* Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674;
Dow Europe, GmbH, Horgen, Switzerland CH-8810; and
Bayer CropScience LP, Stillwell, Kansas 66085
Received May 2, 2003; accepted July 19, 2003
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ABSTRACT |
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Key Words: bisphenol A; BPA-monoglucuronide metabolism, disposition; rats; pregnant; embryo; fetus.
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INTRODUCTION |
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Although there are in vitro data that show that BPA acts as a weak agonist at the estrogen receptors alpha or beta (Brotons et al., 1995; Krishnan et al., 1993
; Olea et al., 1996
), studies of the in vivo estrogenicity of BPA have demonstrated dose- and route-dependence (Bitman and Cecil, 1970
; Bond et al., 1980
; Matthews et al., 2001
; Reel et al., 1985
) or only transient effects (Laws and Carey, 1997
). One apparently critical factor for elicitation of in vivo estrogenicity of BPA is the bioavailability of parent BPA, that is, the internal dose of unchanged BPA that is needed for sufficient estrogen receptor binding to elicit an estrogenic effect. The prior data on pharmacokinetics and metabolism in nonpregnant rats indicated that the bioavailability of parent BPA was very limited following oral gavage administration of BPA (Pottenger et al., 2000
; Upmeier et al., 2000
).
Pregnancy results in major changes in physiological parameters that could have considerable impact on the pharmacokinetics and metabolism of BPA. Examples include overall maternal weight gain and increase in organ size, particularly liver, with an increase in total body water and blood volume. In addition there is increased blood flow to the uterus, a decrease in overall plasma protein concentration with a concomitant increase in specific plasma proteins, altered hormone blood concentrations, decreased gastrointestinal (GI) motility, and increased GI transit time (Miller, 1983). These parameters are not a comprehensive list of pregnancy-related physiological changes, but represent a few that could potentially affect the pharmacokinetics and metabolism of BPA. For example, the increased liver size (40% in rats, Dutton, 1980
) implies an increased metabolic capacity. Therefore, in late-stage pregnancy, BPA may be subject to even greater first pass metabolism by the liver. The increased blood volume may result in lower blood concentrations, whereas extrahepatic glucuronidation may also participate in BPA metabolism. Thus, it could be postulated that elimination of parent BPA would be more rapid in pregnant rats compared with nonpregnant rats, similar to paracetamol plasma clearance in pregnant women (Miners and Mackenzie, 1991
).
Additional factors that could affect BPA pharmacokinetics and metabolism include the impact of pregnancy on expression and activity of the uridine diphosphoglucuronsyltransferases (UGTs), on potentially competing enzymes such as the sulfotransferases, or on the expression of ß-glucuronidase activity. UGT is known to undergo hormonal regulation of expression, demonstrating substrate-specific increases in activity (i.e., oral contraceptive steroids resulting in enhanced glucuronidation of many drugs [Miners and Mackenzie, 1991]; intra-muscular injection of ß-estradiol leading to increased glucuronidation of estrone [Dutton, 1980
]). Alternatively, UGT can also demonstrate substrate-specific decreases in activity (i.e., decreased 2-aminophenol glucuronidation in pregnant women). Thus, the physiological status of pregnancy and its corresponding hormonal milieu could selectively or coordinately increase or decrease the expression/activity of enzymes, and so alter metabolic capacity for clearance of BPA.
Although other studies have investigated BPA kinetics in pregnant animals, this study is the first to examine the pharmacokinetics and metabolism of BPA at three different stages of pregnancy in Sprague-Dawley rats. This study is also unique in that the concentrations of both BPA and BPA-glucuronide in maternal plasma, placenta, or yolk sac, and embryonic or fetal tissue were determined.
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MATERIALS AND METHODS |
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Authentic standards of BPA-glucuronide were synthesized at The Dow Chemical Company or obtained from ULTRAFINE Chemicals (Batch No.: 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 spectroscopy (NMR).
To assess the stability of BPA-glucuronide during collection, storage, and processing of tissues, a "stock solution" consisting of a mixture of 14C-BPA-glucuronide and 14C-BPA (14C-BPA-glucuronide/14C-BPA standard mixture) was obtained by collecting urine from 024 h postdosing from two female Fischer 344 rats administered 100 mg 14C-labeled BPA/kg bw by a single po administration. The urine from both rats was pooled and was determined by liquid scintillation spectrometry (LSS) to contain 2921 dpm/µl urine (5.4 ng equivalents [eq] BPA/µl). As determined by high performance liquid chromatography with radioactivity monitor (HPLC/RAM) analysis, the radioactivity in the pooled urine was about 97% 14C-BPA-glucuronide and about 3% 14C-BPA as observed in previous studies (Pottenger et al., 2000).
Animals.
Female CD Sprague-Dawley (S-D) rats, 11 weeks of age at mating, and 1114 weeks at dosing, were purchased from Hilltop Laboratories (Scottdale, PA). Serial blood specimens were collected via in-dwelling jugular vein cannulae implanted by the animal supplier. Care and husbandry of animals were in accordance with the Guide for the Care and Use of Laboratory Animals (1985). The study was reviewed and approved by the Institutional Animal Care and Use Committee of The Dow Chemical Company. GD 0 of pregnancy was defined in this study as the day when sperm were found in the vaginal washing or a vaginal plug was detected.
