was distributed and eliminated rapidly, but remained in the intestinal contents, liver, and kidney for 72 h. The major metabolite in the plasma and urine was BPA glucuronide, whereas most of the BPA was excreted with the feces as free BPA. A second peak in the time-course of plasma radioactivity suggested enterohepatic recirculation of BPA glucuronide. There was limited distribution of 14C-BPA to the fetus and neonate after oral administration to the dam. Significant radioactivity was not detected in fetuses on gestation days 12 and 15. On day 18, however, radioactivity was detected in the fetal intestine and urinary bladder 24 h after oral dosing of 14C-BPA to the pregnant rats. Part of radioactivity was transferred to neonatal rats from the milk of the treated lactating dam and remained in the intestine of the neonates after 24-h nursing by an untreated dam.

Keywords

Bisphenol-A Distribution Pregnant rat Fetus Neonate 

Introduction

Bisphenol A (BPA; 2,2-bis-(4-hydroxyphenyl)propane), an industrial chemical widely used to make polycarbonate plastic and epoxy resins, has well known estrogen-mimic activity (Dodds and Lawson 1936; Bitman and Cecil 1970; Krishnan et al. 1993). Although its estrogenic activity is several thousands times less than that of estradiol (Schafer et al. 1999; Hiroi et al. 1999; Papaconstantinou et al. 2000), possible endocrine-disrupting activity of BPA came into question because of food-contact applications of BPA-derived materials. The US Environmental Protection Agency (EPA) has established a maximum acceptable dose (reference dose) of BPA as 50 μg kg−1 day−1 (US EPA 2002). By using toxicological indices such as body weight reduction, this reference dose was obtained from the no effective level of chronic BPA exposure in rats and mice (50 mg kg−1 day−1) multiplied by uncertainty factor of 0.001.

Migration of BPA monomer to food or beverages from polycarbonate plastic resins or epoxy can-coatings has been shown to be several parts per billion (ppb), resulting in a dietary intake of BPA of less than 0.118 μg kg−1 day−1 (BAGIG 2003). BPA-derived plastic materials are also used as dental sealants; these cause oral exposure to BPA monomer because of incomplete in-situ polymerization (Pulgar et al. 2000). The amount of BPA released from the sealant was lower than the reference dose (Manabe et al. 2000) particularly when the surface layer of the sealant was treated to reduce the amount of BPA monomer being released (Rueggeberg et al. 1999). Probable human BPA exposure is, therefore, considerably lower than the reference dose. However, several studies have revealed low-dose effects of BPA (ranging from 2 to 500 μg kg−1) on spermatogenesis or prostate weight in fetal or neonatal BPA exposure in mice (Welshons et al. 1999; Gupta 2000).

Biological fate of BPA in experimental animals has been evaluated using much higher doses of 14C-BPA ranging from 10 to 100 mg kg−1 (Snyder et al. 2000; Pottenger et al. 2000). This was mainly owing to the low specific radioactivity of the 14C-BPA used and the low sensitivity of radioactivity determination.

In this study we have examined the disposition of low doses BPA in adult, pregnant, and neonatal rats, by using 14C-BPA with relatively high specific radioactivity and radioluminography (Amemiya and Miyahara 1988) that enables highly sensitive radioactivity determination, to determine whether or not 14C-BPA will be easily distributed to fetal rats.

Experimental

Materials

14C-BPA with specific radioactivity 2.62 GBq mmol−1 and radiochemical purity >99% was obtained from NEN Life Science Products (Boston, MA, USA). Non-radioactive BPA was from Nacalai Tesque (Kyoto, Japan). Liquid scintillation cocktail (Hionicfluor) and tissue solubilizer (Soluene-350) were obtained from Packard Bioscience (Groningen, The Netherlands). All other chemicals used in the study were of reagent grade.

Fischer (F344/DuCrj) SPF rats, widely used in endocrine studies because of their high sensitivity to estrogen, were obtained from Charles River Japan (Kanagawa, Japan). Rats were acclimatized for more than 1 week before administration of 14C-BPA. Before administration, rats were fed food and water ad libitum without fasting.

The bioimaging analyzer (BA; BAS-2500), phosphor imaging plates (IP; SR2040), and lead shield box were obtained from Fuji Photo Film (Tokyo, Japan). The cryo-microtome (PMV Type 450) was from PMV (Stockholm, Sweden). Thin polyester film (Diafoil, 4 μm thick) was purchased from Mitsubishi Chemical Polyester Film (Tokyo, Japan). A series of plastic radioactivity standards (CFQ-7601; 10 different 14C-radioactivity sources, 2×2 cm) was obtained from Amersham Pharmacia Biotech (Tokyo, Japan). The liquid scintillation spectrometer (2500TR) was from Packard Instruments (Meriden, CT, USA). Thin-layer chromatography (TLC) plates (silica gel 60F254) were from E. Merck (Darmstadt, Germany).

