* Department of Xenobiotic Metabolism and Molecular Toxicology, Graduate School of Biomedical Sciences, and Research Institute for Radiation Biology and Medicine, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan; and
Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Arise 518, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan
1 To whom correspondence should be addressed. Fax: +81-82-257-5329. E-mail: siyoshi{at}hiroshima-u.ac.jp.
Received September 24, 2003; accepted November 12, 2003
ABSTRACT
We previously demonstrated that the estrogenicity of either bisphenol A [BPA; 2,2-bis(4-hydroxyphenyl)propane] or bisphenol B [BPB; 2,2-bis(4-hydroxyphenyl)butane] was increased several times after incubation with rat liver S9 fraction (Yoshihara et al., 2001). This metabolic activation, requiring both microsomal and cytosolic fractions, was observed with not only rat liver, but also human, monkey, and mouse liver S9 fractions. To characterize the active metabolites of BPA and BPB, we investigated the structures of the isolated active metabolites by negative mode LC/MS/MS and GC/MS. The active metabolite of BPA gave a negative mass peak at [M-H]- 267 on LC/MS and a single daughter ion at m/z 133 on MS/MS analysis, suggesting an isopropenylphenol dimer structure. Finally, this active metabolite was confirmed to be identical with authentic 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene (MBP) by means of various instrumental analyses. The corresponding peaks of the BPB metabolite were [M-H]- 295 and m/z 147, respectively, suggesting an isobutenylphenol dimer structure. Further, coincubation of BPA and BPB with rat liver S9 afforded an additional active metabolite(s), which gave a negative mass peak at [M-H]- 281 and two daughter ion peaks at m/z 133 and m/z 147 on MS/MS analysis. These results strongly suggest that the active metabolite of either BPA or BPB might be formed by recombination of a radical fragment, a one-electron oxidation product of carbon-phenyl bond cleavage. It is noteworthy that the estrogenic activity of MBP, the active metabolite of BPA, is much more potent than that of the parent BPA in several assays, including two reporter assays using a recombinant yeast expressing human estrogen receptor
and an MCF-7-transfected firefly luciferase plasmid.
Key Words: bisphenol A; bisphenol B; active metabolite; estrogenic activity; liver S9; metabolic activation.
Considerable attention has been focused on environmental chemicals that affect the reproductive function by disrupting the endocrine system (Colborn, 1995). Bisphenol A [BPA, 2,2-bis(4-hydroxyphenyl)propane] is a weak estrogen that is widely used as a component of polycarbonate and epoxy plastics, as well as in the inner plastic lining of food cans and dental sealants. Trace amounts of BPA have been reported to leach from these consumer products (Brotons et al., 1995
; Olea et al., 1996
; Yamamoto and Yasuhara, 1999
), and BPA has been detected not only in the aquatic environment (Khim et al., 2001
; Motoyama et al., 1999
), but also in maternal and umbilical cord blood of humans (Ikezuki et al., 2002
; Schonfelder et al., 2002
). Thus, BPA is regarded as one of the endocrine disrupting chemicals (EDCs) ingested by humans in daily life. Estrogenicity of BPA was first reported in 1936 by Dodds and Lawson and subsequently identified as a result of studies on the release of estrogenic material from autoclaved polycarbonate flasks (Krishnan et al., 1993
). Although the estrogenic activity of BPA is less potent than that of 17ß-estradiol (E2) in in vitro assays (Gaido et al., 1997
; Krishnan et al., 1993
; Kuiper et al., 1997
), BPA has similar potency to E2 in stimulating prolactin release in estrogen-sensitive Fischer 344 rats in vivo (Steinmetz et al., 1997
). This discrepancy between the estrogenic potency of BPA in vitro and in vivo suggests that an active metabolite(s) may be formed in vivo. On the other hand, the results of in vivo studies on the estrogenicity of BPA are conflicting in terms of the effective dose. When pregnant CF-1 mice were fed with 2 or 20 ng/g body weight/day, the weights of male reproductive organs, such as preputial gland and seminal vesicle, were affected in their offspring (Nagel et al., 1997
; vom Saal et al., 1998
). However, other investigators could not reproduce these findings in male offspring at the same dose (Ashby et al., 1999
; Cagen et al., 1999
).
