Abstract
This study investigated the kinetics of glucuronidation of bisphenol A (BPA; 4,4′-dihydroxy-2,2-diphenylpropane) in cryopreserved human hepatocytes (HCs). Incubation conditions were developed using Sprague-Dawley rat HCs. For determination of the kinetic constants of BPA glucuronidation rates with human HCs, viable HCs (0.125 × 106) were incubated with [14C]BPA (1.3-52 μM) for 10 min. The glucuronidation reaction demonstrated Michaelis-Menten kinetics and yielded a mean Km for males and females of 9 ± 3 and 8 ± 2 μM, respectively. The Vmax values of these reactions were 438 ± 129 pmol/min/106 for male HCs and 480 ± 208 pmol/min/106 for female HCs. The scaled intrinsic clearance (CLint) for male human HCs was 149 ± 67 ml/min/kg (range 53-246) and for female HCs was 165 ± 89 ml/min/kg (range 73-336). Overall, there are no apparent gender differences in the glucuronidation of BPA. These CLint values were then extrapolated to estimate total hepatic metabolic clearance (CLmet) using a nonrestrictive well stirred model. The estimated CLmet value for both male and female HCs was 6 ml/min/kg, which represents 30% of hepatic blood flow. Thus, in vivo clearance seems to depend highly on plasma protein binding. These in vitro results correlate well with in vivo studies in humans, which report extensive glucuronidation of BPA.
Bisphenol A (BPA; 4,4′-dihydroxy-2,2-diphenylpropane) has a number of industrial and commercial applications, including its use as a monomer in the manufacture of epoxy resins and polycarbonate plastic. Because of the use of these materials in consumer products, humans may be exposed to low levels of BPA (Kang et al., 2006). Evidence that humans are exposed to BPA is apparent from studies that measured it and/or its metabolites in human urine (Calafat et al., 2005).
BPA is referred to as a xenoestrogen because it can interact with intracellular estrogen receptors. Such binding is functional, since exposure to BPA can result in uterine changes in female rats at sufficiently high oral doses, or at lower doses when administered parenterally (Morrissey et al., 1987; Laws et al., 2000). Unlike 17β-estradiol, this interaction does not affect cell proliferation in the human endometrial cell line, ECC-1 (Gould et al., 1998; Bergeron et al., 1999). However, BPA can cause estrogen-mediated effects at high doses, and low-dose effects have been reported (vom Saal et al., 1997).
The hydroxyl moieties present on BPA provide functional groups for glucuronidation (Fig. 1). Indeed, in rodents and humans, BPA is extensively converted to BPA-glucuronide (BPAG), which is the major circulating metabolite and major excreted metabolite (Knaak and Sullivan, 1966; Pottenger et al., 2000; Snyder et al., 2000; Völkel et al., 2002, 2005). Efficient glucuronidation would serve as a first-pass effect for BPA and would reduce systemic exposure to BPA (Inoue et al., 2001). In general, glucuronidation is considered to be a detoxication mechanism. In addition, BPAG does not bind to the estrogen receptor (Snyder et al., 2000; Matthews et al., 2001).
Because of the critical role glucuronidation plays in the detoxification and elimination of BPA, this study was designed to estimate the kinetic parameters of BPA glucuronidation by human hepatocytes (HCs). Such kinetic parameters can be used in physiologically based pharmacokinetic models to assess tissue exposure to ingested BPA and to estimate boundaries of variability of BPA glucuronidation in humans. Because of the cost and limited availability of human HCs, male Sprague-Dawley (SD) rat HCs were used to develop the incubation conditions for human HCs. Incubation with rat HCs assessed how cryopreservation, cell number, incubation time, and exogenous UDPGA influenced the intrinsic metabolic clearance of BPA via glucuronidation. Based on the results of these experiments, cryopreserved human HCs from males (eight) and females (seven) were incubated with BPA for determination of the kinetic parameters of BPA glucuronidation.
Materials and Methods
Chemicals. [ring-14C(U)]BPA (1.0 μCi/ml; 4.0 mg/ml ethanol) with specific radioactivity of 200 mCi/mmol was provided by Moravek Biochemicals (Brea, CA). Purity of [14C]BPA was determined by reverse phase HPLC to be 98%. Unlabeled BPA was supplied by RTI International (Research Triangle Park, NC; lot 9176-10-02) and was confirmed by HPLC to be 99% pure. The purity of dosing solutions was confirmed by HPLC and reassessed during each incubation. BPAG standard was provided by The Dow Chemical Company (Midland, MI). The following reagents were purchased from the vendors indicated: Flo-Scint III from PerkinElmer Life and Analytical Sciences (Boston, MA); Universol Cocktail from ICN Radiochemicals (Irvine, CA); HPLC-grade acetonitrile from Honeywell Burdick & Jackson, Inc. (Muskegon, MI); Williams' Medium E (WME) without phenol red from Invitrogen (Carlsbad, CA); and fetal bovine serum from HyClone (Logan, UT). Collagenase B was purchased from Boehringer Mannheim, (Mannheim, Germany; lot 83229923).