Preparation of dosing solution

Isotonic phosphate buffer (34 mmol L−1, pH 7.4) was prepared by dissolving one packet of powder for phosphate buffer (RM102-5; Iatron Laboratories, Tokyo, Japan) and 14.8 g sodium chloride in 2 L distilled water. A series of dosing solutions with BPA concentrations of 20 μg (0.23 MBq) mL−1, 100 μg (1.15 MBq) mL−1 and 500 μg (5.75 MBq) mL−1 were prepared by dissolving 14C-BPA in the isotonic phosphate buffer. The dosing solution of 500 μg mL−1 contained 10% ethanol to dissolve BPA. The radiochemical purity of 14C-BPA in each of freshly prepared and stored (at 4°C for 1 week) dosing solutions was confirmed to be >99% by TLC using dichloromethane–methanol, 24:1 (v/v) as mobile phase.

Determination of radioactivity in the plasma

Rats were subjected to bolus i.v. injection and p.o. administration of 14C-BPA at the age of 10 weeks. For i.v. administration, male and female rats were injected with 14C-BPA at doses of 100 and 500 μg mL−1 kg−1 into the foot vein (n=3 for each dose and sex). For p.o. administration, male and female rats were orally administered 14C-BPA at doses of 20, 100 and 500 μg mL−1 kg−1 (n=3 for each dose and sex).

Venous blood samples (ca. 0.1 mL) were taken from the tail vein into heparinized microtubes at designated time points. The tubes were then centrifuged at 8000×g for 5 min at room temperature to separate the plasma. A 20-μL aliquot of each plasma sample was then spotted on to tissue paper support (5 mm×10 mm) attached to a plastic sheet. A series of standard plasma samples containing known concentrations of 14C-radioactivity ranging from 1 to 20,000 dpm was also spotted in the same manner as the plasma samples to be determined. The plastic sheet was dried at room temperature, covered with thin polyester film, and exposed to an IP for 48 h in a lead shield box. The IP was then read by a BA (pixel size 100 μm, sensitivity 30000, latitude 5, gradation 8 bit) to determine the photo-stimulated luminescence (PSL) intensity in each spot area.

The calibration plot was obtained from background-subtracted PSL intensity (Y-axis) and radioactivity (X-axis) of each standard plasma sample (n=3 for each amount of radioactivity). Linear least-squares fitting to Y=aX with weight factor of 1/Y was used to obtain the calibration line. The PSL value of each standard sample was interpolated to the calibration line to obtain the calculated radioactivity. We defined the accuracy rate as the percent ratio of the calculated radioactivity to the actual radioactivity in each standard sample. The lower limit of quantitation (LOQ) was determined as the lowest amount of radioactivity of the standard sample satisfying the average accuracy rate of 100±10% with a coefficient of variation (COV) of less than 20% in triplicate samples for each radioactivity concentration.

The area under the time–radioactivity curve (AUC) was calculated by the trapezoidal method using data obtained from 0 to 72 h. The half life in the terminal phase was determined by the least-squires method.

Excretion study

Male and female rats at the age of 10 weeks were orally administered 100 μg kg−1 14C-BPA (n=3 for each sex) and placed in metabolic cages separately. Urine and feces were collected up to 168 h after administration. Urine samples involving cage wash water were collected in tubes cooled in an ice–water bath, and adjusted to the designated volume with distilled water. Feces were homogenized with approximately three volumes of distilled water and adjusted to the designated volume with distilled water. After sample collection rats were killed by ether anesthesia and solubilized in 500 mL 0.5 mol L−1 sodium hydroxide with 50 mL toluene under reflux for 72 h to determine the residual radioactivity in the carcass. Radioactivity in each of the urinary, fecal, and carcass samples was determined by using a liquid scintillation counter.

Determination of tissue radioactivity distribution

Male and female rats at the age of 10 weeks were orally administered 100 μg kg−1 14C-BPA and killed by ether anesthesia 30 min, 24 h, and 72 h after administration (n=3 for each time point and sex).

