Age and Dose Dependency of the Pharmacokinetics and Metabolism of Bisphenol A in Neonatal Sprague-Dawley Rats Following Oral Administration

J. Y. Domoradzki*,1, C. M. Thornton*, L. H. Pottenger*, S. C. Hansen*, T. L. Card*, D. A. Markham*, M. D. Dryzga*, R. N. Shiotsuka{dagger} and J. M. Waechter, Jr.*

* Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674, and {dagger} Bayer CropScience LP, Stillwell, Kansas 66085

Received July 9, 2003; accepted October 20, 2003

ABSTRACT

Previous studies demonstrated the rapid clearance of bisphenol A (BPA) from blood following oral administration to adult rats with the principal metabolite being BPA-monoglucuronide (BPA-glucuronide). Since the ontogeny of glucuronyl transferases (GT) differs with age, the pharmacokinetics of BPA were studied in neonatal animals. 14C-BPA was administered via gavage at 1 or 10 mg/kg body weight to rats at postnatal day (pnd) 4, pnd 7, pnd 21, or to 11 week old adult rats (10 mg/kg dose only). Blood (neonates and adults) and selected tissues (neonates) were collected at 0.25, 0.75, 1.5, 3, 6, 12, 18, and 24 h postdosing. BPA and BPA-glucuronide in the plasma were quantified by high-performance liquid chromatography; radioactivity in the plasma and tissues was quantified by liquid scintillation spectrometry. The data indicate that neonatal rats at all three ages metabolized BPA to BPA-glucuronide, although an age dependency in the number and concentration of plasma metabolites was observed, consistent with the ontogeny of GT. BPA-glucuronide and BPA concentrations in the plasma were greater in neonates than in adults, except at 24 h postdosing, suggesting an immaturity in the development of hepatic excretory function in neonatal rats. Nevertheless, the half-lives for the elimination of BPA-glucuronide in plasma were more rapid in neonatal animals than in adults, likely due to reduced microflora ß-glucuronidase activity and an absence of enterohepatic recirculation. A dose dependency in the metabolism and pharmacokinetics of BPA administered to neonates was also observed with nearly complete metabolism of BPA to BPA-glucuronide (94–100% of the plasma radioactivity) at a dose of 1 mg/kg. This was in contrast to finding up to 13 different plasma metabolites observed at the 10 mg/kg dose. These data indicate that, from early in neonatal life through pnd 21, there is sufficient GT activity in rats to efficiently metabolize BPA to its nonestrogenic metabolite at low doses.

Key Words: bisphenol A; BPA-glucuronide; pharmacokinetics; rats; neonates.

Bisphenol A (BPA) is a monomer used in the production of polycarbonate plastics, epoxy resins, and other products. Although the weak estrogen-like activity of BPA at higher doses has been known for decades (Dodds and Lawson, 1936Go), a more recent hypothesis that has been advanced is that exposure to extremely low doses of BPA may cause health effects by disrupting endocrine functions (National Toxicology Program, 2001Go). The glucuronidation of BPA, via the UDP-glucuronosyl transferases (GT), has been shown to be the major in vivo metabolic pathway for BPA in the rat (Pottenger et al., 2000Go), nonhuman primates (Kurebayashi et al., 2002Go), and humans (Völkel et al., 2002Go). This route of metabolism results in the production of BPA-monoglucuronide, a metabolite that has been shown to be devoid of estrogenic activity (Matthews et al., 2001Go; Snyder et al., 2000Go).

Since some consumer products composed of BPA-based plastics may come into contact with food, there is some potential for exposure of both adults and children to minute amounts of BPA in the diet. In 2001, the United Kingdom’s Food Standards Agency (FSA) studied the BPA content in canned foods and beverages purchased in the UK (Goodson et al., 2002Go; UK FSA, 2001Go) and estimated an average upper bound BPA level of 21.7 parts per billion (ppb). This results in an estimated BPA dietary intake of 0.36 to 0.38 µg/kg body weight (bw) per day. The European Commission’s Scientific Committee on Food (SCF) has estimated total dietary intake of BPA from all food contact sources to be in the range of 0.48 to 1.6 µg/kg bw/day, which is below the tolerable daily intake set by the SCF of 10 µg/kg bw/day (SCF, 2002Go).

As the ontological pattern of expression of GT activity in fetal/neonatal rat liver varies by the type of substrate (Lucier, 1981Go), fetal and neonatal animals may be more susceptible to BPA toxicity if there is insufficient GT activity relative to the dose administered to effect detoxication. For example, towards the so-called Group I substrates, including 2-aminophenol and 4-nitrophenol, GT activity is detectable about 5 days prior to birth, peaks immediately after birth, and then decreases during the postnatal period until reaching adult levels (Lucier, 1981Go). Activity for Group II substrates, including ß-estradiol and bilirubin, is detectable at about 1 day prior to birth and then increases to adult levels by 1 to 2 weeks after birth. The GT activity toward androsterone appears only in postweanlings but then rapidly increases to adult levels.

UGT2B1 has been identified as the principal isozyme conjugating BPA with glucuronic acid in Wistar rats (Yokota et al., 1999Go). More recently, Matsumoto et al. (2002)Go have studied the ontogeny of GT activity towards BPA in developing Wistar rats and shown an age dependency in liver microsomal activity, with activity increasing as the age of the neonate increased. These data suggest that neonatal rats may be more susceptible to the effects of BPA due to a decreased ability to metabolize BPA to BPA-monoglucuronide. Indeed, studies investigating the toxicity of BPA in neonatal rats using subcutaneous administration at moderate doses showed transient effects in the male reproductive system. Specifically, Fisher et al. (1999)Go reported a transient decrease in cell height of the efferent ducts of the testes in Wistar rats injected subcutaneously with 37 mg BPA/kg/day on days 2–12 of age. Using the identical exposure regimen, Atanassova et al. (2000)Go reported that BPA treatment advanced the time to onset of pubertal spermatogenesis when measured on day 18 of age but not on day 25. In adulthood, these animals showed normal mating and fertility. Nagao et al. (1999)Go observed normal histology and function in the reproductive organs of male and female Sprague-Dawley rats treated subcutaneously on postnatal days 1 to 5 at a dose of BPA more than 100-fold lower than in previously described studies (0.3 mg/kg). These studies support a classical dose-response relationship, rather than effects produced only at very low doses, for the effects of BPA. As these studies all used a route of administration that is not typical of the potential human exposures to BPA, and since the relative bioavailability of BPA is significantly greater following subcutaneous versus oral administration (Pottenger et al., 2000Go), these studies are clearly not relevant for human risk assessment.

