62, 221–227 (2001)
Copyright © 2001 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Metabolic Activation of Bisphenol A by Rat Liver S9 Fraction
Shin’ichi Yoshihara, 1 Misako Makishima, Noriko Suzuki, and Shigeru Ohta
Institute of Pharmaceutical Sciences, Faculty of Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
Received April 24, 2000; accepted May 7, 2001
Bisphenol A (BPA) is a well-known endocrine-disrupting chemical found in the environment. To assess the metabolic modulation
of estrogenic activity of BPA after ingestion, we investigated
whether the incubation of BPA with rat liver S9 fraction results in
metabolic activation or inactivation of estrogenic activity using a
recombinant yeast expressing human estrogen receptor and
MCF-7 transfected firefly luciferase plasmid for a reporter assay.
When 0.1 mM BPA was incubated with rat liver S9 for 1 h, the
estrogenic activity was increased about two to five times compared
with that of the control, in which the S9 was inactivated prior to
incubation. This metabolic activation was inhibited by SKF 525-A,
an inhibitor of cytochrome P450. With increasing incubation time,
the estrogenic activity increased time-dependently. Interestingly,
however, the metabolic activation did not proceed with either
microsomes or cytosol alone and was restored by a recombination
of both fractions. The active metabolite was eluted at later retention time than that of BPA on HPLC with a reversed-phase
column. Bisphenol B and methoxychlor were also activated by
incubation with rat liver S9, whereas 4-tert-octylphenol and 4-nonylphenol, as well as 17␤-estradiol, were metabolically inactivated.
The present results clearly indicate that BPA is metabolically
activated in terms of estrogenicity under the conditions existing
only with combined rat liver microsomes and cytosol.
Key Words: bisphenol A; 17␤-estradiol; estrogenic activity; recombinant yeast assay; MCF-7; rat liver S9; metabolic activation.
In recent years there has been increasing public concern that
chemicals in the environment may affect the endocrine function of humans and wildlife (Colborn, 1995). It is already
known that a wide variety of chemicals have estrogenic activity. These substances include phytoestrogens such as daidzein
and genistein and manmade chemicals such as pesticides,
plasticizers, and breakdown products of surfactants. Bisphenol
A (BPA), 2,2-bis(4-hydroxyphenyl) propane, is a monomer of
epoxy and polycarbonate plastics widely used in consumer
products. BPA was also identified as the estrogenic contaminant released from the coated inner plastic lining of food cans
(Brotons et al., 1995) and from dental sealants and composites
(Olea et al., 1996). Accordingly, BPA is regarded as one of the
endocrine-disrupting chemicals (EDCs) most likely to be ingested in our daily life. Judging from the high LD50 values of
1
To whom correspondence should be addressed. Fax: 82-257-5329. E-mail:
[email protected].
4.24 g/kg for rats and 2.5 g/kg for mice (AIHA, 1967; NIOSH,
1978), the acute toxicity of BPA appears not to be high.
However, there are several reports suggesting hazardous effects of BPA (Hardin et al., 1981; Maguire, 1988; Reel et al.,
1985), including DNA adduct formation (Atkinson and Roy,
1995). Estrogenicity of BPA was found incidentally by Feldman et al. (1984) as a result of autoclaving polycarbonate
flasks. Subsequently, they demonstrated that BPA promotes
cell proliferation and progesterone receptor synthesis in estrogen-responsive MCF-7 human breast cancer cells (Krishnan et
al., 1993). On the other hand, the results of in vivo studies on
the estrogenicity of BPA are very controversial in terms of the
dose affected. The low-dose (2 or 20 ␮g/kg/day) effects of
BPA on male reproductive organs such as prostate glands,
preputial glands, and epididymides 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., 1999;
Cagen et al., 1999).