Biological Materials.Animals. Male SD rats (200 -250 g, 70 days old) were purchased from Harlan (Indianapolis, IN). All animals were maintained in an Association for Assessment and Accreditation for Laboratory Animal Care-approved animal care facility for at least 1 week before experimentation. During acclimatization, four animals were housed per cage in hanging steel cages with a pelleted feed hopper and a water bottle. The animals had free access to food (Harlan Teklad 4% mouse/rat diet; Harlan Teklad, Madison, WI) and water. This feed is routinely analyzed by the manufacturer for nutritional components and environmental contaminants.
Care of the animals was in accordance with the Guide for the Care and Use of Laboratory Animals (ILAR, 1996). Animals were housed in a facility with an air circulation of 15 fresh filtered air changes per hour and with no interchange between rooms. Room temperature was maintained between 68 and 74°F and the relative humidity between 40 and 60%. A light/dark cycle was maintained at 12-h intervals. Animals were provided deionized, reverse osmosis water ad libitum.
Hepatocyte Isolation. To isolate HCs in our laboratory, rats were anesthetized with sodium pentobarbital (i.p., 50 mg/kg), and the portal veins were cannulated and livers perfused as described by Pritchett et al. (2002). Only those cell preparations with >90% viability (as determined by trypan blue exclusion) were used for primary culture.
Rat Hepatocytes (Commercial Sources). To evaluate whether cryopreservation and/or delivery time had an effect on the ability of HCs to glucuronidate BPA, metabolic constants (Vmax and Km) derived from freshly isolated male SD rat HCs were compared with those from noncryopreserved and cryopreserved rat HCs purchased from BD Gentest (Woburn, MA). Suspensions of SD rat HCs isolated by the vendor were shipped (for arrival within 18 -24 h) to our laboratory in 50 ml of shipping media. Upon arrival, HCs were diluted 1:1 with WME (37°C, pH 7.4) and subjected to low-speed centrifugation (50g). In addition, HCs from the same isolations were cryopreserved and then shipped to the laboratory. They were stored in liquid nitrogen until use.
Immediately before experimentation, single vials of cryopreserved HCs were thawed in a 37°C water bath. Suspensions of cells were transferred into two 15-ml centrifuge tubes containing 7 ml of WME (pH 7.4, 37°C). Samples were subjected to low-speed centrifugation (2 min, 50g). The supernatant was removed and the pellets were combined and suspended in 4 ml of WME. Viable cells (as determined by trypan blue dye exclusion) were diluted with WME to appropriate concentrations allocated into 24-well tissue culture plates and allowed to stabilize for 10 min in a 37°C incubator (95% air/5% CO2) incubator.
Human Hepatocytes. Cryopreserved male and female human HCs were obtained from three sources, In Vitro Technologies (Baltimore, MD), BD Gentest, and XenoTech (Lenexa, KS). Vital statistics and health information for donors of human hepatocytes are provided in Supplemental Data. Samples were selected for use on the basis of gender and cell viability, after thawing, of greater than 70%. Human HCs were obtained from either portions of liver or whole livers using a procedure similar to the collagenase perfusion technique used for rodents. Medical histories provided by the vendors are in Supplemental Data Tables 1 and 2. Cryopreserved samples were treated as described above.
Bisphenol A Glucuronidation Reaction. On the basis of previous studies (Pritchett et al., 2002), noncytotoxic concentrations of BPA (<75 μM) were used for metabolism studies. Reactions were terminated by snap-freezing in liquid nitrogen (2 min) and then were stored at -80°C until analysis. For analysis, the HCs in media were thawed and then centrifuged for 4 min at 12,000 rpm to obtain supernatant. Lysis of the HCs by this procedure results in quantification of both BPA and BPA metabolites in both media and cells.
