Disposition of a Low Dose of Bisphenol A in Male and Female Cynomolgus Monkeys

Hideo Kurebayashi*,1, Ryoko Harada{dagger}, Richard K. Stewart{dagger}, Hiroaki Numata{dagger} and Yasuo Ohno*

* Division of Pharmacology, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya, Tokyo 158-8501, Japan; and {dagger} ITR Laboratories Canada Inc., 19601 Clark Graham, Montréal, Québec, Canada H9X 3T1

Received December 31, 2001; accepted March 12, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bisphenol A (BPA) is a weak estrogenic compound mass-produced with potential human exposure. Following a single oral or intravenous (iv) dose of 100 µg/kg [ring-14C(U)] radiolabeled bisphenol A (14C-BPA) to male and female cynomolgus monkeys, 79–86% of the administered radioactivity was excreted in urine over 7 days, and most of the urinary excretion was recovered by 24 h after dosing, a large part of this occurring within 12 h. The fecal excretion of radioactivity over 7 days was minimal (1.8–3.1%). Toxicokinetic parameters obtained from plasma 14C-BPA–derived radioactivity during 48 h were Cmax = 104–107 ng-eq/ml between 0.25 and 2 h, and AUCoral = 244–265 ng-eq•h/ml after oral dosing. In the case of the iv dose, AUCiv was 377–382 ng-eq•h/ml, and the bioavailability was 0.66–0.70. The terminal elimination half-life was larger post-iv dose (t1/2iv = 13.5–14.7 h) than post-oral dose (t1/2oral = 9.63–9.80 h). After iv dose, the fast-phase half-life (t1/2f) of total radioactivity was 0.61–0.67 h. The t1/2f of unchanged14C-BPA for females (0.39 h) was smaller than that for males (0.57 h). These results suggested the distribution of lipophilic 14C-BPA in adipose tissue after iv dose, in contrast to first pass metabolism after oral dose. 14C-BPA–derived radioactivity was strongly bound to plasma protein (fp = 0.055). Radio-HPLC analysis suggested the predominant plasma and urinary metabolites were mono- and diglucuronide of 14C-BPA and unchanged 14C-BPA was very low (<=1.5%) after oral dose. These results indicate that the intestinal absorption and metabolism of BPA was rapid and extensive, and the major metabolites, glucuronide conjugates of 14C-BPA, were rapidly excreted into urine in monkeys.

Key Words: bisphenol A; monkeys; toxicokinetics; absorption; metabolism; excretion; glucuronide.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, much scientific as well as public attention has been focused on chemicals that might be capable of mimicking endogenous hormone action and thus interfering with normal endocrine function. Such chemicals, now designated as endocrine disrupters, are postulated to bind to hormone receptors and mimic hormone action and may have the potential to alter normal hormonal function in humans and wildlife. Suspected effects of the disruption of endocrine function may include reduced fertility and increased incidence of cancer in estrogen-responsive tissues (Colborn et al., 1993Go; Davis et al., 1993Go).

Considerable evidence (Field et al., 1990Go) exists indicating that many classes of environmental contaminants, including dioxins, polychlorobiphenyls (PCBs), polycyclic aromatic hydrocarbons, and 4-alkylphenols, have the ability to interfere with normal hormonal activity by mimicking or blocking the action of natural hormones (McLachlan, 1993Go). Thus, such chemicals may give rise to a range of toxic effects including developmental abnormalities and disruption of endocrine function. The primary consideration for assessing the impact of a given environmental contaminant is the potential to produce a biological effect, as this criterion can be used to eliminate inactive compounds from the risk assessment process.

Bisphenol A (BPA), mass-produced for a monomer component of polycarbonate plastics, is used in many consumer products, including lacquers applied as food can linings (Brotons et al., 1995Go) and dental composite fillings and sealants (Olea et al., 1996Go), and it leaches in small amounts from polycarbonate flasks when autoclaved (Krishnan et al., 1993Go). In vitro, BPA exhibits weak estrogenic activity, compared with 17ß-estradiol, by binding to and activating estrogen receptors (Gaido et al., 1997Go; Krishnan et al., 1993Go). On the other hand, the results of in vivo studies on the estrogenicity of BPA are very controversial in terms of the dose-related effects. Low-dose (2 or 20 µg/kg/day) effects of BPA on male reproductive organs such as prostate gland, preputial gland, and epididymis in CF-1 mice exposed during prenatal development were reported by Nagel et al. (1997) and vom Saal et al. (1998). However, other investigators have not observed such effects of BPA administered to pregnant CF-1 mice even at the same dose (Ashby et al., 1999Go; Cagen et al., 1999Go). Increased uterine levels of estrogen-responsive proteins and other adverse effects can be seen in vivo after oral administration of high doses of BPA ranging from 50 to 1000 mg/kg (Atkinson and Roy, 1995Go; Bond et al., 1980Go; Gould et al., 1998aGo; Morrissey et al., 1987Go). Steinmetz et al. (1997) reported that BPA induced hyperprolactinemia in Fischer 344 rats but not in Sprague-Dawley rats.

Determination of the absorption, tissue distribution, metabolism, and excretion of potentially biologically active contaminants is a prerequisite for risk assessment purposes, as these parameters are fundamental in establishing in vivo activity and lend further biological significance to environmental abundance data. There are a few reports of the biological fate of BPA in rats using much higher doses of [ring-14C(U)] radiolabeled bisphenol A (14C-BPA) ranging from 10 to 800 mg/kg (Knaak and Sullivan, 1966Go; Pottenger et al., 2000Go; Snyder et al., 2000Go). The use of high doses was mainly due to the low specific radioactivity of the 14C-BPA used. We are concerned with low doses of endocrine-disrupting chemicals by human food intake, and we are more concerned with the species differences in biological responses. We need the reliable extrapolation of relevant animal toxicity data to humans. Risk estimation based on results of animal experiments is valid only when species differences in metabolism and disposition are recognized and taken into account. We have selected the species most likely to be metabolically similar to humans and have examined disposition of a low dose (100 µg/kg) of 14C-BPA in cynomolgus monkeys by using 14C-BPA with relatively high specific radioactivity.