Upon arrival in the laboratory (fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International), each animal was evaluated by a laboratory veterinarian and found in good health, then acclimated to the laboratory environment and to glass Roth-type metabolism cages for at least 24 h. The animal rooms were maintained on a 12-h light/dark cycle and at 2123°C and 4070% relative humidity. Animals were provided LabDiet® Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO). Feed and municipal water were provided ad libitum, except when feed was withdrawn approximately 14 h before dosing and returned approximately 4 h postdosing.
Data analysis and quality assurance.
Means and SDs were calculated with Microsoft Excel® using full precision mode accuracy. Area under the curve (AUC) for plasma radioactivity concentration-time curves as well as BPA-glucuronide concentration-time curves were estimated based on the linear trapezoidal rule as described by Gibaldi and Perrier (1982) and calculated using WinNonLin® noncompartmental analysis (Scientific Consulting, Inc., Cary, NC).
This study was conducted in accordance with EPA Toxic Substance Control Act (EPA-TSCA, 1984) Good Laboratory Practice Standards.
Analytical conditions.
Typical HPLC conditions are described in Pottenger et al.(2000) with slight variations for the analysis of samples from dams dosed with 0.5 mCi/animal. The variations from the published method included: the use of an Agilent 1100 series pump, autosampler, and UV detector. Chromatography was performed with mobile phases containing 0.1% acetic acid in water (A) and 0.1% acetic acid in acetonitrile (B). The gradient differed slightly with a 20-min linear gradient from 95% A to 95% B followed by a 5-min hold at 95% B. Fractions were collected at 10-s intervals.
Study of the Disposition of 14C-BPADerived Radioactivity in Nonpregnant and Pregnant Rats
Study overview.
Radiolabeled 14C-BPA was administered by gavage in a corn oil vehicle (Acros Organics, Geel, Belgium, Lot #A31) at a dose of 10 mg BPA/kg bw and a dosing volume of 5 ml/kg bw. Groups of four pregnant rats were dosed on either GD 6, 14, or 17 along with one group of nonpregnant rats.
These gestational stages represented early, middle, and late stages of pregnancy. GD 6 corresponds with an embryonic stage of development and the beginning of organogenesis. At GD 610, the placenta is not established and therefore placental metabolism would not potentially alter the pharmacokinetics and metabolism at this stage. The hormonal changes occurring during pregnancy are established on GD 6 and any potential hormonal effects on enzyme expression/activity would likely be evident. GD 14 corresponds with the beginning of the fetal stage of development and the presence of an established placenta. This stage of pregnancy corresponds with late organogenesis in the fetus. Placental tissue has been shown to demonstrate both glucuronidation and glucuronidase activity, and therefore may affect the in vivo pharmacokinetics and metabolism of BPA in pregnant rats. In addition, females at GD 1418, have considerable weight gain, increases in organ size and blood volume, and changes in hemodynamics, all of which might be expected to have impact on pharmacokinetic parameters.
Females at GD 17 were chosen to examine BPA pharmacokinetics at the final stages of gestation. Over GD 1721, the majority of weight gain and increases in organ size, total body water, and glomerular filtration rate occur. The placenta is large and metabolically active, and the fetal metabolic activity is also increased, both potentially affecting BPA pharmacokinetics.
Confirmation of the concentration and homogeneity of the dosing solutions were analytically determined using HPLC analysis and LSS. The dose level was selected based on previous studies (Pottenger et al., 2000). The gestation periods were selected to study early, middle, and late gestation to determine the potential impact of each stage on the disposition and metabolism of BPA. Rats were placed in Roth-type metabolism cages immediately postdosing to allow for the separation and collection of urine and feces through 96 h postdosing. Blood samples were collected via an in-dwelling jugular vein cannula at selected time points over a 96 h period postdosing. BPA and BPA-glucuronide were quantified in pooled plasma samples by reverse-phase HPLC using fraction collection (20 s) and LSS. Pooled excreta samples from selected dose groups and collection intervals were analyzed and quantified by HPLC for BPA and BPA-glucuronide. Structural confirmation of BPA and BPA-glucuronide peaks in HPLC profiles was obtained using LC/MS for selected plasma, urine, and fecal samples. Selected tissues were collected at sacrifice and analyzed for radioactivity. A final cage wash was collected and the mass balance of administered radioactivity determined.
Sample collection and preparation/disposition study.
Heparinized blood specimens were collected at 0.25, 0.75, 1.5, 3, 6, 12, 18, 24, 48, 72, and 96 h postdosing. Plasma from individual animals was obtained by centrifugation and analyzed for radioactivity by LSS. Plasma samples were also pooled by equal volume according to time point and stage of gestation and analyzed for BPA and BPA-glucuronide by HPLC.
