Disposition of a Low Dose of 14C-Bisphenol A in Male Rats and Its Main Biliary Excretion as BPA Glucuronide

Hideo Kurebayashi1, Hiroshi Betsui and Yasuo Ohno

Division of Pharmacology, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya, Tokyo 158-8501, Japan

Received November 14, 2002; accepted January 7, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bisphenol A (BPA) is a weak xenoestrogen mass-produced with potential human exposure. The disposition of bisphenol A in male Fischer-344 (F344) rats dosed orally (100 or 0.10 mg/kg) or intravenously (0.10 mg/kg) was determined. Smaller amounts of the dose appeared in the urine. The main excretion route was feces in rats irrespective of dose and administration route. The biliary excretion during 6 h was 58–66% after iv dosing and 45–50% after oral dosing at 0.10 mg 14C-BPA/kg. Toxicokinetic parameters obtained from 14C-BPA-derived radioactivity in blood were the terminal elimination half-life, t1/2ß = 39.5 h, and total body clearance, CLtot = 0.52 l/h/kg after iv dosing of 0.10 mg 14C-BPA/kg to male rats. The blood concentration reached its maximum of 5.5 ng-eq/ml at 0.38 h after oral dose. AUC(0–6 h), AUC(0–48 h), and AUCinf of 14C-BPA-derived radioactivity, were 34, 118, and 192 ng-eqh/ml for the iv dose and 18, 102, and 185 ng-eqh/ml for the oral dose, respectively. The oral bioavailability of F(0–6 h), F(0–48 h), and Finf were 0.54, 0.86, and 0.97, respectively. The 14C-BPA-derived radioactivity was strongly bound to plasma protein (free fraction, fu = 0.046) and preferentially distributed to the plasma with a blood/plasma ratio of 0.67. From the bile of male rats orally dosed at 100 mg/kg, we have isolated and characterized BPA glucuronide (BPA-gluc) by ESI/MS, 1H and 13C NMR spectroscopy. HPLC analysis showed that BPA-gluc was the predominant metabolite in bile and urine. Unchanged BPA was mostly detected in feces. These results suggest that BPA is mainly metabolized to BPA-gluc and excreted into feces through the bile and subject to enterohepatic circulation in rats irrespective of dose and administration route.

Key Words: bisphenol A; xenoestrogens; absorption; excretion; biliary metabolite; BPA glucuronide; enterohepatic circulation; rats.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, considerable attention has been focused on a wide variety of endocrine disrupting chemicals in the environment that have estrogenic activity, variously referred to as environmental estrogens, xenobiotic estrogens, or xenoestrogens (Colborn et al., 1993Go; Davis et al., 1993Go).

Bisphenol A (4,4'-isopropylidene-2-diphenol, BPA) is a monomer for the synthesis of polycarbonate plastics and epoxy resins. Because of the use of BPA in the production of materials used in contact with food and potable water (Brotons et al., 1995Go) as well as sealant and restorative materials used in dentistry (Olea et al., 1996Go), there could be potential for oral exposure of humans to trace amounts of BPA.

Generally, BPA has shown a low order of toxicity in extensive studies of its potential to produce adverse effects (BIBRA, 1989Go). Although it has been known for several decades that BPA produced weak responses in uterotropic assays (Bitman and Cecil, 1970Go; Dodds and Lawson, 1936Go), there has recently been renewed attention to the estrogenic potential of BPA. Specifically, Krishnan et al.(1993)Go found that BPA leaching from autoclaved polycarbonate flasks was confounding studies to determine if S cerevisiae produced estrogens. More recently, Gaido and coworkers (1997)Go confirmed the weak estrogenicity of BPA in vitro, showing BPA to be approximately 15,000 times less potent than 17ß-estradiol, and Kuiper et al.(1997)Go demonstrated that BPA could interact with both the {alpha}- and ß-estrogen receptors. In addition, BPA was fed to pregnant mice at low doses (2 or 20 µg/kg/day) and the exposure of male mouse fetuses to either dose of BPA significantly increased their adult prostate weight relative to control males (Nagel et al., 1997Go; vom Saal et al., 1998Go).

In rats, there were strain differences in sensitivity to many classes of chemical compounds, including estrogens such as BPA. F344 rats were shown to be more sensitive to BPA than Sprague-Dawley (SD) rats. Ovariectomized (OVEX) F344 rats administered 40–45 µg BPA/day or 1.2–1.5 µg estradiol/day subcutaneously from silastic capsule implants responded with increased prolactin release and prolactin regulating factor activity; however, at equal doses no effect was seen in OVEX SD rats (Steinmetz et al., 1997Go). The reproductive tract of F344 rats may also be more sensitive to BPA than that of SD rats. Continuous subcutaneous delivery of BPA from silastic capsules at doses of 0.3 mg/kg/day resulted in increased uterine wet weight and cellular alterations in OVEX F344 rats, but there was no response in OVEX SD rats (Steinmetz et al., 1998Go).