The first report concerning the metabolic fate of BPA was presented by Knaak and Sullivan (1966), who showed that in the urine of rats administered 14C-BPA orally, the major metabolite of BPA was the glucuronide, and considerable amounts of unchanged BPA and hydroxylated BPA were detected in the feces. Recently, several groups of investigators have confirmed that BPA monoglucuronide is the predominant in vivo metabolite in rats (Pottenger et al., 2000; Snyder et al., 2000
), monkeys (Kurebayashi et al., 2002
), and humans (Volkel et al., 2002
). Further, in isolated hepatocytes from rats (Nakagawa and Tayama, 2000
; Pritchett et al., 2002
), mice (Pritchett et al., 2002
), and humans (Pritchett et al., 2002
), a BPA monoglucuronide has also been identified as the predominant metabolite, and a sulfate conjugate or a glucuronide/sulfate diconjugate is a minor metabolite. On the other hand, 3-hydroxy BPA (BPA catechol) and BPA o-quinone were detected as cytochrome P450 (CYP)-dependent metabolites in rat liver S9 fraction (Yoshihara et al., 2001
). Among these reported metabolites, BPA monoglucuronide is almost completely devoid of estrogenic activity (Matthews et al., 2001
) and BPA catechol and BPA o-quinone also show little activity (Yoshihara et al., 2001
). We previously reported a uniquely potent estrogenic metabolite of BPA that seems to be formed only in the presence of both microsomal and cytosolic fractions (Yoshihara et al., 2001
), with a greater molecular weight (by 40 mass units) than that of BPA, and we suggested an isopropenylphenol dimer structure for it.
In the present study, therefore, we investigated the chemical structure of this unique active metabolite of BPA and examined its estrogenic potency using several in vitro assay methods. In addition, we examined the structure of the active metabolite of bisphenol B (BPB).
MATERIALS AND METHODS
Chemicals.
The sources of materials used were as follows: BPA and BPB were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan); E2, NADP, and glucose-6-phosphate were purchased from Sigma Chemical Co. (St. Louis, MO); chlorophenol red-ß-D-galactopyranoside (CPRG) and ELISA kits for human estrogen receptors (hER
) and ß (hERß) ligand screening were obtained from Boehringer Mannheim GmbH (Germany) and Toyobo Co., Ltd. (Osaka, Japan), respectively. ICI182,780 was supplied by Tocris Cookson Ltd. (Bristol, U.K.). Zymolyase 20T was obtained from Seikagaku Co. (Tokyo, Japan). We synthesized 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene (MBP), as described below. Other chemicals used were of the highest quality commercially available.
Incubation of BPA and BPB with liver S9 fraction.
Male Wistar rats (5-6 weeks of age) and male ddy mice (5 weeks of age) were purchased from CLEA Japan, Inc. (Tokyo, Japan) and Japan SLC, Inc. (Shizuoka, Japan), respectively. Frozen livers of Japanese monkey bred in the Primate Research Institute, Kyoto University, were donated by Dr. K. Asaoka. The liver S9 fraction, which contained 250 mg liver/ml was prepared by centrifugation of whole homogenate at 9000 x g for 20 min. Human liver S9 fraction prepared from equal amounts of seven different individual donors was purchased from Daiichi Chemicals (Tokyo, Japan). In the experiments concerning species difference, the incubation mixture consisted of 0.1 µmol of BPA in 2 µl acetonitrile, 0.5 µmol of NADP, 5 µmol of glucose-6-phosphate, 5 µmol of MgCl2, and 0.2 ml of liver S9 (50 mg liver equivalent) in a final volume of 1 ml of 100 mM potassium phosphate buffer (pH 7.5) containing 0.1 mM EDTA. The incubation was carried out at 37°C for 60 min. The reaction was quenched with 1 ml of 20% trichloroacetic acid (TCA), and the metabolites were extracted by a solid-phase extraction (SPE) method using a Sep-Pak Plus C18 cartridge (Waters Associates Inc., Milford, MA). Estrogenic activity was measured with the yeast estrogen screening (YES) assay as described previously (Yoshihara et al., 2001). The incubation system inactivated by addition of 20% TCA prior to incubation was used as the denatured control. For structural analysis of metabolites formed, the incubation mixture consisted of 0.5 µmol of BPA or/and BPB in 10 µl of acetonitrile, 2.5 µmol of NADP, 25 µmol of glucose-6-phosphate, 25 µmol of MgCl2, and 1 ml of rat liver S9 (250 mg liver equivalent) in a final volume of 5 ml of 100 mM potassium phosphate buffer (pH 7.5) containing 0.1 mM EDTA. The incubation was carried out at 37°C for 60 min. The reaction was quenched by addition of 5 ml of 20% TCA, and the metabolites were extracted by the SPE method using a Sep-Pak Plus C18 cartridge as described previously (Yoshihara et al., 2001
). The extract taken up in 200 µl of acetonitrile was subjected to preparative HPLC.