This study investigated possible gender dependency in the bioavailability of 14C-BPA by determining the radioactivity concentration-time courses in plasma following oral and iv administration of 14C-BPA to young adult cynomolgus monkeys at a low dose level along with elimination kinetics of 14C-BPA–derived radioactivity in urine and feces, and with additional studies of 14C-BPA–derived metabolites using radio-HPLC. Therefore, this study was initiated to obtain toxicokinetic information on BPA in monkeys, which allows interspecies comparison with the kinetics of BPA in rats.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
14C-BPA (4,4`-isopropylidene-2-diphenol, 2,2-bis-(p-hydroxyphenyl)-2-propane) was obtained from NEN Life Science Products (Boston, MA) with a specific activity of 2.62 GBq/mmole and a radiochemical purity of 99% (TLC). Nonradiolabeled BPA was supplied by Aldrich Chemical Co. (Milwaukee, WI). D-saccharic acid 1,4-lactone was from Sigma Chemical Co. (St. Louis, MO). A ß-glucuronidase/arylsulfatase (ß-G/Aryl) solution from Helix pomatia was obtained from Roche Molecular Biochemicals (Laval, Québec, Canada). All other chemicals used in the present study were of reagent grade.

The authentic standard of monoglucuronide of BPA (BPA-gluc) was purified on a semipreparative HPLC from the bile of male rats orally dosed with 100 mg/kg BPA. The structure was confirmed by liquid chromatography/mass spectroscopy (LC/MS) and nuclear magnetic resonance (NMR) analysis. BPA sulfate was synthesized according to the method of Hoiberg and Mumma (1969) based on the reaction of BPA and sulfuric acid with dicyclohexylcarbodiimide.

Animal experiments.
Six young adult cynomolgus monkeys, 3 males (4.2, 4.3, and 2.7 kg) and 3 females (3.1, 2.3, and 2.3 kg), were transferred from the ITR spare colony to individual stainless steel wire mesh bottom cages with a water bottle. The source for these animals was HRP Inc., Alice, TX. The animal room environment was controlled (temperature 21 ± 3°C; relative humidity 50 ± 20%; 10–15 changes of air per hour). Lighting was set for 12 h light (from 0700 h to 1900 h) and 12 h dark.

During acclimation and throughout the study, a standard certified commercial primate chow (Teklad Certified Primate Chow #8726C, Harlan Teklad, Madison, WI) was available ad libitum to each monkey except for 4 h after oral dosing. Municipal tap water purified by reverse osmosis and exposed to ultraviolet light (further treated by filtration at 0.2 µm) was provided to the animals ad libitum. Throughout the study period, Primatreat® (Bio Serv, French Town, NJ) was given once daily to enrich the diet.

In a repeated measures design, the original animals were administered 14C-BPA. On Day 1, the test article was administered once by iv bolus to each animal (100 µg/kg). On Day 15, after a 2-week washout period, the test article was administered once orally to each animal by gavage (100 µg/kg). The animals were released to ITR's spare colony 2 weeks after completion of this study.

All animals used in this study were cared for in accordance with the principles outlined in the current Guide to the Care and Use of Experimental Animals, published by the Canadian Council on Animal Care, and the Guide for the Care and Use of Laboratory Animals, a National Institutes of Health publication.

Preparation of dosing solution and administration of test articles.
A dosing solution with BPA concentration of 100 µg (1.15 MBq)/ml was prepared by dissolving 14C-BPA in 1/15 M isotonic phosphate buffer (pH 7.4). On Day 1, the test article was given to the animals once by iv injection as a bolus, at a dose volume of 1.0 ml/kg. The animals were not fasted prior to dosing. On Day 15, the test article was given to the animals orally by gavage using a tube attached to a plastic syringe at a dose volume of 1.07 ml/kg. The animals were not fed for 4 h postdose.

Excreta sample.
Immediately after dosing, each animal was returned to its cage (equipped with a metal mesh separator for feces collection) and was held thus for 7 consecutive days. Urine and cage wash were collected between 0–12, 12–24, 24–48, 48–72, 72–96, 96–120, 120–144, and 144–168 h. Feces samples were collected every 24 h for 7 days. Urine samples were collected into ice-chilled containers. Cage wash samples were obtained by rinsing each cage with 350 or 500 ml of purified water, which was collected using a rubber scraper (squeegee) into a suitable container. Collected excreta samples were weighed in preweighed vials immediately after collection.

Determination of radioactivity of excreta.
Each fecal sample was mixed with twice its weight of purified water and was then homogenized. Aliquots (200 µl) of fecal homogenate and aliquots (200 µl) of urine or cage wash samples without dilution were kept in a vacuum desiccator with biphosphate pentoxide at room temperature in the dark overnight. Samples were combusted with the sample oxidizer (Biological Oxidizer Model 307, Canberra-Packard Canada, Mississauga, Ontario, Canada), then mixed with a carbon dioxide–absorbing chemical (Carbo-Sorb® E, Packard Instrument Co., Meriden, CT) and a scintillation cocktail (Permafluor® E+, Packard Instrument Co., Meriden, CT).

Radioactivity measurement.
Radioactivity measurements were conducted using a Beckman LS 6000TA liquid scintillation counter (LSC). The counting efficiency was determined by the instrument using a set of standards provided by the manufacturer. Chemical and color quenching and chemiluminescence were automatically corrected by the instrument. When samples had radioactivity levels less than twice the background levels, they were considered below the level of quantitation (BLQ).

Plasma sample.
Blood samples (1.0 ml/time point) were collected into tubes (lithium heparin as anticoagulant) from the femoral vein of each monkey by venipuncture following each dosing at the following time points: 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 h post-iv dose; 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h post-oral dose. Following collection, each blood sample was centrifuged for plasma separation. Aliquot plasma samples (100 µl) were mixed with a 10-ml scintillation cocktail (ReadyGel, Beckman, Fullerton, CA).