Recently, a novel metabolic pathway of BPA in MV1, a
Gram-negative aerobic bacterium isolated from enriched
sludge taken from a wastewater treatment plant, was demonstrated by Spivack et al. (1994). The major route of metabolism
(⬎80%) in this bacterial strain was oxidative cleavage of an
intermediary metabolite, 4,4⬘-dihydroxy-␣-methylstilbene, to
4-hydroxybenzaldehyde and 4-hydroxyacetophenone. It is
noteworthy that the chemical structure of 4,4⬘-dihydroxy-␣methylstilbene is similar to that of diethylstilbestrol, a potent
synthetic estrogen. Knaak and Sullivan (1966) first reported the
metabolic fate of BPA in rats, showing that the major metabolite in urine was the glucuronide of BPA; considerable
amounts of free BPA and hydroxylated BPA were found in
feces. Recently, two groups of investigators reconfirmed that
the predominant metabolite in urine of rats is BPA monoglucuronide (Pottenger et al., 2000; Snyder et al., 2000). On the
contrary, Miyakoda et al. (1999; 2000) have shown that BPA,
not the glucuronide, was detected in fetuses after oral administration of BPA to the pregnant rats. This observation is very
suggestive. The fetus must be a primary target of BPA; nevertheless, fetal liver may lack the ability to inactivate BPA to
the glucuronide, which is essentially inactive as an estrogen
(Snyder et al., 2000). In this connection, Steinmetz et al.
(1997) found that although the potency of BPA in stimulating
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YOSHIHARA ET AL.
prolactin gene expression and release in vitro was 1000- to
5000-fold lower than that of 17␤-estradiol (E2), BPA showed
similar potency to E2 in stimulating prolactin release in vivo in
estrogen-sensitive Fisher 344 rats. The discrepancy between
the estrogenic potency of BPA in vitro and in vivo may suggest
the formation of active metabolite(s) in vivo.
Needless to say, in any assessment of the in vivo impact of
EDCs, the metabolic modulation of estrogenic activity must be
considered. Therefore, we investigated how the metabolism of
EDCs (especially BPA) by rat liver S9 fraction affects their
estrogenic activity, other than the conjugation reaction, using
recombinant yeast and MCF-7 reporter assays.
MATERIALS AND METHODS
Chemicals. The sources of materials used were as follows: BPA, bisphenol B (BPB), 4-tert-octylphenol, and 4-nonylphenol were obtained from Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan); 17␤-estradiol (E2), tamoxifen,
NADP, and glucose-6-phosphate were purchased from Sigma Chemical Co.
(St. Louis, MO); methoxychlor and chlorophenol red ␤-D-galactopyranoside
(CPRG) were obtained from Aldrich Chemical Company, Inc. (Milwaukee,
WI) and Boehringer Mannheim GmbH (Germany), respectively. SKF 525-A
was a gift from Smith Kline & French Laboratories (Philadelphia, PA). BPA
catechol and BPA o-quinone were synthesized according to the method reported by Atkinson and Roy (1995). Other chemicals used were of the highest
quality commercially available.