Following CO2 euthanasia, the rats were sacrificed by exsanguination via cardiac puncture. Maternal blood, brain, fat, liver, kidneys, ovaries, placenta, uterus, skin, embryo/fetus, and remaining carcass were collected. When possible, six individual embryos/fetuses were collected (three from each uterine horn at specific positions) from each dam, along with their corresponding individual placenta (with chorion and amnion). Any remaining embryos/fetuses were collected and combined for analysis as a pooled sample, as were their corresponding placentae (with chorion and amnion). Fetuses were euthanized via sublingual deposition of sodium pentobarbital. The maternal blood, fat, ovaries, uterus, and skin samples were directly solubilized using Soluene® 350 (Packard Instrument Company, Inc., Downers Grove, IL) or tetraethylammonium hydroxide 20% (aqueous). Aqueous homogenates (2533% w/w) were prepared from the remaining tissues and weighed aliquots were solubilized as described above and analyzed for radioactivity by LSS.
Urine voided during successive 12-h intervals through 24 h and subsequent 24-h intervals through 96 h was collected in dry ice-chilled containers. Weighed aliquots of urine were mixed with Aquasol® liquid scintillation fluid (New England Nuclear, Boston, MA) and analyzed for radioactivity by LSS. Feces were collected at 24-h intervals over 96 h postdosing in dry ice-chilled containers. Aqueous homogenates (25% w/w) were prepared and weighed aliquots were solubilized using Soluene® 350 and analyzed for radioactivity by LSS using Hionic-Fluor® (Packard Instrument Company, Inc., Downers Grove, IL).
Preparation of pooled plasma samples for HPLC radiochemical analysis/disposition study.
Plasma specimens were pooled at the time of collection. Prior to analysis, pooled samples were stored at -80°C. For analysis the specimens were thawed and mixed well, prior to extraction. Approximately 20500 µl of pooled plasma was weighed and extracted with ~ 100500 µl of acetonitrile (containing 1% acetic acid, v/v) by brief vortex mixing. Approximately 3001200 µl of 50/50 acetonitrile/water (containing 1% acetic acid, v/v) was then added, the samples 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 HPLC. HPLC system recoveries were 93 ± 16% and extraction efficiencies were 98 ± 12%. Selected samples of maternal plasma were analyzed by HPLC-electrospray-ionization-mass spectrometry (ESI-MS) to identify the major radiochemical peaks.
Preparation of urine samples for HPLC radiochemical analysis/disposition study.
Prior to analysis, individual urine specimens were stored at -80°C. For analysis the specimens were thawed, mixed well, and an equal volume aliquot pooled for the following time intervals (012, 1224, 2448 h). Pooled urine samples were centrifuged for ~10 min (~600 x g) to remove any particulate matter and an aliquot was directly injected into the HPLC system for analysis. HPLC system recoveries were 96 ± 8%. Selected samples of urine were analyzed by HPLC-ESI-MS to identify the major radiochemical peaks.
Preparation of fecal samples for HPLC radiochemical analysis/disposition study.
Fecal homogenates prepared from 024 and 2448 h fecal samples were removed from the -80°C freezer, thawed, mixed well, and an equal volume aliquot from each pooled for analysis. The pooled specimens were mixed well and a sample (~ 2 g) was weighed, extracted three times with ~6 ml of acetonitrile:acetic acid (99:1) and centrifuged (~600 x g) for ~10 min. The supernatants from each extraction were combined and then concentrated under a stream of dry N2 to ~ 4 ml and aliquots were directly injected into the HPLC system for analysis. Extraction efficiencies were 99 ± 11% and HPLC system recoveries were 99 ± 2%. Selected samples of fecal extracts were analyzed by HPLC-ESI-MS to identify the major radiochemical peaks.
Study to Determine the Concentrations of BPA and BPA-Glucuronide in Maternal Plasma, Yolk Sac/Placenta, and Embryo/Fetus at Different Stages of Gestation
Study overview.
Radiolabeled 14C-BPA was administered by gavage in a vehicle of corn oil at 10 mg BPA/kg bw (0.2 mCi/rat) to rats at three different days of gestation (GD 11, embryonic stage; GD 13, embyro/fetal stage; GD 16, fetal stage). GD 11 rather than GD 6 was chosen as the embryonic stage to investigate since the tissue sample size was critical for analysis of radioactivity, BPA, and BPA-glucuronide. GD 16 was chosen instead of GD 17 since at the 96 h sacrifice the animals would be at GD 20 instead of 21 and the chance of delivery would be minimized. GD 14 was chosen as a midpoint between GD 11 and GD 16. Five rats per group were dosed at each stage of gestation and sacrificed at 0.25 h postdosing (the previously observed Cmax for plasma radioactivity in maternal animals), 12 h postdosing (the time of the secondary maximum [Cmax2] of plasma radioactivity in maternal animals), and 96 h postdosing (when BPA was anticipated to be nonquantifiable in the maternal blood). All animals were euthanized by CO2 and exsanguinated via cardiac puncture.