The sensitivity of target tissues for estrogens and estrogenic chemicals may depend on the dose levels of the specific chemicals as well as routes of administration. Further, risk estimation based on results of animal experiments is valid only when metabolism and disposition are recognized, and taken into account.

There are several reports on the biological fate of BPA in rats using relatively high doses of 14C-BPA ranging from 10 to 800 mg/kg. Knaak and Sullivan (1966)Go reported that most radioactivity was recovered in feces of rats after a high dose of 800 mg BPA/kg. These observations are consistent with results reported by Pottenger et al.(2000)Go and Snyder et al.(2000)Go after oral dose of 100 mg/kg of 14C-BPA. The higher fraction excreted via the feces might be likely due to the saturation of the metabolic or excretory mechanisms responsible for the elimination of BPA via the urinary route. The larger fraction found in feces might be a reflection of the differences in the doses. The use of high doses was mainly due to the low specific radioactivity of 14C-BPA used, and the results have a limited value for risk assessment that are near dose in animal experiments. The doses used in these studies were much higher than those used by Nagel et al.(1997)Go and vom Saal et al.(1998)Go in toxicity studies. The pharmacokinetic studies of relatively high dose of BPA have been conducted in rats; however, there was no experimental data on the biliary excretion. We are concerned with the toxicokinetics of the low dose of the human environmental exposure level for endocrine disrupting chemicals.

We have directly shown the biliary excretion and metabolite in rats dosed with BPA and determined the toxicokinetic parameters from blood 14C-BPA–derived radioactivity concentration profiles, following oral and iv administration of 14C-BPA at a low dose of 0.10 mg/kg in male F344 rats, along with elimination of the radioactivity in bile, urine, and feces. Additionally, we have isolated and characterized BPA glucuronide (BPA-gluc) as the main biliary metabolite of BPA from the bile of male rats orally dosed at 100 mg/kg.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
[Ring-14C(U)] radiolabeled bisphenol A (4,4'-isopropylidene-2-diphenol, 2,2-bis-(p-hydroxyphenyl)-2-propane), 14C-BPA with specific radioactivity of 2.62 GBq/mmol and radiochemical purity of more than 99% (TLC) was obtained from NEN Life Science Products (Boston, MA). A stable isotope BPA substituted with 16 deuterium atoms, D16-BPA (99 atom % D) was obtained from C/D/N Isotopes (Québec, Canada). Nonradioactive BPA, liquid scintillation cocktail (Cleasol I) were purchased from Nacalai Tesque Co. (Kyoto, Japan). The tissue solubilizer (Soluene-350) was obtained from Packard Bioscience B.V. (Groningen, The Netherlands). ß-Glucronidase from E.coli. IX-A(Sigma,G-7396) and sulfatase H-1(Sigma,S-9626) from Helix pomatia were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals used in the present study were of reagent grade.

BPA sulfate was synthesized according to the method of Hoiberg and Mumma (1969)Go based on the reaction of BPA and sulfuric acid with dicyclohexylcarbodiimide.

Animals and administration of test articles.
Male and female Fischer 344 (F344/N Slc) rats 8–9 weeks of age were purchased from SLC Japan Co., Ltd. (Shizuoka, Japan). Rats were acclimatized for more than one week before use. Prior to the administration, rats were fed food and water ad libitum without fasting. Each group of three rats was subjected to bolus iv injection or oral administration of BPA at the age of 10 weeks.

A dosing solution with 14C-BPA concentration of 0.10 mg (1.15 MBq)/ml was prepared by dissolving 14C-BPA in 1/15M isotonic phosphate buffer (pH 7.4). The test solution was given to the animals once by gavage (po) or by a bolus intravenous (iv) injection via tail vein at a dose volume of 1.0 ml/kg. Immediately following dosing, two groups of male rats were housed individually in metabolic cages to collect for urine and feces.

After iv or oral administration of 14C-BPA to male F344 rats, another group of three rats was lightly anesthetized with ethyl ether for each time and venous blood samples (ca. 0.1 ml) were serially collected from jugular vein at designated time points (0.25, 0.5, 1, 2, 4, 6, 24, 32, and 48 h) by heparinized syringes.

Additional studies were carried out with male and female F344 rats with exteriorized bile flow. Three rats for each group were anesthetized by ip injection (50 mg/kg) of Nembutal sodium solution (Abbott Laboratories, North Chicago, IL), and the midline incision was made. The bile duct was cannulated with a polyethylene tube PE-10. After surgery, the incision was sutured, 14C-BPA dissolved in buffer was administered orally or by iv via tail vein at low dose of 0.10 mg/ml/kg. The rats were restrained for the collection of bile in Bollman cages, but had access to drinking water containing 0.9% w/v sodium chloride. The bile was collected every 2 h for up to 6 h after dose of 0.10 mg 14C-BPA/kg.

At the high dose of 100 mg/kg, BPA or D16-BPA dissolved in corn oil was orally administered by gavage to the other group of male F344 rats, as BPA was insoluble at 100 mg/ml in 1/15M isotonic phosphate buffer (pH 7.4). The bile was collected over 18 h. The bile was stored at -20°C until analysis by HPLC.