Recombinant yeast estrogenicity assay. The recombinant yeast strain
transfected with the human estrogen receptor (ER␣) gene, together with
expression plasmids carrying the reporter gene lac-Z preceded by an estrogenresponsive element (ERE) was provided with permission by Prof. Sumpter
(Brunel University, U.K.). The yeast estrogenicity assay was conducted as
described by Routledge and Sumpter (1996, 1997), with some minor modifications. In brief, to check an availability of the yeast cultured, 10-␮l aliquots
of ethanol solutions of E2 used as standards ranging from 2.9 ⫻ 10 –13 to 5 ⫻
10 –7 M as well as other EDCs tested, including BPA, ranging from 2.4 ⫻ 10 –9
to 4.4 ⫻ 10 – 4 M were transferred to 96-well plastic microtiter plates (Becton
Dickinson, Falcon 3072 Microtest) to obtain dose-response curves. For an
assay of estrogenic activity after incubation of EDCs with rat liver S9 fraction,
10-␮l aliquots of ethanol solution of solid-phase extracts from incubation
mixtures prepared as described below were transferred to wells. After evaporation of the solvent to dryness, 200-␮l aliquots of the yeast assay medium
containing recombinant yeast and CPRG, the chromogenic substrate of ␤-galactosidase reporter enzyme, were dispensed into each well. If needed, 1 ␮M
tamoxifen was added to wells as an antiestrogen. The plates were sealed with
autoclave tape and incubated at 32°C in a dry incubator without shaking to
avoid contamination between wells. After 24 – 48 h, the red color of the
hydrolysis product of CPRG by ␤-galactosidase was read using a microplate
reader (Bio-Rad, Model 550) in terms of the absorbance at 540 nm. All assays
were carried out at least in duplicate using a blank well containing the yeast
assay medium alone. The data were corrected for turbidity using the absorbance reading at 620 nm, and values were calculated as follows: Net OD540 ⫽
(OD540 for test – OD620 for test) – (OD540 for blank – OD620 for blank).
The concentration of EDC indicated, containing both the unchanged substrate
and its metabolite(s), was expressed as a concentration of the substrate added
in the respective incubation mixture.
ERE-luciferase reporter assay in MCF-7 cells. ERE-luciferase reporter
assay using MCF-7 cells was carried out by the method described previously
(Sugihara et al., 2000). In brief, MCF-7 cells in 12-well plates at 1 ⫻ 10 5
cells/well were transiently transfected with 1.9 ␮g of p(ERE) 3-SV40-luc and
0.1 ␮g of pRL/CMV (Promega Co., Madison, WI) as an internal standard
using Transfast (Promega Co.) according to the manufacturer’s protocol.
Following the treatment of the cells with the test sample in 10 ␮l of ethanol for
24 h, the assay was performed with a Dual Luciferase assay kit (Promega Co.).
The final concentration of the BPA equivalent, consisting of unchanged BPA
and its metabolites in the assay well, was estimated by the original amount of
BPA as the substrate.
Incubation of EDCs with rat liver subcellular fractions. Male Wistar rats
(5– 6 weeks of age) were purchased from CLEA Japan, Inc. (Tokyo, Japan).
The liver S9 fraction, consisting of both microsomal and cytosolic fractions,
was obtained by centrifugation of whole-liver homogenate at 9000 ⫻ g for 20
min. The S9 fraction was further centrifuged at 105,000 ⫻ g for 60 min to
separate the microsomes and cytosolic fraction (Yoshihara and Ohta, 1998).
The incubation mixture consisted of the indicated concentrations of EDCs
(usually 0.1 mM) in 5 ␮l ethanol, an NADPH-generating system (0.5 mM
NADP ⫹, 5 mM glucose-6-phosphate, 5 mM MgCl 2), and 50 mg liver equivalent of S9 fraction or microsomes and/or cytosolic fraction in a final volume
of 1 ml 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM
EDTA. For the inhibition study of P450, 0.1 mM SKF 525-A was also added
to the reaction mixture. The incubation was carried out at 37°C for 60 min with
shaking. After addition of 1 ml of 20% trichloroacetic acid (TCA), the
quenched reaction mixture was allowed to stand for 15 min on ice, then was
centrifuged at 2500 rpm for 10 min. The incubation system without an
NADPH-generating system, or inactivated by addition of TCA prior to incubation, was used as the control. A 1.8-ml aliquot of the resultant supernatant
was passed through a Sep-Pak Plus C18 cartridge (Waters Associates Inc.,
Milford, MA) preconditioned with 10 ml methanol and 20 ml water for
solid-phase extraction. The cartridge was washed with 10 ml water and the
remaining water was purged by flushing with nitrogen gas. The adsorbed
substances were eluted with 3 ml ethanol, and the eluate was evaporated to
dryness in vacuo. The residue was dissolved in 200 ␮l ethanol, then 10 ␮l of
the sample solution was transferred to a well of a microtiter plate for estrogenicity assay as described above. All the experiments were conducted in
duplicate.
