Metabolism and Disposition of Bisphenol A in Female Rats

Toxicology and Applied Pharmacology 168, 225–234 (2000) doi:10.1006/taap.2000.9051, available online at http://www.idealibrary.com on

Metabolism and Disposition of Bisphenol A in Female Rats Rodney W. Snyder, Susan C. Maness, Kevin W. Gaido, Frank Welsch, Susan C. J. Sumner, and Timothy R. Fennell 1 Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709 Received May 8, 2000; accepted August 24, 2000

Metabolism and Disposition of Bisphenol A in Female Rats. Snyder, R. W., Maness, S. C., Gaido, K. W., Welsch, F., Sumner, S. C. J., and Fennell, T. R. (2000). Toxicol. Appl. Pharmacol. 168, 225–234. Bisphenol A (BPA), which is used in the manufacture of polycarbonates, elicits weak estrogenic activity in in vitro and in vivo test systems. The objectives of this study were to compare the patterns of disposition of radioactivity in adult female F-344 and CD rats after oral administration of 14C BPA (100 mg/kg), to isolate the glucuronide of BPA and to assess its estrogenic activity in vitro, and to evaluate the transfer of radioactivity to pups from lactating dams administered 14C BPA. Over 6 days, F-344 rats excreted more radioactivity in urine than CD rats. The major metabolite in urine was identified as bisphenol A glucuronide (BPA gluc) by incubation with ␤-glucuronidase and 1H and 13C NMR spectroscopy. In lactating CD rats administered 14C BPA (100 mg/kg) by gavage, only a small fraction of the label was found in milk, with 0.95 ⴞ 0.66, 0.63 ⴞ 0.13, and 0.26 ⴞ 0.10 ␮g equiv/ml (mean ⴞ SD) from dams collected 1, 8, and 26 h after dosing, respectively. Radioactivity in pup carcasses indicated exposure in the range of microgram equivalents per kilogram; those values ranged from 44.3 ⴞ 24.4 for pups separated from their lactating dams at 2 h to 78.4 ⴞ 10.9 at 24 h. BPA gluc was the prominent metabolite in milk and plasma. In test systems for activation of in vitro estrogen receptors ␣ and ␤, BPA gluc did not show appreciable efficacy at concentrations up to 0.03 mM, indicating that metabolism via glucuronidation is a detoxication reaction. © 2000 Academic Press

Key Words: bisphenol A; disposition; lactation; rat; Sprague– Dawley; Fischer 344; estrogen receptor.

Recently, much scientific as well as public attention has been focused on chemicals that might be capable of mimicking endogenous hormone action and thus interfering with normal endocrine function. Such chemicals, now summarily designated as endocrine disruptors, are postulated to bind to hormone receptors and mimic hormone action and may have the potential to alter normal hormonal function in humans and wildlife. Suspected effects of the disruption of endocrine func1 To whom correspondence should be addressed at Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, NC 27709. Fax: (919) 558-1300. E-mail: [email protected]

tion may include reduced fertility and increased incidence of cancer in estrogen-responsive tissues (Colborn et al., 1993; Davis et al., 1993). Bisphenol A (BPA), a monomer component of polycarbonate plastics, is used in many consumer products, including lacquers applied as food-can linings and dental composite fillings and sealants (Brotons et al., 1995; Olea et al., 1996), and leaches in small amounts from polycarbonate flasks when autoclaved (Krishnan et al., 1993). In vitro, BPA exhibits weak estrogenic activity, compared with 17␤-estradiol, by binding to and activating estrogen receptors (Krishnan et al., 1993; Gaido et al., 1997). Increased uterine levels of estrogen-responsive proteins and other adverse effects can be seen in vivo after oral administration of high doses of BPA ranging from 50 to 1000 mg/kg (Bond et al., 1980; Morrissey et al., 1987; Atkinson and Roy, 1995; Gould et al., 1998a). In rats, strains differ in sensitivity to many classes of chemical compounds, including estrogens such as BPA. Fischer 344 (F-344) rats are shown to be more sensitive to BPA than Sprague–Dawley (SD) rats in a number of responses (Steinmetz et al., 1997, 1998). Ovariectomized (OVEX) F-344 rats administered 40 – 45 ␮g BPA/day or 1.2–1.5 ␮g 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., 1997). The reproductive tract of F-344 rats may also be more sensitive to BPA than the reproductive tract 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 F-344 rats, but there was no response in OVEX SD rats (Steinmetz et al., 1998). In previous studies with Elias rats, the major metabolite in urine was the glucuronide of BPA, and free BPA was identified in feces (Knaak and Sullivan, 1966). Recent studies on the disposition of 14C BPA conducted in F-344 rats (Pottenger et al., 2000) indicated that orally administered BPA undergoes first-pass metabolism primarily to a glucuronide and that the route of entry significantly affects the fate of the chemical. These observations are consistent with in vitro data showing that in male rat liver microsomes BPA conjugates with glucuronic acid (Yokota et al., 1999). Much speculation and concern have been raised about the