Nevertheless, further study of the metabolism and pharmacokinetics of BPA may provide key information on the bioavailability and/or the dose delivered to target tissues that will aid in the interpretation of toxicity observations from different studies. The purpose of our work was to determine the potential effect of age on the pharmacokinetics and metabolism of BPA following its oral administration to Sprague-Dawley rats at three neonatal ages. The hypothesis that we investigated was that the metabolism of BPA in neonates is both age and dose dependent.

MATERIALS AND METHODS

Test materials and standards.
Two lots of 14C-radiolabeled bisphenol A (4,4'-isopropylidene-2-diphenol or 2,2-bis-(p-hydroxyphenyl)-2-propane), uniformly labeled in both phenol rings, were obtained from Moravek Biochemical (Brea, CA). BPA with a specific activity of 56 mCi/mmole was used to prepare the 10 mg BPA/kg bw dosing solution, and BPA with a specific activity of 200 mCi/mmole was used to prepare the 1 mg BPA/kg bw dosing solution. Radiochemical purities of 98.8% and 99.0% (lots 129-032-056 and 233A-261-200, respectively) were confirmed by reversed-phase high-performance liquid chromatography (HPLC) analysis with flow-through radioactivity detection. The concentration of BPA in the dosing solutions was confirmed using the same method of analysis. Nonradiolabeled BPA was supplied by the Dow Chemical Company (Midland, MI) with a purity of 99.7%.

An authentic standard of the monoglucuronide of BPA was synthesized at the Dow Chemical Company or obtained from Ultrafine Chemicals (batch 073/65, purity 96.7%; Manchester, UK). The structure was confirmed as the BPA-glucuronide by liquid chromatography/mass spectrometry (LC/MS) and/or nuclear magnetic resonance spectrometry (NMR).

Animals and husbandry.
Male and female Sprague-Dawley (S-D) rats, 4, 7, and 21 days old at dosing, were purchased from Hilltop Laboratories (Scottdale, PA). Male and female adult rats with in-dwelling cannula in the jugular veins and 11 weeks old at dosing were obtained from the same supplier. Neonates arrived from the supplier already cross-fostered by age and gender with 12–15 neonates per dam. Care and husbandry of animals were in accordance with the Guide for the Care and Use of Laboratory Animals (1996). The study was reviewed and approved by the Institutional Animal Care and Use Committee of the Dow Chemical Company.

Upon arrival in the laboratory, each animal was evaluated by a laboratory veterinarian and found in good health. (Fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.) The cross-fostered pups, one sex per dam, were housed in polypropylene littering tubs with corncob bedding and were placed on study the day after arrival. Adults with in-dwelling jugular vein cannulas were acclimated to Roth cages and dosed the day after receipt to ensure patency of the cannulas. After dosing, adults were housed in glass Roth-style metabolism cages. The animal rooms were maintained on a 12 h light/dark cycle at 21–23°C and 40–70% relative humidity. Dams and adult animals were provided LabDiet® Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO) and municipal water ad libitum, except when feed was withdrawn (except for one pellet) from adults at approximately 14 h before dosing until ~4 h postdosing.

Study design: dose and justification.
Three neonates/age/sex/dose level/sacrifice time were dosed by gavage with radiolabeled 14C-BPA in a corn oil vehicle (Acros Organics, Geel, Belgium) at doses of 1 or 10 mg BPA/kg bw with a dosing volume of 5 g/kg bw. A glass disposable syringe with glass plunger and stainless steel feeding needle (22 gauge, straight, 1 in. long; Popper & Sons, Inc., New Hyde Park, NY) was used for dosing the neonates. Confirmation of the concentration and homogeneity of the dosing solutions was analytically determined using HPLC analysis and liquid scintillation spectrometry (LSS). The 10 mg/kg dose was selected to represent a dose that was several orders of magnitude greater than expected human oral exposures. The 1 mg/kg dose was selected to investigate the potential for dose dependency in the metabolism and pharmacokinetics of BPA in neonatal rats. The ages of the neonates were selected to study the potential impact of early, middle, and late postnatal development on the disposition and metabolism of BPA.

Sample collection and preparation.
Blood samples were collected in capillary tubes from neonatal rats at 0.25, 0.75, 1.5, 3, 6, 12, 18, and 24 h postdosing. For blood collection, the neonates were anesthetized by a subcutaneous injection of a solution of ketamine, xylazine, and heparin (100 mg/kg bw, 32 mg/kg bw, and 2000 units/kg bw, respectively). Deep anesthesia was reached within ~5 min and the heparinized blood specimens were collected from the cranial vena cava. Following blood collection and sacrifice, the esophagus of each animal was inspected for integrity by injecting a solution of blue dye into the mouth. Perforations of the esophagus as a result of dosing were not observed in this study. Plasma was obtained by centrifugation and weighed aliquots were mixed with Aquasol® liquid scintillation fluid (New England Nuclear, Boston, MA) and analyzed for radioactivity by LSS. The remaining plasma from each animal was pooled for each time point, and BPA and BPA-glucuronide were quantified by reversed-phase HPLC. Structural confirmation of BPA and BPA-glucuronide peaks in HPLC profiles was carried out using LC/MS for selected plasma samples. Selected tissues were collected from each animal and analyzed for radioactivity by LSS. Blood from adults was collected via the jugular vein cannula at 0.25, 0.75, 1.5, 3, 6, 12, 18, 24, 48, 72, and 96 h, directly solubilized using Soluene 350 (Packard Instrument Company, Inc., Downers Grove, IL) or tetraethylammonium hydroxide 20% (aqueous), and then analyzed for radioactivity.

Brain, liver, kidneys, gonads, uterus, skin, and the remaining carcass were collected from the neonates at each time of sacrifice to determine relative distribution to the tissues involved in the metabolism and excretion of BPA and those that are known to be sensitive to estrogens. Radioactivity in the carcass was determined for comparison, as a nonestrogen-sensitive tissue, to the other tissues analyzed. The tissues were directly solubilized using Soluene 350 or tetraethylammonium hydroxide 20% (aqueous) and then analyzed for radioactivity by LSS. The same tissues were collected from adults at 96 h postdosing, solubilized, and analyzed by LSS as described above.