HPLC analysis of BPA metabolites. For an analytical HPLC, 40 ␮l of the
sample solution prepared as above was applied to a Beckman Gold Nouveau
HPLC system (Beckman Instruments, Inc., Fullerton, CA) equipped with a
UV/VIS diode array detector and an analytical reversed-phase column (Supelcosil ABZ ⫹ Plus; 4.6 ⫻ 150 mm, 5 ␮m; Supelco, Bellefonte, PA). The
separation of metabolites was performed with a linear gradient of 0 –70%
acetonitrile in 0.06% acetic acid over 15 min, then held for 10 min at a flow
rate of 2 ml/min; the chromatogram was monitored at 275 nm.
For a preparative HPLC, 5 ml of the incubation mixture consisting of 250
mg liver equivalent of rat liver S9, the same final concentration of an NADPHgenerating system as in the usual scale, and 0.5 mM BPA was incubated for 90
min at 37°C. The reaction was quenched by addition of 5 ml of 20% TCA, and
the metabolites were extracted using a Sep-Pak Plus C18 cartridge as described
above. The extract taken up in 50 ␮l acetonitrile was applied to a preparative
reversed-phase column (Supelcosil ABZ ⫹ Plus; 10 ⫻ 250 mm, 5 ␮m; Supelco,
Bellefonte, PA). The separation of metabolites was performed with a linear
gradient of 0 –70% acetonitrile in 0.06% acetic acid over 20 min, then held for
10 min at a flow rate of 2 ml/min; the chromatogram was monitored at 275 nm.
The eluate was collected at every 1 min interval using a fraction collector and
again extracted by a Sep-Pak Plus C18 cartridge. The extract from each
fraction was dissolved in 100 ␮l acetonitrile; 10 ␮l of each sample solution
was applied for the yeast estrogenicity assay and the remainder for GC/MS or
LC/MS analysis.
RESULTS
Metabolic Activation of BPA by Rat Liver S9
The estrogenic activity of BPA in the yeast assay was
enhanced by incubation with rat liver S9 fraction more than
three times compared with that of the control, in which the S9
was denatured with TCA prior to incubation (Fig. 1). This
enhancement was partially inhibited by SKF 525-A, a well-
METABOLIC ACTIVATION OF BPA
FIG. 1. Metabolic activation of estrogenic activity of BPA by rat liver S9
fraction using yeast estrogenicity assay. In a complete system, BPA (0.1 mM)
was incubated with native rat liver S9 fraction for 1 h at 37°C with or without
0.1 mM SKF 525-A, as described in the Materials and Methods section. In a
denatured system, the incubation was carried out with the S9 fraction denatured by TCA prior to addition of BPA. The estrogenicity of solid-phase
extracts from the incubation mixture was assayed using a recombinant yeast
screen. In the assay testing antagonist, 1 ␮M tamoxifen was added in the well
of titer plate. Data represent the mean ⫾ SE of four experiments.
known inhibitor of cytochrome P450 (P450). The estrogenic
activity generated with S9 was almost completely inhibited by
inclusion of tamoxifen, an antagonist of estrogens, in the assay
well. To test whether this enhancement of the estrogenicity is
due to the increase of permeability of BPA into the yeast cell,
which may be caused by the metabolite(s), we examined the
effects of the extract containing metabolite(s) from the complete system on the estrogenicity of the extract containing no
metabolite but much more unchanged BPA from the denatured
system by combination of each other. The results clearly demonstrated that the estrogenicity by the combined samples was
somewhat decreased or at least not synergistically increased,
suggesting no effect on permeability; the yeast assay also gave
a good dose response under the conditions used (Fig. 2). The
metabolic activation catalyzed by S9 was not only substrate
concentration– dependent (Fig. 3), but also incubation time–
dependent (Fig. 4). To further confirm the metabolic activation
of BPA, we assessed the estrogenic activity of the extracts
from the respective incubation mixtures using an alternative
assay system with the mammalian cell line MCF-7. Almost
similar enhancement of the estrogenicity of BPA with S9 was
exhibited in the reporter assay using MCF-7 cells (Fig. 5).