Preparative HPLC of BPA and BPB metabolites.
Preparative HPLC was performed in a Beckman Gold Nouveau HPLC system (Beckman Instruments, Inc., Fullerton, CA) equipped with a preparative reversed-phase column (Supelcosil ABZ+ Plus; 10 x 250 mm, 5 µm; Supelco, Bellefonte, PA) as described previously (Yoshihara et al., 2001). The separation of metabolites was performed with a linear gradient of 0-80% acetonitrile in 0.06% acetic acid over 25 min, followed by a hold for 14 min at a flow rate of 2 ml/min; the chromatogram was monitored at 275 nm. The eluate was collected at 1-min intervals, and after dilution of each fraction with 18 ml of water, the eluted metabolite was again extracted onto a Sep-Pak Plus C18 cartridge. The final extract from each fraction was dissolved in 200 µl of acetonitrile; 10 µl of each sample solution was used for the YES assay and the remainder for LC/MS/MS or GC/MS analysis.
Analysis of BPA and BPB metabolites by LC/MS/MS.
A Waters Alliance liquid chromatograph coupled to a Quattro-Ultima mass spectrometer fitted with negative mode ESI was used. Separation was performed on a reversed-phase Supelcosil ABZ+ Plus column (150 x 4.6 mm, 5 µm; Supelco, Bellefonte, PA) using a mobile phase of 90% MeOH in water at a flow rate of 0.6 ml/min.
Analysis of BPA metabolites and MBP by GC/MS.
GC/MS analysis was performed on a Shimadzu GC-17A/QP-5000. The GC conditions were as follows: DB-5 capillary column (30 m x 0.2 mm inside diameter, 0.25 µm film thickness; J & W Scientific, Folsom, CA), helium as carrier gas at a flow rate of 1.3 ml/min, splitless injection at 220°C injection port temperature, temperature program of 100°C for 1 min, to 250°C over 14 min, and hold for 10 min.
Yeast estrogen screening (YES) assay.
For the YES assay, a recombinant yeast strain transfected with the hER gene and expression plasmids carrying the reporter gene lac-Z preceded by an estrogen-responsive element (ERE) was kindly provided by Prof. J. Sumpter (Brunel University, U.K.). The yeast estrogenicity assay was conducted as described by Routledge and Sumpter (1997), with some minor modifications (Yoshihara et al., 2001
). In brief, 10-µl aliquots of each sample in acetonitrile were incubated with 200-µl aliquots of the yeast assay medium containing recombinant yeast and CPRG, a chromogenic substrate of the ß-galactosidase reporter enzyme, in a 96-well plastic microtiter plate at 32°C. After 24-48 h, the absorbance of the red color due to the hydrolysis product of CPRG was read using a microplate reader (Model 550; Bio-Rad Laboratories, Hercules, CA) at 540 nm. All assays were carried out at least in duplicate using a blank well containing the same amounts of acetonitrile and the yeast assay medium alone. The data were corrected for turbidity using the absorbance at 620 nm, and the values were calculated as follows: Net OD540 = (OD540 for test - OD620 for test) - (OD540 for blank - OD620 for blank).