The plasma protein binding of 14C-BPA–derived radioactivity was determined as follows. At some time points during 0.25–4 h, the plasma was directly applied to Microcon YM-10 ultrafiltration tube (Millipore Corporation, Bedford, MA). The tube was centrifuged at 15,000 rpm for 15 min, and the radioactivity concentration in the filtrate was determined by LSC as unbound concentration. The free fraction in plasma (fp) was determined as the ratio of unbound concentration in the filtrate to the total plasma concentration.

Methanol extraction and radioactivity recovery of 14C-BPA and its metabolites.
Methanol (1 ml) was added to each plasma and urine sample (0.2 ml each), followed by thorough vortexing. The samples were then centrifuged at 3000 rpm at 4°C for 10 min, and the supernatant removed. For each sample, the extraction procedure was repeated 3 times. The supernatants from each sample were combined and evaporated at 75°C under vacuum and redissolved in 200 µl methanol. This solution was then transferred into a 1.5-ml Eppendorf tube and centrifuged at 10,000 rpm for 2 min at room temperature. Subsequent to methanol extraction, a 75-µl aliquot of each supernatant solution was analyzed by HPLC, and 50 µl of the supernatant was analyzed by LSC to determine the radioactivity concentration.

The recovery of radioactivity from monkey plasma samples by methanol extraction was found to be 96.6 ± 1.0% (n = 42 for 21 male and 21 female samples). The recovery of radioactivity from the monkey urine samples by methanol extraction was 99.2 ± 0.8% (n = 16 for 8 male and 8 female samples).

Hydrolysis of conjugate metabolites of 14C-BPA.
Aliquots (200 µl) of selected plasma or urine were subjected to enzymatic digestion to obtain a final 0.3 ml of 50 mM Tris-HCl buffer (pH 7.4) containing 20 µl of ß-G/Aryl, with and without the 6.7 mM D-saccharic acid 1,4-lactone. Incubations were performed for 2 h at 37°C, and enzymatic reactions were terminated by placing the samples on ice, followed by methanol extraction and HPLC analysis.

HPLC analysis and quantitation of 14C-BPA and its metabolites.
The selected urine sample (0–12 or 12–24 h after iv and oral dosing) and the plasma sample (0.083–2 h after iv dosing and 0.5 or 2 h after oral dosing) were analyzed by radio-HPLC. The composition of BPA and its metabolites was determined by the area percentage method. Subsequent to methanol extraction, a 75-ml aliquot of each reconstituted solution was analyzed by gradient HPLC as follows: Waters HPLC System with Multisolvent delivery system 600E, Automatic Injector WISP M715, Absorbance Detector 486 tuneable at UV254 nm (Milford, MA), and Radioactivity Monitor LB 507A (EG & G Berthold, Aliquippa, PA) operated with a solid phase scintillator (Yttrium Glass, 0.15-ml cell volume).

The chromatographic system consisted of a TSKgel ODS-80TS QA column (4.6 x 150 mm, 5 µm; Tosoh, Tokyo, Japan) using a linear gradient mobile phase ranging from 90% A/10% B to 10% A/90% B over 10 min and maintained at 10% A/90% B for a further 5 min (A:10% acetonitrile, 0.5% acetic acid in 89.5% water; B:99.5% acetonitrile in 0.5% acetic acid) at a flow rate of 1.0 ml/min.

Radioactive BPA was coincident with the same RT of unlabeled reference compound detected by UV at 254 nm (Absorbance Detector 486 tuneable).

For each sample, relative retention time (RRT) of peaks in HPLC chromatogram was calculated as follows:


Toxicokinetic parameters.
Toxicokinetic parameters were determined from the individual plasma 14C-BPA–derived radioactivity concentration-time curves from the same dose (100 µg/kg) of oral and iv-administered 14C-BPA. Peak plasma concentrations (Cmax) and the time to reach Cmax (Tmax) were obtained from the observed data. The plasma 14C-BPA–derived radioactivity concentration profiles were analyzed by noncompartmental methods (Gibaldi and Perrier, 1982Go) to obtain total body clearance (CLtot), apparent volume of distribution at steady state (Vss), and mean residence time (MRT) using WinNonlin noncompartmental analysis (Scientific Consulting, Inc., Cary, NC). The area under the plasma 14C-BPA–derived radioactivity concentration-time curve (AUC) was calculated by the linear trapezoidal method and extrapolated to infinity by C/ß, where C is the concentration of the last measured time point and ß is the slope of the terminal phase determined by least-square linear regression analysis. The terminal half-1ife (t1/2) was calculated as 0.693/ß. The fast-phase half-life (t1/2f) was calculated similarly by the least-square linear regression analysis. The bioavailability (F) of 14C-BPA–derived radioactivity was determined by comparing AUC following oral and iv administration of 14C-BPA at the same dose level. F = (AUCoral/Doseoral)/(AUCiv/Doseiv).

Parametric Student t-test was employed to determine statistic significance, and a p value < 0.05 was considered to be significant.

Good laboratory practices.
This study was conducted in compliance with the Good Laboratory Practice Regulations of the United States Food and Drug Administration (21 CFR Part 58 and subsequent amendments), the OECD principles of Good Laboratory Practice, and the Specifications of the Japanese Ministry of Health and Welfare, Ordinance No. 21, March 26, 1997.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Disposition of 14C-BPA in Monkeys
Excretion results after a single oral and iv administration of 100 µg/kg 14C-BPA in monkeys are presented in Table 1Go. The mean recoveries of the administered dose in the excreta of male and female monkeys ranged from 81 to 88% of total radioactivity for both routes of administration. Most of the radioactivity was found in urine and cage wash, with a small amount in the feces (1.8–3.1%). Taking the cage wash sample as part of the urine, the results of the mean cumulative urinary and fecal excretion of radioactivity were graphically presented in Figures 1 and 2GoGo.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Disposition of 14C-Bisphenol A-Derived Radioactivity following Oral and iv Administration to Male and Female Cynomolgus Monkeys at 100 µg/kg over 168 Hours
 


View larger version (21K):
[in this window]
[in a new window]
 
FIG. 1. Cumulative excretion of radioactivity in excreta after iv administration of 14C-BPA at 100 µg/kg to cynomolgus monkeys. Data represent the mean ± SD of 3 animals. No error bar indicates the error was included within the symbol. Squares, male urine; diamonds, female urine; circles, male feces; crossbars, female feces.