Preparation of pooled plasma samples for HPLC radiochemical analysis.
Prior to analysis, individual samples were stored at -80°C. Plasma specimens were thawed, mixed well, and pooled by combining equal volume aliquots. For the animals dosed on pnd 4 with 1 mg BPA/kg bw, the total amount of plasma was pooled due to the limited sample volume. From 20 to 300 µl of pooled plasma were weighed and extracted with ~100 µl acetonitrile (containing 1% acetic acid, v/v) by briefly (~30 s) vortex mixing. Approximately 300 µl of 50/50 acetonitrile/water (containing 1% acetic acid, v/v) was added; the samples were then thoroughly mixed by vortexing for 2 to 3 min and centrifuged for ~10 min (~600 x g). Aliquots of the supernatant were directly injected for analysis by reversed-phase HPLC. Typical HPLC conditions are described in Pottenger et al. (2000)Go. Radioactivity was detected using an online radioactivity detector (LB509 model, Berthold, Bad Wildbad, Germany) and using LSS of eluent fractions collected every 20 s during the run. The disintegrations per minute values produced from LSS analysis of each set of eluent fractions were converted into chromatographic data files using TurboChrom (Boston, MA). Metabolite profiles were produced by manual integration of those LSS-derived, "reconstructed" chromatograms. Plasma extraction efficiency was 91 ± 14% for samples collected from the neonates dosed with 10 mg BPA/kg bw. Extraction efficiencies for neonates dosed with 1 mg BPA/kg bw were 104.6 ± 14.4%, 101.9 ± 8.4%, and 97.0 ± 3.7% for pnd 4, 7, and 21 neonates, respectively. Plasma HPLC system recoveries were 94 ± 10%.

Data analysis and quality assurance.
Means and standard deviations were calculated with Microsoft Excel (Microsoft Corp., Redmond, WA) using full precision mode accuracy. Pharmacokinetic parameters were estimated for blood plasma and tissues, including area under the curve (AUC), Tmax, Cmax, and elimination rate constants and half-lives, using a computer modeling program (PK Solutions, Summit Research Services, Montrose, CO) or WinNonLin noncompartmental analysis (Scientific Consulting, Inc., Cary, NC). Concentrations of BPA-glucuronide were calculated as microgram equivalents (µg eq) of BPA based on the specific activity of the parent compound and were expressed as µg eq BPA (as glucuronide) per gram plasma.

This study was conducted in accordance with EPA Toxic Substance Control Act (EPA-TSCA, 1984) Good Laboratory Practice Standards.

RESULTS

Plasma Radioactivity Time Course
14C-BPA–derived radioactivity in plasma from individual pups (serial sacrifice of three pups/time point) and adults following oral administration of either 1 (pups only) or 10 mg (pups and adults) 14C-BPA/kg bw was somewhat variable from animal to animal (Table 1Go). In general, peak values (Cmax) were reached early, either 0.25 or 0.75 h postdosing. The time course of radioactivity in plasma for male and female animals was similar except for males dosed with 10 mg/kg on pnd 4 at 0.25 h, where the mean was 56.75 µg eq BPA/g plasma, about 4-fold higher from the pnd 4 females at that dose. By 24 h postdosing, mean plasma radioactivity values ranged from about 4- to 100-fold lower for all groups. A trend was noted such that the concentration of radioactivity tended to be higher in pnd 4 pups and then decreased with increasing age. Plasma radioactivity concentrations in adult animals peaked early, were about equivalent between males and females, showed a secondary maximum at about 24 h, and remained quantifiable in male rats at 96 h postdosing.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Concentration of Radioactivity (µg Equivalents BPA/g) in Plasma Following a Single Oral Administration of 1 or 10 mg BPA/kg bw to Postnatal Days 4, 7, or 21 Sprague-Dawley Rats and 10 mg BPA/kg bw to 11-Week-Old Sprague-Dawley Rats
 
Plasma Metabolite Profiles
Representative radiochemical profiles from the HPLC analysis of pooled plasma collected at 0.25 h postdosing from female and male rats dosed on pnd 4, 7, and 21 with 10 mg 14C-BPA/kg bw are shown in Figure 1Go. There were a total of up to 13 radioactive peaks or radioactive "peak areas" (one or more unresolved peaks) detected in plasma of pnd 4 female rats at 0.25 h postdosing (Fig. 1AGo). The retention time for peak 8 matched that of an authentic standard of BPA. Peak 6 was the major metabolite derived from 14C-BPA with a retention time matching that of an authentic standard of BPA-glucuronide. There were no major qualitative differences in the plasma metabolite profiles observed between female and male rats at any age (Fig. 1Go).



View larger version (26K):
[in this window]
[in a new window]
 
FIG. 1. Reconstructed HPLC radiochemical profiles (response in mV) of pooled plasma from female and male Sprague-Dawley rats, postnatal day 4, 7, or 21, at 0.25 h postdosing of a single oral administration of 10 mg 14C-BPA/kg bw. Plasma was pooled from three animals/age group/sex.

 
Except for the pnd 4 animals, BPA-glucuronide consistently was the major component of plasma radioactivity at 0.25 h postdosing. In the youngest animals (pnd 4), the concentration of BPA-glucuronide in plasma was approximately 5-fold less than the concentration of BPA at the earliest sample collection time of 0.25 h. As the age of the animals increased, the concentrations of BPA-glucuronide found in the plasma were higher than the plasma concentrations of BPA and any of the other minor metabolites (Fig. 1Go). This age-dependent increase in plasma BPA-glucuronide concentrations corresponded with a concomitant decrease in the concentrations of BPA found in the plasma. For animals dosed on pnd 7, BPA-glucuronide increased approximately 2.5-fold from the plasma concentrations in pnd 4 animals, whereas the BPA concentrations were from about 10- to 50-fold less for males and females, respectively. At pnd 21, the concentrations of BPA-glucuronide were about equivalent or slightly increased from those in the plasma at pnd 7, but BPA concentrations were from 10- to 18-fold lower.

Representative HPLC profiles of plasma analyzed from female rats that were dosed with either 10 or 1 mg BPA/kg bw on pnd 4 and sacrificed at 0.25, 1.5, and 12 h postdosing are shown in Figure 2Go. Up to 13 peaks or peak areas were observed in the plasma of females dosed with 10 mg BPA/kg bw and sacrificed at 0.25 h postdosing. However, only two peaks were observed at 0.25 h postdosing in the plasma of females dosed with 1 mg BPA/kg bw. The larger of these two peaks observed at the lower dose was BPA-glucuronide and the other was BPA. At 0.25 h postdosing, the plasma concentrations of BPA following administration of a 10 mg BPA/kg dose to pnd 4 female rats were approximately 170 times greater than the plasma concentrations of BPA following the 1 mg/kg dose (10.2 vs. 0.06 µg BPA/g plasma). A comparison of the concentrations of BPA-glucuronide between these same groups shows that plasma concentrations at the 10 mg/kg dose were only three times greater than the concentrations found following the 1 mg BPA/kg bw dose. By 1.5 h after administration of the 10 mg/kg dose (Fig. 2BGo), the plasma metabolite profile was reduced to four peaks and two peak areas. The concentrations of BPA in plasma decreased about 60-fold the concentrations of BPA found at 0.25 h postdosing. Concomitantly, the concentration of BPA-glucuronide increased about 3-fold of that observed at 0.25 h postdosing and the concentrations of BPA-glucuronide were approximately 60 times that of the concentrations of BPA. By 12 h postdosing at 10 mg/kg, only one peak was present, BPA-glucuronide, at a concentration of 1.37 µg eq BPA (as glucuronide)/g plasma.