Yeast two-hybrid assay.
The yeast two-hybrid assay system with rat ER or rat ERß and the coactivator, TIF2, was used in this study as described previously (Kawagoshi et al., 2000
), with a minor modification. Briefly, 1-µl aliquots of acetonitrile solution of test chemicals in a 96-well plastic microtiter plate were incubated with 100 µl of the precultured yeast suspension at 30°C for 18 h. The absorbance was read at 620 nm; then the cell wall of the yeast was digested enzymatically by incubation with 25 µl of Z-buffer containing Zymolyase 20T (5 mg/ml) at 30°C for 30 min. The lysate was mixed with 25 µl of CPRG (0.5 mg/ml) in 0.1 M sodium phosphate buffer (pH 7.0) and incubated at 30°C for 1 h. Following addition of 25 µl of 2 M sodium carbonate to quench the hydrolysis, the red color that developed was read at 540 nm using a microplate reader. The data were corrected as described in the case of YES assay.
ERE-luciferase reporter assay in MCF-7 cells.
ERE-luciferase reporter assay using MCF-7 cells was carried out by the method described previously (Yoshihara et al., 2001). In brief, MCF-7 cells in 12-well plates at 1 x 105 cells/well were transiently transfected with p(ERE)3-SV40-luc and phRL/CMV (Promega Co.) as an internal standard. Following the treatment of the cells with the test sample for 24 h, the assay was performed with a Dual Luciferase Assay Kit (Promega Co.).
ERE-luciferase reporter assay in NIH/3T3 cells.
The construction of reporter and expression plasmids was described previously (Maruyama et al., 2001). NIH/3T3 cells in 48-well plates at 2 x 104 cells/well were transiently transfected with 0.3 µg of (ERE)3-SV40-luc and 0.1 µg of pSG5-hER
or hERß along with 0.01 µg of phRL-CMV (Promega Co., Madison, WI) with TransFast transfection reagent containing a synthetic cationic lipid (Promega Co.), following the supplier's protocol. After incubation with the test sample for 24 h, cells were harvested with 30 µl of cell lysis buffer (Promega Co.), and the firefly and renilla luciferase activities were determined with the Dual Luciferase Assay Kit (Promega Co.). Firefly luciferase reporter activity was normalized to renilla luciferase activity from phRL-CMV.
Competitive binding assay using an ELISA kit.
The binding affinity of BPA and MBP for hER and hERß was assayed using ELISA Kits (Ligand Screening Systems-ER
and -ERß; Toyobo Co., Ltd., Osaka, Japan) according to the method described in the manufacturer's protocol. In principle, the assay is based on competition of the estrogenic ligand with E2 for binding to the receptor in the first step. In the second step, free E2 binds to an immobilized anti-E2 antibody in competition with horseradish peroxidase (HRP)-labeled E2. Finally, the amounts of HRP-labeled E2 bound to the antibody were measured based on the peroxidase activity. In this assay, diethylstilbestrol (DES) was used as a positive standard.
Chemical synthesis of MBP.