Maternal blood was collected at sacrifice and the radioactivity in individual maternal blood and maternal blood plasma was determined by LSS. For the animals dosed on GD 11, all embryos, yolk sacs, and placentae were collected at each sacrifice time and pooled for the analysis of radioactivity. Likewise, all embryos and yolks sacs were collected and pooled for analysis from animals dosed and sacrificed on GD 13 at 0.25 h and 12 h postdosing. For the rats dosed on GD 13 but sacrificed at 96 h postdosing, the fetuses and placentae were collected and pooled by litter for the analysis of radioactivity. Fetuses and placentae were collected and pooled by litter for animals dosed on GD 16 and sacrificed at 0.25 h, 12 h, and 96 h postdosing. Blood, liver, and brain were also collected from each fetus at the 96 h postdosing sacrifice of animals dosed on GD 16 and pooled by litter for the analysis of radioactivity. If there was sufficient radioactivity found in individual or pooled specimens, subsequent HPLC analysis for parent compound and/or metabolites was carried out, including the quantitation of BPA-glucuronide.
In addition, five pregnant S-D rats (GD 16) were dosed by gavage with 14C-labeled BPA in corn oil at a dose of 10 mg/kg bw but with a greater amount of radioactivity (0.5 mCi/rat) to improve the limit of detection for analytes. Animals were sacrificed at 0.25 h postdosing and maternal blood, placentae, and fetuses collected from each animal for HPLC analysis. Placentae and fetuses were pooled by litter for analysis of radioactivity, BPA, and BPA-glucuronide. Prior to dosing, samples were prepared to study the stability of BPA-glucuronide in placenta and fetal homogenates obtained from control (not dosed with BPA) S-D rats. Results showed that the hydrolysis of BPA-glucuronide to BPA was initiated within 60 min by contact with placental and fetal tissue homogenates at room temperature. Thus, careful attention was taken to ensure that tissues were removed and processed within 10 min postmortem to minimize hydrolysis of BPA-glucuronide to BPA during specimen handling and analysis. Tissues were homogenized, solubilized, and analyzed for radioactivity by LSS as described above.
Preparation of maternal plasma, yolk sac/placenta, and embryo/fetus samples for HPLC radiochemical analysis.
The pooled yolk sac/placenta and embryo/fetal homogenates (weighed aliquots of ~ 0.75.0 g) were extracted twice with 5-ml aliquots of acetonitrile/water (60/40 v/v, 1% acetic acid) using vortex mixing (2 min) followed by centrifugation. The extracts were combined and concentrated to ~500 µl under a stream of dry N2, diluted with ~400 µl of acetonitrile (containing 1% acetic acid), mixed and analyzed.
Plasma was prepared for HPLC analysis as previously described except that for the 12 h postdosing groups, the supernatant was reduced to ~500 µl under a stream of dry N2 to provide sufficient radioactivity for analysis. Aliquots of the supernatant were analyzed by HPLC using fraction collection and LSS.
For animals dosed on GD 16 with 0.5 mCi radioactivity/animal, tissues were immediately collected, homogenized on ice, and stored at -80°C following sacrifice until their extraction and analysis. Plasma samples from these animals were prepared from blood as described above and were immediately stored at -80°C. Aliquots (0.5 g) of placenta and fetal homogenates were extracted with 1 ml of 1% acetic acid in acetonitrile/water (60/40, v/v) by vortex mixing for ~5 min. Each aliquot of plasma (1 g) was extracted with 1 ml of 1% acetic acid in acetonitrile. The samples were centrifuged at 1322 x g for 15 min and the supernatant analyzed via HPLC. Concurrent with the analysis of homogenates from dosed animals, solvent controls and matrix controls were prepared and analyzed. The matrix controls consisted of tissue homogenates prepared from control (undosed) animals that were fortified with known amounts of the 14C-BPA-glucuronide/14C-BPA standard mixture (see Materials and Methods). These controls were analyzed along with tissue homogenates of dosed animals to determine if the extraction and analysis technique resulted in the hydrolysis of BPA-glucuronide to BPA. Results showed that the analytical method used for plasma or tissues did not result in the hydrolysis of BPA-glucuronide.
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RESULTS |
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Tissues (brain, fat, liver, kidney, ovary, uterus, and skin), carcass, embryo/fetus, and placenta contained from 16% of administered dose at 96 h postdosing, with SDs typically greater than 50% of the mean (Table 1). The embryo/fetus samples accounted for <0.1% of administered dose for the GD 1721 group and were nonquantifiable (NQ) for the GD 1418 dose group. Placenta had quantifiable radioactivity only at GD 1721, representing 0.01% of the administered dose. The radioactivity distributed to the fetal and placental tissue from the GD 1721 group was equivalent to 0.024 and 0.027 µg eq BPA/g tissue, respectively. The disposition of 14C-BPAderived radioactivity in pregnant S-D rats was similar to nonpregnant animals for all stages of pregnancy studied.
Blood plasma radioactivity concentration-time profiles.
14C-BPAderived radioactivity was quantifiable in the plasma from 0.25 h postdosing through 48 h postdosing for all dose groups (Table 2). Selected pharmacokinetic parameters estimated for the plasma data are summarized in Table 3
. The plasma radioactivity in all groups was highest at 0.25 h postdosing (Cmax1) but reached a secondary peak (Cmax2) between 12 and 24 h postdosing. All dose groups showed considerable interanimal variability in plasma radioactivity concentrations resulting in large standard deviations for many time-points. The pattern of a primary peak (Cmax1) and a secondary peak (Cmax2) in total plasma radioactivity, which was reproducible across dose groups, is evidence of enterohepatic circulation of 14C-BPAderived radioactivity in pregnant and nonpregnant female S-D rats. By 72 h postdosing, radioactivity was not quantifiable in the plasma of nonpregnant rats or pregnant rats dosed on GD 14, with the limit of detection (LOD) ranging from 0.0110.039 µg eq BPA/g plasma. Radioactivity was nonquantifiable in the plasma from rats dosed on GD 17 by 96 h postdosing (LOD = 0.008 µg eq BPA/g plasma). Plasma from rats dosed on GD 6 contained quantifiable levels of radioactivity at terminal sacrifice (96 h postdosing, thus the time-to-NQ was not determined).