Measurement of radioactivity.
The feces were homogenized with 30 ml methanol by a Polytron homogenizer (Kinematica, Switzerland) and centrifuged at 3000 rpm for 10 min. The supernatant was used for analysis. The blood sample was decolorized by several drops of 30% H2O2 and dissolved by Soluen 350.

Aliquots of the solubilized blood (50–100 µl), plasma (20–100 µl), urine (0.1–0.2 ml), fecal homogenate samples (50 µl), and bile (20 µl) were mixed with 10 ml of liquid scintillator, Cleasol I (Nacalai Tesque Co., Kyoto, Japan) and counted for radioactivity to determine the total concentration with liquid scintillation counter (LSC3500, Aloka, Tokyo, Japan). Counting efficiency was corrected by external standard procedure. The recovery of added BPA from feces by methanol extraction was more than 85% (n = 3).

Toxicokinetic parameters.
Toxicokinetic parameters were determined from the individual blood 14C-BPA–derived radioactivity concentration-time curves from the same dose (0.10 mg/kg) of orally and iv administered 14C-BPA. The area under the blood 14C-BPA–derived radioactivity concentration-time curve, AUC(0–6 h), AUC(0–24 h), and AUC(0–48 h) was calculated by the linear trapezoidal method.

The blood 14C-BPA–derived radioactivity concentration profiles after iv dose were fitted to a two-compartment, first-order elimination model to obtain estimates for AUCinf, half-1ives (t1/2), total body clearance (CLtot), apparent volume of distribution at steady state (Vss), and mean residence time (MRT) using the WinNonlin nonlinear estimation program (Scientific Consulting, Inc., Cary, NC).

Additionally, the blood 14C concentration-time curves after oral dosing were fit to a two-compartment first-order input, first-order elimination model using the nonlinear estimation program WinNonlin to obtain estimates for the absorbance rate (ka), AUCinf, t1/2, CLtot, peak blood concentrations (Cmax), and the time to Cmax (Tmax).

The oral bioavailability F of 14C-BPA–derived radioactivity was determined by comparing AUC following oral and iv administration of 14C-BPA at the same dose level. F = (AUCoral/Doseoral)/(AUCiv/Doseiv).

Determination of blood/plasma distribution ratio and protein binding in plasma.
The partitioning of 14C-BPA–derived radioactivity between blood and plasma, and its plasma protein binding was determined in rats as follows. Briefly, 0.10 mg/kg of 14C-BPA was administered by iv to rats and blood collected 30 min later by cardiac puncture following ether anesthetization. The plasma was immediately separated from the whole blood by centrifugation at 3000 rpm for 20 min, and 100 µl of whole blood was decolorized by 100 µl of 30% H2O2, and incubated with Soluene 350. The parts of them were dissolved by LS cocktail (Cleasol I) to determine blood and plasma distribution (CB/Cp) by liquid scintillation counter (LSC). The remaining plasma was directly applied to Microcon YM-10 ultrafiltration tube (Millipore Corporation, Bedford, MA). The ultrafiltration tube was centrifuged at 15,000 rpm for 15 min, and BPA concentration in the filtrate was determined by LSC as unbound concentration. The free fraction in plasma (fu) was determined as the ratio of unbound concentration in the filtrate to the total plasma concentration.

HPLC instrumentation.
HPLC analyses were performed using a Shimadzu LC-10A (Kyoto, Japan) system with SPD-M10A photodiode array detector and SIL-10A autosampler. Mobile phase A was water:acetonitrile (90:10; v/v) containing 0.2% acetic acid and mobile phase B was acetonitrile. The flow rate was 1.0 ml/min. The detector was set to monitor absorbance at 278 nm, and UV spectral data were collected from 220 to 340 nm at 640-msec intervals. In the gradient-elution program, the solvent composition was held at 100% solvent A for 1 min and then changed linearly to 90% solvent B in 20 min. The analytical column was TSKgel ODS-80Ts (5 µm, 150 x 4.6 mm I.D. Tosoh, Tokyo, Japan) or Fluofix-120N (5 µm, 250 x 4.6 mm I.D. Neos, Kobe, Japan). The column temperature was maintained at 35°C. The concentration of BPA was calculated from the peak areas of different concentrations of BPA. In a case of 0.10 mg 14C-BPA/kg, the radioactivity eluted from the HPLC was collected in scintillation vials and analyzed by liquid scintillation counting. Recovery of the radioactivity from the HPLC was almost complete (>98%) in all samples analyzed. All samples were filtered through a 0.45-µm filter prior to HPLC analysis.

Electrospray ionization/mass spectroscopy (ESI/MS) analysis.
The samples were also subjected to HPLC/negative electrospray ionization/mass spectroscopy (LC/–ESI/MS) analyses with Finnigan MAT LCQ, equipped with a Finnigan electrospray source in the negative ion monitoring mode. The LC system consisted of Hitachi L-7100 pumps (Tokyo, Japan) and a Waters 996 photodiode array detector. Analytes were resolved on similar columns described above with the same solvents. The flow rate was 0.3 ml/min. Nitrogen was used as the nebulizing and drying gas. The interface temperature was 220°C; the spray voltage 4.2 kV. The mass spectrometer acquired spectra between m/z 100 to 800 over a negative ion scan duration of 1 s.