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potential adverse effects of exposure to low doses of estrogenic chemicals in utero and in early postnatal life (Sharpe and Skakkebaek, 1993). The outcomes of some experiments conducted in pregnant mice administered BPA have caused controversy. Investigators have reported effects of BPA such as changes in prostate, preputial gland, and epididymis size and decreased efficiency of sperm production in the male offspring of pregnant dams that were administered BPA for 7 consecutive days (Nagel et al., 1997; Vom Saal et al., 1998). However, others have been unable to reproduce or extend these observations (Cagen et al., 1999). Other preliminary observations revealed that male CD rats exposed to BPA in their diet from conception to 70 days postpartum had dose-dependent decreases in body weights, while seminal vesicle and epididymis weights were increased (Fritz and Lamartiniere, 1999). Pilot experiments suggest that low levels of BPA are present in milk of lactating Sprague–Dawley rats administered low levels of BPA throughout pregnancy and lactation in drinking water (Gould et al., 1998b). Whether exposure of rat conceptuses or pups to BPA transplacentally or through milk might have long-term reproductive consequences is unknown. Concern has also been raised over exposure of babies during a sensitive period of development to low levels of BPA in formula prepared in polycarbonate baby bottles. Multigenerational reproductive studies are used to examine the potential effects of exposure to chemicals throughout various critical phases of development. An understanding of the disposition of chemicals administered to dams and their transfer via the placenta or in milk to the developing offspring may aid in the interpretation of studies in which chemicals are administered over a broad time range in neonatal and postnatal development (Kwon et al., 2000). Three major objectives were addressed in the present study: (1) to compare the patterns of disposition of radioactivity in adult female F-344 and Sprague—Dawley rats after oral bolus administration of 14C BPA and to create a bridge between pharmacokinetic studies conducted with F-344 rats (Pottenger et al., 2000) and studies carried out with Sprague—Dawley rats (Kwon et al., 2000); (2) to isolate and characterize the glucuronide of BPA and to establish whether conjugation results in a loss of estrogenic activity; and (3) to evaluate the transfer of radioactivity to pups upon administration of 14C BPA to lactating dams.

METHODS Chemicals. [Ring- 14C]BPA was synthesized by Wizard Laboratories (West Sacramento, CA; specific activity ⫽ 41 mCi/mmol). Unlabeled BPA was obtained from Aldrich Chemical Co. (Milwaukee, WI), while 35% tetraethyl ammonium hydroxide (TEAH) in water was purchased from Sachem (Austin, TX). Ecolume was obtained from ICN (Costa Mesa, CA), and all other chemicals were from Fisher Scientific (Pittsburgh, PA) or Sigma Chemical Co. (St. Louis, MO), except where noted. ␤-Glucuronidase Type 10-B was purchased from Sigma.

Animals. This study was conducted under federal guidelines for the use and care of laboratory animals (National Research Council, 1996) and was approved by the Institutional Animal Care and Use Committee of the Chemical Industry Institute of Toxicology (CIIT). Animals were housed in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. Female Crl:CD(SD)BR rats (CD) and female CDF(F-344)/CrlBR rats (F-344) were obtained from Charles River Labs. (Raleigh, NC). The animals were approximately 10 weeks old at the time of dosing. Lactating CD dams with litters were purchased from the same source, and the pups were 14 days old when their mothers received a single dose of BPA. All animals were housed in filter-capped cages containing Alpha-Dri contact bedding (Shepherd Specialty Papers, Inc., Kalamazoo, MI). Rats were housed singly, and bedding was changed twice per week. Individual dams were housed with their litters. The room temperature was maintained at approximately 72 ⫾ 7°F, and relative humidity was maintained at approximately 30 –70%. Automatic light controls provided fluorescent lighting for a photoperiod of approximately 12 h. Animals were fed pelleted standard NIH-07 rodent chow (Zeigler Bros., Gardners, PA) ad libitum. Water was filtered through a Hydro Picosystem filter system and was provided ad libitum from water bottles. Animal treatment. The target dose of 100 mg BPA/kg body weight was dissolved in propylene glycol (1 ml/kg). The specific activity of 14C BPA in the dosing solution for the disposition studies in female rats was approximately 0.5 and 1.0 mCi/mmol when given to lactating rats. The individual doses were calculated by differential weighing of the dosing syringe before and after administration of the syringe contents. Immediately following 14C BPA gavage, two groups of four rats from each strain were placed for 144 h into glass metabolism cages for collection of urine and feces on dry ice (collection time intervals were 24 h each). Four lactating female CD rats (pnd 14) were administered 14C BPA and placed into plastic cages for approximately 1 h. The animals were then anesthetized immediately prior to sample collection (ketamine, 40 mg/kg; xylazine, 5 mg/kg) and injected with oxytocin (0.1– 0.2 ml of a 10 U/ml solution, ip) to facilitate milk release. Milk and blood were collected 1 h after BPA dosing. Blood, liver, abdominal and subcutaneous fat, kidneys, lungs, intestines ⫹ contents, and carcass were collected from all dams for analysis of 14 C content. Another four lactating CD rats received 100 mg 14C BPA/kg by gavage and then were returned to their home plastic cages for up to 6 h with their pups (10 pups per dam). Groups of four pups were removed 2 and 4 h later, and blood was collected by cardiac puncture under CO 2 anesthesia. The two remaining pups were removed 6 h following dosing, and blood was collected by cardiac puncture under CO 2. Two additional lactating female CD rats were administered 14C BPA and placed into plastic cages for 24 h with their pups (10 pups per dam). At 24 h following dosing, five pups were removed, and blood was collected by cardiac puncture under CO 2. The other five pups were killed, and no tissues were collected. Carcasses from all the other pups were analyzed for radioactivity content. At 8 and 26 h following administration of BPA, the dams were anesthetized as described above, and oxytocin was administered. Milk, blood, and tissues were collected from four rats at 8 h after dosing and from two rats at 26 h after dosing. Blood was centrifuged, and plasma was separated. Analysis of radioactivity. Aliquots of urine, milk, and plasma were weighed in duplicate, and scintillation fluid was added directly to the sample prior to counting in a Packard Tri-Carb 2200CA scintillation counter (Packard, Meriden, CT). Feces were softened by adding 1.0% (v/v) Triton X-100 to the total amount collected. Once softened, weighed aliquots were digested with 25% (w/w) TEAH in methanol. The digests were decolorized with 200 ␮l of concentrated HCl and 500 ␮l of 30% (v/v) H 2O 2. Liquid scintillation fluid (10 ml) was added, and radioactivity was determined after chemiluminescence ceased. Aliquots of blood, tissues, and carcasses were dissolved in 35% (v/v) aqueous TEAH, decolorized, mixed with scintillant, and counted on the scintillation counter after chemiluminescence ceased. HPLC analysis. Analysis of plasma, milk, and excreta samples was conducted by HPLC on a Hewlett–Packard 1100 LC Chemstation HPLC System consisting of a binary pump with diode array detector and thermostatted