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 2. Reconstructed HPLC radiochemical profiles (response in mV) of pooled plasma from female Sprague-Dawley rats, postnatal day 4, at 0.25, 1.5, and 12 h postdosing of a single oral administration of (A–C) 10 or (D–F) 1 mg 14C-BPA/kg bw. Plasma was pooled from three animals/time point/dose level.

 
By 1.5 h postdosing, a dose of 1 mg/kg bw also resulted in markedly lower plasma BPA concentrations relative to those found at 0.25 h postdosing. By 12 h postdosing, BPA could no longer be detected in the plasma of animals dosed with 1 mg/kg bw (Fig. 2FGo). Generally, similar metabolite profiles were obtained from the analysis of plasma from pnd 4 male rats dosed with either 10 or 1 mg BPA/kg bw (data not shown). The exceptions were at 0.25 h postdosing, following the 10 mg/kg dose, when the concentration of BPA in the plasma from males was 5-fold greater than that found in females, and at 12 h postdosing, when BPA was still detectable in plasma at approximately twice the limit of detection (0.052 µg BPA/g plasma, data not shown).

Concentration-Time Profiles of BPA and BPA-Glucuronide in Plasma
BPA.
The concentration-time profiles of BPA and BPA-glucuronide, determined by HPLC analysis of plasma (pooled by time point, gender, and dose group) for female and male rats dosed with 1 or 10 mg BPA/kg bw on pnd 4, 7, 21, and for adults dosed at 11 weeks of age (10 mg/kg bw, only), are shown in Figure 3Go.



View larger version (20K):
[in this window]
[in a new window]
 
FIG. 3. BPA (squares) and BPA-monoglucuronide (triangles) plasma time courses, expressed as µg BPA/g plasma and µg equivalents (eq) of BPA (as glucuronide)/g plasma, in the plasma of female and male neonatal (postnatal days 4, 7, and 21) and adult Sprague-Dawley rats following a single oral administration of 1 (solid symbols) or 10 (open symbols) mg BPA/kg bw. (A) Females at pnd 4; (B) females at pnd 7; (C) females at pnd 21; (D) females at 11 weeks; (E) males at pnd 4; (F) males at pnd 7; (G) males at pnd 21; and (H) males at 11 weeks. Plasma was pooled from three animals/sex/time point/dose level. The range of the mean limit of detection for groups (sex/age) ranged from 0.014 to 0.048 for animals dosed with 10 mg BPA/kg bw; the range was 0.006 to 0.010 for animals dosed with 1 mg BPA/kg bw. In female adult animals dosed with 10 mg/kg and sacrificed at 96 h postdosing, the concentration of radioactivity in brain and ovaries was nonquantifiable and the concentration in carcass was 0.142 ± 0.168. In male animals dosed with 10 mg/kg, the concentration of radioactivity in brain was nonquantifiable and was 0.874 ± 0.879 and 0.012 ± 0.008 µg eq BPA/g tissues in carcass and testes, respectively.

 
In female neonates dosed with 1 mg BPA/kg bw, BPA was detectable in plasma of pnd 4 and pnd 7 animals at similar concentrations ranging between 0.010 and 0.076 µg BPA/g plasma (range of limit of detection [LOD] was 0.005–0.012 µg BPA/g plasma, Fig. 3A and BGo). In these animals, BPA was no longer detectable in the plasma by 6 h postdosing. In the plasma of pnd 21 female rats dosed with 1 mg BPA/kg bw, BPA was detected at only one time point just at the limit of detection (0.006 µg BPA/g plasma, Fig. 3DGo). Similar concentrations of BPA in plasma were found in male rats dosed with 1 mg BPA/kg bw on pnd 4, 7, and 21 (Fig. 3E–GGo).

Female neonates of all ages dosed with 10 mg BPA/kg bw had higher plasma concentrations of BPA at the earlier times postdosing than adult female rats (Fig. 3A–DGo). However, BPA was rapidly cleared from plasma so that, by 12, 18, and 12 h postdosing, BPA was nondetectable in plasma of female rats dosed on pnd 4, 7, and 21, respectively. Male pups showed a similar pattern, with early time points higher than adult male plasma BPA and nondetectable levels of BPA reached by 24, 24, and 18 h postdosing for pnd 4, 7, and 21, respectively (Fig. 3E–GGo). In adult female rats dosed with 10 mg BPA/kg bw, BPA was only detected at 0.75 h postdosing at a mean concentration of 0.063 µg BPA/g plasma (Fig. 3DGo). In male adult rats dosed with 10 mg/kg, BPA was detectable in the plasma up to 1.5 h postdosing with concentrations ranging from 0.011–0.024 µg BPA/g plasma (Fig. 3HGo).

Values for pharmacokinetic parameters derived from plasma concentration–time profiles of BPA and BPA-glucuronide, including Cmax, t1/2, and area under the curve (AUC), are shown in Table 2Go.


View this table:
[in this window]
[in a new window]
 
TABLE 2 Pharmacokinetic Parameters of BPA and BPA-Monoglucuronide in Plasma Following a Single Oral Administration of 1 or 10 mg BPA/kg bw to Postnatal Days 4, 7, or 21 Sprague-Dawley Rats and 10 mg BPA/kg bw to 11-Week-Old Sprague-Dawley Rats
 
At a dose of 10 mg/kg bw, the maximum concentrations of BPA in plasma (Cmax) decreased as the age of the animals increased (Table 2Go). For pnd 4 rats, BPA concentrations were 48.3 and 10.2 µg BPA/g plasma for males and females, respectively; at pnd 7, the Cmax values were 1.1 and 1.4 µg BPA/g plasma in male and female rats, respectively. By pnd 21, peak BPA concentrations were 0.2 µg BPA/g plasma for both male and female rats. At a dose of 1 mg/kg, the Cmax values of BPA in plasma for animals dosed on pnd 4 or 7 were similar; the Cmax values in male and female rats ranged from 0.03 to 0.08 µg BPA/g plasma. BPA in the plasma at pnd 21 was detectable at only 3 h postdosing (at the LOD of 0.006 µg BPA/g plasma).