The purified sample was subjected to electrospray ionization/high resolution mass spectroscopy (ESI/HRMS) analyses with AccuTOFMS (JMS-T100LC, JEOL, Tokyo, Japan) using sodium trifluoroacetate as the internal standard.

Isolation of main biliary metabolite on semipreparative HPLC.
The main biliary metabolite was purified on a semipreparative HPLC using the bile of male rats orally dosed with BPA, at 100 mg/kg. The rat bile collected over 18 h was added by two volumes of methanol, vortexed, and subjected to centrifugation at 15,000 rpm for 10 min at 4°C. The supernatant was filtrated through an ultrafree-MC tube (Millipore Corporation, Bedford, MA). The filtrate was subjected to semipreparative HPLC.

The chromatographic system consisted of a Bondapak C18 semi-preparative column (PrePak 25 mm x 10 cm, Waters, Milford, MA, USA) and a mobile phase of MeCN/water, 33:67 (v/v) containing 0.2% (v/v) acetic acid; the flow rate was 3.9 ml/min at room temperature. The column effluent was monitored by SPD-M10A diode-array detector described above. The fractions of the main peak were subjected to semipreparative HPLC several times and collected to confirm no existence of other peaks.

Enzymatic characterization of conjugates.
Bile solution (total, 100 µl, 0.1 M phosphate buffer, pH 6.0) was added to 1000 units ß-glucuronidase from E.coli. IX-A (Sigma, G-7396) or two units sulfatase H-1 (Sigma, S-9626) with 30 mM D-saccharic acid 1,4-lactone. The mixture was incubated for 3 h at 37°C before analysis. These samples were chromatographed as described above.

NMR analysis.
1H-NMR and 13C-NMR spectra were recorded on a JEOL {alpha}-500 spectrometer at 500 MHz and at 125 MHz, respectively, using tetramethylsilane as an internal standard. CD3OD (Aldrich, Milwaukee, WI) was used as solvent.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Disposition of a Low Dose of 14C-BPA (0.1 mg/kg): Excretion Routes
Following a single iv or oral administration of 0.10 mg 14C-BPA/kg to male rats, the urinary excretion of the radioactivity during 24 h was 8.4 ± 1.8% after the iv dose and 6.3 ± 1.1% for the oral dose (Table 1Go). The cumulative excretion in the urine for two days was 12.5 ± 0.9% for iv dosing and 10.1 ± 1.6% for oral dosing. Fecal elimination of 14C-BPA-derived radioactivity represented the major elimination route in male rats. The fecal excretion of the radioactivity during two days was 77.6 ± 1.8% of the administered dose for iv dosing, and 81.6 ± 3.7% for oral dosing. The total radioactivity recovered in the excreta for two days was 90.1 ± 2.7% and 91.8 ± 5.0% for iv and oral dosing, respectively. Over two days, rats excreted more radioactivity in feces than in urine.


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TABLE 1 Excretion of 14C-BPA–Derived Radioactivity in Urine and Feces in Male Rats after Oral and Intravenous Administration of 0.10 mg/kg of 14C-BPA
 
Biliary Excretion
Following a single oral or iv administration of 0.10 mg 14C-BPA/kg to the bile duct-cannulated F344 rats, the biliary excretion amounted to about 45–66% for 6 h (Table 2Go). The mean cumulative biliary excretion of radioactivity in rats during 6 h was 50% for males and 45% for females post-oral dose, and 66% for males and 58% for females post-iv dose, respectively. There was a tendency for the radioactivity to be more rapidly excreted into bile in male rats post-iv dose than females or post-oral dose. The results showed the biliary excretion was a very important excretion route in rats oral or iv dosed at 0.10 mg 14C-BPA/kg.


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TABLE 2 Biliary Excretion (%) of Radioactivity and Ratio (%) of the Main Metabolite BPA-Gluc in Male and Female Rats after Intravenous and Oral Administration of 14C-BPA at a Dose of 100 µg/kg
 
We have analyzed the metabolites of bile samples of F344 rats dosed orally or by iv at 0.10 mg 14C-BPA/kg using HPLC and the authentic standard BPA-gluc that we isolated from the bile of male rats orally dosed at 100 mg BPA/kg described below. HPLC analysis showed that 14C-BPA-gluc was the predominant metabolite in bile (Table 2Go). The excretion as 14C-BPA-gluc amounted to 84–88% of the biliary radioactivity during 6 h. The biliary excretion of the radioactivity as 14C-BPA-gluc within 6 h was 43% of the total dose for males and 40% for females post-oral dose, and 55% for males and 50% for females post-iv dose, respectively. We have detected BPA-gluc as the main biliary metabolite of rats dosed at 0.10 mg 14C-BPA/kg.