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DISPOSITION OF BISPHENOL A IN FEMALE RATS autosampler. Elution of radioactivity was monitored with a Packard 525TR Flow Scintillation Analyzer. The HPLC effluent was mixed with Flo-Scint II (Packard) at a flow rate of 4.0 ml/min. Samples of urine and solvent extracts of feces were analyzed using a Beckman Ultrasphere ODS column (C18, 4.6 ⫻ 250 mm, 5 ␮m, Beckman Coulter, Inc., Fullerton, CA) with Ultrasphere All-Guard Cartridge (C18, 7.5 mm ⫻ 4.6 mm, 5 ␮m). Elution was conducted with a 20-min linear gradient from 5–95% acetonitrile in water at a flow rate of 1.0 ml/min (System A). For analysis of plasma, milk, and ␤-glucuronidase incubates, samples were chromatographed on a Supelcosil LC-Hisep shielded hydrophobic phase column (4.6 ⫻ 250 mm, 5 ␮m, Supelco, Bellefonte, PA) and Hisep guard cartridge (4.0 mm ID). This column enables the direct injection of proteincontaining samples without prior cleanup. Elution was conducted with 15% acetonitrile in 180 mM ammonium acetate for 5 min followed by a 15-min linear gradient to 60% acetonitrile at a flow rate of 1.0 ml/min (System B). Metabolite isolation. Metabolites in urine from the CD and F-344 rats were analyzed by reverse-phase HPLC with radiochemical detection. The major radioactive metabolite peak was isolated by solid-phase extraction (SPE) with LC-18 Supelclean 3-ml reverse-phase SPE tubes (Supelco) and HPLC. Samples of 24-h urine from F-344 rats were pooled, acidified with 10 ␮l formic acid/milliliter, transferred to conditioned LC-18 SPE tubes, and slowly passed through the column by positive pressure. The column was eluted with increasing concentrations of methanol (0, 40, 50, 60, 70, and 100%) in water; eluate fractions were collected, and radioactivity in aliquots was determined by scintillation counting. Fractions with the most radioactivity were analyzed by HPLC using the system described above for urine analysis. Fractions were collected and those containing the metabolite of interest were dried under nitrogen to remove acetonitrile and then lyophilized on a Labconco Freeze Dryer 4.5 (Kansas City, MO). Metabolites in feces were determined by reverse-phase HPLC with radiochemical detection to compare samples from the CD and F-344 rats. Representative samples of 24- and 72-h feces from both F-344 and CD rats were extracted with water followed by an acetonitrile extraction. The major radioactive peak was extracted by SPE with a Supelco LC-18 Supelclean SPE tube and HPLC. The extracts were added to conditioned LC-18 SPE tubes and slowly passed through the column by positive pressure. Elution was conducted with 80% methanol in water. The extracts were dried, reconstituted in acetonitrile, and then analyzed by HPLC. Incubation with ␤-glucuronidase. An aliquot of the major metabolite isolated from urine was incubated with ␤-glucuronidase Type 10-B (200 units/␮l) in sodium acetate buffer (0.2 M, pH 4.57) or with sodium acetate buffer alone at 37°C for approximately 15 h. After incubation, the samples were centrifuged (Eppendorf 5414 Microcentrifuge, Hamburg, Germany) for 1 min; the supernatant was removed and kept on ice until HPLC analysis. NMR analysis. NMR spectra were acquired on a Varian VXR 300 spectrometer (Palo Alto, CA) equipped with a 5-mm 1H/ 19F broadband probe. D 2O (Aldrich, Milwaukee, WI) was used as solvent. 1H NMR spectra were acquired with a 10-␮s pulse width and a 60-s relaxation delay and using 30 K data points and a 4000-Hz sweep width (3.7-s acquisition time). 13C NMR spectra were acquired in the double precision mode with a 9-␮s pulse width and a 30-s relaxation delay and using 30 K data points and a 16,502-Hz sweep width (0.91-s acquisition time). Calculated values of 13C chemical shift were generated using Specinfo (STN International, Columbus, OH). Plating and transfection. HepG2 human hepatoma cells (ATCC, Rockville, MD) were plated in triplicate in 24-well plates (Falcon Plastics, Oxnard, CA) at a density of 10 5 cells/well in complete medium consisting of phenol red-free Eagle’s Minimal Essential Medium (Gibco/BRL, Grand Island, NY) supplemented with 10% resin-stripped fetal bovine serum (Hyclone, Logan, UT), 2% L-glutamine, and 0.1% sodium pyruvate. Cells were incubated overnight at 37°C in a humidified atmosphere of 5% CO 2/air and then transfected following the SuperFect procedure (Qiagen, Valencia, CA) with three plasmids (Gould et al., 1998a): (1) a receptor plasmid encoding either human ER␣ (40 ng/well) or ER␤ (50 ng/well), (2) a 405-ng/well C-3 luc reporter plasmid, which contains an estrogen-responsive human complement factor 3 (C3)