To optimize the concentration of HCs to be used for subsequent incubations with human HCs, viable male SD rat HCs (62,500, 125,000, or 375,000 cells/0.5 ml) were suspended in serum-free WME, stabilized (10 min, 5% CO2, 95% air), and then incubated with [14C]BPA at 37°C. For all experiments, metabolism was initiated by the addition of a stock solution of [14C]BPA dissolved in dimethyl sulfoxide.
For concentration-dependent metabolism studies, [14C]BPA concentrations (0.6 -52 μM with 0.45 μCi/incubation) were used. Every treatment solution contained 100 μl of [14C]BPA stock solution (1.0 mCi/ml; 4.0 mg/ml ethanol) with a specific radioactivity of 200 mCi/mmol, and various amounts of unlabeled BPA and dimethyl sulfoxide, resulting in six different specific activities (mCi/mmol). Rates of BPA metabolism were determined at 10 min. For the determination of BPAG formation over time, suspensions containing viable cells (as determined by trypan blue dye exclusion) were incubated with [14C]BPA (2.6 μM) for 5 to 60 min.
Analysis of Samples. After storage at -80°C, the lysed samples were transferred to 2-ml centrifuge tubes, vortexed for 30 s, and then centrifuged for 4 min at 12,000 rpm. Aliquots of the supernatant were injected (50 μl) onto a Phenomenex (Torrance, CA) Luna 5 μm C-18 analytical column (250 mm × 4.6 mm) and eluted with a mobile phase consisting of water/acetonitrile, both containing 0.1% acetic acid. The HPLC system was composed of an Agilent 1100 quaternary pump, refrigerated autosampler, and a diode array detector (Agilent Technologies, San Jose, CA). The column effluent was monitored in tandem with the diode array detector at a wavelength of 280 nm and radiochemically with a β-RAM Flow-Through Monitor System (IN/US Systems, Inc., Tampa, FL). The flow rate was 1 ml/min with a 10-min run time. The mobile phase gradient started with 40% acetonitrile and 60% water from 0 to 5 min, increased to 95% acetonitrile from 5 to 7 min, and decreased to 75% from 7 to 10 min. The column was then brought back to initial conditions (40%:60%) for a period of 10 min before injection of the next sample. Bisphenol A eluted at approximately 6 min under these conditions. For radiometric analysis, a flow cell with a volume of 500 μl and a scintillation fluid flow rate of 3 ml/min was used. The efficiency of the β-RAM Flow-Through Monitor System was 92% and monitored throughout the course of these experiments by collection of column effluent, which was counted directly on an LS5000TD Beckman liquid scintillation counter (Beckman Coulter, Fullerton, CA).
LC-MS/MS. For structural confirmation of the BPAG, unlabeled BPA was incubated with cryopreserved human HCs, and the samples were prepared and analyzed as described above. The HPLC procedure was similar to the method described above except for an additional 5 min of 40% acetonitrile and 60% water at the beginning and end of the run, which resulted in a run time of 20 min with an elution time of 9.7 min for BPAG.
Initial screening of each sample was done in the first quadrupole based on MS measurements only (Q1 MS mode). The Q1 MS parameters were full-scan type between 50 and 550 m/z; negative polarity. After the Q1 MS scan, single ion monitoring and MS/MS analyses were performed using flow injection at a rate of 0.5 ml/min. The center masses in single ion monitoring mode were as follows: 402.7 to 403.7 for BPAG and 227.30 for BPA; 403.2 was selected as the parent mass for MS/MS at a collision energy of 20 eV, which collected all product ions between 50 and 450 m/z. The MS/MS spectrum of a BPAG standard was compared with that obtained for the biologically derived samples.
Data Analysis. The data obtained were subjected to analysis based on Michaelis-Menten kinetics. A software program for hyperbolic regression analysis of enzyme kinetic data (copyright 1992-1993, J. S. Rasterby) was used. This program determines the Km and Vmax values from enzyme-kinetic data using nonlinear regression analysis. The results from the hyperbolic regression were used to determine the intrinsic clearance (CLint = Vmax/Km), which is expressed in volume/time.
To model CLint to in vivo hepatic clearance (CLmet), the data were subjected to analysis using the nonrestrictive well stirred model described by Ito and Houston (2004). This model incorporates the following: the free fraction of BPA in plasma [0.06, (fu); Teeguarden et al., 2005], the fraction of BPA unbound to microsomal protein [calculated as 0.94 (fuinc); Hallifax and Houston 2006], hematocrit (0.4 for human; Guyton, 1991), and human hepatic blood flow [20 ml/min/kg body weight; (Qh); Soars et al., 2002]. Physiological parameters were human liver weight (20 g/kg body weight) and hepatic cellularity (120 × 106 cells/kg body weight) from Soars et al. (2002, 2003).