 


View larger version (21K):
[in this window]
[in a new window]
 
FIG. 2. Cumulative excretion of radioactivity in excreta after oral administration of 14C-BPA at 100 µg/kg to cynomolgus monkeys. Data represent the mean ± SD of 3 animals. No error bar indicates the error was included within the symbol. Squares, male urine; diamonds, female urine; circles, male feces; crossbars, female feces.

 
Within the first 12 h after iv administration of 100 µg/kg C-BPA to monkeys, 73.7 ± 8.6% of the administered dose for males and 66.2 ± 17.4% for females were recovered in the urine in all animals (Fig. 1Go). By 24 h postdose, the recoveries in the urine increased to 81.7 ± 8.8% of the administered dose for males and 74.3 ± 17.0% for females. The total radioactivity recovered in the urine over 7 days was 85.8 ± 9.1% for males and 78.8 ± 15.7% for females. The cumulative excretion in feces during 7 days was 1.84 ± 1.45% for males and 2.07 ± 0.77% for females. The total radioactivity recoveries in the excreta over 7 days after iv dosing were 87.6 ± 9.7% and 80.9 ± 15.0% of the administered dose for males and females, respectively.

Following a single oral administration of 100 µg/kg 14C-BPA to monkeys, the radioactivity excreted mainly in the urine over 7 days was 84.8 ± 8.6% of the administered dose for males and 81.9 ± 7.8% for females, and was comparable to the excretion after iv administration (Figs. 1 and 2GoGo). The urinary excretion was essentially completed by 24 h postdose. The mean cumulative recoveries in feces for 7 days were 2.14 ± 0.67% of the administered dose for males and 3.08 ± 1.53% for females. The total radioactivity recovered in the excreta over 7 days was 87.0 ± 8.0% of the administered dose for males and 85.0 ± 6.7% for females.

Following oral or iv administration, 14C-BPA–derived radioactivity was excreted principally in the urine during 24 h, especially within the first 12 h. The recovery in the urine for the third-day period was below 1% for male and female monkeys. The recovery for the seventh-day period was 0.07% for males and 0.11% for females in the urine and was below 0.01% for males and females in the feces. It appeared that almost all of the radioactivity was excreted from the body in 7 days, although the residual radioactivity remaining in the body, if any, was not determined.

Urinary excretion of 14C-BPA–derived radioactivity represented the major elimination pathway for all groups. Because the fecal excretion was very small after oral administration, it is considered that the absorption should be complete. There was no obvious difference in rate or route of excretion of radioactivity between genders after oral and iv dosing.

Plasma Radioactivity Toxicokinetics
The concentration-time profiles of 14C-BPA–derived radioactivity in plasma concentration over 48 h post iv and oral dose are shown in Figures 3 and 4GoGo for male and female animals, respectively. The plasma levels of 14C-BPA–derived radioactivity were not quantifiable in blood after 72 h postdose for either gender (limit of quantitation, LOQ = 0.3 ng-eq BPA/g plasma for 100 µg/kg dose). Toxicokinetic parameters estimated from each of 3 plasma time-course data for both genders are summarized in Table 2Go.



View larger version (16K):
[in this window]
[in a new window]
 
FIG. 3. Mean plasma concentration of radioactivity after iv and oral dosing of 14C-BPA at 100 µg/kg to male cynomolgus monkeys. Data represent the mean ± SD of 3 animals. No error bar indicates the error was included within the symbol. Circles, iv dosing; diamonds, oral dosing.

 


View larger version (15K):
[in this window]
[in a new window]
 
FIG. 4. Mean plasma concentration of radioactivity after iv and oral administration of 14C-BPA at 100 µg/kg to female cynomolgus monkeys. Data represent the mean ± SD of 3 animals. No error bar indicates the error was included within the symbol. Circles, iv dosing; diamonds, oral dosing.

 

View this table:
[in this window]
[in a new window]
 
TABLE 2 Toxicokinetic Parameters of 14C-BPA-Derived Radioactivity in Male and Female Cynomolgus Monkeys after Intravenous and Oral Administration of 14C-BPA at Dosage of 100 µg/kg
 
Following a single iv administration of 100 µg/kg 14C-BPA to monkeys, the terminal elimination half-life (t1/2iv) of the radioactivity in plasma was 13.5 h for males and 14.7 h for females. The AUCiv was 377 ng-eq•h/ml for males and 382 ng-eq • h/ml for females. Total body clearance (CLtot) was 0.27 l/h/kg for males and 0.28 l/h/kg for females. The apparent volume of distribution at steady state (Vss) was 1.58 l/kg for males and 1.82 l/kg for females. Mean residence time (MRT) was 5.93 h in males, and 6.68 h in females.

Following a single oral administration of 100 µg/kg 14C-BPA to monkeys, the plasma concentration reached its maximum (Cmax) of 104 ng-eq/ml for males at 0.5 or 2.0 h postdose and 107 ng-eq/ml for females at 0.25 or 0.5 h postdose. The t1/2oral of the radioactivity in plasma was 9.63 h in males and 9.80 h in females. The AUCoral was 265 ng-eq•h/ml for males and 244 ng-eq•h/ml for females. Based on individual AUC values, the bioavailability (F) of the orally administered material was 0.70 for males and 0.66 for females.