14C-BPA Toxicokinetics in Blood
The concentration-time profiles of 14C-BPA-derived blood radioactivity concentration over 48 h post-iv and oral dose at 0.10 mg 14C-BPA/kg are shown in Figure 1Go. The blood levels of 14C-BPA-derived radioactivity became unquantifiable after 48 h postdosing (limit of quantitation [LOQ] = 1 ng BPA/g blood). Toxicokinetic parameters estimated from each of three blood time-course data for iv and oral administration are summarized in Table 3Go.



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FIG. 1. Blood levels of 14C radioactivity in male F344 rats after iv or oral administration of 14C-BPA at 0.10 mg/kg. Data represent the mean ± SD of three animals. No error bar indicates that the error was included within the symbol.

 

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TABLE 3 Toxicokinetic Parameters of 14C-BPA–Derived Radioactivity in Blood after Intravenous and Oral Administration of 14C-BPA at 0.10 mg/kg to Male Rats
 
Following a single iv administration of 0.10 mg 14C-BPA/kg to rats, in the toxicokinetic parameters obtained from 14C-BPA-derived radioactivity in blood, the elimination half-lives were t1/2{alpha} = 0.59 ± 0.09 h and t1/2ß = 39.5 ± 2.1 h. Total body clearance (CLtot) was 0.52 ± 0.01 l/h/kg. The apparent volume of distribution at steady state (Vss) was 27.0 ± 0.7 l/kg. Mean residence time (MRT) was 51.7 ± 2.4 h.

Following a single oral administration of 0.10 mg 14C-BPA/kg to rats, the 14C-BPA was rapidly absorbed with ka of 3.6/h and Cmax of 5.5 ng-eq/ml with Tmax of 0.38 h. The terminal elimination half-life, t1/2ß of the radioactivity in blood was 44.5 ± 4.1 h.

AUC(0–6 h), AUC(0–48 h), and AUCinf of 14C-BPA-derived radioactivity were 34, 118, and 192 ng-eqh/ml for iv dose and 18, 102, and 185 ng-eqh/ml for oral dose, respectively. The oral bioavailability of F(0–6 h), F(0–48 h), and Finf were 0.54, 0.86, and 0.97, respectively.

During 6 h postdosing, the blood levels of 14C-BPA-derived radioactivity demonstrated marked route-dependent differences. The AUC(0–6 h) (34 ng-eqh/ml) for the iv dose was twofold greater than AUC(0–6 h) (18 ng-eqh/ml) for the oral dose, but the difference gradually decreased after 24 h. In summary, the bioavailability of the radioactivity after oral administration demonstrated a clear dependence on the period of determination.

Blood/Plasma Distribution Ratio and Plasma Protein Binding
The 14C-BPA–derived radioactivity preferentially distributed to the plasma with a blood/plasma distribution ratio of 0.67 ± 0.05 (mean ± SD, n = 11) with the blood radioactivity concentration at 80 ± 29 nM.

At 6–31 ng-eq/ml (27–135 nM) of 14C-BPA–derived radioactivity distributed in the blood, the plasma protein binding was relatively high with 95.4 ± 0.3% of 14C-BPA–derived radioactivity bound to plasma protein (free fraction, fu = 0.046).

Identification of a Main Metabolite of BPA in the Bile of Rats Orally Dosed at 100 mg BPA/kg
We have analyzed the metabolite in the bile of rats orally dosed in corn oil at 100 mg BPA/kg by HPLC. Representative HPLC chromatograms of rat bile collected during 18 h after oral administration of BPA are presented in Figure 2AGo. The nature of the main peak, P-1 (retention time, RT = 13.1 min), was confirmed by incubation of bile samples with glucuronidase, resulting in the disappearance of the peak P-1 and a corresponding increase of the peak P-2 (RT = 14.9 min) in the chromatogram (Fig. 2BGo). The effect of the glucuronidase could be blocked by coincubation of bile samples with the specific inhibitor saccharic acid 1,4-lactone (30 mM). P-2 from the HPLC profile had the same retention time and UV spectra as the standard of BPA when analyzed using separate injections and when employing HPLC co-chromatography. The incubation of bile sample with sulfatase and saccharic acid 1,4-lactone (30 mM) caused no change in the chromatogram (Fig. 2CGo). These analyses by HPLC suggested that the main biliary metabolite was a glucuronide conjugate of BPA (BPA-gluc).



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FIG. 2. HPLC chromatograms of biliary metabolites of male rats orally dosed at 100 mg/kg of BPA. The sample of bile was treated by none (A), ß-glucuronidase (B), or sulfatase (C) for 3 h. HPLC analyses were performed using a Shimadzu SCL-10A system with a photodiode array detector and Fluofix-120N (5 µm, 150 x 4.6 mm I.D.). The details are described in Materials and Methods.

 
Conjugation was also confirmed by the negative ion LC/–ESI/MS (Table 4Go). The analyses by LC-MS yielded the metabolites with retention times of 12.3 and 13.1 min; unmetabolized BPA had a retention time of 14.9 min. The -ESI/MS spectrum of the most prominant metabolite (P-1) showed a base peak at m/z = 403, corresponding to M–H, and a peak at m/z = 227, which corresponds to a loss of 177 (glucuronide = 177 amu; Fig. 3Go, left panel). BPA-gluc fragmented by the loss of the glucuronic acid moiety to give the aglycone – H (mz 227).