Rats were shaved, frozen, and embedded in 5% carboxymethylcellulose paste. The rats in the paste were immersed in dry ice–acetone mixture to make frozen blocks. Whole-body sections 30 μm thick were then cut by use of a cryo-microtome. The sections were freeze-dried, covered with thin polyester film, and exposed to an IP together with a series of plastic radioactivity standards, calibrated for 30μm thick tissue sections containing 14C-radioactivity, for from 1 to 16 days in a lead shield box. The IP was then read by the BA (pixel size 50 μm, sensitivity 30,000, latitude 5, gradation 8 bit).

The calibration plot was obtained from background-subtracted PSL intensity per mm2 (Y-axis) and tissue equivalent radioactivity (X-axis) of each plastic radioactivity standard as described above. Nine replicates of regions of interest (ROI) of different size were taken in each area of plastic radioactivity standard. The COV of average PSL value of nine replicates was expressed as a function of the PSL value and the size of ROI to determine LOQ.

Transfer of radioactivity to fetal rats, neonatal rats, and milk

In the following transfer study, 9-week-old virgin female rats mated with male rats for pregnancy were used.

Pregnant rats were orally administered 500 μg kg−1 14C-BPA on gestation days 12, 15, and 18, and killed 30 min and 24 h after administration (n=1 for each time point and pregnancy period). The rats were subjected to quantitative whole-body radioluminography as described above.

A lactating rat on postnatal day 11 was orally administered 500 μg kg−1 14C-BPA and kept in a cage together with five neonatal rats for 24 h. Two of the five neonatal rats (one male and one female) were then killed and subjected to quantitative whole-body autoradiography. The other three neonatal rats were kept in a cage together with another lactating rat without BPA administration for the next 24 h. Two of the three remaining neonatal rats (one male and one female) were then killed and subjected to quantitative whole-body radioluminography.

Milk and venous blood samples were taken from lactating rats (n=3) on postnatal day 11 at designated time points under light anesthesia with ether after oral administration of 500 μg kg−1 14C-BPA. The blood samples were treated to obtain the plasma as described above. For milk sampling, intraperitoneal injection of oxytocin (0.5 IU mL−1 saline kg−1) was performed about 30 min before milk sampling. A 1-mL aliquot of each of plasma and milk samples was solubilized in 2 mL tissue solubilizer followed by liquid scintillation counting.

Metabolite analysis in plasma, urine and feces

Plasma samples were obtained from the whole blood taken from the descending aorta of male rats 15 min, 6 h, and 24 h after oral administration of 500 μg kg−114C-BPA. Urine and fecal samples (0–24 h) were those obtained in the excretion study of male rats. Part of each urine sample (1 mL) was subjected to hydrolysis of possible glucuronides by incubating with 40 μL β-glucuronidase solution (from Helix pomatia, Sigma, St Louis, MO, USA) and 1 mL 0.5 mol L−1 acetate buffer (pH 5.0) at 37°C for 1 h. A 1-mL (hydrolyzed urine sample) or 0.5-mL (other samples) aliquot of each sample was extracted three times with 5 vol methanol. The residue from the methanol extract was solubilized in 2 mL tissue solubilizer. Recovery from the methanol extraction was determined by liquid scintillation counting of a portion of each of methanol extract and solubilized residual samples.

The methanol extract was evaporated in vacuo, redissolved in a small volume of methanol, and subjected to TLC on silica gel 60F254 with chloroform–methanol–acetic acid–distilled water, 50:20:1:4 (v/v), as mobile phase. The TLC plate was covered with thin polyester film and exposed to an IP for 24 h in a lead shield box. The IP was then read by the BA (pixel size 200 μm, sensitivity 10,000, latitude 4, gradation 8 bit). Relative amounts of BPA and its metabolites were determined from the ratio of PSL in each spot area compared with the PSL in the whole developing area.

Results

Determination of radioactivity in the plasma

Table 1 shows the average accuracy and COV for triplicate analysis of the concentration of radioactivity in each standard plasma sample. The average accuracy for radioactivity from 2–20000 dpm/20μL was 97.8 to 105.3% with a COV of 10–18.3%. The LOQ was 2 dpm/20μL in the conditions used for plasma radioactivity determination.
Table 1

Accuracy rate and COV of radioactivity concentration determined by radioluminography in triplicate standard plasma samples

Radioactivity (dpm/20 μL)

Average accuracy (%)

COV of accuracy (%)