Based on the results, it was concluded that when 14C-BPA was given orally to monkeys at a dose of 100 µg/kg, the oral absorption of this compound was rapid and high. The terminal elimination half-life (t1/2iv) following iv dosing was larger than t1/2oral following oral dosing; this may be due to deposition of lipophilic 14C-BPA in adipose tissue after iv dose, in contrast to first-pass metabolism after oral dose.

HPLC Analysis of 14C-BPA and Its Metabolites in Monkey Urine with Hydrolysis of Conjugates
Representative HPLC radiochromatograms of 14C-BPA and its metabolites in 0–12-h urine of male monkey after iv and oral administration of 100 µg/kg of 14C-BPA following nonhydrolysis, in vitro hydrolysis, and enzyme inhibition are presented in Figure 5Go. Radiochemical peaks were numbered consistently across the different samples, with a total of five peaks with different retention times resolved, and one peak region as the peaks P-1 (RRT = 0.16–0.19), P-2 (RRT = 0.71–0.73), P-3 (RRT = 0.79–0.82), P-4 (RRT = 0.90–0.93), and P-5 (BPA; RRT = 0.99–1). The results of radio-HPLC analyses of the 0–12-h and 12–24-h urine of male and female monkeys after iv or oral administration of 14C-BPA are summarized in Table 3Go. The major peaks found in nonhydrolyzed urine were two well-resolved peaks, P-1 and P-3, with relative peak areas ranging from 14 to 16% and 79 to 81% for 0–12-h urine, and 19 to 25% and 73 to 80% for 12–24-h urine, respectively. BPA peak was infrequently present at low levels between 0.0 and 1.5% in both genders (iv and oral dosing). Two additional minor peaks (P-2 and P-4) were observed at RRT = 0.71–0.73 and 0.90–0.93, with relative peak areas of 0.0–0.7% and 0.0–3.9%, respectively.



View larger version (20K):
[in this window]
[in a new window]
 
FIG. 5. HPLC radio-chromatograms of Bisphenol A and its metabolites in 0–12-h urine of male monkey after iv and oral administration of 100 µg/kg of 14C-BPA: nonhydrolysis, in vitro hydrolysis and enzyme inhibition of samples. The details were described in Materials and Methods.

 

View this table:
[in this window]
[in a new window]
 
TABLE 3 Composition of 14C-BPA and Its Metabolites in Monkey 0–12 h and 12–24 h Urine before and after in Vitro Hydrolysis
 
The nature of the major peaks, P-1 and P-3, was confirmed by incubation of urine samples with ß-G/Aryl, resulting in the disappearance of the peaks P-1 and P-3 and a corresponding increase of the P-5(BPA) in the chromatogram (Fig. 5Go and Table 3Go). From the HPLC profile, P-5 had the same retention time as the authentic standard of BPA when analyzed using separate injections and when employing HPLC co-chromatography. The effect of the glucuronidase (ß-G) was blocked by coincubation of urine samples with the specific inhibitor saccharic acid 1,4-lactone. The results suggest that both of the major metabolite peaks, P-1 and P-3, are glucuronide conjugates of 14C-BPA ,and the bigger one, P-3, corresponding to a relatively less polar metabolite, is monoglucuronide of BPA (BPA-gluc). This RRT was coincident with that of the standard BPA-gluc that was isolated in our laboratory from the bile of male rats orally dosed at 100 mg/kg BPA. It was suggested that another prominent peak (P-1) eluting at RRT = 0.16–0.19 corresponded to a relatively more polar metabolite, diglucuronide of BPA.

There was another possiblity that BPA sulfate was present in the monkey urine and its RRT was near that of P-4 (Fig. 5Go and Table 3Go). In 0–12-h urine of monkeys, the ratio of P-4 decreased by the hydrolysis of ß-G/Aryl with the specific ß-G inhibitor saccharic acid 1,4-lactone, suggesting it was BPA sulfate (Table 3Go). On the other hand, the P-4 in 12–24-h urine, the ratio of which increased after hydrolysis of ß-G/Aryl, might be librated 5-hydroxybisphenol A, as its RRT was near that of P-4. The remaining minor and infrequent peaks observed in monkey urine remained unidentified.

Radio-HPLC analysis of metabolites in urine samples suggested that the predominant urinary metabolites were mono- and diglucuronide conjugates of 14C-BPA in both genders and routes of administration.

HPLC Analysis and Quantitation of 14C-BPA and Its Metabolites in Monkey Plasma
The results of the HPLC analysis of BPA and its metabolites in the monkey plasma sample after iv and oral administration of 14C-BPA (100 µg/kg) are summarized at Table 4Go. There were clear differences in the concentration-time profiles of BPA in plasma, based on route of administration.


View this table:
[in this window]
[in a new window]
 
TABLE 4 Composition of BPA and Its Metabolites in Monkey Plasma after iv and Oral Administration of 14C-BPA
 
After iv (0.083–2 h) dosing, most of the plasma radioactivity was P-3, BPA-gluc (57–80% for males and 61–82% for females). Even at 5 min post-iv dose, the ratios of unchanged 14C-BPA (RRT = 1) in plasma were 27% for males and 29% for females. P-1 was not prominent in monkey plasma of either gender. The plasma levels of 14C-BPA–derived radioactivity were unquantifiable by radio-HPLC after 4-h (limit of quantitation, LOQ = 3 ng-eq. BPA/ml).

The plasma samples (0.5 and 2 h) of oral dosing showed that almost all of the plasma radioactivity was P-3, BPA-gluc (95 ± 8% for males and 92 ± 10% for females in Table 4Go). P-3, BPA-gluc was more predominant in plasma than in urine. The unchanged 14C-BPA was very low (0.0–1.4%) at 0.5 and 2 h following oral dosing. The other metabolite peaks were low in monkey plasma of both genders.