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TABLE 4 LC/–ESI/MS Data of Metabolites of BPA in Rats
 


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FIG. 3. Negative electrospray mass spectra of BPA glucuronide (left panel), D16-BPA glucuronide (center), and BPA-sulfate (right panel). LC/–ESI/MS was performed with Finnigan MAT LCQ. The details are described in Materials and Methods.

 
The -ESI/MS spectrum of a peak of D16-BPA metabolite (Fig. 3Go, center) showed a base peak at m/z = 417, corresponding to the M–H [M–1]-, and a peak at m/z = 241, which corresponds to a loss of 177 [M – C6H9O6]-. The -ESI/MS spectrum showed the spectrum of the monoglucuronide conjugate of D16-BPA (Fig. 3Go, center).

The main biliary metabolite P-1 from the HPLC profile was tentatively identified as the BPA-gluc using HPLC/–ESI/MS.

We have purified and isolated the main metabolite P-1 on a semipreparative HPLC from the bile of male rats orally dosed at 100 mg BPA/kg to get one peak on HPLC. The structure of the isolated metabolite was confirmed as the monoglucuronide of BPA by electrospray ionization/high resolution mass spectroscopy (ESI/HRMS) and nuclear magnetic resonance (NMR) analysis (Fig. 4Go).



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FIG. 4. Structure of BPA glucuronide.

 
The negative (–ESI/HRMS) analysis determined the molecular formula C21H23O8 [M–H]-; m/z 403.1400 (calcd. m/z: 403.1393) and the positive (+ESI/HRMS) analysis gave quasi-molecular ions [M+Na]+ and [M+K]+, determined the molecular formula C21H24O8Na; m/z 427.1373 (calcd. m/z: 427.1369) and C21H24O8K; m/z 443.1114 (calcd. m/z: 443.1108).

The 1H NMR spectrum (Table 5Go) of the isolated metabolite in CD3OD had signals consistent with the BPA portion of a glucuronide located between 6.65 and 7.12 ppm (ring protons; integration 8) and near 1.58 ppm (CH3, integration 6). Signals consistent with glucuronide protons were present between 3.47 and 3.76 ppm (2'-5', integration 4) and near 4.9 ppm (1', integration 1). A monosubstituted BPA-glucuronide is indicated by a 1:1 integration for the BPA:glucuronide portion of the molecule.


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TABLE 5 1H and 13C NMR Chemical Shifts of BPA Glucuronide in CD3OD
 
The 13C NMR spectrum (Table 5Go) had signals consistent with the BPA portion of a glucuronide located near 31.6 ppm (2CH3, broad signal), 42.7 ppm (C5), 115.6 and 117.4 ppm (C2 and C9 ring carbons), 128.7 ppm (resolved C3 and C8 ring carbons), 143.1 and 146.6 ppm (ring carbons C4 and C7), and 156.1 and 156.9 ppm (ring carbons C1 and C10). Additional signals were present at shifts consistent with the glucuronide portion of the molecule located near 73.5–77.7 ppm (2'-5'), 102.6 ppm (1'), and 175.6 ppm (6'). These chemical shifts are similar, but different at (6') from those observed for BPA-gluc using D2O (Table 5Go).

A synthesized standard of BPA sulfate conjugate was available for this comparison of mass spectrum and retention time with an authentic standard (Fig. 3Go, right panel, and Table 4Go). The -ESI/MS spectrum showed a base peak at m/z = 307, corresponding to the [M–1]-. This spectrum also showed a peak at m/z = 227 [M – HSO3] which corresponds to a loss of 81 (sulfate = 81 amu).

The nature of BPA sulfate was also confirmed by incubation of the sample with sulfatase, resulting in the disappearance of the peak and a corresponding increase of the BPA peak in the chromatogram. From liberated BPA, we calculated the amount of BPA sulfate.

Metabolism and Routes of Excretion after an Oral Dose of 100 mg BPA/kg
We measured the metabolites in the urine, feces, and bile of male rats orally dosed in corn oil at 100 mg BPA/kg. HPLC analysis of the urine from selected collection intervals (0–24, 24–48 or 48–72 h) allowed the quantitation of BPA and its metabolites (Table 6Go). The identity was based on matching retention times with authentic standard for BPA, BPA-gluc, and BPA-sulfate. In addition, selected urine samples were subjected to LC/–ESI/MS to structurally confirm the presence of BPA-gluc and BPA-sulfate. HPLC analysis of urine sample across 72 h resulted in an estimate of BPA-gluc comprising 6.5% of the dose. HPLC analysis of urine showed little BPA (1.1%) or BPA-sulfate (0.3%) after oral dose of BPA in male F344 rats.