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Plasma BPA-glucuronide concentration-time profiles.
The BPA-glucuronide concentration-time profiles in pooled plasma for both nonpregnant and pregnant rats are shown in Figure 1. In all groups, an initial peak (Cmax1) in the plasma concentrations of BPA-glucuronide occurred at 0.25 h postdosing and a secondary peak (Cmax2) was observed at 12, 18, or 24 h postdosing. The BPA-glucuronide concentration-time profiles were nearly identical to the plasma radioactivity concentration-time profiles since BPA-glucuronide represented the vast majority of 14C-BPAderived radioactivity found in the plasma as evidenced by the similarity of the area-under-the plasma concentration-time curves (AUCs) for radioactivity and BPA-glucuronide (Table 3
). The AUCs for the BPA-glucuronide concentration-time profiles ranged from 9599% of the AUC values obtained for radioactivity concentration-time profiles for all groups.
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Fecal metabolite profiles.
Up to seven separate peaks were identified in the pooled fecal homogenates analyzed by HPLC from the 024 and 2448 h postdosing collection intervals. The majority of fecal radioactivity was identified as BPA based on co-chromatography and retention time match with authentic standard. Assuming the metabolite profiles for the 4872 h and 7296 h collection intervals were similar to those observed for the earlier intervals, BPA comprised approximately 8389% of the radioactivity recovered from the feces for all dose groups over the 96-h collection period (data not shown). Approximately 2 to 3% of the radioactivity recovered from the feces over the 96-h collection period was found to be BPA-glucuronide (data not shown). No marked differences were observed between the fecal metabolite profiles in non-pregnant and pregnant animals.
Concentrations of BPA and BPA-Glucuronide in Maternal Plasma, Yolk Sac/Placenta, and Embryo/Fetus at Different Gestation Stages
Concentrations of BPA and BPA-glucuronide in maternal plasma.
The concentrations of BPA-glucuronide and BPA found in maternal plasma following administration to pregnant S-D rats of a single dose of 10 mg BPA/kg bw are presented in Table 4. At 0.25 h postdosing, the average peak maternal plasma concentrations of BPA-glucuronide for the animals dosed on GD 11 or 13 were similar, whereas, the BPA-glucuronide plasma concentration in rats dosed on GD 16 were approximately 1.72 times higher. By 12 h postdosing, the concentration of BPA-glucuronide was about 7- to 11-fold less than was seen than at 0.25 h postdosing for all groups. At 96 h postdosing, there was insufficient radioactivity in the plasma samples for HPLC analysis. Maternal plasma concentrations of BPA-glucuronide in rats dosed on GD 16 were approximately 1.8 times higher than observed in rats dosed on GD 11 or 13. Similar to the animals dosed on GD 11 or 13, the concentration of BPAglucuronide at 12 h was approximately 10-fold less than that found at 0.25 h postdosing. The average maternal plasma concentration of BPA-glucuronide in animals dosed on GD 16 with 0.5 mCi 14C-BPA and sacrificed at 0.25 h was similar to animals dosed with 0.2 mCi. BPA was only detected in maternal plasma samples from 3 of 20 dams dosed on GD 11 and 13 with 0.2 mCi 14C-BPA and sacrificed at 0.25 and 12 h postdosing. For animals dosed on GD 16 with 0.2 mCi 14C-BPA and sacrificed at 0.25 h, only two rats out of five had detectable levels of BPA in plasma. Nondetectable concentrations of BPA were observed in animals dosed on GD 16 and sacrificed at 12 h postdosing. The concentration of BPA at 0.25 h postdosing in the maternal plasma of rats dosed on GD 16 with 0.5 mCi of 14C-BPA was found to be 0.064 ± 0.025 µg BPA/g plasma. These concentrations were similar to the BPA concentrations that were occasionally observed in the maternal plasma of nonpregnant and pregnant S-D rats dosed to determine the disposition of BPA.
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For GD 13 animals, BPA-glucuronide and BPA were detected in the pooled yolk sacs at 0.25 h and 12 h postdosing. The concentrations of BPA-glucuronide were approximately 9 to 24-fold lower than the BPA-glucuronide concentrations found in maternal plasma. BPA concentrations were roughly in the same range as the plasma concentrations that were found in only 2 of 10 maternal animals dosed on GD 13. BPA-glucuronide and BPA were not detected in embryonic tissue of GD 13 animals except for BPA at 0.25 h postdosing, which was detected at a concentration of 0.013 µg BPA/g tissue.