20,000

101.2

1.0

5000

102.4

3.4

1000

100.2

2.0

200

101.0

4.5

50

105.3

3.9

10

96.4

15.1

5

108.3

18.3

2

97.8

16.8

1a

75.3

46.3

aBelow LOQ

Figure 1 shows time-courses of radioactivity concentration in rat plasma after i.v. injection and p.o. administration. Several peaks were observed in the time–radioactivity curves for both dosing routes. The AUC up to 72 h was almost linearly correlated with the dose of BPA (Table 2). The apparent absorption ratio of radioactivity after p.o. dosing calculated from the relative AUC as compared with i.v. dosing was 60–82% for male rats and 35–50% for female rats. Half-lives of plasma radioactivity were determined by the terminal phase data depending on dose, administration route, and gender (Table 2). They were from 13 to 21 h after i.v. administration and from 18 to 22 h after p.o. administration (except for male rats dosed p.o. with 20 μg kg−1, for which the estimate of half-life was variable, because plasma radioactivity was close to the detection limit).
Fig. 1

Time-course of plasma radioactivity in male (upper) and female (lower) F344 rats after p.o. administration (20, 100, 500 μg kg−1) and i.v. injection (100, 500 μg kg−1) of 14C-BPA. Data are means±SD from three rats

Table 2

Elimination half-life and AUC of plasma radioactivity in male and female rats after p.o. administration (20, 100, 500 μg kg−1) and i.v. injection (100, 500 μg kg−1) of 14C-BPA

Parameters

Dosing route and amount (μg kg−1)

p.o.

i.v.

20 

100

500

100

500

Male rats

Elimination half life (h)

78±52

18±3

21±3

19±2

21±3

AUC (ng eq h mL−1)

36±6

178±44

663±164

266±46

865±97

Average AUC a

219a

Apparent absorption (%)

82

81

60

Female rats

Elimination half life (h)

20±7

22±13

18±8

13±3

16±2

AUC (ng eq h mL−1)

14±5

99±19

500±43

190±45

1029±81

Average AUCa

198a

Apparent absorption (%)

35

50

50

Data are mean±SD from 3 rats

aAverage AUC was calculated as {AUC100μg/kg i.v.+(AUC500μg/kg i.v.)/5}}/2 and used to calculate the apparent absorption ratio in oral dosing. One fifth of this value was used as AUC (0–72 h) for i.v. dose of 20 μg kg−1

Excretion study

Cumulative excretion of radioactivity in the urine and feces in male and female rats was studied after oral administration of 100 μg kg−1 14C-BPA (Table 3). The preliminary study showed no excretion of radioactivity in the expired air. In male rats urinary excretion was only 10% in 7 days whereas for female rats it was about 35%. The major excretion route was fecal excretion both in male rats (88%) and female rats (64%).
Table 3

Cumulative excretion of radioactivity in urine and feces of male and female rats after oral administration of 100 μg kg−1 14C-BPA

Time (h)

Excretion of radioactivity (% of dose)

Urine

Feces

Total

Male rats

0–4

1.0±0.6

4–8

2.4±0.5

8–24

7.7±0.3

57.9±9.9

65.6±9.8

24–48

9.1±0.5

82.1±6.7

91.2±6.1

48–72

9.5±0.6

86.2±4.1

95.7±3.5

72–96

9.5±0.7

87.5±2.9

97.0±2.3

96–120

9.6±0.7

87.9±2.5

97.5±1.8

120–144

9.6±0.7

88.0±2.3

97.7±1.7

144–168

9.6±0.7

88.1±2.3

97.7±1.6

Carcass (168 h)

0.1±0.2

Female rats

0–4

0.3±0.1

4–8

8.4±4.7

8–24

20.5±9.3

17.9±17.1

38.4±21.8

24–48

28.8±13.3

48.7±9.5

77.5±9.7

48–72

31.5±13.3

58.4±9.0

89.8±4.8

72–96

32.8±12.8

62.2±11.2

95.0±1.6

96–120

33.3±12.8

63.5±11.5

96.8±1.3

120–144

33.5±12.8

63.8±11.4

97.3±1.4

144–168

33.6±12.8

64.0±11.4

97.6±1.5

Carcass (168 h)

0.0±0.0

Data are means±SD from 3 rats

Determination of tissue radioactivity distribution

In order to create a function to calculate LOQ, the relationship between average PSL value and its COV obtained from nine ROIs in the homogeneous plastic standard areas was examined for variable ROI sizes. Figure 2A shows an example of the analysis for 24-h exposure. The COV of the PSL value was lower when PSL value was larger. There were significant linear relationships between the average PSL value and its COV in logarithmic XY scales for all four ROI sizes tested (r2>0.995, P<0.001). The slopes of four regression lines ranged from −0.472 to −0.502 with average slope of −0.490. The PSL value giving the COV of 20%, i.e. the LOQ, was determined from each regression line. Figure. 2B shows the relationship between LOQ and corresponding ROI size. There was also a significant linear relationship between LOQ and ROI size in logarithmic XY scales (r2=0.990, P<0.001). For each exposure condition in this quantitative whole-body radioluminography, the regression line between LOQ and ROI size was determined to judge whether or not the PSL value of each tissue ROI with variable ROI size exceeded the LOQ.
Fig. 2A–B