Results
The initial experiments focused on the metabolic profile of BPA upon incubation with cryopreserved human or freshly isolated rat hepatocytes. Under the conditions of these experiments, human and rat HCs formed only a single metabolite (Fig. 2). The formation of this metabolite was linear from 10 to 60 min and stoichiometric with the disappearance of BPA for both human (Fig. 3) and rat (data not presented) HCs.
This metabolite coeluted with the synthetic standard for BPA-monoglucuronide. To further confirm the chemical nature of this metabolite, cryopreserved human HCs were incubated with unlabeled BPA. LC-MS/MS analysis of the peak that eluted at 5.5 min revealed a fragmentation pattern identical to that previously reported for BPA-monoglucuronide (Pritchett et al., 2002) Fragmentation of the parent ion, m/z 402 (BPAG), produced ions of 174 (glucuronide) and m/z 227 (BPA) (Fig. 4).
Male SD rat HCs were used to further develop incubation conditions to be used for kinetic analysis of BPA glucuronidation by human HCs. Specifically, the effects of cryopreservation, cell concentration, and exogenous UDPGA were investigated. SD rat HCs that were prepared by the vendor and shipped overnight had a reduced Vmax for glucuronidation of BPA compared with that of HCs isolated in-house and used immediately (Table 1). Cryopreserved HCs prepared from the same livers as nonfrozen, shipped HCs had a Vmax for glucuronidation of BPA similar to that of freshly isolated HCs. Based on these results, cryopreserved human HCs were used for kinetic analysis of BPA glucuronidation.
Cell concentration was found to affect the Vmax and CLint of BPA (Table 2). Although experimental conditions were similar in Tables 1 and 2, variability was observed in the Vmax of HCs isolated in-house with 375,000 HCs/ml. As cell concentration increased from 62,500 to 375,000 HCs/0.5 ml, Vmax and CLint decreased. Although Vmax and CLint were highest at 62,500 HCs/0.5 ml, a concentration of 125,000 HCs/0.5 ml was used for kinetic analysis. This cell concentration provided greater analytical sensitivity for analysis of BPAG. Finally, addition of 1000 μM UDPGA to rat HCs greatly increased BPA glucuronidation (2-fold increase in Vmax; data not shown).
Glucuronidation of BPA by Human Hepatocytes. The conditions for determination of kinetic constants of BPA glucuronidation with suspensions of cryopreserved human HCs were as follows. HCs (125,000) were incubated with [14C]BPA (0.66 -52 μM) for 10 min in a shaking water bath (70 oscillations/min, 37°C). HCs were incubated with or without the addition of UDPGA (0, 50, or 1000 μM).
As shown in Fig. 5, when various concentrations of [14C]BPA were incubated with cryopreserved human HCs, the formation of the BPAG followed Michaelis-Menten kinetics. Identical data sets were generated for HCs from eight males and seven females. The Vmax for the human HCs, derived from male donors, ranged from 266 to 616 pmol/min/106 HCs (mean ± S.D. of 438 ± 129). The Km values ranged from 6 to 14 μM with a mean of 9 ± 3 μM (Table 3). The CLint (Vmax/Km) values for each individual specimen ranged from 20 to 93 μl/min/106 HCs. To extrapolate CLint to a more realistic assessment of in vivo metabolic clearance, the data were subjected to analysis using the nonrestrictive well stirred model described by Soars et al. (2002). This model incorporates such physiological parameters as human liver weight, hepatocellularity, hepatic blood flow, and plasma protein binding to model CLint to CLmet. These extrapolations resulted in a scaled CLint with a mean ± S.D. of 149 ± 67 ml/min/kg andaCLmet with a mean ± S.D. of 6 ± 2 ml/min/kg.
The metabolic constants of BPA glucuronidation by human HCs from female donors are summarized in Table 4. The Vmax for female HCs ranged from 234 to 840 pmol/min/106 cells and the Km of these HCs ranged from 5 to 12 with a mean ± S.D. of 8 ± 2 μM. The scaled CLint of the samples from human females ranged from 73 to 336 ml/min/kg with a mean ± S.D. of 165 ± 89 ml/min/kg. The mean CLmet was 6 ± 2 ml/min/kg for HCs obtained from either male or female donors and reflects a liver extraction ratio of 0.3.