The half-lives for the elimination of BPA from the plasma of males and females dosed with 1 mg BPA/kg bw on pnd 4 and 7 ranged from 7 to 22 h. At a dose of 10 mg BPA/kg bw, the half-lives for the elimination of BPA from plasma could be determined for all ages except adults and were 4–17 h (Table 2Go). Two calculated values for BPA half-lives at 1 mg/kg bw were higher than the others, male pnd 4 and pnd 7, with half-lives of 17 and 22 h, respectively. Given that the data for pups was only collected over a 24-h period and that the plasma BPA concentrations were near the LODs for samples collected at the end of the 24-h period, these half-lives may not be reliable.

The ratios of the peak BPA plasma concentrations at the two doses administered (Cmax 10/Cmax 1) were markedly different at each age of animal studied. This ratio was significantly greater at pnd 4 than at pnd 7, for both male and female animals (1610 and 170 on pnd 4 for male and female rats, respectively; 28 and 18 on pnd 7 for males and females, respectively). The Cmax 10/Cmax 1 could not be calculated for animals dosed on pnd 21 or adults since BPA was not detectable in these animals at a dose of 1 mg/kg bw or no data were available (adults).

The AUC values calculated for BPA in plasma are shown in Table 2Go; they generally decreased with increasing age and were not dose proportionate. The AUCs for BPA in plasma following the 10 mg/kg bw dose divided by the AUCs for BPA in plasma following the 1 mg/kg bw dose (AUC 10/AUC 1) also showed marked differences between animals of different ages (Table 2Go). For animals dosed on pnd 4, the AUC 10/AUC 1 for BPA was 115 and 72 for males and females, respectively. At pnd 7, this ratio was lower, 19 and 17 for males and females, respectively, but could not be calculated for animals dosed on pnd 21 since BPA was not detected in plasma at the lower dose.

BPA-glucuronide.
The peak concentrations of BPA-glucuronide in the plasma of neonatal animals ranged from 9 to almost 22 times higher than in adult animals following administration of 10 mg BPA/kg bw (Table 2Go). At the lower dose of 1 mg BPA/kg bw, the peak concentrations of BPA-glucuronide in the plasma were similar between sexes and at all of the ages studied. However, the half-lives of BPA-glucuronide elimination in plasma were significantly shorter in the older, pnd 21 rats, suggesting an age dependency for the elimination of BPA-glucuronide from plasma.

In neonatal animals receiving a 1 mg/kg dose, differences among the BPA-glucuronide AUCs at different ages were also apparent, with AUCs decreasing as age increased. Peak BPA-glucuronide concentrations were roughly proportionate to those observed at the 10 mg/kg dose, ranging from about 3- to 13-fold less than the Cmax values for 10 mg BPA/kg bw dose. Comparison of the ratios of plasma AUCs for BPA-glucuronide at 10 mg/kg to that at 1 mg/kg (AUC 10/AUC 1) shows a roughly proportionate relationship to dose, with the AUC 10/AUC 1 ranging from 5 to 17.

After a dose of 10 mg/kg, peak BPA-glucuronide concentrations in the plasma of adult female rats occurred at 0.25 h postdosing, followed by a rapid decline and secondary peaks at 18 and 24 h postdosing in females and males, respectively (Fig. 3D and HGo). The half-lives for the elimination of BPA-glucuronide from plasma were 10.8 and 22.5 h in female and male adult rats, respectively.

For neonatal rats at all ages studied, the plasma concentrations of BPA-glucuronide exceeded the concentrations of BPA in plasma except at the earliest sampling time in animals dosed on pnd 4 (Fig. 3A–C and E–GGo). At both doses, BPA-glucuronide was rapidly eliminated from the plasma in an apparent monophasic manner with half-lives for elimination ranging from 4.4 to 9.8 h in females and from 4.4 to 9.1 h in males (Table 2Go). The half-lives for the elimination of BPA-glucuronide (4.4 h) in both male and female neonates dosed on pnd 21 were significantly shorter than the half-lives in younger animals.

Neonatal rats at all of the ages studied were clearly capable of glucuronidating BPA, with the majority of plasma radioactivity, at most times postdosing, identified as BPA-glucuronide (Table 3Go). Calculation of the percentage of total radioactivity AUC demonstrates that, following the administration of 1 mg BPA/kg bw, from 94–100% of the 14C-BPA–derived radioactivity in plasma of female and male pups was BPA-glucuronide. Following a dose of 10 mg BPA/kg bw, 71–97% of total plasma radioactivity AUC was present as BPA-glucuronide; for adults this ratio was essentially 100%.


View this table:
[in this window]
[in a new window]
 
TABLE 3 Percentage of Plasma AUC for Radioactivity That Was BPA-Glucuronidea in Male and Female Sprague-Dawley Rats Administered 1 or 10 mg Bisphenol A/kg bw on Postnatal Day 4, 7, or 21
 
14C-BPA–Derived Radioactivity in Tissues and Plasma
The concentration-time profiles of 14C-BPA–derived radioactivity in selected tissues (brain, carcass, and gonads) from females and males dosed on pnd 4 with 1 or 10 mg/kg bw are shown in Figures 4A–4DGo. At both doses, 14C-BPA–derived radioactivity was distributed to all of the tissues analyzed and increased in a manner roughly proportionate to the dose administered. The concentration of radioactivity in brain was at least 10-fold lower than the other tissues analyzed at both doses at all times postdosing. The concentrations of 14C-BPA–derived radioactivity in ovaries, which were about 20- to 40-fold greater than those found in brain, were 1- to 3-fold less than those concentrations found in carcass except for the three earliest times postdosing at 10 mg/kg. In males, the testes concentrations of 14C-BPA–derived radioactivity were about 3- to 7-fold less than those found in carcass and about 10-fold greater than those found in brain at both dose levels.



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 4. 14C-BPA–derived radioactivity, expressed as µg equivalents (eq) of BPA/g tissue, in tissues (carcass, triangles; brain, squares; and gonads, diamonds) collected from postnatal day 4 Sprague-Dawley rats at 24 h postdosing of a single oral administration of 1 (solid symbols) or 10 (open symbols) mg BPA/kg bw. Each point represents the mean value for three individual tissues analyzed/time point/dose/sex.

 
The concentration-time profiles for 14C-BPA–derived radioactivity in the kidney and liver of female and male neonates and in the uterus were similar to those observed for other tissue, although concentrations from tissue to tissue varied (data not shown). 14C-BPA–derived radioactivity concentrations in adult tissues at 96 h following the administration of 10 mg BPA/kg bw were lower than neonatal tissues concentrations at 24 h postdosing (data not shown). However, since a more rapid elimination of BPA and its metabolite in neonates was observed, as demonstrated in the plasma concentration–time profiles (Figs. 3A–3DGo), the concentration of radioactivity in neonatal tissues would be expected to reach adult levels or lower by 96 h postdosing.