BPA-glucuronide and BPA were detected in placental tissue at 0.25 h and 12 h postdosing in GD 16 animals dosed with BPA at 0.2 mCi/rat (Table 4) and average placental concentrations of BPA-glucuronide were 0.223 and 0.025 µg eq BPA (as glucuronide)/g tissue. At 96 h postdosing, there was insufficient radioactivity in tissue to carry out HPLC analysis. The average placental concentration of BPA-glucuronide in animals dosed on GD 16 with 0.5 mCi 14C-BPA was 0.342 µg eq BPA (as glucuronide)/g tissue for animals sacrificed at 0.25 h postdosing. Average concentrations of 0.116 and 0.034 µg BPA/g placenta were detected at 0.25 h and 12 h postdosing, respectively. Similarly, an average concentration of 0.095 µg BPA/g placenta was detected at 0.25 h postdosing in rats dosed with 0.5 mCi 14C-BPA. At 96 h postdosing there was insufficient radioactivity for HPLC analysis. BPA and BPA-glucuronide concentrations found in placental tissue at 12 h were about 4.9- and 9-fold lower, respectively, than the concentrations observed at 0.25 h postdosing. The concentrations of BPA-glucuronide in placenta were approximately 8- to 7-fold lower than the BPA-glucuronide concentrations found in maternal plasma at 0.25 and 12 h postdosing, respectively. The average BPA concentration detected in placental tissue at 0.25 h postdoing was similar to the lowest maternal plasma concentration that was found in 2 of 10 dams dosed on GD 16. Although BPA was detected in placental tissue at 12 h postdosing, BPA was not detected in maternal plasma at this time.
In animals dosed with 0.2 mCi of 14C-BPA on GD 16, BPA-glucuronide and BPA were detected in two pooled specimens of fetal tissue at 0.25 h postdosing. Pooled specimens of fetal tissue from the three other dams did not contain sufficient radioactivity for HPLC analysis. The concentrations of BPA-glucuronide observed were 0.031 and 0.009 µg eq BPA (as glucuronide)/g tissue at 0.25 h postdosing. Concentrations of BPA observed at 0.25 h postdosing were 0.122 and 0.020 µg BPA/g tissue. At 12 h postdosing, insufficient radioactivity in the pooled specimens from individual dams did not allow for HPLC analysis. There was insufficient activity in fetal tissue to quantify at 96 h, except in one pooled fetal specimen from one dam where the concentrations were 0.016 µg eq BPA (as glucuronide)/g tissue and 0.008 µg BPA/g tissue. Average concentrations of BPA-glucuronide and BPA detected in fetal tissue at 0.25 h postdosing in animals dosed with 0.5 mCi were 0.013 µg eq BPA (as glucuronide)/g tissue and 0.018 µg BPA/g tissue. These values are similar to those observed in fetal tissue from two of five dams dosed with 0.2 mCi/rat on GD 16.
As previously noted, maternal plasma, placental, and fetal concentrations of BPA-glucuronide and BPA were measured after GD 16 animals received a higher amount of radioactivity (~0.5 mCi per animal, 10 mg/kg bw). In that experiment, the ratio of BPA-glucuronide found in maternal plasma to that observed in the placenta was approximately five. The concentrations of BPA in the maternal plasma and placenta were comparable. In addition, approximately 26-fold greater BPA-glucuronide concentrations were found in placenta as compared to fetal tissue. The concentration of BPA detected in fetal tissues was about 5-fold less than the concentration detected in placental tissue and 4-fold less than the concentration in maternal plasma.
The radioactivity distributed to fetal plasma, liver, or brain from GD 16 dams dosed with 0.2 mCi of 14C-BPA did not contain sufficient radioactivity to produce meaningful radiochemical profiles (less than 0.016 µg eq BPA/g tissue; data not shown).
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DISCUSSION |
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A previous study of BPA pharmacokinetics and metabolism in F344 rats provided evidence for the enterohepatic recirculation of 14C-BPAderived radioactivity (Pottenger et al., 2000). In the present study, the pronounced pattern of a primary and a secondary Cmax in plasma BPA-glucuronide was clear evidence that enterohepatic recirculation occurred in both pregnant and nonpregnant S-D rats (Fig. 1
). It is important to note that at the time of the secondary Cmax, the concentrations of BPA in the blood were still below the limit of detection. No marked strain differences between S-D rats and Fischer 344 rats were observed in the disposition of BPA when these data were compared to the prior study of Pottenger et al.(2000)
. Nevertheless interanimal variability for the data derived from S-D rats was clearly greater than in Fischer 344 rats; this was the only observed difference between these two strains and a possible explanation for the variability in data is that S-D rats are outbred.
The disposition of BPA in pregnant S-D rats in this study was generally consistent with data generated by other investigators using pregnant rats. Miyakoda et al.(1999) reported a mean maternal plasma concentration of BPA of 34 ng/g at 1 h postdosing in pregnant Wistar rats dosed on GD 19 with 10 mg BPA/kg bw by gavage. These results were consistent with the data from the present study where the peak mean maternal plasma concentration of BPA was 64 ± 25 ng/g at 0.25 h postdosing in S-D rats dosed on GD 16. Takahashi and Oishi (2000)
administered 1000 mg BPA/kg bw by gavage to pregnant Fischer 344 rats on GD 18 and reported that the peak maternal plasma concentration was 14.7 µg/g at 20 min postdosing. Comparison of this finding with the data from studies with an oral dose of 10 mg/kg bw indicates that the plasma concentration for the high dose was roughly twice as high as would be expected based on the 100-fold increase in dose. This observation suggests that the pharmacokinetics of BPA may be nonlinear at higher doses.