Toxicokinetics of 14C-BPA and Its Metabolites in Monkey Plasma at Fast Phase
At fast phase during 2 h after iv injection, the plasma levels of 14C-BPA declined more rapidly than those of 14C-BPA-gluc (P-3) and the other metabolites in males (Fig. 6Go) and females (Fig. 7Go). The fast-phase half-life (t1/2f) of total radioactivity was 0.61–0.67 h and the t1/2f of P-3 corresponding to BPA-gluc was 0.79–0.82 h (Table 5Go). The t1/2f of unchanged 14C-BPA (t1/2f = 0.39 h) for females was smaller than that (t1/2f = 0.57 h) for males (p < 0.05). This may be due to preferential distribution of 14C-BPA in lipophilic and/or estrogenophilic organs of females after iv dose.



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 6. Mean plasma levels of 14C-BPA and its metabolites in fast-phase post-iv dose of 14C-BPA 100 µg/kg in male cynomolgus monkeys. Data represent the mean ± SD of 3 animals. No error bar indicates the error was included within the symbol. Squares, total radioactivity; diamonds, BPA; circles, Peak 3; triangles, Peak 4; asterisks, Peak 1.

 


View larger version (19K):
[in this window]
[in a new window]
 
FIG. 7. Mean plasma levels of 14C-BPA and its metabolites in fast-phase post-iv dose of 14C-BPA 100 µg/kg in female cynomolgus monkeys. Data represent the mean ± SD of 3 animals. No error bar indicates the error was included within the symbol. Squares, total radioactivity; diamonds, BPA; circles, Peak 3; triangles, Peak 4; asterisks, Peak 1.

 

View this table:
[in this window]
[in a new window]
 
TABLE 5 Fast Phase Half Lives (t1/2f) of 14C-BPA, BPA-gluc, and Total Radioactivity in Male and Female Cynomolgus Monkeys after iv Dose of 14C-BPA at 100 µg/kg
 
At 11–160 ng-eq/ml (45–700 nM) of 14C-BPA–derived radioactivity distributed in the blood of both genders, the plasma protein binding was relatively strong, with 94.5 ± 0.6% of 14C-BPA–derived radioactivity bound to plasma protein (fp = 0.055).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Following iv or oral administration of 100 µg/kg 14C-BPA to male and female monkeys, 79–86% of the administered radioactivity was excreted in the urine over 7 days, and most of the urinary excretion was recovered by 24 h, especially within the first 12 h. The fecal excretion of radioactivity over 7 days was minimal (1.8–3.1% of the administered dose). Because the fecal excretion was very small after oral dose, it is considered that the absorption should be complete.

The current studies on the metabolism and toxicokinetics of BPA showed some notable differences between the results from monkeys and the works on rats (Pottenger et al., 2000Go; Snyder et al., 2000Go). In the current study, regardless of iv or oral route of administration, a much smaller fraction of the dose was excreted via the feces in monkeys than in rats. The fraction of the administered dose excreted via the urine or feces was dependent upon species without any marked gender differences. One possible explanation is that the monkey, with a larger fraction of the dose in systemic circulation, had a larger amount of the administered dose available for renal excretion. Cmax, systemic plasma concentration, and AUC of 14C-BPA–derived radioactivity appeared to be much higher in monkeys than in rats following oral administration of 14C-BPA.

Knaak and Sullivan (1966) reported that 56% of the orally administered dose was excreted in feces of rats by 8 days after a high dose of 800 mg BPA/kg. Snyder, et al. (2000) reported that female CD rats excreted 70% of the radioactivity in feces, whereas F-344 rats excreted 50% by 6 days after oral dose of 100 mg/kg 14C-BPA, and reported that free BPA accounted for most of the radioactivity recovered in feces and other metabolites accounted for no more than 2% of the total radioactivity. These observations are consistent with results reported by Pottenger et al. (2000). As free BPA is the major component detected in feces, one possibility is that the radiolabel may not have been absorbed, and passed through the gut unchanged into the feces. Alternately, the higher fraction excreted via the feces in these works with relatively high doses of 100–800 mg BPA/kg is likely due to the saturation of metabolic or excretory mechanisms responsible for the elimination via the urinary route. The other possibility is that BPA-gluc may be transported into the intestine via bile and hydrolyzed, resulting in the fecal excretion of free BPA. Pottenger et al. (2000) proposed a binding to plasma proteins or an enterohepatic circulation of 14C-BPA–derived radioactivity to account for the longer elimination phase of plasma radioactivity compared with the parent compound in rats. The plasma protein binding of radioactivity was relatively strong, with 94.5 ± 0.6% for monkeys and 93.2 ± 1.9% for rats after iv dose of 100 µg/kg 14C-BPA (Kurebayashi et al., in preparation). There was no significant difference between them.

Radio-HPLC analysis demonstrated that the most predominant metabolite (P-3) in urine and plasma was 14C-BPA-gluc after iv or oral dosing to male and female monkeys (Fig. 5Go and Table 3Go). A more polar metabolite (P-1), diglucuronide of BPA, was not so prominent in plasma as in urine (0–12-and 12–24-h urine in Tables 3 and 4GoGo), and this metabolite might be formed slowly. 14C-BPA and other metabolites were detected in monkey urine at levels much lower than 14C-BPA-gluc.

After iv dose in monkeys, the fast-phase half-life (t1/2f) of unchanged 14C-BPA was smaller than those of total radioactivity and 14C-BPA-gluc (Figs. 6 and 7GoGo; Table 5Go). The plasma levels of 14C-BPA more rapidly declined by distribution and metabolism, and 14C-BPA-gluc was a major metabolite, formed from 14C-BPA and decreased by metabolism to P-1 and excretion into urine. The t1/2f of 14C-BPA for females(0.39 h) was smaller than t1/2f for males (= 0.57 h; Table 5Go). The plasma levels of 14C-BPA declined more rapidly in females than in males. This may be due to rapid preferential distribution of 14C-BPA into lipophilic and/or estrogenophilic organs of females after iv dose. On the other hand, the terminal t1/2iv of the radioactivity was larger following iv dosing than t1/2oral following oral dosing (Table 2Go). This may be due to the deposition of lipophilic 14C-BPA in adipose tissue after iv administration, in contrast to first-pass metabolism to 14C-BPA-gluc after oral administration.