An example of the determination of LOQ for quantitation of tissue radioactivity by radioluminography (for 24 h exposure). (A) The X-axis shows the average PSL of nine ROIs taken in homogenous areas of the plastic standards and the Y-axis shows the COV. The relationship was determined in different ROI sizes: (filled circles) 0.33mm2 (log[COV]=−0.502 log[PSL]+1.465, r2=0.999, P<0.001); (filled squares) 0.95mm2 (log[COV]=−0.497 log[PSL]+1.305, r2=0.995, P<0.001); (empty circles) 2.64mm2 (log[COV]=−0.490 log[PSL]+1.114, r2=0.999, P<0.001); and (empty squares) 9.00mm2 (log[COV]=−0.472 log[PSL]+0.847, r2=0.997, P<0.001). The X value for crossing point of each regression line and the horizontal dotted bar (showing COV=20%) is the LOQ for each ROI size. (B) The relationship between LOQ and ROI size. The regression line was linear (log[LOQ]=−0.899 log[ROI size]−0.056, r2=0.990, P<0.001)

Figure 3 shows whole-body images showing radioactivity distribution obtained from male rats after oral administration of 100 μg kg−1 14C-BPA. Relatively high radioactivity was seen in the intestinal contents, liver, and kidney. Table 4 summarizes tissue radioactivity in male and female rats after oral administration of 100 μg kg−1 14C-BPA. Although radioactivity in other tissues was relatively low and eliminated quickly, radioactivity remained in intestinal contents, liver, and kidney 72 h after oral administration. Radioactivity in some tissues of females was similar to those of males at 30 min, and became lower than the males at 24 and 72 h.
Fig. 3

Whole-body images showing radioactivity distribution in male rats orally administered 100 μg kg−1 14C-BPA. The whole-body images of rats killed 30 min after administration were obtained by exposure of sections to an imaging plate for 8 days. Other images were obtained by exposure for 16 days

Table 4

Tissue radioactivity distribution in male and female rats after oral administration of 100 μg kg−1 14C-BPA

Tissue

Radioactivity concentration (ng BPA equivalent g−1 or mL−1)

30 min

24 h

72 h

Male rats

Adrenal gland

6.74±2.20

NQ

NQ

Blood

5.84±1.08

3.13±2.65

NQ

Bone marrow

2.18±0.92

NQ

ND

Brain

NQ

NQ

ND

Brown fat

1.53±0.54

ND

ND

Epididymis

NQ

ND

ND

Eyeball

NQ

NQ

ND

Heart

NQ

NQ

ND

Kidney cortex

18.08±3.15

17.02±2.28

11.58±1.56

Kidney medulla

50.82±33.61

10.05±6.51

NQ

Liver

37.04±13.40

22.24±4.12

7.97±0.64

Lung

4.45±0.20

NQ

NQ

Mandibular gland

1.65±1.04

ND

ND

Pituitary gland

6.85 (n=2)

ND

ND

Prostate gland

NQ

ND

ND

Skeletal muscle

0.64±0.19

NQ

ND

Skin

2.74±0.57

NQ

ND

Testis

0.57±0.30

NQ

ND

Thyroid gland

3.61±1.99

NQ

ND

Female rats

Adrenal gland

5.12±0.83

NQ

NQ

Blood

6.57±1.41

NQ

NQ

Bone marrow

NQ

ND

ND

Brain

NQ

ND

ND

Brown fat

NQ

ND

ND

Eyeball

NQ

ND

ND

Kidney cortex

12.83±4.66

4.07±0.86

1.49±0.26

Kidney medulla

17.87±18.49

3.73±0.28

NQ

Liver

45.22±10.29

13.25±3.66

2.68±0.74

Lung

3.97±1.11

NQ

NQ

Mandibular gland

NQ

ND

ND

Skeletal muscle

NQ

ND

ND

Skin

2.35±1.06

NQ

ND

Uterus

4.14 (n=2)

ND

ND

Data are means±SD from 3 rats

NQ—Nonquantifiable (below LOQ)

ND—Not determined (indistinguishable)