Effects of Exogenous UDPGA on the Glucuronidation of BPA by Human Hepatocytes. When exogenous UDPGA (0, 50, or 1000 μM) was added to human cryopreserved HCs from male donors before incubation with [14C]BPA, the CLint (Vmax/Km) increased from 53 ± 26 to 151 ± 122 and 215 ± 88 μl/min, for 0, 50, and 1000 μM, respectively. Similar increases in CLint were seen in HCs obtained from females (59 ± 31 to 67 ± 32 and 172 ± 103 μl/min). These increases in CLint resulted in an increase in CLmet from 30% (no exogenous UDPGA) of hepatic blood flow (20 ml/min) to 63 and 59% (1000 μM UDPGA) for males and females, respectively (Tables 3 and 4).
Discussion
Because of the important role of hepatic UGTs in the biotransformation of BPA, the goal of these studies was to characterize the rate of BPA glucuronidation in HCs derived from rats and humans. The work reported herein defines BPAG as the only metabolite formed during the initial disappearance of BPA from the incubation medium in suspensions of HCs derived from rats and humans. Similar results were reported by Pritchett et al. (2002) using monolayers of rat and human HCs. Although a sulfate and a diglucuronide of BPA were formed by monolayers, BPAG was the only metabolite detected in the first 15 min of incubation. The in vitro results presented for suspensions of human HCs also correlate directly with in vivo studies of BPA performed in humans. Völkel et al. (2002, 2005) reported that BPAG was the exclusive metabolite of BPA detected in the plasma and urine of these human subjects.
In all rat and human samples, the formation of BPA-glucuronide followed Michaelis-Menten kinetics. Because of the similarity in glucuronidation of BPA between freshly isolated and cryopreserved SD rat HCs and the availability of cryopreserved suspensions of human HCs, cryopreserved HCs were developed as the model system for kinetic analyses. Certain technical parameters were shown to affect the rate of glucuronidation in HC suspensions. These include cell concentration, shipping of nonfrozen HC preparations, and addition of UDPGA to HC incubations.
The inverse relationship of increasing cell concentration on the rate of glucuronidation of BPA is most likely a result of a decrease in the per cell concentration of BPA. At lower intracellular concentrations of BPA (as occurs when cell density increases), saturation of the enzyme would not be maintained, particularly as glucuronidation proceeds. Higher concentrations of cells could also result in some cellular clumping. Overall, as hepatocellularity increases, a decrease in the HC surface area available for diffusion of BPA into the cells could occur. An increase in cell number could also promote nonspecific binding of BPA. Nonspecific binding to other proteins would decrease the amount of free BPA available for binding to and metabolism by UGTs. In fact, in both humans and rats, BPA has been reported to be bound to plasma proteins, resulting in an unbound free fraction of 0.06% (Teeguarden et al., 2005). Similar cell concentration-dependent decreases in scaled CLint of triazolam, diazepam, and clonazepam by dog HCs were reported by Jones and Houston (2004). It was shown that increasing cell concentrations from 0.5 × 106 to 4 × 106 cells/ml resulted in a 2-fold reduction in scaled CLint. Interestingly, Jones and Houston (2004) recommended similar incubation times and HC concentrations to yield statistically better predictions of in vivo values of 35 drugs. Thus, the kinetic calculations presented herein were derived from incubations that contained 125,000 HCs/0.5 ml.
Since the goal of these studies was to determine the glucuronidation of BPA in commercially available human HCs, the ability of commercially available SD rat HCs to glucuronidate BPA was investigated. Both cryopreserved and nonfrozen shipped SD rat HCs maintained their ability to glucuronidate BPA. However, shipped nonfrozen HCs had a decreased Vmax with respect to BPA glucuronidation. This decreased glucuronidation capacity may have resulted from a loss in endogenous glucuronic acid during the 16- to 18-h transit time. It also could have resulted from an alteration in enzyme activity. In vitro studies with microsomes and HCs have shown that a carrier-mediated transporter of UDPGA plays a regulatory role in hepatic glucuronidation (Bossuyt and Blanckaert, 1997). Whether or not this transporter was affected during transit is not known. Importantly, these studies demonstrated that cryopreserved rat HCs maintained rates of glucuronidation similar to those observed in freshly isolated HCs. Therefore, it appears that cryopreserved human HCs are an appropriate model for evaluating BPA glucuronidation in an intact cellular system.