TABLE 1 Disposition of Radioactivity Over 144 hours in Urine, Feces, and Carcass from CD and F-344 Female Rats Administered [U-ring- 14C] BPA (100 mg/kg) by Gavage % of Administered Dose

a b

Sample

CD

F-344

Urine Feces Carcass Total

21 ⫾ 1.8 a 70 ⫾ 9.1 1.4 ⫾ 1.2 93 ⫾ 12

42 ⫾ 4.1 b 50 ⫾ 4.9 b 1.1 ⫾ 0.6 93 ⫾ 9.7

Mean ⫾ SD (n ⫽ 4). Significantly different from CD rats, p ⬍ 0.05.

promoter, and (3) a 10-ng/well pCMV ␤-gal plasmid (transfection control). Transfected cells were then rinsed with phosphate-buffered saline and treated with various concentrations of test chemical or dimethyl sulfoxide (vehicle control, Sigma) in complete medium. After a 24-h incubation, treated cells were rinsed with phosphate-buffered saline and lysed with 65 ␮l of lysing buffer (25 mM Tris–phosphate pH 7.8; 2 mM 1,2-diaminocyclohexaneN,N,N⬘,N⬘-tetraacetic acid; 10% glycerol; 0.5% Triton X-100; 2 mM dithiothreitol). Lysate was divided into two 96-well plates for luciferase and ␤-galactosidase determination. Luciferase assay. Luciferase assay reagent (100 ␮l, Promega, Madison, WI) was added to 20 ␮l of lysate, and luminescence was determined immediately thereafter using a ML3000 microtiter plate luminometer (Dynatech Labs., Chantilly, VA).

␤-Galactosidase assay. Twenty microliters of a 4 mg/ml solution of chlorophenol red–␤-D-galactopyranoside (CPRG; Sigma) and 150 ␮l CPRG buffer (60 mM Na 2HPO 4, 40 mM NaH 2PO 4, 10 mM KCl, 1 mM MgSO 4, 50 mM ␤-mercaptoethanol, pH 7.8) were added to 30 ␮l of lysate. Absorbance at 570 nm was determined over a 30-min period using a V max kinetic microplate reader (Molecular Devices, Menlo Park, CA). BPA metabolism by HepG2 cells. HepG2 cells were plated as described above, and a known amount of 14C BPA was added. Samples were incubated for 24 h and then analyzed by HPLC; elution of radioactivity was monitored as described above. Hydrolysis of BPA gluc by the cells was investigated by analyzing the culture media from the plating and transfection experiment by HPLC and monitoring elution of radioactivity. Statistical analysis. The percentage of dose data were statistically analyzed following ArcSin transformation of the square root of the mean using a two-sample t test in Minitab software (State College, PA). The 95% confidence level (p ⬍ 0.05) was selected to indicate significant differences. The values presented here for estrogen receptor activity represent the means ⫾ SE resulting from three separate experiments with triplicate wells for each treatment concentration. Concentration–response data were analyzed using the sigmoidal dose-response function of the graphical and statistical program Prism (Graph Pad, San Diego, CA).

RESULTS

Disposition of 14C-BPA in female rats. Of the total radioactivity administered, 93% was recovered from both the female CD rats and F-344 rats (Table 1). Most of the label was found in urine and feces (⬎91%) with a small amount in the carcass (approximately 1%). Urinary excretion of 14C was twice as high in F-344 rats compared with CD rats (Table 1 and Fig.

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only observed at 24 h. Also, there were no strain differences in percentage of dose recovered from the carcasses. Representative HPLC chromatograms of urine from CD and F-344 rats using System A are shown in Fig. 2. A peak with the retention time of BPA was identified as a minor component in some of the urine samples. A major peak with a retention time of 7.0 min and a number of small peaks between 3 and 6 min were detected. The major metabolite was isolated from urine by solid-phase extraction and HPLC to confirm its identity. Incubation of this product with buffer alone (Fig. 3A) caused no change in retention time (retention time ⫽ 16.8 min, System B). Incubation with ␤-glucuronidase (Fig. 3B) resulted in the shift of the radioactive peak to the retention time of authentic 14 C-BPA (retention time ⫽ 19.8 min, System B). This observation suggested that the major metabolite was a glucuronide. The 1H NMR spectrum (Fig. 4) of the reconstituted isolated metabolite had signals consistent with the BPA portion of a glucuronide located between 6.8 and 7.4 ppm (ring protons; integration 8) and near 1.8 ppm (CH 3, integration 6). Signals consistent with glucuronide protons were present between 3.6 and 4.1 ppm (2⬘–5⬘, integration 4) and near 5.1 ppm (1⬘,

FIG. 1. Cumulative percentage of dose in urine (A), feces (B), and total urine plus feces (C) versus time in female F-344 and CD rats administered 14C BPA (100 mg/kg) by gavage.