Table 4Go shows the AUC values for 14C-BPA–derived radioactivity for both sexes and dose levels. At 1 mg/kg, the AUC values for ovaries and uterus were lower than for kidney and liver, except at pnd 4. In males dosed with 1 mg/kg, AUC values for testes were always lower than the AUC values for liver and kidneys (Table 4Go). AUC values for 14C-BPA–derived radioactivity in ovaries and uterus were less than plasma AUCs at both dose levels, except for animals dosed on pnd 4 and 7 with 10 mg/kg. AUC values for testes were always less than plasma AUCs at both dose levels.


View this table:
[in this window]
[in a new window]
 
TABLE 4 Half-Lives and Area-under-the-Curve (AUC) of 14C-BPA–Derived Radioactivity in Tissues and Plasma Following Oral Administration of 1 or 10 mg BPA/kg bw to Female and Male Sprague-Dawley Rats on Postnatal Days 4, 7, and 21
 
Half-lives for the elimination of 14C-BPA–derived radioactivity in plasma and tissues are also presented in Table 4Go for female and male rats dosed on pnd 4, 7, or 21 with either 1 or 10 mg BPA/kg bw. In general, half-lives for the elimination of radioactivity in each of the tissues decreased as the age of the animal increased. For example, half-lives of radioactivity in brain tissue for female rat pups dosed with 1 mg BPA/kg were 11.7, 6.7, and 3.6 h for pnd 4, 7, and 21, respectively.

The calculated AUC values for brain tissue were the lowest of all tissues and ranged from 0.1–0.4 and 1.7–4.7 µg-h/g for the 1 and 10 mg/kg dose levels, respectively. AUC values for carcass were the highest of all tissue AUCs and ranged from 8.3–22.2 and 95.2–135.5 µg-h/g for the low and high doses, respectively. Some evidence of dose dependency in the pharmacokinetics of 14C-BPA–derived radioactivity was observed based on a disproportionate increase in certain tissue AUCs as the dose was raised from 1 to 10 mg/kg. The AUC 10/AUC 1 ratio for brain was almost double that expected (17), but only for pnd 21 males and females. Also, the AUC 10/AUC 1 ratios for ovary and uterus were about 1.5- to 3-fold higher than expected for all ages, ranging from 15.3 to 36.2. The AUC 10/AUC 1 ratios for all other tissues were roughly proportionate to the increase in dose, although several ratios tended to be slightly lower than expected.

DISCUSSION

Glucuronidation of BPA represents the major in vivo metabolic pathway for BPA in the adult rat (Pottenger et al., 2000Go; Upmeier et al., 2000Go). Glucuronidation of BPA was catalyzed by an isoform of UDP-glucuronosyltransferase, UGT2B1, in the in vitro studies of Yokota et al. (1999)Go, who studied BPA metabolism in Wistar rat liver microsomes. Matsumoto et al. (2002)Go studied the ontogeny and activity toward BPA of GT in developing Wistar rats and found an age dependency in liver microsomal activity, with activity increasing as the age of the neonate increased.

Consistent with the observations of Matsumoto et al. (2002)Go, the data from this study clearly demonstrated an age dependency in BPA metabolism, likely due to the ontogeny of GT in neonates. Age-related differences were observed in the plasma metabolite profiles as well in the pharmacokinetics of BPA and BPA-glucuronide.

At a dose of 10 mg/kg, an age dependency in BPA metabolism was indicated by the BPA plasma concentrations increasing at least an order of magnitude in the neonatal pnd 4 and 7 rats as compared to the peak plasma concentrations of BPA in adult rats (Table 2Go and Fig. 3Go). This also resulted in higher AUCs for parent compound in neonates (Table 1Go). BPA-glucuronide concentrations in the plasma of all neonates given a dose of 10 mg/kg were also greater than those found in adult animals, resulting in higher AUCs for BPA-glucuronide in neonates (Table 2Go and Fig. 3Go).

Age-related differences in the relative plasma concentrations of BPA to BPA-glucuronide were also observed. In animals dosed on pnd 4, the plasma concentrations of BPA at 0.25 h postdosing were higher than the plasma concentrations of BPA-glucuronide. In animals dosed on pnd 7, BPA-glucuronide concentrations at the first plasma time point were roughly four times greater than the BPA plasma concentrations, and animals dosed on pnd 21 were found to have 80-fold more BPA-glucuronide than BPA in plasma (Fig. 3Go). At a dose of 10 mg/kg, additional evidence of an age dependency in BPA metabolism was a clear decrease in the number of plasma metabolites as neonatal age increased (Fig. 1Go).

An age dependency in the half-lives for the elimination of BPA from the plasma was also observed. BPA in the plasma of adult rats was barely detectable and reached concentrations that were nondetectable by 0.75 h postdosing. Plasma concentrations of BPA in neonates declined to the levels found in adults in 12 h or less but did not reach nondetectable concentrations until 12–24 h postdosing (t1/2: 4.3–17 h; Table 2Go and Fig. 3Go). Age dependency for the elimination of BPA-glucuronide was also observed with more rapid elimination of BPA-glucuronide from the plasma of neonates (t1/2: 4.4–9.8 h) as compared with adult animals (t1/2: 10.8–22.5 h; Table 2Go).

In addition to the longer half-life for the elimination of BPA-glucuronide from adult plasma, a secondary maximum in plasma was observed for this metabolite, suggesting enterohepatic recirculation in adults. This concept is supported by the study of Kurebayashi et al. (2003)Go who reported that about 40% of an oral dose of either 0.1 or 100 mg/kg bw was excreted as BPA-glucuronide in the bile within 6 h postdosing. Examination of the plasma BPA-glucuronide curves for animals dosed with BPA on pnd 7 and 21 shows a tendency toward a secondary maximum that was not as prominent as that observed in adult animals. However, these data could be adequately described using an apparent first-order rate for the elimination of BPA-glucuronide, suggesting a decreased biliary excretion and/or enterohepatic recirculation of BPA may occur in neonates. Indeed, studies by Klaassen (1972Go, 1973)Go reported a diminished biliary excretion for some pharmaceuticals in newborn rats, suggesting an immaturity in the hepatic excretory function. Alternately, neonates may not have completely hydrolyzed BPA-glucuronide in the gastrointestinal tract, resulting in a decreased resorption of BPA. This is supported by the work of Chang et al. (1994)Go, who reported about a 15-fold increase in ß-glucuronidase activity in the intestinal microflora of Fischer 344 rats as their age increased from 7 to 35 days.