MBP, a candidate active metabolite of BPA, was chemically synthesized according to the method described by Dai et al. (1985) with minor modifications (Farr, 1999
). A mixture of 5 g of BPA and 20 mg of NaOH was heated under vacuum at 175°C until most of the solids had melted. The melted mixture was heated by increasing temperature to 220°C under a reduced pressure of 350 mm Hg for 2 h, followed by 2.5 h at 250°C under a pressure of 50 mm Hg. From the yellow solid (3.45 g) obtained, phenol was removed by distillation at 90-140°C under a reduced pressure of 5 mm Hg. The resultant yellowish solid (0.96 g) dissolved in a small amount of ethyl acetate was applied to a silica gel column and fractionated by eluting with a mixture of ethyl acetate:n-hexane (2:3). The fractions containing MBP were pooled, and the organic solvent was evaporated to dryness. The crude MBP (0.78 g) was purified by recrystallization with a mixed solvent of dichloromethane/n-hexane. Finally, 65.2 mg of MBP was obtained as a white powder giving a melting point at 130.5-132°C. The 1H NMR data (400 MHz, CDCl3) of authentic MBP (Fig. 1) were as follows:
1.20 (s, 6H, CH3), 2.74 (s, 2H, CH2), 4.64 (s, 1H, OHc or OHd), 4.71 (d, 1H, Hb, J = 2.0 Hz), 4.74 (s, 1H, OHc or OHd), 5.07 (d, 1H, Ha, J = 2.0 Hz), 6.67 (d, 2H, Hf or Hh, J = 8.8 Hz), 6.67 (d, 2H, Hf or Hh, J = 8.4 Hz), 7.09 (d, 2H, He or Hg, J = 8.8 Hz), 7.12 (d, 2H, He or Hg, J = 8.8 Hz).
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Species Differences in Metabolic Activation of BPA by Liver S9
The metabolic activation of either BPA or BPB essentially required both microsomal and cytosolic fractions (Yoshihara et al., 2001). Therefore, the liver S9 fractions of mice, monkeys, and humans, as well as rats, were adopted as enzyme sources in this experiment. As shown in Figure 2, in addition to rat liver S9, all other S9 fractions tested, including human, were able to generate a 2- to 4-fold enhancement of the estrogenicity of the metabolites extracted after incubation.
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This is the first report to elucidate the structure of an estrogenically active metabolite of BPA formed by incubation with rat liver S9. This active metabolite was identified as MBP, 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene, by comparison with an authentic sample synthesized by alkaline fusion of BPA (Dai et al., 1985). MBP is an isopropenylphenol dimer, so its formation might involve the isopropenylphenol radical, formed by oxidative cleavage of carbon-phenyl bond, as an intermediate (Scheme 1). The involvement of this mechanism is strongly supported by the observations that M-3/M-4, thought to be isobutenylphenol dimers, are formed in the case of incubation of BPB (Fig. 5), and M-5/M-6, which may be combination products of the isopropenylphenol and isobutenylphenol moieties, are formed in the case of coincubation of BPA and BPB (Fig. 6). Formation of the radical as an intermediate was further supported by the disappearance of both M-1 and M-2 when
-tocopherol, a radical scavenger, was added to the incubation system with BPA (unpublished observation). As demonstrated previously, this metabolic activation depends on the coexistence of both microsomal and cytosolic fractions, namely S9; neither microsomes nor cytosol alone are effective (Yoshihara et al., 2001
). Concerning the factors in these fractions, involvement of CYP as a microsomal factor is suggested, but the cytosolic factor(s), which seems to be protein(s) precipitated between 50 and 70% ammonium sulfate (data not shown), have not yet been identified.
It is important that MBP exhibited much more potent estrogenic activity compared with BPA itself (Figs. 9 and 10). Although its estimated potency varied somewhat among the assay methods used, the estrogenicity of MBP appeared to be several-fold to several thousand-fold stronger than that of BPA (Table 1). For example, in ELISA assay MBP gave almost the same IC50 value for inhibition of E2 binding to hER as DES, a positive control. The actual reason why the estrogenic activity of MBP should be much more potent compared to BPA is not fully understand yet. To obtain, however, some structural similarities of MBP with E2, we carried out a preliminary computational study employing restrained energy minimization using a Chem 3D Pro. The results demonstrated that, unlike BPA itself, both compounds show almost similar bulkiness with coplanar conformation, giving the distances of 10.7 Å and 9.3 Å between two hydroxy moieties of E2 and MBP, respectively (data not shown). This 1.4 Å difference in oxygen-oxygen distances might be allowable upon ligand binding to ER (Anstead et al., 1997
). These structural similarities of both estrogens might be one of the possible explanations for a potent estrogenicity of MBP. Based on the results shown in Figure 2, it seems very likely that the metabolic activation of BPA by liver S9 fraction is a common phenomenon observed across a wide range of animal species.