1A), with significant differences in cumulative excretion observed at all time points. F-344 rats excreted 42% of the 14 C-BPA dose in urine by 6 days, whereas CD rats excreted only 21% by this route. When analyzing individual time intervals, only the collections from 0 –24 and 48 –72 h were significantly different between CD and F-344 rats. The feces from CD rats contained 70% of the dose, while 50% of the 14C was excreted in F-344 rats (Fig. 1B). Significant differences in cumulative excretion in feces were observed at 120 and 144 h following dosing. Significant differences in the cumulative percentage of dose excreted in urine and feces combined were

FIG. 2. Typical reverse-phase HPLC chromatograms of urine collected 24 h after dosing from female CD and F-344 rats administered 14C BPA (100 mg/kg) by gavage. Urine was chromatographed on a Beckman Ultrasphere C 18 column as described under Methods for System A.

DISPOSITION OF BISPHENOL A IN FEMALE RATS

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85 ⫾ 3.2% of the 14C label in the urine was found in the BPA gluc peak while 2 ⫾ 1% was associated with the BPA peak. In F-344 rats at 24 h, 81 ⫾ 3.7% of the radioactivity was present in the BPA gluc peak, while 10 ⫾ 6.1% was found in the BPA peak. A similar distribution was observed in 48-, 72-, and 96-h urine samples (Table 2). Additional peaks on the radiochromatograms from 24-h urines were present at 3.0 ⫾ 0.3, 4.4 ⫾ 0.7, and 5.6 ⫾ 0.4 min and overall accounted for about 10% of the 14C-BPA given to CD rats and approximately 5% of that administered to F-344 rats. In both rat strains, free BPA was the major peak found in feces at 24 and 72 h (data not shown) where BPA accounted for 98% of the radioactivity.

FIG. 3. HPLC chromatograms of the major urinary metabolite of BPA incubated with buffer (A) and incubated with ␤-glucuronidase (B). HPLC was conducted on a Supelco Hisep column as described under Methods for System B.

integration 1). A monosubstituted BPA-glucuronide is indicated by a 1:1 integration for the BPA:glucuronide portion of the molecule. The 13C NMR spectrum (Fig. 5) had signals consistent with the BPA portion of a glucuronide located near 33 ppm (2CH 3, broad signal), 45 ppm (C), 120 ppm (C2 and C9 ring carbons), 131 ppm (resolved C3 and C8 ring carbons), 146 and 149 ppm (ring carbons C4 and C7), and 156 and 158 ppm (ring carbons C1 and C10). Additional signals were present at shifts consistent with the glucuronide portion of the molecule located near 75–79 ppm (2⬘–5⬘), 104 ppm (1⬘), and 198 ppm (6⬘). These chemical shifts are similar to those calculated for the BPAglucuronide using Specinfo (Fig. 5, table inset). For comparison of urinary metabolites between strains and at different time points, the metabolite peaks detected on HPLC were quantitated as a percentage of the total radioactivity detected (Table 2). BPA glucuronide (BPA gluc) was the major peak in urine samples from both rat strains (retention time ⫽ 7.0 min); at all collection intervals, both strains showed some free BPA (retention time ⫽ 14.4 min). In CD rats at 24 h,

Disposition of BPA in lactating rats. The disposition of 14C BPA in lactating CD rats and transfer of radioactivity to the pups via milk was investigated following a single gavage dose (100 mg/kg). At 1, 8, and 26 h after dosing, most of the dose (83, 75, and 26%, respectively) in all three groups of dams was found in the intestine and intestinal contents (Table 3). A small fraction of the dose (0.4 – 0.6%) was recovered from the carcass. Among the organs, the second highest fraction of the 14C administered was found in the liver. Similar tissue distributions were seen at 1 and 8 h after dosing. As anticipated, much less (26%) of the 14C label was found in intestine ⫹ contents 26 h after dosing. Throughout the observation times, the second highest 14C levels were consistently found in the liver, where the value reached a peak at 8 h. At all three time points, the concentration of radioactivity in maternal plasma, blood, and milk was ranked plasma ⬎ blood ⬎ milk. The level of radioactivity in milk corresponded to 4.2 ␮M equivalents, or approximately 1.0 ␮g equiv/ml, 1 h after 14C-BPA gavage. That level had declined to 2.8 ␮M equiv (0.6 ␮g equiv/ml) at 8 h and had fallen to 1.1 ␮M equiv (0.3 ␮g equiv/ml) by 26 h. Radioactivity amounting to less than 0.01% of the dose administered to the dam was detected in pup carcasses (Table 3). The quantity of 14C label/kg pup weight tended to increase over the 24 h (Table 4). At 2 h following dosing, the radioactivity corresponded to 44 ␮g equiv/kg pup and increased to 63 ␮g equiv/kg pup at 4 h. Pup 14C content fell slightly to 54 ␮g equiv/kg pup at 6 h but had increased to 78 ␮g equiv/kg pup by 24 h. The source of this 14C was presumably the mother’s milk and not urine or other contaminants. Radioactivity with the retention time of BPA gluc made up the major radioactive peak in plasma 1, 8, and 26 h after dosing (Fig. 6A). Free BPA was detected in plasma of 4 of the 10 animals dosed. The radiochromatogram of milk collected at 1 h after gavage is shown in Fig. 6B. The major radioactive peak in milk was BPA gluc. Free BPA was detected in the milk of two animals. This free BPA found in both milk and plasma was not likely derived from the hydrolysis of BPA gluc since the biological materials were stored frozen and maintained in the autosampler at 4°C. However, it cannot be excluded that ␤-glu-