As previously reported (Pottenger et al., 2000Go; Snyder et al., 2000Go), BPA-gluc is the major metabolite of 14C-BPA in urine and plasma of rats. In the present study, bolus gavage of 14C-BPA resulted in BPA-gluc as the major 14C-labeled product in plasma of monkeys. As BPA-gluc is thought to be biologically inactive, rapid conversion of BPA to BPA-gluc results in low bioavailability of free BPA, which is thought to be the active form. Taken together, these findings suggest that glucuronidation is the primary phase II biotransformation pathway for BPA, both in rats and in nonhuman primates.

In this study, systemic plasma concentration of free BPA was very low in monkeys after oral treatment with 100 µg/kg. As 14C-BPA was rapidly metabolized to BPA-gluc, first-pass metabolism is the likely explanation for the low systemic bioavailability of 14C-BPA following oral treatment. This high metabolic clearance by first-pass metabolism could be mediated by both intestinal and hepatic enzymes. The contribution of the hepatic UDP-glucuronosyl transferase is supported by the rapid clearance of plasma 14C-BPA and the formation of BPA-gluc after iv dosing (Tables 4 and 5GoGo). The in vitro formation of BPA-gluc was shown by incubation of BPA with male rat liver microsomes (Yokota et al., 1999Go).

From plasma concentration profiles of total radioactivity and because plasma levels of radioactivity seemed much higher in monkeys than in rats after oral dosing with 14C-BPA, we thought that the biliary excretion of 14C-BPA-gluc was quantitatively unimportant in monkeys compared with rats. This species difference in the biliary excretion of BPA metabolite is a possible explanation. 14C-BPA was metabolized principally to 14C-BPA-gluc in monkeys and rats. 14C-BPA-gluc appeared to be excreted mainly into the bile in rats via the first-pass effect, but not in monkeys. One reason for this could be the well-known difference between rats and primates in molecular threshold for biliary excretion of xenobiotics. According to Hirom et al. (1977), for organic anions such as glucuronides, the minimum molecular weight at which biliary elimination becomes appreciable is about 325 ± 50 for rats and about 500 for humans. Therefore BPA-gluc, with a molecular weight of 404, may be eliminated in primates mainly through the kidney rather than in bile. It was suggested that in rodents, BPA-gluc undergoes enterohepatic recirculation; thus the enterohepatic circulation of 14C-BPA–derived radioactivity may explain the longer elimination phase of plasma radioactivity.

Pottenger et al. (2000) also showed the differences in plasma radioactivity profiles between oral, ip, and sc administration at high doses of 10–100 mg BPA/kg in rats. The bioavailability of 14C-BPA–derived radioactivity demonstrated a clear dependence on the route of administration, with oral administration resulting in the lower bioavailability of both parent compound and 14C-BPA–derived radioactivity. Parent BPA was the major component at Tmax in rat plasma following sc and ip administration, but the profile was very different from oral dosing where BPA-gluc was the major peak. This route dependency in the relative bioavailability likely offers a toxicokinetic explanation for apparent route differences in estrogenic potency (i.e., uterine changes) that have been observed for BPA in female rats. Specifically, recent studies have demonstrated a clear route dependency in the magnitude of the uterotropic response to BPA, where significantly larger oral doses were required to produce an estrogenic response than those required subcutaneously (Laws and Carey, 1997Go; Twomey, 1998aGo,bGo). The route dependency in parent compound bioavailability explains the route dependency in the uterotropic response to BPA, based on the assumption that free parent compound is responsible for the estrogenic effects of BPA. Recently, it has been reported that the BPA-gluc was not a ligand in vitro for either the human or mouse estrogen receptor, nor did it have estrogenic activity as measured in a reporter cell assay that measures gene expression from chimeric estrogen receptors transiently transfected into MCF-7 cells (Matthews et al., 2001Go).

Estrogenic potency values derived from in vitro assays are dependent on the principles of the assay, and as such, may not take into account species-specific parameters such as biotransformation or tissue distribution of the test compound. Determination of the toxicokinetic parameters of potentially biologically active contaminants is a prerequisite for risk assessment purposes, as these parameters are fundamental in establishing in vivo activity and lend further biological significance to environmental data. After absorption, the lipophilic nature of many persistent environmental contaminants predisposes to accumulation, whereas intrinsic hormonal activity may be appreciably modulated by biotransformation. The potency of the natural estrogens is significantly reduced by conjugation to glucuronic and sulfuric acids (Klein et al., 1994Go). The findings in this study suggest that glucuronidation is the primary phase II biotransformation pathway for BPA in the rat and in nonhuman primates, and the primary detoxication pathway of BPA.

Based on the results, it was concluded that when 14C-BPA was given orally to monkeys, the intestinal absorption of this compound was rapid and high. Following oral or iv administration, 14C-BPA was easily metabolized to glucuronide conjugates of 14C-BPA and principally excreted in urine during the first 24 h. The route-dependent difference (t1/2iv > t1/2oral) and gender-dependent difference of t1/2f of 14C-BPA suggested the preferential distribution of lipophilic 14C-BPA in adipose tissue after iv dosing, in contrast to first-pass metabolism after oral dosing.


    ACKNOWLEDGMENTS
 
This study was supported in part by a grant of the Japanese Ministry of Health and Welfare.


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


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ashby, J., Tinwell, H., and Haseman, J. (1999). Lack of effects for low dose levels of bisphenol A and diethylstilbestrol on the prostate gland of CF1 mice exposed in utero. Regul. Toxicol. Pharmacol. 30, 156–166.[ISI][Medline]

Atkinson, A., and Roy, D. (1995). In vivo DNA adduct formation by bisphenol A. Environ. Mol. Mutagen. 26, 60–66.[ISI][Medline]

Bond, G. P., McGinnis, P. M., Cheever, K. L., Harris, S. J., Platnick, H. B., and Neimeier, R. W. (1980). Reproductive effects of bisphenol A. Toxicologist 19, A23 (Abstract).