Transfer of radioactivity to fetal rats, neonatal rats, and milk

Table 5 summarizes tissue radioactivity in pregnant, fetal, and neonatal rats 0.5 and 24 h after oral administration of 500 μg kg−1 14C-BPA to dams. Distribution of radioactivity in the maternal rat was essentially identical for 12, 15, and 18 days gestation each time. Relatively high radioactivity was seen in the liver and kidneys and levels were dose-dependent.
Table 5

Tissue radioactivity in pregnant, fetal, and neonatal rats after oral administration of 500 μg kg−1 14C-BPA to dams

Radioactivity concentration (ng BPA eq. g−1 or mL−1)

Dam and fetal tissues

12 days of gestation

15 days of gestation

18 days of gestation

30 mina

24 h

30 min

24 h

30 min

24 h

Amniotic fluid

ND

ND

NQ

NQ

NQ

NQ

Blood

43.32

4.33

37.51

3.83

30.99

10.79

Kidney cortex

NDb

NDb

55.42

17.06

34.41

NDb

Kidney medulla

NDb

NDb

270.80

NDb

138.18

NDb

Liver

219.59

61.44

260.68

54.88

317.26

78.03

Lung

37.74

4.00

24.23

2.61

10.81

4.59

Ovary

21.94

3.96

13.91

NQ

15.67

3.49

Placenta

15.43

NQ

18.12

NQ

9.91

3.86

Skin

12.27

NQ

8.85

NQ

11.89

NQ

Uterus

22.68

ND

NQ

NQ

15.31

NQ

Fetus

NQ

NQ

NQ

NQ

NQ

3.28

Fetal membrane

NQ

NQ

NQ

NQ

NQ

10.87

Yolk sac

NQ

ND

ND

ND

NQ

54.14

Neonatal tissues

Immediately after 24 h lactationc

24 h after 24 h lactationd

Male

Female

Male

Female

Gastric contents

2.36

2.35

ND

ND

Intestinal contents

45.83

30.03

10.44

11.94

Urinary bladder

9.70

ND

ND

ND

NQ—Nonquantifiable (below LOQ)

ND—Not determined (indistinguishable)

aEach time shows the sacrifice time after oral administration of 14C-BPA to each pregnant rat

bND—Not determined because of flare effect due to high radioactivity of intestinal contents

cPut together with a lactating rat orally administered 14C-BPA for 24 h followed by sacrifice

dPut together with a lactating rat orally administered 14C-BPA for 24 h, then nursed by untreated rat for 24 h followed by sacrifice

Figure 4 shows the distribution of radioactivity in fetal rats after oral administration of 500 μg kg−1 14C-BPA to pregnant on rats gestation days 12, 15, and 18. Radioactivity in fetal tissues was undetectable in pregnant rats on gestation days 12 and 15. On gestation day 18, radioactivity in fetal tissues was not seen at 30 min, but was seen 24 h post-dose in the fetal urinary bladder and intestine. The level of radioactivity in fetal tissues was about 30% of than in the maternal blood (Table 5). The fetal membrane contained the same level and the level of radioactivity in the yolk sac was much higher than in the maternal blood. The amniotic fluid contained a negligible level of 14C-BPA.
Fig. 4

Fetuses in pregnant rats 30 min and 24 h after oral administration of 500 μg kg−114C-BPA. The images 30 min after administration were obtained by exposure for 24 h. The images 24 h after the administration were obtained by exposure for 8 days. The brightness and contrast are controlled to equalize the background level. (The characters “F”, “P”, and “B” in the images denote fetus, placenta and background areas, respectively.)

In neonatal rats nursed by a 14C-BPA-treated dam for 24 h, most of radioactivity was located in the intestinal contents immediately after the 24 h lactation, and decreased to one third after 24 h when the pups were nursed by an untreated dam (Table 5).

Figure 5 shows the transfer of radioactivity to the milk in lactating rats orally administered 500 μg kg−1 14C-BPA. The maximum milk concentration of 4.46 ng BPA equivalent mL−1 was observed 8 h after administration; this was followed by elimination of radioactivity with a half-life of about 26 h. AUC(0–48 h) was 156 ng BPA equivalent h mL−1. The maximum plasma radioactivity of 27.2 ng BPA equivalent mL−1 was observed at 4 h after administration, followed by elimination of radioactivity with a half-life of about 31 h in lactating rats. AUC(0–48 h) was 689 ng BPA equivalent h mL−1.
Fig. 5

Time-course of radioactivity in plasma and milk of lactating rats (postnatal day 11) after oral administration of 500 μg kg−1 14C-BPA. Data are means±SD from three rats