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FIG. 4.

1

H NMR (300 MHz) spectrum of the major urinary metabolite in D 2O.

curonidases in plasma and milk may have hydrolyzed the glucuronide to its parent compound. Comparative ER␣ and ER␤ Activity of BPA and BPA Glucuronide. The estrogen receptor (ER) binding activities of 17-␤ estradiol, BPA, and BPA gluc were compared in HepG2 cells cotransfected with either ER␣ or ER␤ receptor plasmid along with the estrogen-responsive reporter plasmid C3-Luc (Fig. 7). BPA was a complete agonist with both ER␣ and ER␤. The EC50 values for ER␣ induction of luciferase activity by estradiol and BPA were 1.9 ⫻ 10 ⫺ 9 and 6.4 ⫻ 10 ⫺ 7 M, respectively and 1.0 ⫻ 10 ⫺ 8 and 8.9 ⫻ 10 ⫺ 7 M, respectively,

FIG. 5.

13

for ER␤. In contrast to results obtained with BPA, BPAglucuronide at the highest concentration tested (3 ⫻ 10 ⫺ 5 M) induced minimal activity on either ER␣ or ER␤ activation. The ability of HepG2 cells to metabolize BPA and BPA gluc was determined. Metabolism was minimal for BPA; over 95% of the radioactivity detected remained as BPA (retention time: 20.5 min). Only approximately 0.3% of the radioactivity was found in the BPA gluc peak (retention time: 16.8), and approximately 3% was found in an unknown peak with a retention time of 23.4 min (not shown). No hydrolysis of the BPA gluc was detectable upon incubation with HepG2 cells (not shown).

C NMR (75 MHz) spectrum of the major urinary metabolite in D 2O.

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TABLE 2 Metabolite Peak Distribution in Urine from Female F-344 or CD Rats Administered 14C BPA (100 mg/kg) Urinary metabolites as percentage of total peak area of CD Peak

Retention time (Name)

24 h a

48 h

72 h

96 h

1 2 3 4 5

3.0 ⫾ 0.3 min 4.4 ⫾ 0.7 min 5.6 ⫾ 0.4 min 7.6 ⫾ 1.2 min (BPA glucuronide) 14.4 ⫾ 0.1 min (BPA)

1.5 ⫾ 0.2 b 4.5 ⫾ 1.6 3.8 ⫾ 1.0 85 ⫾ 3.2 2.2 ⫾ 1.4

0.4 ⫾ 0.5 2.3 ⫾ 1.2 2.5 ⫾ 1.3 85 ⫾ 6.3 4.6 ⫾ 5.8

0.16 ⫾ 0.3 2.8 ⫾ 0.7 3.0 ⫾ 0.8 88 ⫾ 1.3 2.5 ⫾ 1.3

ND c 2.6 ⫾ 2.0 1.9 ⫾ 0.6 86 ⫾ 6.0 2.7 ⫾ 0.7

Urinary metabolites as percentage of the total peak area of F-344 1 2 3 4 5

3.0 ⫾ 0.3 min 4.4 ⫾ 0.7 min 5.6 ⫾ 0.4 min 7.6 ⫾ 1.2 min (BPA glucuronide) 14.4 ⫾ 0.1 min (BPA)