Brotons, J. A., Olea-Serrano, M. F., Villalobos, M., Pedraza, V., and Olea, N. (1995). Xenoestrogens released from lacquer coatings in food cans. Environ. Health Perspect. 103, 608–612.[ISI][Medline]

Cagen, S. Z., Waechter, J. M., Jr., Dimond, S. S., Breslin, W. J., Butala, J. H., Jekat, F. W., Joiner, R. L., Shiotsuka, R. N., Veenstra, G. E., and Harris, L. R. (1999). Normal reproductive organ development in CF-1 mice following prenatal exposure to bisphenol A. Toxicol. Sci. 50, 36–44.[Abstract]

Colborn, T., vom Saal, F. S., and Soto, A. M. (1993). Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ. Health Perspect. 101, 378–384.[ISI][Medline]

Davis, D. L., Bradlow, H. L., Wolff, M., Woodruff, T., Hoel, D. G., and Anton-Culver, H. (1993). Medical hypothesis: Xenoestrogens as preventable causes of breast cancer. Environ. Health Perspect. 101, 372–377.[ISI][Medline]

Field, B., Selub, M., and Hughes, C. L. (1990). Reproductive effects of environmental agents. Semin. Reprod. Endocrinol. 8, 44–54.[ISI]

Gaido, K. W., Leonard, L. S., Lovell, S., Gould, J. C., Babai, D., Portier, C. J., and McDonnell, D. P. (1997). Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. Toxicol. Appl. Pharmacol. 143, 205–212.[ISI][Medline]

Gibaldi, M., and Perrier, D. (1982). Pharmacokinetics, 2nd ed. Marcel Dekker, New York.

Gould, J. C., Leonard, L. S., Maness, S. C., Wagner, B. C., Conner. K., Zacharewski, T., Safe, S., McDonnell, D. P., and Gaido, K. W. (1998a). Bisphenol A interacts with the estrogen receptor in a distinct manner from estradiol. Mol. Cell. Endocrinol. 142(Suppl. 1-2), 203–214.[ISI][Medline]

Hirom, P. C., Idle, J. R., and Millburn, P. (1977). Comparative aspects of the biosynthesis and excretion of xenobiotic conjugates by non-primate mammal. In Drug MetabolismFrom Microbe to Man (D. V. Parke, and R. L. Smith, Eds.). pp. 299–329. Taylor & Francis, London.

Hoiberg, C. P., and Mumma, R. O. (1969). Preparation of sulfate esters. Reactions of various alcohols, phenols, amines, mercaptans and oximes with sulfuric acid and dicyclohexylcarbodiimide. J. Am. Chem. Soc. 91, 4273–4278.[ISI]

Klein, K. O., Baron, J., Colli, M. J., McDonnell, D. P., and Cutler, C. B., Jr. (1994). Estrogen levels in childhood determined by an ultrasensitive recombinant cell bioassay. J. Clin. Invest. 94, 2475–2480.[ISI][Medline]

Knaak, J. B., and Sullivan, L. J. (1966). Metabolism of bisphenol A in the rat. Toxicol. Appl. Pharmacol. 8, 175–184.[ISI][Medline]

Krishnan, A. V., Stathis, P., Permuth, S. F., Tokes, L., and Feldman, D. (1993). Bisphenol A: An estrogenic substance is released from polycarbonate flasks during autoclaving. Endocrinology 132, 2279–2286.[Abstract]

Laws, S. C., and Carey, S. A. (1997). Comparison of the estrogenic activity of 4-tertoctylphenol, nonylphenol, bisphenol A and methoxychlor in Long Evans female rats. Toxicologist 36, 359.

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 beta. Chem. Res. Toxicol. 14, 149–57.[ISI][Medline]

McLachlan, J. A. (1993). Functional toxicology: A new approach to detect biologically active xenobiotics. Environ. Health. Perspect. 101, 386–387.[ISI][Medline]

Morrissey, R. E., George, J. D., Price, C. J., Tyl, R. W., Marr, M. C., and Kimmel, C. A. (1987). The developmental toxicity of bisphenol A in rats and mice. Fundam. Appl. Toxicol. 8, 571–582.[ISI][Medline]

Nagel, S. C., vom Saal, F. S., Thayer, K. A., Dhar, M. G., Boechler, M., and Welshons, W. V. (1997). Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol. Environ. Health Perspect. 105, 70–76.[ISI][Medline]

Olea, N., Pulgar, R., Pérez, P., Olea-Serrano, F., Rivas, A., Novillo-Fertrell, A., Pedraza, V., Soto, A. M., and Sonnenschein, C. (1996). Estrogenicity of resin-based composites and sealants used in dentistry. Environ. Health Perspect. 104, 298–305.[ISI][Medline]

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]

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.[ISI][Medline]

Steinmetz, R., Brown, N. G., Allen, D. L., Bigsby, R. M., and Ben-Jonathan, N. (1997). The environmental estrogen bisphenol A stimulates prolactin release in vitro and in vivo. Endocrinology 138, 1780–1786.[Abstract/Free Full Text]

Twomey, K. (1998a). Bisphenol A: Uterotropic assay in immature rats (subcutaneous dosing). Report to the Bisphenol A Global Industry Group from Central Toxicology Laboratory, Cheshire, U. K. [CTL Report No. CTL/P/5943].

Twomey, K. (1998b). Bisphenol A: Uterotropic assay in immature rats (oral dosing). Report to the Bisphenol A Global Industry Group from Central Toxicology Laboratory, Cheshire, U. K. [CTL Report No. CTL/P/6029].

vom Saal, F. S., Cooke, P. S., Buchanan, D. L., Palanza, P., Thayer, K. A., Nagel, S. C., Parmigiani, S., and Welshons, W. V. (1998). A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm production, and behavior. Toxicol. Ind. Health 14, 239–260.[ISI][Medline]

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