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TABLE 6 Excretion (%) of Metabolites of Bisphenol A in Male Rats after Oral Administration at Dose of 100 mg/kg
 
HPLC analysis of fecal extracts through 72 h post-oral dose of 100 mg BPA/kg allowed quantitation of the amount of BPA metabolites in feces. The majority (61 ± 11%) was recovered as free BPA based on retention time and UV spectra match with an authentic BPA. BPA-gluc or BPA-sulfate was not detected in feces.

HPLC analysis of bile samples found the predominant biliary metabolite was BPA-gluc. Following oral administration of BPA to male F344 rats at 100 mg/kg, the biliary excretion as BPA-gluc amounted to 41 ± 7% of the dose within 18 h. There was little BPA or BPA-sulfate.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bisphenol A, which is used in the manufacture of polycarbonates, elicits weak estrogenic activity in in vitro and in vivo test systems. This study of disposition of BPA in rats showed that the main excretion route was the feces in rats over a wide range of dosages of BPA. The amounts in the excreta of rats after a single oral dose of 0.10 or 100 mg/kg body weight are compared. After iv or oral administration of 0.10 mg 14C-BPA/kg in a buffer to male F344 rats, its principal route of excretion was feces (78–82%). Urinary excretion was small, 10–13% of dose during 48 h. We have detected free BPA as the majority of the methanol extracts from feces of rats orally dosed in corn oil at 100 mg BPA/kg.

Knaak and Sullivan (1966)Go reported that 56% of the orally administered dose was excreted in feces of rats by eight days after a high dose of BPA in propylene glycol at 800 mg/kg. Snyder et al.(2000)Go reported that female CD rats excreted 70% of the radioactivity in feces, while F344 rats excreted 50% by six days after oral dose of 14C-BPA in propylene glycol at 100 mg/kg, and said free BPA accounted for most of radioactivity recovered in feces, and other metabolites account for no more than 2% of the total radioactivity. These observations are consistent with results of rats dosed in corn oil reported by Pottenger et al.(2000)Go. Since free BPA is the major component detected in feces, one possibility is that the radiolabeled compound may not have been absorbed and passed through the gut unchanged into the feces. Alternately, the higher fraction excreted via the feces in these works with relatively high doses of BPA 100–800 mg/kg is likely due to the saturation of metabolic or excretory mechanisms responsible for the elimination via the urinary route. The other possibility is that BPA is absorbed, metabolized in liver, and excreted into the intestine via bile and hydrolyzed, resulting in the fecal excretion of free BPA.

We have isolated the main biliary metabolite as BPA-gluc and detected free BPA as the major metabolite of the feces from rats orally dosed in corn oil at 100 mg BPA/kg. This study has shown the biliary excretion as BPA-gluc was a very important excretion route in rats orally dosed at 100 and 0.10 mg/kg BPA. Further, this study also has shown that the main excretion route was the feces in F344 rats after oral or iv dose of 14C-BPA at 0.10 mg/kg. The biliary excretion amounted to about 45–66% for 6 h in male and female rats orally or by iv dosed at 0.10 mg 14C-BPA/kg. A part of them might be reabsorbed from the guts of the intact rats. These results have shown the fecal and biliary excretions are the main excretion routes of BPA in rats in either route or dose of administration.

These results were consistent with the time-dependent bioavailability of 14C-BPA–derived radioactivity such F(0–6 h), F(0–48 h), and Finf as 0.54, 0.86, and 0.97, respectively, in this study (Table 3Go). The AUC(0–6 h) for the iv dose was twofold greater than AUC(0–6 h) for the oral dose, but the difference decreased after 48 h. Once the initial phase of absorption and elimination was completed, it appeared that 14C-BPA–derived radioactivity demonstrated similar kinetics across these routes of administration.

There is a significant difference in the excretion route of 14C-BPA–derived radioactivity between rats and monkeys (Kurebayashi et al., 2002Go). Following an oral or iv dose of 14C-BPA 0.10 mg/kg to male and female monkeys, the majority (79–86%) of excretion was in urine and the fecal excretion of radioactivity was minimal (1.8–3.1%). The difference in renal clearance may be attributed to their blood/plasma distribution partitioning or their plasma protein binding.

We have shown that there was no significant difference in plasma protein binding of 14C-BPA–derived radioactivity between rats and monkeys. The plasma protein binding of the radioactivity was relatively strong with 95.4 ± 0.3% for rats in this study and 94.5 ± 0.6% for monkeys at a dosage of 0.10 mg 14C-BPA/kg (Kurebayashi et al., 2002Go). Pottenger et al.(2000)Go proposed a binding to plasma proteins or an enterohepatic circulation of 14C-BPA–derived radioactivity to account for the longer elimination phase of plasma radioactivity compared to the parent compound in rats. We have shown that the proposed enterohepatic circulation of 14C-BPA–derived radioactivity should account for the longer elimination phase of blood radioactivity in rats.