These results strongly suggest that the enhancement of estrogenicity of BPA by incubation with S9 fraction might be due
to the formation of active metabolite(s) by P450.
Requirement of Microsomes and Cytosol for Metabolic
Activation of BPA
Unexpectedly, however, the incubation of BPA with rat liver
microsomes alone as well as cytosolic fraction was unable to
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FIG. 2. Effects of combination of extracts from complete and denatured
systems on estrogenicity by yeast estrogenicity assay. The indicated amounts
of the solid-phase extracts in ethanol from the incubation system containing
native (C) or denatured (D) S9 and 0.1 mM BPA were assayed by yeast screen
as described in the Materials and Methods section. In some experiments, the
extracts from complete and denatured systems were combined in the well and
then assayed. Data represent the mean of two experiments.
increase the estrogenic activity (Fig. 4). The ability to activate
the estrogenicity was restored by recombination of both microsomes and cytosolic fraction. It is noteworthy that although
unchanged, BPA was decreased time-dependently by incubation with S9, but the estrogenicity of the extract was increased
during incubation. Figure 6 shows the analytical HPLC profiles
obtained by incubation of BPA with S9 or microsomes and
cytosolic fraction alone. Peaks 1 and 2, eluted faster than
unchanged BPA, were common metabolites by either S9 or
microsomes, whereas no metabolite was formed with cytosol
alone. The metabolite giving peak 2 showed a tendency to
convert to that giving peak 1 during a storage of the extract at
FIG. 3. Effects of substrate concentration on metabolic activation of BPA
by rat liver S9 fraction using yeast estrogenicity assay. Various concentrations
of BPA were incubated with rat liver S9 fraction for 1 h at 37°C. The control
incubation was carried out with the acid-denatured S9 fraction. The concentrations indicated are expressed as a substrate concentration in the incubation
system. Other details are the same as in the legend to Figure 1. No error bar
indicates the error was included within the symbol.
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YOSHIHARA ET AL.
FIG. 4. Requirement of microsomes and cytosol for metabolic activation
of BPA. BPA (0.1 mM) was incubated with rat liver S9 (S9) or microsomes
(Ms) and/or cytosol (Cs) in the presence of an NADPH generating system for
various time periods at 37°C as described in the Materials and Methods
section. The estrogenicity of the reaction products was assayed using a recombinant yeast screen. The concentration of BPA remained after incubation with
the S9 was determined by HPLC. Data represent the mean ⫾ SE of four
experiments. No error bar indicates the error was included within the symbol.
room temperature. On the contrary, peaks 3 and 4, eluted later
than BPA, were detected only in the extract from S9 system,
but from neither microsomes nor cytosolic fraction alone.
Therefore, these results strongly suggest that the metabolite(s)
FIG. 6. HPLC chromatograms of BPA metabolites by rat liver S9, microsomes, and cytosol. BPA (0.1 mM) was incubated with the respective subcellular fraction of rat liver for 1 h at 37°C. Analytical HPLC was performed
using a reversed-phase column with monitoring at 275 nm as described in the
Materials and Methods section.
giving either peak 3 or peak 4 must be a candidate as an active
metabolite (see the following section).
Estrogenic Activity of BPA Metabolites
FIG. 5. Metabolic activation of BPA by rat liver S9 fraction using MCF-7
estrogenicity assay. The same samples assayed by yeast screen in Figure 2
were assessed by ERE-luciferase reporter assay as described in the Materials
and Methods section. The concentrations are indicated as the amount of BPA
equivalent in the assay well. Estrogenic activity was expressed as a relative
activity with respect to the control using MCF-7 cells. Data represent the
mean ⫾ SE of four experiments.