On the other hand, it is well known that in several animal species, including humans, the predominant metabolite of BPA in vivo (Kurebayashi et al., 2002; Pottenger et al., 2000
; Snyder et al., 2000
; Volkel et al., 2002
) or in isolated hepatocytes (Nakagawa and Tayama, 2000
; Pritchett et al., 2002
) is BPA monoglucuronide, which is inactive as an estrogen (Matthews et al., 2001
). Thus, metabolic activation to MBP may not be significant, at least in usual circumstances. However, under circumstances where glucuronidation is unable to work efficiently as a detoxification pathway of BPA, such metabolic activation might occur. With regard to glucuronidation, fetal livers of rats (Coughtrie et al., 1988
; Yokota et al., 2000
) and humans (Coughtrie et al., 1988
; Pacifici et al., 1993
) show little or no activity. In accordance with the poor glucuronide conjugation capacity of the fetus, similar levels of unconjugated BPA, but not the glucuronide, were detected in the fetal plasma of rats (Miyakoda et al., 2000
; Takahashi and Oishi, 2000
) and humans (Ikezuki et al., 2002
; Schonfelder et al., 2002
). Although the rodent fetal liver has a limited capacity for CYP-dependent xenobiotic metabolism compared with the adult liver, some forms of CYP are expressed in late gestation (Cresteil et al., 1986
; Raucy and Carpenter, 1993
). Additionally, unlike rodent species, the human fetal liver exhibits a significant metabolizing activity for xenobiotics (Hakkola et al., 1998
; Ring et al., 1999
) and expresses certain isoforms of CYP, such as CYP 3A7(Kitada et al., 1987
). If this kind of metabolic activation of BPA is realized in vivo, especially in the fetus, which is one of the most important targets of EDCs, the MBP formed may be a significant EDC. This would provide a possible explanation for the discrepancy between the estrogenic potency of BPA in vitro and in vivo (Steinmetz et al., 1997
) and the low-dose effects of BPA in mice exposed during prenatal development (Nagel et al., 1997
; vom Saal et al., 1998
).
In conclusion, we have shown that MBP, 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene, is a very potent estrogenic metabolite of BPA formed by liver S9 fractions of several animal species, including human. Further study of the precise mechanism of this unique metabolic activation, including the factors involved, is currently under way. We are also trying to elucidate whether this kind of metabolic activation actually occurs in the fetal liver, and also to detect MBP as an in vivo metabolite in the fetus. Additionally, the in vivo assay of estrogenic potency of MBP in mice is in progress.
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This work was supported by a Grant-in-Aid for Scientific Research on a Priority Area (13027256) from the Japanese Ministry of Education, Science and Culture. We would like to thank Prof. John Sumpter, Brunel University, U.K. and Associate Prof. Jun-ichi Nishikawa, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan, for providing the recombinant yeast for the YES assay and the two-hybrid assay, respectively. We also thank Dr. Kazuo Asaoka, Primate Research Institute, Kyoto University, Inuyama, Japan, for donating frozen blocks of monkey liver.
REFERENCES
Anstead, G. M., Carlson, K. E., and Katzenellenbogen, J. A. (1997). The estradiol pharmacophore: Ligand structure-estrogen receptor binding affinity relationships and a model for the receptor binding site. Steroids 62, 268303.[CrossRef][ISI][Medline]
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 CF-1 mice exposed in utero. Regul. Toxicol. Pharmacol. 30, 156166.[CrossRef][ISI][Medline]
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, 608612.[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., Veenstr, G. E., and Harris, L. R. (1999). Normal reproductive organ development in CF-1 mice following prenatal exposure to bisphenol A. Toxicol. Sci. 50, 3644.[Abstract]
Colborn, T. (1995). Environmental estrogens: Health implications for humans and wildlife. Environ. Health Perspect. 103, 135136.[ISI][Medline]
Coughtrie, M. W. H., Burchell, B., Leakey, J. E. A., and Hume, R. (1988). The inadequacy of perinatal glucuronidation: Immunoblot analysis of the developmental expression of individual UDG-glucuronosyltransferase isoenzymes in rat and human liver microsomes. Mol. Pharmacol. 34, 729735.[Abstract]
Cresteil, T., Beaune, P., Celier, C., Leroux, J. P., and Guengerich, F. P. (1986). Cytochrome P450 isoenzyme content and monooxygenase activities in rat liver: Effect of ontogenesis and pretreatment by phenobarbital and 3-methylcholanthrene. J. Pharmacol. Exp. Ther. 236, 269276.[Abstract]
Dai, S. H., Lin, C. Y., Rao, D. V., Stuber, F. A., Carleton, P. S., and Ulrich, H. (1985). Selective indirect oxidation of phenol to hydroquinone and catechol. J. Org. Chem. 50, 17221725.[ISI]
Dodds, E. C., and Lawson, W. (1936). Synthetic estrogenic agents without the phenanthrene nucleus. Nature 137, 996.