An age dependency in the BPA pharmacokinetics was also found in the differences between the tissue radioactivity-concentration AUCs for the ovaries, uterus, and testes, tissues that are estrogen sensitive. Generally, as neonatal age increased to pnd 21, the AUCs decreased to be about an order of magnitude lower than those observed in animals dosed on pnd 4 (Table 4Go). This indicates that the bioavailability of either BPA or BPA-derived metabolites was greater in these tissues in the youngest neonates studied.

The age-dependent metabolism of BPA and the longer half-lives for the elimination of BPA in neonates observed at a dose of 10 mg/kg suggests that younger neonatal rats might be more susceptible to the toxicity of BPA at doses that saturate the available metabolic capacity at those ages. These observations potentially offer an explanation of data from both Fisher et al. (1999)Go and Atanassova et al. (2000)Go. These investigators reported only transient effects in the testes of neonatal rats injected subcutaneously with 37 mg/kg/day BPA on days 2–12 of age, a dose that likely saturated the metabolic capacity of the GT pathway in the earliest days of treatment. As the age of the animals increased so did their capacity to metabolize BPA to the nonestrogenic metabolite BPA-glucuronide. Hence, only transient effects of BPA might be expected in the testes or other estrogen-sensitive tissues.

The present study also demonstrated a dose dependency in BPA pharmacokinetics and metabolism in neonates. Only BPA and its glucuronide were found in plasma at the dose of 1 mg/kg, whereas, at 10 mg/kg, up to 13 additional plasma metabolites were observed at sampling times of less than 3 h. At 1 mg/kg, the percentage of the plasma AUC for radioactivity that was BPA-glucuronide was nearly 100 at all ages; but, it was somewhat lower at 10 mg/kg (Table 3Go). Additional evidence for dose dependency was observed in the ratio of the plasma concentration–time curve AUCs for BPA-glucuronide/BPA (Table 2Go). For animals dosed on pnd 4 or 7 with a dose of 1 mg/kg, the AUC for plasma concentration–time profiles of BPA-glucuronide were up to 96 times greater than the AUC for the plasma BPA concentration–time profiles. By pnd 21, only BPA-glucuronide was detectable in the plasma. For animals dosed on pnd 7 or 21 with a dose of 10 mg/kg, the AUC for plasma concentration–time profiles of BPA-glucuronide were only up to 56 times greater than the AUC for the plasma BPA concentration–time profiles. The greatest difference between the two doses was observed in the younger animals dosed on pnd 4: the ratios were only 3.5 and 7 for males and females, respectively, at the dose of 10 mg/kg, and 45 and 96 for males and females, respectively, at the dose of 1 mg/kg. As the dose of 1 mg/kg was clearly below the level where available GT activity was saturated, the much lower doses of BPA that are typical of environmental exposures would also be within the range of linear pharmacokinetics.

This clear dose dependency in metabolism likely explains the absence of even transient effects of BPA on the reproductive organs seen in the study by Nagao et al. (1999)Go, who used a dose of 0.3 mg/kg/day that is clearly in the range of linear pharmacokinetics for neonates. Dose dependency in BPA metabolism is also consistent with the absence of low-dose effects following the oral administration of BPA to both adult and neonatal Sprague-Dawley rats in multigeneration reproduction studies (Ema et al., 2001Go; Tyl et al., 2002Go). Thus, our study, along with the negative results observed at low doses in well-conducted reproduction studies, supports a classical dose response for the effects of BPA and does not support the hypothesis of low-dose effects.

No marked sex-dependent differences were observed in plasma metabolite profiles or in plasma concentration–time curves for BPA and BPA-glucuronide. The AUC for BPA and BPA-glucuronide were similar for males and females of the same age. However, the AUC for BPA in males dosed on pnd 4 with 10 mg BPA/kg bw was three times that found in females (Table 2Go). This somewhat higher AUC observed in males at this dose for BPA plasma concentration–time profiles was a result of a markedly higher plasma concentration at only the initial sampling time postdosing. Additional support for an absence of sex differences in BPA disposition was the generally similar half-lives for the elimination of 14C-BPA–derived radioactivity in both plasma and tissues in neonates of the same age (Table 4Go).

Generally, BPA or its metabolites did not appear to be preferentially distributed to specific tissues in neonatal animals. The 14C-BPA–derived radioactivity was distributed to all of the tissues collected and brain concentrations were consistently about 10-fold lower than the concentrations of other tissues. Additionally, testes concentrations of 14C-BPA–derived radioactivity were about 10-fold less than those found in carcass, but greater than those found in the brain. For females dosed on pnd 4 or 7 with a dose of 10 mg BPA/kg bw, the AUC for ovaries or uterus was higher than the AUC for plasma. However, these tissue AUCs were similar to the AUC for carcass on pnd 4 and pnd 7. This is additional evidence for a dose dependency in BPA tissue disposition that was only apparent in younger animals. Within age groups, the half-lives for the elimination of 14C-BPA–derived radioactivity in tissues generally followed the half-lives for the elimination of radioactivity in plasma, further suggesting that there was roughly equal distribution to all tissues. For animals dosed on pnd 4 or 7, the tissue and plasma half-lives were longer than for animals dosed on pnd 21, suggesting an age dependency in the pharmacokinetics of BPA in neonates. 14C-BPA–derived radioactivity concentrations in adult tissues at 96 h were approximately equivalent to neonatal tissue concentrations at 24 h, consistent with the more rapid elimination of BPA and its metabolites in neonates.

In conclusion, our research has demonstrated that phase II metabolism of BPA to its nonestrogenic monoglucuronide conjugate occurred as early as pnd 4 in rats. Evidence of age dependency in the glucuronidation of BPA was observed, a finding consistent with the recent study of GT ontogeny in neonatal rats by Matsumoto et al. (2002)Go. No marked sex differences were observed in BPA metabolism and pharmacokinetics for neonatal to weanling rats. Importantly, BPA pharmacokinetics in neonates were found to be dose-dependent from 1 to 10 mg/kg, as evidenced by a larger fraction of the administered dose being metabolized to the monoglucuronide at the lower dose. This dose dependency in BPA metabolism is consistent with the absence of low-dose effects following the oral administration of BPA to neonatal Sprague-Dawley rats in multigeneration reproduction studies (Ema et al., 2001Go; Tyl et al., 2002Go). Clear evidence for a dose dependency in the metabolism and pharmacokinetics of BPA administered to neonates was provided by the differences between the dose levels in the number and concentration of plasma metabolites. At the dose of 1 mg/kg, metabolism of BPA to BPA-glucuronide was nearly complete, suggesting that lower doses would result in essentially the sole production of this nonestrogenic metabolite.