0.6 ⫾ 0.6 2.6 ⫾ 0.5 2.6 ⫾ 0.5 81 ⫾ 3.7 10 ⫾ 6.1

0.2 ⫾ 0.2 1.7 ⫾ 1.1 1.5 ⫾ 9.2 87 ⫾ 5.1 8.1 ⫾ 5.7

ND 1.8 ⫾ 1.0 1.2 ⫾ 0.3 88 ⫾ 4.2 8.0 ⫾ 3.7

ND 1.9 ⫾ 0.8 0.8 ⫾ 0.6 89 ⫾ 2.6 5.8 ⫾ 1.5

a

Hours after dose. Mean ⫾ SD. c ND ⫽ no peaks detected. b

DISCUSSION

As expected, BPA gluc was the major urinary metabolite of C-BPA given by gavage. Free BPA and other metabolites were detected but at levels much lower than those of the BPA gluc. In addition, free BPA accounted for most of radioactivity recovered in feces, and other metabolites account for no more than 2% of the total 14C label detected. These observations are consistent with results reported by Pottenger et al. (2000). Since free BPA is the major component detected in feces, one possibility is that the radiolabel may not have been absorbed and passed through the gut unchanged into the feces. Another possibility is that the glucuronide may be transported into the intestine via bile and hydrolyzed, resulting in the excretion of free BPA. Pottenger et al. (2000) proposed either a binding to plasma proteins or an enterohepatic circulation of 14C-BPAderived radioactivity to account for the longer elimination phase of plasma radioactivity compared to parent compound. Strain differences were found in the disposition of radioactivity in urine and feces of female rats administered BPA. In F-344 rats, more of the total 14C dose was excreted in urine compared with CD rats. The metabolite profiles detected in urine from the two strains were similar, and the distribution of radioactivity among the peaks differed mainly in the amount of free BPA. In F-344 rats, more free BPA may be reaching the kidney, or additional hydrolysis of conjugates in urine may result in the additional amount of free BPA. In feces, however, free BPA was the only peak detected in both strains. These differences in excretion and distribution in urine may be attributable to strain differences in absorption or metabolism of BPA. In F-344 rats, approximately 34% of the dose is excreted in urine as BPA gluc, compared with 18% of the dose in CD 14

rats. Based on the total amount of BPA gluc in urine, F-344 rats exhibited an increased capacity for glucuronidation. The UDP– glucuronyltransferase activity in male Sprague–Dawley rats was higher than that observed in Long Evans, Wistar King, and Donryu rats for androsterone and testosterone. Activities with bilirubin, p-nitrophenol, or phenolphthalein as substrate were similar among the strains (Matsui et al., 1979). Data to enable direct comparison of glucuronyl transferase activity in F-344 and CD female rats to our knowledge are not available. As previously reported (Pottenger et al., 2000), BPA gluc is the major metabolite of 14C-BPA in plasma after oral dosing. In the present study, bolus gavage of BPA resulted in BPA gluc as the major 14C-labeled product in plasma. Since BPA gluc is thought to be biologically inactive, rapid conversion to the BPA gluc on oral administration of BPA results in low bioavailability of free BPA, which is thought to be the active form. Pottenger et al. (2000) also showed that there were differences in plasma radioactivity profiles between oral, intraperitoneal, and subcutaneous administration. Parent BPA was the major metabolite in plasma following intraperitoneal administration, and following subcutaneous dosing BPA gluc was the major peak, but the profile was very different from oral dosing. This makes it difficult to compare the results of this study conducted with relatively high doses, 100 mg/kg/day by gavage, with those of Steinmetz et al. (1997), who administered approximately 40 or 300 ␮g BPA/day by silastic implant to OVEX rats. The data presented do not distinguish between a difference in susceptibility to the same internal dose of active estrogenic chemical and differences in internal dose. However, in a recent publication, Long et al. (2000) described further investigations of strain differences of BPA effects on rat vaginal

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TABLE 3 Radioactivity in Milk, Blood, Plasma, and Tissues and in Offspring Carcasses following Administration of 100 mg/kg 14C BPA to CD Rats Dams on Postnatal Day 14 Radioactivity recovered in tissues and body fluids at: 1 h a,b

Dam Milk g Blood g Plasma g Tissues Carcass Liver Intestine ⫹ contents Kidney Lung Subcutaneous fat Abdominal fat Pups 2-h carcass 4-h carcass 6-h carcass 24-h carcass Total

8 hc

24 h d

% Dose e

␮M e

% Dose e

␮M e

% Dose f

␮M f

0.0031 ⫾ 0.0022 h 0.0059 ⫾ 0.0040 0.0106 ⫾ 0.0100

4.2 ⫾ 2.9 h 7.7 ⫾ 4.9 13.8 ⫾ 12.5

0.0020 ⫾ 0.0005 0.0056 ⫾ 0.0025 0.0104 ⫾ 0.0033

2.75 ⫾ 0.55 7.55 ⫾ 2.82 14.1 ⫾ 3.6

0.0008 0.008 0.0015

1.1 1.17 2.13

0.64 ⫾ 0.33 0.38 ⫾ 0.13 83 ⫾ 59 0.019 ⫾ 0.011 0.005 ⫾ 0.003 0.0024 ⫾ 0.0014 0.0018 ⫾ 0.0010

3.6 ⫾ 1.9 28 ⫾ 9 2242 ⫾ 275 9.5 ⫾ 5.3 5.3 ⫾ 3.7 3.21 ⫾ 1.73 2.31 ⫾ 1.18

0.59 ⫾ 0.14 0.74 ⫾ 0.08 75 ⫾ 7.9 0.021 ⫾ 0.004 0.007 ⫾ 0.002 0.0023 ⫾ 0.0001 0.0037 ⫾ 0.0039