Farr, I. V. (1999). Synthesis and characterization of novel polyimide gas separation membrane material systems. Ph.D. Dissertation, Virginia Polytechnic Institute and State University, Blacksburg, VA.
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, 205212.[CrossRef][ISI][Medline]
Hakkola, J., Pelkonen, O., Pasanen, M., and Raunio, H. (1998). Xenobiotic-metabolizing cytochrome P450 enzymes in the human feto-placental unit: Role in intrauterine toxicity. Crit. Rev. Toxicol. 28, 3572.[ISI][Medline]
Ikezuki, Y., Tsutsumi, O., Takai, Y., Kamei, Y., and Taketani, Y. (2002).Determination of bisphenol A concentrations in human biological fluids reveals significant early prenatal exposure. Human Reprod. 17, 28392841.
Kawagoshi, Y., Fukunaga, I., Nishikawa, J., and Nishihara, T. (2000). Study on simplication of yeast two-hybrid based assay for estrogenic activity. J. Environ. Chem. 10, 6572.
Khim, J. S., Lee, K. T., Kannan, K., Villeneuve, D. L., Giesy, J. P., and Koh, C. H. (2001). Trace organic contaminants in sediment and water from Ulsan Bay and its vicinity, Korea. Arch. Environ. Contam. Toxicol. 40, 141150.[CrossRef][ISI][Medline]
Kitada, M., Kamataki, T., Itahashi, K., Rikihisa, T., and Kanakubo, Y. (1987). P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, is the 16-hydroxylase of dehydroepiandrosterone 3-sulfate. J. Biol. Chem. 262, 1353413537.
Knaak, J. B., and Sullivan, L. J. (1966). Metabolism of bisphenol A in the rat. Toxicol. Appl. Pharmacol. 8, 175184.[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, 22792286.[Abstract]
Kuiper, G., Carlson, B., Grandien, K., Enmark, E., Haggblad, J., Nilsson, S., and Gustafsson, J. A. (1997). Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptor and ß. Endocrinology 132, 22792286.[CrossRef]
Kurebayashi, H., Harada, R., Stewart, R. K., Numata, H., and Ohno, Y. (2002). Disposition of a low dose of bisphenol A in male and female cynomolgus monkeys. Toxicol. Sci. 68, 3242.