ACKNOWLEDGMENTS

This study was supported by the PC/BPA Industry Group of the American Plastics Council, Arlington, VA. The authors gratefully acknowledge the contributions of the following individuals who assisted during these studies: Ashley Liberacki, Ed Carney, Bruce Kropscott, Jason Whalen, Amanda Aultman, Jane Lacher, DVM, Erica Mullen, Marcy Knoerr, Amy Clark, Alan Mendrala, Shakil Saghir, Sandy Eddy, Jan Beyers, Glorious Cummings-Tolbert, Janice Hammond, Frank Lee, Wendy Kempf, Beth Stieve, and Tim Bell.

NOTES

1 To whom correspondence should be addressed at The Dow Chemical Company, Toxicology and Environmental Research and Consulting, Building 1803, Midland, MI 48674. Back

REFERENCES

Atanassova, N., McKinnell, C., Turner, K. J., Walker, M., Fisher, J. S., Morley, M., Millar, M. R., Groome, N. P., and Sharpe, R. M. (2000). Comparative effects of neonatal exposure of male rats to potent and weak environmental estrogens on spermatogenesis at puberty and the relationship to adult testis size and fertility: Evidence for stimulatory effects of low estrogen levels. Endocrinology 141, 3898–3907.[Abstract/Free Full Text]

Chang, J., Chadwick, R. W., Allison, J. C., Hayes, Y. O., Talley, D., and Autry, C. E. (1994). Microbial succession and intestinal enzyme activities in the developing rat. J. Appl. Bacteriol. 77, 709–718.[ISI][Medline]

Dodds, E. C., and Lawson, W. (1936). Synthetic oestrogenic agents without the phenanthrene nucleus. Nature 137, 996.

Ema, M., Fujii, S., Furukawa, M., Kiguchi, M., Ikka, T., and Harazzono, A. (2001). Rat two generation reproduction toxicity study of bisphenol A. Reprod. Toxicol. 15, 505–523.[CrossRef][ISI][Medline]

Fisher, J. S., Turner, K. J., Brown, D., and Sharpe, R. M. (1999). Effect of neonatal exposure to estrogenic compounds on development of the efferent ducts of the rat testis through puberty to adulthood. Environ. Health Perspect. 107, 397–405.[ISI][Medline]

Goodson, A., Summerfield, W., and Cooper, I. (2002). Survey of bisphenol A and bisphenol F in canned foods. Food Addit. Contam. 19, 796–802.[CrossRef][ISI][Medline]

Klaassen, C. D. (1972). Immaturity of the newborn rat’s hepatic excretory function for ouabain. J. Pharmacol. Exp. Ther. 183, 520–526.[ISI][Medline]

Klaassen, C. D. (1973). Hepatic excretory function in the newborn rat. J. Pharmacol. Exp. Ther. 184, 721–728.[ISI][Medline]

Kurebayashi, H., Betsui, H., and Ohno, Y. (2003). Disposition of a low dose of 14C-bisphenol A in male rats and its main biliary excretion as BPA glucuronide. Toxicol. Sci. 73, 17–25.[Abstract/Free Full Text]

Kurebayashi, H., Harada, R., Stewart, R. K., Numata, H., and Ohno, Y. (2002). Disposition of a low dose of bisphenol A in male and female Cynomolgus monkeys. Toxicol. Sci. 68, 32–42.[Abstract/Free Full Text]

Lucier, G. W. (1981). Developmental aspects of drug conjugation. In Developmental Toxicology (C. A. Kimmel and J. Buelke-Sam, Eds.), pp. 101–114. Raven Press, New York.

Matsumoto, J., Yokota, H., and Yuasa, A. (2002). Developmental increases in rat hepatic microsomal UDP-glucuonosyltransferase activities toward xenoestrogens and decreases in pregnancy. Environ. Health Perspect. 110, 193–196.[ISI][Medline]

Matthews, J. B., Twomey, K., and Zacharewski, T. R. (2001). In vitro and in vivo interactions of bisphenol A and its metabolite, bisphenol A glucuronide, with estrogen receptors {alpha} and ß. Chem. Res. Toxicol. 14(2), 149–157.[CrossRef][ISI][Medline]

Nagao, T., Saito, Y., Usumi, K., Kuwagata, M., and Imai, K. (1999). Reproductive function in rats exposed neonatally to bisphenol A and estradiol benzoate. Reprod. Toxicol. 13, 303–311.[CrossRef][ISI][Medline]

National Toxicology Program (2001). National Toxicology Program’s Report of the Endocrine Disruptors Peer Review.

Pottenger, L. H., Domoradzki, J. Y., Markham, D. A., Hansen, S. C., Cagen, S. Z., and Waechter, J. M., Jr. (2000). The relative bioavailability and metabolism of bisphenol A in rats is dependent upon the route of administration. Toxicol. Sci. 54, 3–18.[Abstract/Free Full Text]

Scientific Committee on Food (SCF) (2002). Opinion of the Scientific Committee on Food on Bisphenol A, April 17. http://europa.eu.int/comm/food/fs/sc/scf/out128_en.pdf.

Snyder, R. W., Maness, S. C., Gaido, K. W., Welsch, F., Sumner, S. C. J., and Fennell, T. R. (2000). Metabolism and disposition of bisphenol A in female rats. Toxicol. Appl. Pharmacol. 168, 225–234.[CrossRef][ISI][Medline]

Tyl, R. W., Myers, C. B., Marr, M. C., Thomas, B. F., Keimowitz, A. R., Brine, D. R., Veselica, M. M., Fail, P. A., Chang, T. Y., Seely, J. C., et al. (2002). Three-generation reproductive toxicity study of dietary bisphenol A in CD Sprague-Dawley rats. Toxicol. Sci. 68, 121–146.[Abstract/Free Full Text]

United Kingdom Food Standards Agency (UK FSA) (2001). Survey of Bisphenols in Canned Foods, March 2001. http://www.food.gov.uk/science/surveillance/fsis-2001/bisphenols.

Upmeier, A., Degen, G. H., Diel, P., Michna, H., and Bolt, H. M., (2000). Toxicokinetics of bisphenol A in female DA/Han rats after a single iv and oral administration. Arch. Toxicol. 74, 431–436.[CrossRef][ISI][Medline]

Völkel, W., Colnot, T., Csanády, G. A., Filser, J. G., and Dekant, W. (2002). Metabolism and kinetics of bisphenol A in humans at low doses following oral administration. Chem. Res. Toxicol. 15, 1281–1287.[CrossRef][ISI][Medline]

Yokota, H., Iwano, H., Endo, M., Kobatashi, T., Inoue, H., Ikushiro, S., and Yuasa, A. (1999). Glucuronidation of the environmental oestrogen bisphenol A by an isoform of UDP-glucuronosyltransferase, UGT2B1, in the rat liver. Biochem. J. 340, 405–409.[CrossRef][ISI][Medline]