Metabolite analysis in plasma, urine, and feces

Recovery of radioactivity from plasma and urine samples was >95% whereas that from fecal samples was about 70%. For the presentation of metabolites, “M” was used for common metabolites in any two or all of plasma, urine, and fecal samples from male rats after oral administration of 500 μg kg−1 14C-BPA (Table 6). “P” and “U” denote plasma- and urine-specific metabolites, respectively. The common presence of “M” metabolites in different samples was confirmed by co-TLC.
Table 6

Composition of metabolites in plasma, urine, and fecal samples from male rats after oral administration of 500 μg kg−1 14C-BPA

Metabolite

Composition of radioactivity (% of total)

Plasma

Urine

Feces

15 min

6 h

24 h

Before hydrolysis

After hydrolysisa

0–24 h

Origin

9.3±0.7

9.1±2.1

7.2±3.7

11.5±4.0

6.8±1.0

2.0±0.5

M1

11.3±1.1

8.0±1.3

3.4±0.8

M2

77.4±1.0

59.3±13.2

74.2±7.6

39.8±3.1

3.8±0.3

M3

4.4±0.4

7.5±2.3

4.0±1.6

1.6±0.4

U1

10.2±0.9

8.9±1.5

P1

2.4±0.0

U2

7.2±0.9

6.9±1.2

U3

3.2±1.7

BPA

2.3±0.2

1.7±0.3

0.3±0.5

1.6±0.7

47.1±5.7

77.2±1.7

M4

4.7±0.6

5.9±0.7

Others

4.2±1.4

29.9±10.9

18.3±9.4

10.8±5.3

6.6±1.1

9.9±2.1

Data are means±SD from 3 rats

aUrine samples (0–24 h) incubated with β-glucuronidase from Helix pomatia

Figure 6 shows TLC images obtained from radioluminography of 14C-BPA metabolites after oral administration of 500 μg kg−1 to male rats. The composition of metabolites in plasma samples after 15 min, 6 h, and 24 h showed the major metabolite was M2 (60–80% of plasma radioactivity); parent BPA was present in only minor amounts (0.3–2.3%) in the plasma samples (Table 6). The major metabolite in the 24 h-urine was also M2 (ca. 40%). BPA (1.6%) and several other metabolites (7–12%) were seen in the urine. Hydrolysis of urine samples with β-glucuronidase increased free BPA with almost equivalent reduction of M2, suggesting that M2 is BPA glucuronide. In the feces, the major fraction was free BPA (ca. 80%) with several other metabolites (1.5–6%).
Fig. 6

TLC images showing separation of BPA metabolites in plasma, urine, and fecal extracts from male rats after oral administration of 500 μg kg−1 14C-BPA. The methanol extracts were subjected to TLC with chloroform–methanol–acetic acid–distilled water 50:20:1:4 (v/v) as mobile phase

Discussion

Radioluminographic determination of radioactivity is superior to liquid scintillation counting when a small amount of sample is to be determined. It enabled quantitation of radioactivity as low as 2 dpm in 20 μL plasma (Table 1). For liquid scintillation counting the LOQ was about 30 dpm for 2 min counting (data not shown). In tissue radioactivity determination the latter sensitivity is sometimes greater than the former for larger organs because of the larger amount of sample for counting. In this study we used the radioluminographic determination because of much higher radioactive sensitivity for small tissues such as fetuses. We examined the disposition of orally administered 14C-BPA in pregnant rats and placental transfer to fetuses to determine whether or not 14C-BPA could be easily distributed to fetuses.

The time-course of plasma radioactivity (Fig. 1) showed several peaks after both i.v. injection and p.o. administration, suggesting enterohepatic recirculation of 14C-BPA. The half-lives of radioactivity in the terminal phase were 13–22 h for females and 18–21 h for males (except p.o. 20 μg kg−1 dose) (Table 2). They were longer in rats than monkeys. After a single oral dose of 100 μg kg−1 14C-BPA to male and female cynomolgus monkeys, the terminal elimination half-lives of radioactivity were 9.6 h for males and 9.8 h for females; they were 13.5 h for males and 14.7 h for females post i.v. dose (Kurebayashi et al. 2002). The proposed enterohepatic circulation of 14C-BPA-derived radioactivity should account for the longer elimination phase of blood radioactivity in rats (Kurebayashi et al. 2003).

BPA metabolites accounted for most of radioactivity in the plasma and urine, and parent BPA in the plasma was detected in trace amounts only (Table 6). M2, the major metabolite in both plasma and urine, is quite likely to be BPA glucuronide because glucuronidase treatment reduced the fraction of M2 and increased the fraction of BPA in the urine. BPA glucuronide transferred to circulating blood is likely to be excreted rapidly in the urine as the major urinary metabolite. Therefore, the enterohepatic recirculating material was thought to be glucuronide of BPA that was shown to be the major metabolite of BPA in the plasma and urine after oral administration (Snyder et al. 2000; Pottenger et al. 2000; Kurebayashi et al. 2003).