Maruyama, S., Fujimoto, N., Asano, K., and Ito, A. (2001). Suppression by estrogen receptor ß of AP-1 mediated transactivation through estrogen receptor . J. Steroid Biochem. Mol. Biol. 78, 177184.[CrossRef][ISI][Medline]
Matthews, J. B., Twomey, K., and Zacharewski, T. R. (2001). In vitro and in vivo interactions of bisphenol A and its metabolite, bisphenol A glucuronide, with estrogen receptors and ß. Chem. Res. Toxicol. 14, 149157.[CrossRef][ISI][Medline]
Miyakoda, H., Tabata, M., Onodera, S., and Takeda, K. (2000). Comparison of conjugative activity, conversion of bisphenol A to bisphenol A glucuronide, in fetal and mature male rat. J. Health Sci. 46, 269274.[ISI]
Motoyama, A., Suzuki, A., Shirota, O., and Namba, R. (1999). Direct determination of bisphenol A and nonylphenol in river water by column-switching semi-microcolumn liquid chromatography/electrospray mass spectrometry. Rapid Commun. Mass Spectrom. 13, 22042208.[CrossRef][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, 7076.[ISI][Medline]
Nakagawa, Y., and Tayama, S. (2000). Metabolism and cytotoxicity of bisphenol A and other bisphenols in isolated rat hepatocytes. Arch. Toxicol. 74, 99105.[CrossRef][ISI][Medline]
Olea, N., Pulgar, R., Perez, P., Olea-Serrano, F., Rivas, A., Novillo-Fertrell, A., Pedraza, V., Soto, A. M., and Sonnenshein, C. (1996). Estrogenicity of resin-based composites and sealants used in dentistry. Environ. Health Perspect. 104, 298305.[ISI][Medline]
Pacifici, G. M., Kubrich, M., Giuliani, L., de Vries, M., and Rane, A. (1993). Sulphation and glucuronidation of ritodrine in human fetal and adult tissues. Eur. J. Clin. Pharmacol. 44, 259264.[ISI][Medline]
Pottenger, L. H., Domoradzki, J. Y., Markham, D. A., Hansen, 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, 318.
Pritchett, J. J., Kuester, R. K., and Sipes, I. G. (2002). Metabolism of bisphenol A in primary cultured hepatocytes from mice, rats, and humans. Drug Metab. Dispos. 30, 11801185.
Raucy, J. L., and Carpenter, S. J. (1993). The expression of xenobiotic-metabolizing cytochromes P450 in fetal tissues. J. Pharm. Toxicol. Methods 29, 121128.[CrossRef][ISI][Medline]
Ring, J. A., Ghabriel, H., Ching, M. S., Smallwood, R. A., and Morgan, D. J. (1999). Fetal hepatic drug elimination. Pharmacol. Ther. 84, 429445.[CrossRef][ISI][Medline]
Routledge, E. J., and Sumpter, J. P. (1997). Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environ. Toxicol. Chem. 15, 241248.[ISI]
Schonfelder, G., Wittfoht, W., Hopp, H., Talsness, C. E., Paul, M., and Chahoud, I. (2002). Parent bisphenol A accumulation in the human maternal-fetal-placental unit. Environ. Health Perspect. 110, A703A707.[ISI][Medline]
Snyder, R. W., Maness, S. C., Gaido, K. W., Welsch, F., Sumner, S. C. J., and Fennel, T. R. (2000). Metabolism and disposition of bisphenol A in female rats. Toxicol. Appl. Pharmacol. 168, 225234.[CrossRef][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, 17801786.
Takahashi, O., and Oishi, S. (2000). Disposition of orally administered 2,2-bis(4-hyroxyphenyl)propane (bisphenol A) in pregnant rats and the placental transfer to fetuses. Environ. Health Perspect. 108, 931935.[ISI][Medline]
Volkel, W., Colnot, T., Csanady, G. A., Filser, J. G., and Dekant, W. (2002). Metabolism and kinetics of bisphenol A in humans at low doses following oral administration. Chem. Res. Toxicol. 15, 12811287.[CrossRef][ISI][Medline]
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, 239260.[ISI][Medline]
Yamamoto, T., and Yasuhara, A., (1999). Quantities of bisphenol A leached from plastic waste samples. Chemosphere 38, 25692576.[CrossRef][ISI][Medline]
Yokota, H., Inoue, H., Matsumoto, J., Shibata, N., Kato, S., and Yuasa, A. (2000). Glucuronidation of environmental estrogens in rat liver. The 3rd Annual Meeting of Japanese Society of Endocrine Disruptors Research. Yokohama, Japan. Abstract B-1-4.
Yoshihara, S., Makishima, M., Suzuki, N., and Ohta, S. (2001). Metabolic activation of bisphenol A by rat liver S9 fraction. Toxicol. Sci. 62, 221227.