More recently, Shin et al.(2002) reported peak maternal BPA concentrations of 927.3 ± 194.3 ng/ml at 5 min after iv administration of a 2 mg/kg bw dose to anesthetized, pregnant S-D rats on GD 1719. The half-lives for elimination of BPA in this study ranged from 2.2 to 3.9 h, depending on the tissue measured. These half-lives were significantly longer than those in unanesthetized rats from other studies published by the same investigators, which ranged from 38 to 62 min. The shorter half-lives in unanesthetized rats are consistent with the work of Upmeier et al.(2000)
, as well as the data of the present study, because BPA was typically at nondetectable levels in the blood at most times postdosing. These data suggest a very limited bioavailability of BPA after oral dosing due to first pass metabolism and to the rapid elimination of BPA from the blood. Extensive first pass metabolism for BPA is supported by the glucuronidation of BPA in metabolism studies conducted in vitro with hepatocytes from rats, mice, and humans (Elsby et al., 2001
; Nakagawa and Tayama, 2000
; Pritchett et al., 2002
) as well as studies using isolated rat intestine (Inoue et al., 2001
). The rapid appearance in plasma of the major metabolite, BPA-glucuronide, also supports the notion of limited systemic bioavailability of unchanged parent BPA.
Shin et al.(2002) also reported that the BPA concentrations in the placenta following a single 2 mg/kg iv dose were slightly higher (approximately 1.5 times greater) than the concentrations of BPA found in the maternal plasma. Data from the present study are in agreement, where the peak concentrations of unchanged BPA in the placenta were also about 1.5 times greater than the concentrations of BPA found in maternal plasma although these data were variable (Table 4
). Specifically, in the rats dosed with 0.5 mCi of 14C-BPA, the mean maternal plasma BPA concentrations were 64 ± 25 ng/g and average BPA concentrations in the placenta were 95 ± 31 ng/g. These data are consistent with the in vitro data of Csanády et al.(2002)
who determined the human tissue:blood partition coefficients for BPA (including placenta). The partition coefficient for placenta was 1.43 and was similar to other well-perfused tissues. Therefore the ratio of the concentration of BPA in maternal plasma and placenta would be expected to be similar.
BPA-glucuronide and BPA were the only 14C-BPAderived moieties observed by the HPLC analysis of the maternal plasma, placental, or fetal tissue homogenates (data not shown). In animals dosed on GD 16 with 0.5 mCi of 14C-BPA, the sum of the BPA-glucuronide and BPA mean concentrations shows that the distribution of the total 14C-BPAderived material to the placenta was only about one-fourth of that found in the maternal plasma. In the same animals, the total 14C-BPAderived material distributed to fetal tissue was about one-tenth of that found in the placenta (Table 4). These data also suggest that neither BPA-glucuronide nor BPA were preferentially distributed to the placenta or the fetus since the total concentration of 14C-BPAderived material found in these tissues at the time of Cmax was lower than that in the maternal plasma. The ratios of BPA-glucuronide to BPA in the maternal plasma, placenta, and fetal tissue were 26.5, 3.6, and 0.7, respectively. The decrease in the ratio of BPA-glucuronide to BPA concentrations in the placenta as compared with the maternal plasma may have been a result of the ample ß-glucuronidase activity found in the rat placenta (Bulmer, 1967
; Schultz and Jacques, 1971
) rather than the distribution of BPA (as parent compound) from maternal plasma.
Alternatively, the rate of transfer of BPA from maternal plasma to the placenta may have been greater than the rate of transfer for BPA-glucuronide. However, the most likely explanation for the greater percentage of BPA as total radioactivity in the placenta as compared to that same percentage in maternal plasma is probably based on the relative rates of BPA-glucuronide hydrolysis to BPA glucuronidation. In addition, the transfer rates for each compound out of the placenta, either through the return to the maternal plasma or transfer to the fetus, may affect the ratio of BPA to BPA-glucuronide.
The fetal disposition of BPA in this study shows many similarities and reasonable consistency with data generated by other investigators. Miyakoda et al.(1999) reported that the concentrations of BPA in fetal rats were 11, 4.4, and 7.5 ng/g at 1, 3, and 24 h postdosing, respectively, following an oral 10 mg/kg bw dose to pregnant Wistar rats on GD 19. In the current study, the peak mean fetal tissue concentration of BPA at 0.25 h postdosing, using 0.5 mCi of radioactivity per animal was similar at 18 ± 11 ng/g in pregnant S-D rats dosed with 10 mg/kg bw by gavage on GD 16.