When exogenous UDPGA was added to suspensions of male and female human HCs, an increase in the glucuronidation rates of BPA was observed. It is not known whether these increased rates, caused by addition of UDPGA, are reflective of the in vivo conditions or whether they are artifactual. The isolation procedure may result in some loss of UDPGA and/or some UDPGA may leak from the cells during incubation. Dependence of glucuronidation rate on UDPGA levels has been reported in isolated HCs. A decreased glucuronidation of benzo(a)pyrene has been reported in HCs after a reduction in UDPGA (Singh and Schwarz, 1981). When sufficient human HCs were available, incubations were conducted with (50 and 1000 μM) and without addition of UDPGA. In those studies, the 50 μM UDPGA should be considered more realistic because it reflects the intracellular concentration of UDPGA (Croci and Williams, 1985). The 1000 μM UDPGA would reflect glucuronidation in the presence of an unlimited supply of cofactor. Although the addition of exogenous UDPGA (1000 μM) increased the Vmax, it also increased the Km. This result may suggest that under these conditions, other glucuronosyltransferases may participate in the glucuronidation of BPA. It is also possible that this high concentration of UDPGA could promote nonenzymatic glucuronidation of BPA by a bimolecular nucleophilic substitution reaction (Yin et al., 1994). The relative result of incubating HCs with exogenous UDPGA was an increase in CLint. However, when modeled to reflect hepatic blood flow, the overall effects in CLmet were minimal. Thus, the glucuronidation of BPA in this in vitro cell system is dependent on endogenous levels of UDPGA. All cryopreserved HCs contained sufficient UDPGA to glucuronidate BPA efficiently.
The Km values observed in male SD rat HCs (11 ± 6 μM; Table 2) and in male and female human HCs (9 ± 3 μM; Tables 3 and 4) are comparable to the Km of 10 μM used by Teeguarden et al. (2005) to construct a physiologically based pharmacokinetic model for BPA. The use of this Km observed in vitro simulated plasma BPA concentrations at Tmax and during distribution and elimination for blood sampling times after i.v. administration of 10 mg/kg to rats. Furthermore, this Km accurately predicted the plasma BPAG level, as well as levels of BPAG in urine in humans who ingested BPA (Völkel et al., 2002).
The mean value for the scaled CLmet for both male and female human HCs was 6 ml/min/kg. This value represents approximately one-fourth of the estimated human liver blood flow of 20 ml/min/kg. The intersubject variations in CLmet ranged from 14 to 42% hepatic blood flow for males and 18 to 50% for females. CLmet values in this range suggest a liver extraction ratio of 0.3. Drugs having an extraction ratio of 0.3 or less are considered to be cleared restrictively due to the influence of plasma protein binding (Rowland and Tozer, 1995).
The estimated hepatic CLmet for BPA reported here most likely underestimates total metabolic clearance of BPA in humans. In rats, extensive intestinal glucuronidation of BPA has been reported to occur (Inoue et al., 2003). Such metabolism would reduce BPA available for absorption into the systemic circulation. Intestinal glucuronidation of BPA may also occur in humans, since it is known that human intestine contains UGT (Fisher et al., 2002). Intestinal and hepatic glucuronidation of BPA most likely explains why Völkel et al., (2002, 2005) did not detect BPA in the plasma of human subjects who were orally administered 5 mg per person. It was subjected to extensive presystemic metabolism as evidenced by the presence of BPAG as the predominant circulating, as well as urinary, metabolite of BPA. In fact, the entire dose of BPA had been eliminated in the urine as BPAG by 32 h.
In summary, the results reported here support the conclusions of other investigators who found that, after oral ingestion, BPA undergoes extensive hepatic metabolism and the predominant route of metabolism is glucuronidation. This glucuronidation by the liver, coupled with that known to occur in the intestine, most likely explains the relative lack of estrogen-mediated effects observed in animals administered BPA by the oral route (Tyl et al., 2002).
Footnotes
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This research was supported in part by the America Plastic Council and Southwest Environmental Health Science Center (ES 06694).
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doi:10.1124/dmd.107.014787.
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ABBREVIATIONS: BPA, bisphenol A; BPAG, bisphenol A glucuronide; HC, hepatocyte; SD, Sprague Dawley; Vmax, maximum velocity; CL, clearance (Vmax/Km); CLint, intrinsic clearance; CLmet, metabolic clearance; WME, William's Medium E; Km, Michaelis-Menten constant; UDPGA, uridine diphosphoglucuronic acid; HPLC, high-performance liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MS, mass spectrometry; Q1, first quadrupole; Rt, retention time.
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↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
- Received January 24, 2007.
- Accepted July 20, 2007.
- The American Society for Pharmacology and Experimental Therapeutics