3.05 ⫾ 0.69 62 ⫾ 15 1967 ⫾ 235 9.21 ⫾ 1.65 5.97 ⫾ 0.74 3.11 ⫾ 0.36 4.78 ⫾ 4.68

0.40 0.1438 26 0.0057 0.0011 0.0003 0.0003

2.04 12.7 687 2.74 0.96 0.44 0.39

— — — — 84 ⫾ 6.2

— — — —

0.0049 ⫾ 0.0028 k 0.0070 ⫾ 0.0031 k 0.0061 ⫾ 0.0029 l — 77 ⫾ 7.9

0.0002 ⫾ 0.0001 k 0.0003 ⫾ 0.0001 k 0.0002 ⫾ 0.0001 l —

— — — 0.0082 m 27

— — — 0.003 m

a

Hours after dosing. Dose administered ⫽ 133 ⫾ 4.4 ␮Ci, pups removed at time of dosing. c Dose administered ⫽ 137 ⫾ 10.2 ␮Ci. d Dose administered ⫽ 145 ⫾ 13.1 ␮Ci. e Mean ⫾ SD (n ⫽ 4). f Mean (n ⫽ 2). g % dose/g. h Mean ⫾ SD (n ⫽ 3). i % dose/g tissue. j No data collected. k Data for individual pups (n ⫽ 16). l Data for individual pups (n ⫽ 8). m Data for individual pups (n ⫽ 10). b

epithelium. Intraperitonal injection of BPA to F-344 and SD rats caused an increase in DNA synthesis in the vaginal epithelium of F-344 rats with an ED50 of 37.5 mg/kg body weight, whereas no effect was found in SD rats at any dose. The elimination of total radioactivity from plasma following an iv dose of 2.9 ␮g 3H BPA/kg was similar in both strains. The authors suggest that intermediate effects, rather than metabolic clearance and early events that lead to the proliferative response, may be involved in the strain difference. Further investigation of the comparative pharmacokinetics of BPA in F-344 and SD rats in the dose range that produces effects in F-344 rats would seem warranted. The glucuronide of BPA was also the major metabolite in milk. Although radioactivity was present as the BPA gluc, ␤-glucuronidases in milk and intestine (Heringova et al., 1965; Gaffney et al., 1983) may hydrolyze the glucuronide, resulting in BPA exposure of pups. A very small fraction of the total

14

C-labeled dose administered to dams was transferred to the pups via milk. Radioactivity in pup carcasses indicated exposure in the micrograms of BPA equivalents per kilogram range. Urine contamination of pups was not controlled for; however, based on the concentration of radioactivity in milk at 8 h (0.63 ␮g equiv/ml milk) and assuming that a 14-day-old pup weighs about 35 g and consumes 5 ml of milk per day (Grigor and Thompson, 1987), an exposure to approximately 3.15 ␮g equiv/day would occur per pup. At 24 h, 2.7 ␮g equiv were determined to be present per pup (Table 4). Therefore, the assumption that 14C intake via milk contributed the radioactivity found in the pup carcass appears reasonable. Since the glucuronide may be hydrolyzed in milk or in the pup’s intestine, the pup may conceivably be exposed to very low levels of free BPA. The BPA gluc essentially failed to activate the HEPG2 cell assay for either ER␣ or ER␤. Either the glucuronide of BPA

233

DISPOSITION OF BISPHENOL A IN FEMALE RATS

TABLE 4 Radioactivity in Pup Carcasses at 2, 4, 6, and 24 h after Dosing Dams with 14C BPA (100 mg/kg) Radioactivity in pups Time of separation (h)

␮g Radioactivity/kg pup

␮g equivalents/total pup

2a 4 6 24

44 ⫾ 24 63 ⫾ 24 54 ⫾ 24 78 ⫾ 11

1.5 ⫾ 0.8 b 2.2 ⫾ 0.9 b 1.9 ⫾ 0.9 c 2.7 ⫾ 0.4 d

a

Postdose. Mean ⫾ SD (n ⫽ 16). c mean ⫾ SD (n ⫽ 8). d mean ⫾ SD (n ⫽ 10). b

cannot interact with the receptor or is excluded from entry into the cell to reach the receptor. Furthermore, these cells did not hydrolyze the BPA gluc to the parent compound, BPA, which exhibits weak estrogenic activity in HEPG2 cells in vitro and weak estrogenicity in vivo. The lack of activity of a tamoxifen

FIG. 7. Activity of 17␤-estradiol (E2), BPA, and BPA gluc with estrogen receptor ␣ (ER␣) and ER␤. HEPG2 cells were transiently transfected with expression plasmids for ER␣ (upper panel) or ER␤ (lower panel) plus C3 luciferase reporter (C3-Luc) and a constitutively active ␤-galactosidase expression plasmid (transfection control). Cells were treated with increasing concentrations of E2 or BPA or BPA-glucuronide. Following a 24-h incubation, cultures were assayed for both luciferase and ␤-galactosidase activity. Luciferase activity was normalized to ␤-galactosidase activity. Values represent the means ⫾ SE of three separate experiments and are presented as percentage response with 100% activity defined as the activity achieved with 3 ⫻ 10 ⫺ 8 M (ER␣) or 3 ⫻ 10 ⫺ 7 M (ER␤) E2.

glucuronide metabolite has been demonstrated in a similar manner (McCague et al., 1990). The extensive conversion of BPA to its glucuronide in vivo may explain the lack of activity observed in some studies in vivo. ACKNOWLEDGMENTS The authors thank Timothy Moore and Donald Stedman for assistance with milk collection, Paul Ross, Carol Bobbitt, and the staff of the CIIT Animal Care Unit, and Dr. Barbara Kuyper for editorial review. FIG. 6. Typical HPLC chromatograms for plasma (A) and milk (B) collected 1 h after dosing rats with 14C BPA. HPLC analysis was conducted on a Supelco Hisep column with elution monitored by detection of radioactivity as described under Methods for System B. Retention time of BPA was 17.6 min.

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