The preparative HPLC profiles of BPA metabolism catalyzed by rat liver S9 and the estrogenic activity of each fraction
separated are shown in Figure 7. At least four metabolites,
which were not found in the chromatogram of the acid-denatured control other than unchanged BPA eluted at 22.0 min,
were detected in the extract from the native S9 system. Among
them, peak 2, with a retention time of 20.7 min, was tentatively
identified as authentic BPA catechol on the basis of its retention time on HPLC, UV spectrum (␭max 280 nm), and [M-H] –
⫽ 242.98 by negative-mode LC/MS. Peak 1, eluted at a
retention time of 18.7 min, was regarded as the secondary
product of peak 2 because this peak increased with decreasing
peak 2 during storage, as described above. Judging from its
identity of the retention time and [M-H] – ⫽ 241.15 by negative-mode LC/MS with the compound found in the authentic
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METABOLIC ACTIVATION OF BPA
bolically activated by S9. However, the estrogenic activities of
4-tert-octylphenol and 4-nonylphenol, as well as an endogenous estrogen, E2, were dramatically decreased by the S9catalyzed metabolism.
DISCUSSION
FIG. 7. Preparative HPLC of BPA metabolites by rat liver S9 and estrogenic activity of separated fractions using yeast estrogenicity assay. BPA (0.5
mM) was incubated with the S9 fraction for 90 min at 37°C as described in the
Materials and Methods section. Preparative HPLC was performed using a
reversed-phase column with monitoring at 275 nm. The estrogenicity of the
extract from each eluate collected at every 1-min interval was assayed using a
recombinant yeast screen as described in the Materials and Methods section.
sample of BPA catechol, this metabolite might be BPA oquinone. Peak 3 (retention time of 26.0 min) and peak 4
(retention time of 26.7 min), less-polar metabolites than BPA,
seemed to be structurally isomeric compounds, showing almost
the same mass numbers of [M-H] – ⫽ 267.05 and [M-H] – ⫽
267.04, respectively, by negative-mode LC/MS. But the former
metabolite gave two fragmented mass peaks of [M-H] – ⫽
251.85 and [M-H] – ⫽ 132.85 by negative-mode LC/MS/MS,
whereas the latter gave only [M-H] – ⫽ 132.83. With respect to
estrogenicity of these metabolites, only peak 4, not peak 3,
exhibited a potent estrogenic activity comparable to that of
BPA recovered (Fig. 7). Both BPA catechol and o-quinone
showed no estrogenic activity at the concentrations tested.
Thereby, the metabolite giving peak 4 should be an active
metabolite as estrogen.
Metabolic Modulation of Other EDCs by Rat Liver S9
The effects of metabolism by rat liver S9 on the estrogenic
activity of other EDCs, as well as E2, are shown in Figure 8.
The metabolic activation of methoxychlor, which has no intrinsic estrogenic activity, by incubation with S9 was confirmed. BPB, 2,2-bis(4-hydroxyphenyl)butane, was also meta-
The metabolic fate of BPA in adult rats is affected by many
factors, such as the route of administration, the doses administered, the sex of rats (Pottenger et al., 2000), and the strains
used (Snyder et al., 2000). In any case, the most predominant
metabolite of BPA in rats in vivo has been attributed to the
monoglucuronide of BPA (Knaak and Sullivan, 1966; Pottenger et al., 2000; Snyder et al., 2000). These observations
were further supported by the evidence that BPA is rapidly
converted to the glucuronide as a major metabolite in isolated
rat hepatocytes (Nakagawa and Tayama, 2000). In contrast,
however, Miyakoda et el. (1999; 2000) recently reported that
about 50 ppb of free BPA was detected in fetuses 1 h after oral
administration to the pregnant rats at a dose of 10 mg/kg. In
this case, no glucuronide was detected in the fetuses, whereas
the glucuronide was again a major metabolite in the dam.