After oral administration of 100 μg kg−1 14C-BPA, urinary excretion amounted to 10% in male rats for 7 days whereas for female rats urinary excretion was about 35%. Differences between urinary excretion of 14C-BPA after oral dose were observed by Pottenger et al. (2000) who showed that urinary elimination of 14C-BPA-derived radioactivity was 13–16% and 21–34% of administered dose in males and females, respectively, for all doses and routes. The AUC of plasma radioactivity in female rats was considerably lower than that in male rats (Table 2), suggesting lower absorption and/or higher urinary excretion of radioactivity in female rats than in male rats (Table 3).

The apparent absorption ratio of radioactivity after p.o. dosing calculated from the relative AUC as compared with i.v. dosing was 60–82% for male rats and 35–50% for female rats (Table 2). Despite considerable absorption of 14C-BPA, the bioavailability of parent BPA should be quite low because only trace amounts of free BPA were present in the plasma (Table 6 and Fig. 6). After oral administration of 100 μg kg−1 14C-BPA, the radioactivity in some tissues of females might have been eliminated more rapidly and become lower than in the males at 24 and 72 h (Table 4). After the disappearance of most of the radioactivity from other tissues, radioactivity remained in the intestinal contents, liver, and kidney at higher levels in males than in females (Fig. 3). The suggested enterohepatic recirculation of 14C-BPA might be more active in male rats than in female rats.

Immediately after and 24 h after nursing with an untreated dam, radioactivity was detected in the intestinal contents of neonatal rats kept for 24 h by a lactating dam orally dosed 500 μg kg−1 14C-BPA (Table 5). This was owing to significant excretion of radioactivity to the milk (Fig. 5). It has been reported that the major molecular species in the milk after oral administration of 14C-BPA is BPA glucuronide (Snyder et al. 2000).

The distribution pattern of radioactivity in pregnant rats was essentially the same as that in non-pregnant female rats (Tables 4 and 5). The distribution levels were dose-dependent in most of the tissues. There was limited distribution of 14C-BPA to the fetus. Radioactivity in fetal tissues was undetectable except for the pregnant rat on gestation day 18 in the fetal urinary bladder and intestine (Fig. 4). On gestation day 18 the amount of radioactivity in fetal tissues at 24 h was about 30% that in maternal blood, and the yolk sac contained a much higher level of radioactivity than the maternal blood (Table 5). Domoradzki et al. (2003) reported that the placenta and/or yolk sac contained higher levels of BPA and BPA glucuronide than the fetus 0.25 and 12 h after oral administration of 10 mg kg−1 14C-BPA to pregnant rats on gestation day 16. On the other hand, Shin et al. (2002) reported that after i.v. injection (2 mg kg−1) of BPA to pregnant rats the AUC of BPA for the fetus was twice that for the maternal blood and the AUC for the placenta was four times that for the maternal blood. We thought these differences were a consequence of the routes of administration, i.v. or p.o., because only trace amounts of parent BPA dosed orally appeared in the plasma.

We are interested in the molecular species transferred to fetuses. We think that BPA glucuronide passed through the placental barrier because drug conjugates are known to pass through the placental barrier at the late stage of pregnancy in rats (Dickinson et al. 1989). We thought that most of the radioactivity transferred from the dams to fetal and neonatal rats was BPA glucuronide, although the actual molecular species present in the fetal and neonatal rats were unclear and hard to identify in this low-dose study. We are also interested in the limited distribution of radioactivity in the intestine of the neonatal and fetal rats. This might be owing to biliary excretion and/or enterohepatic circulation of the radioactivity in neonatal and fetal rats, similar to adult rats.

These results suggested that the absorption, distribution, metabolism, and excretion of 14C-BPA orally administered to rats were rapid and that there was limited distribution of 14C-BPA to fetal tissues after oral administration to pregnant rats.

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Hideo Kurebayashi
    • 1
  • Shin-Ichiro Nagatsuka
    • 2
  • Hiroyuki Nemoto
    • 2
  • Hideyo Noguchi
    • 2
  • Yasuo Ohno
    • 1
  1. 1.Division of PharmacologyNational Institute of Health SciencesTokyoJapan
  2. 2.ADME/TOX Research InstituteDaiichi Pure Chemicals Co. LtdIbarakiJapan

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