Following oral administration of 14C-BPA at 0.10 mg/kg, plasma concentration of the radioactivity was much higher in monkeys than in rats (Kurebayashi et al., 2002Go). This could be due to a higher absorption rate in monkeys. As 14C-BPA was rapidly metabolized to BPA-gluc, the first-pass metabolism and the biliary excretion of BPA-gluc is the likely explanation for the low systemic bioavailability of 14C-BPA post-oral dose in rats. We think that the biliary excretion of BPA-gluc is more important pathway in rats compared with monkeys. This species difference in the biliary excretion of BPA metabolite is a possible explanation. 14C-BPA was metabolized principally to BPA-gluc in monkeys and rats. BPA-gluc has been excreted mainly into the bile in rats via the first-pass effect, but might be lesser in monkeys. One reason for this could be the well-known difference between rats and primates in molecular threshold for biliary excretion of xenobiotics. According to Hirom et al.(1977)Go, for organic anions such as glucuronides, the minimum molecular weight at which biliary elimination becomes appreciable is about 325 ± 50 for rats and about 500 for humans. Therefore BPA-gluc, with a molecular weight of 404, may be eliminated mainly through the kidney in primates, but into bile in rodents. It is suggested that in rats, BPA-gluc undergoes enterohepatic recirculation and this may explain the longer elimination phase of plasma radioactivity, but the quantitative importance of this in man appears to be less than in rodents.

The significant differences in urinary excretion of 14C-BPA after oral dose were observed according to gender and strains of rats. Pottenger et al.(2000)Go proposed that the urinary elimination of 14C-BPA–derived radioactivity was consistently about twofold greater in females than in males, across all doses and routes, representing 13–16% and 21–34% of administered dose in males and females, respectively. Snyder et al.(2000)Go reported that the female F344 rats excreted 42% of the radioactivity in urine by six days, whereas female CD rats excreted 21% by this route after oral administration of 100 mg 14C-BPA/kg to female CD and F344 rats. The feces from CD rats contained 70% of radioactivity, while those from F344 rats had 50%. One possible explanation may be a larger fraction of the dose in the systemic circulation of female rats. The different strain or sex of rats used may explain the difference in the metabolic or excretory mechanisms responsible for the elimination of BPA via the urinary route.

Pottenger et al.(2000)Go also showed the differences in plasma radioactivity profiles between oral, ip, and sc administration at some doses of BPA in corn oil at 10–100 mg/kg in rats. The bioavailability of both parent BPA and plasma radioactivity was clearly route-dependent with oral administration resulting in the lower bioavailability of both. Parent BPA was the major component in plasma following sc and ip administration, but the profile was very different from oral dosing where BPA-gluc was the major peak.

The route dependency in the relative bioavailability likely offers a toxicokinetic explanation for apparent route differences in estrogenic potency (i.e., uterine changes) that have been observed for BPA in female rats. Specifically, recent studies have demonstrated a clear route dependency in the magnitude of the uterotrophic response to BPA, where significantly larger oral doses were required to produce an estrogenic response than those required subcutaneously (Laws and Carey, 1997Go; Twomey, 1998aGo,bGo). The route dependency in parent compound bioavailability explains the route dependency in the uterotrophic response to BPA based on the assumption that free parent is responsible for the estrogenic effects of BPA.

Recently, it has been reported that the BPA-gluc was not a ligand in vitro for either the {alpha}(human) or ß(mouse) estrogen receptor, nor did it have estrogenic activity as measured in a reporter cell assay that measures gene expression from chimeric estrogen receptors transiently transfected into MCF-7 cells (Matthews et al., 2001Go). As previously reported (Pottenger et al., 2000Go; Snyder et al., 2000Go), BPA-gluc is the major metabolite of 14C-BPA in urine and plasma of rats. In the present study, most of BPA resulted in BPA-gluc as the major biliary metabolite in rats. Since BPA-gluc is thought to be biologically inactive, the conversion to BPA-gluc on oral administration of BPA results in low bioavailability of free BPA. This metabolic clearance by first-pass metabolism is likely to be mediated by both intestinal and hepatic enzymes. The contribution of the hepatic UDP-glucuronosyl transferase is supported by the rapid clearance of plasma 14C-BPA and the formation of BPA-gluc after iv dosing at this report. The in vitro formation of BPA-gluc was shown by incubation of BPA with male rat liver microsomes (Yokota et al., 1999Go) and human liver microsomes (Elsby et al., 2001Go). The in situ formation of BPA-gluc was shown by circulation of BPA with male rat liver (Inoue et al., 2001Go).

We conclude that the biliary excretion of BPA-gluc is important in the metabolism of BPA in rats. As 14C-BPA was rapidly absorbed and metabolized to BPA-gluc, this first-pass metabolism and the biliary excretion of BPA-gluc were the likely explanation for the low systemic bioavailability of 14C-BPA post-oral dose in rats. Once BPA-gluc is excreted into bile and enters the intestine, BPA-gluc can be reabsorbed; otherwise, intestinal microflora hydrolyze BPA-gluc, making free BPA sufficiently lipophilic for reabsorption. Repeated enterohepatic recirculation leads to long half-life of BPA in rats at a low level of BPA.


    NOTES
 
1 To whom correspondence should be addressed. Fax: +81-3-3707-6950. E-mail: kurebaya{at}nihs.go.jp. Back


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 DISCUSSION
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