These results suggest that BPA easily passes through the placental barrier and the fetus might lack the ability of glucuronide formation. Subsequently, in fact, Yokota et al. (2000)
have demonstrated that the activity of UGT2B1, a predominant
isoform to catalyze the glucuronidation of BPA (Yokota et al.,
1999), was not detected in the fetus of rat. It is true that the
rodent fetal liver has a limited ability to metabolize xenobiotics
compared to the adult liver. Currently available data, however,
revealed that several forms of P450 are expressed in fetal rat
liver, especially in late gestation (Cresteil et al., 1986; Neubert
and Tapken, 1988; Raucy and Carpenter, 1993). In addition,
unlike rodent species, the human fetal liver has a significant
capacity for xenobiotic metabolism (Hakkola et al., 1998; Ring
FIG. 8. Metabolic modulation of estrogenic activity of E2 and EDCs by
rat liver S9 fraction using yeast estrogenicity assay. Indicated concentrations of
E2 and EDCs were incubated with rat liver S9 for 1 h at 37°C. The control
incubation was carried out with the acid-denatured S9 fraction. Other details
are the same as in the legend to Figure 1. Abbreviations used: BPB, bisphenol
B; methoxy, methoxychlor; E2, 17␤-estradiol; octylphenol, 4-tert-octylphenol;
nonylphenol, 4-nonylphenol.
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YOSHIHARA ET AL.
et al., 1999). With regard to the glucuronosyltransferase activity in human fetal liver, there is an interesting report that the
glucuronidation of ritodrine, a ␤2-adrenoceptor having a bisphenol structure similar to BPA, is little developed compared
to the adult liver (Pacifici et al., 1993). Therefore, to understand the effects of BPA on the fetus, a target of EDCs, it is
indispensable to investigate the metabolism of BPA other than
glucuronidation.
Rat liver S9 fraction is often used as the source of the
microsomal P450-dependent monooxygenase system for metabolic activation of promutagens in the Ames assay (Ames et
al., 1975). Therefore, the rat liver S9 fraction was also adopted
in this experiment to examine the effects of metabolic conversion of EDCs on their estrogenic activity. When BPA was
incubated with rat liver S9, its estrogenic activity assessed by
the yeast reporter assay increased with both a time- and substrate concentration-dependent manner (Figs. 3 and 4). Based
on the result showing no synergistic increase of estrogenicity
from the combination of the extracts, compared to the complete
and denatured systems in the yeast assay, it seems very likely
that the enhanced activity is due to the formation of true
estrogenic metabolite(s), but not to the metabolite(s) causing
an increase of permeability of BPA which remained across the
yeast cell wall (Fig. 2). Furthermore, this metabolic activation
was also confirmed by the results obtained with breast cancer
cell line MCF-7 cells (Fig. 5). Similar metabolic activation of
BPA was also observed by using human liver S9 (data not
shown). The active metabolite contributing to the enhancement
of estrogenicity was eluted at a retention time later than unchanged BPA on a reversed-phase HPLC (Figs. 6 and 7).
Judging from the peak sizes detected, if the extinction coefficient of the unknown metabolite is similar to that of BPA, the
estrogenicity of this minor metabolite must be much more
intense than that of a parent BPA (Fig. 7). Unfortunately, with
respect to the structure of this active metabolite, at present little
is known except a) its molecular weight might be 40 mass
greater than that of BPA; b) it gives a mass peak of [M-H] – ⫽
132.83, suggesting a dimer of propenylphenol structure on
negative-mode LC/MS/MS; and c) it possesses two hydroxyl
groups to be trimethylsilylated (data not shown). Much more
interesting evidence about this active metabolite is that the
formation of this metabolite required both microsomes and
cytosol (Figs. 4 and 6). In this metabolic conversion, because
the reaction required NADPH and was inhibited with SKF
525-A, microsomal P450 should be involved at least as a
primary enzyme. The function of cytosol was lost by boiling
but still retained after dialysis (data not shown), suggesting an
involvement of certain enzyme.