Although the sample collection times used in the present study were not closely spaced (0.25, 12, and 96 h postdosing), the 12 h time point should have demonstrated an increase in the concentration of BPA in the embryo/fetus had this tissue shown a higher affinity than the maternal tissues. In contrast to this study, Miyakoda et al.(1999) was able to detect BPA in the fetus at 24 h postdosing at a concentration of 7.7 ng/g. These investigators concluded that BPA transfer to fetuses was rapid and that BPA has a tendency to remain in fetuses longer than in maternal blood. Recent studies in our laboratory (Waechter, J. M., personal communication) have demonstrated that BPA-glucuronide can be hydrolyzed during sample preparation and analysis of biological specimens. Hence, the release of BPA from BPA-glucuronide during analysis may lead to erroneous conclusions regarding the concentrations of "free" BPA in tissues.
Less consistent with the results of the present study are the findings of Takahashi and Oishi (2000). These investigators reported that the peak fetal concentrations of BPA were approximately 9 µg/g at 20 min postdosing following a gavage dose of 1000 mg/kg bw to pregnant F344 rats dosed on GD 18. These concentrations are about 10-fold higher than would be expected based on the 100-fold increase in the dose above the 10 mg/kg bw dose used in the present study. Again, these results support a nonlinearity of the pharmacokinetics of BPA in pregnant rats at high doses likely due to metabolic saturation. It appears that metabolic saturation was observed in the study by Knaak and Sullivan (1966)
at a dose administered to male rats of 800 mg BPA/kg bw. A higher fraction of the dose was excreted via the urine, 28%, than observed in this study with nonpregnant rats where 15% of the dose was excreted in the urine. Further evidence of metabolic saturation at 800 mg/kg was the presence of a hydroxylated metabolite of BPA in the feces that represented ~ 35% of the fecal radioactivity.
More recently, Shin et al.(2002) reported peak fetal BPA concentrations of 794 ng/g at 5 min postdosing following the iv administration of a 2 mg/kg bw dose to pregnant S-D rats on GD 1719. The AUC for BPA concentrations found in the fetuses were about 2-fold greater than the AUC for BPA concentrations found in maternal serum. In the present study, both BPA-glucuronide and BPA were found in the fetuses, although at about 26-fold and 5-fold lower than the concentration found in the placenta, respectively, and 131-fold and 3.6-fold lower than the concentrations found in maternal plasma, respectively. In the animals dosed with 0.5 mCi, the ratio of BPA-glucuronide to BPA in the fetus was 0.7 whereas the ratio of metabolite to parent in the placenta was 3.6. This study did not attempt to directly address whether the BPA-glucuronide found in the fetus was transferred from the BPA-glucuronide present in the placenta or generated by the fetus in situ after distribution of parent BPA to the fetus. However, Matsumoto et al.(2002)
have reported that fetal microsomes prepared from Wistar rats on GD 19 were found to have negligible UGT activity for BPA, suggesting that the BPA-glucuronide found in the fetus may have been directly transferred from the placenta.
The transfers of BPA-glucuronide and BPA from the placenta to the fetus were likely first-order kinetic processes (Fig. 5). The placental concentration of BPA-glucuronide was greater than the concentration of BPA, but the reverse was true in the fetus, thus the rate for the transfer of BPA to the fetus likely exceeded the rate for BPA-glucuronide transfer (Fig. 5
). Within the fetus, the ratio of free BPA and BPA-glucuronide may be based in part on the relative rates of BPA-glucuronide hydrolysis to BPA glucuronidation in the fetus, as well as any glucuronidation of free BPA which may occur late-term. In addition, the transfer rates for each compounds transport out of the fetus and return to the maternal plasma via the placenta could have affected the ratio of parent to metabolite in the maternal blood. However, the distribution of BPA to the fetus (or placenta) did not alter the overall pharmacokinetic fate of BPA in pregnant rats as compared to nonpregnant rats. Therefore, the rates of transfer in and out of the fetus were apparently not the rate-limiting processes in determining the overall elimination half-life of BPA from the maternal-fetal "unit."
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Comparison of the pharmacokinetics across species may allow important insights into the extrapolation of the treatment-related effects of BPA across species. A recent in vivo study in human volunteers (Völkel et al., 2002) found that BPA was rapidly eliminated as BPA-glucuronide via the urine with a half-life of 3.36 h in both men and nonpregnant women. The present study demonstrated that there were no significant differences in the pharmacokinetic fate of BPA in pregnant (three gestational stages) vs. nonpregnant rats. Hence, the bioavailability of orally ingested BPA in pregnant humans might be expected to be similar to that observed in the study of Völkel et al.(2002)
.
In summary, the pharmacokinetics and metabolism of BPA in pregnant rats were not different from that found for non-pregnant rats at any stage of pregnancy investigated. The overall transfer of 14C-BPAderived material found in the placenta or embryo/fetus was limited to BPA-glucuronide and BPA; the sum of these delivered to either the placenta or fetus was lower than that found in maternal plasma. Therefore, no selective affinity of either yolk sac/placenta or embryo/fetus for BPA or BPA metabolites relative to maternal plasma or tissues was observed in this study. Furthermore, based on the low BPA concentrations found in the fetus, the half-life for the elimination of BPA from the fetus will likely be driven by the maternal half-life for BPA elimination.
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ACKNOWLEDGMENTS |
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REFERENCES |
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