It is not clear, however, whether such a factor in cytosol acts
as a secondary enzyme, converting the primary metabolite by
microsomal P450 to the active one. Atkinson and Roy (1995)
have demonstrated that DNA adduct formation in vitro with
BPA is catalyzed by rat liver microsomes only in the presence
of NADPH. Based on these results, they speculated that BPA
might be activated by P450 to form a reactive BPA o-quinone
via 5-hydroxy BPA (BPA catechol). We first demonstrated
directly the formation of these monooxygenated metabolites,
but they were almost inactive as an estrogen (Fig. 7). We could
not identify a peak corresponding to 4,4⬘-dihydroxy-␣-methylstilbene, an intermediary metabolite in bacteria (Spivack et
al., 1994) that exhibits estrogenicity about 100 times more
potent than that of BPA (our unpublished data).
BPB, a weak estrogen having a quite similar structure with
BPA, was also activated by rat liver S9 (Fig. 8). The HPLC
profile of BPB metabolites was similar to that of BPA metabolites (data not shown), indicating the same type of metabolic
activation might occur. We also reconfirmed the metabolic
activation of methoxychlor, which is known to be activated by
O-demethylation to 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane by P450 (Bulger et al., 1978). In contrast, 4-tert-octylphenol and 4-nonylphenol, alkylphenolic EDCs widely distributed in the environment, as well as an endogenous estrogen,
E2, were metabolically inactivated in terms of estrogenicity by
S9 (Fig. 8).
In conclusion, both BPA and BPB are metabolically activated in terms of estrogenicity by rat liver S9 fraction. This
might account, at least in part, for the discrepancy between the
estrogenic activities of BPA observed in vitro and in vivo
(Steinmetz et al., 1997). Even though the major metabolic
route of BPA in vivo could be glucuronidation rather than
P450-dependent metabolism (Pottenger et al., 2000; Snyder et
al., 2000), the formation of small amounts of unconjugated
metabolites have been demonstrated in vivo (Knaak and Sullivan, 1966; Pottenger et al., 2000) and in vitro (Nakagawa and
Tayama, 2000). The most intensive enhancement, 22.8 ppm,
was observed at a substrate concentration of 0.1 mM BPA. At
the concentrations lower than 0.01 mM BPA (2.28 ppm),
which are comparable to the values reported in vivo Cmax
levels in the blood of female rats following administration of
100 mg/kg (Pottenger et al., 2000), there was no enhancement
of estrogenic activity. However, the metabolic activation by S9
must be taken into account in order to assess BPA as an in vivo
estrogen in the fetus, one of the most important targets of
EDCs, because the fetus has a poor ability to conjugate BPA
with glucuronic acid (Miyakoda et el., 1999; 2000; Yokota et
al., 2000) as a detoxification reaction. Further studies on the
mechanism of this unique metabolic activation of BPA by S9,
as well as the structural elucidation of the active metabolite, are
in progress.
ACKNOWLEDGMENTS
This work was supported in part by a grant for Scientific Research from the
Japanese Ministry of Education. We would like to thank Prof. John Sumpter,
Brunel University, U.K., who provided the recombinant yeast for estrogenicity
assay. We also thank Dr. T. Matsuda, Research Center for Environmental
Quality Control, Kyoto University, Japan, for his helpful advice on carrying
out the yeast screen, Mr. S. Sanoh in our laboratory for MCF-7 reporter assay
and Dr. K. Fukuhara, Nihon Bruker Daltonics K. K., Tokyo, Japan, for LC/MS
analysis.
METABOLIC ACTIVATION OF BPA
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