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
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ABSTRACT |
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Key Words: bisphenol A; 17ß-estradiol; estrogenic activity; recombinant yeast assay; MCF-7; rat liver S9; metabolic activation.
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INTRODUCTION |
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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 prolactin gene expression and release in vitro was 1000- to 5000-fold lower than that of 17ß-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.
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MATERIALS AND METHODS |
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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 estrogen-responsive 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-µl aliquots of ethanol solutions of E2 used as standards ranging from 2.9 x 1013 to 5 x 107 M as well as other EDCs tested, including BPA, ranging from 2.4 x 109 to 4.4 x 104 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-µl 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-µl aliquots of the yeast assay medium containing recombinant yeast and CPRG, the chromogenic substrate of ß-galactosidase reporter enzyme, were dispensed into each well. If needed, 1 µM 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 2448 h, the red color of the hydrolysis product of CPRG by ß-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 x 105 cells/well were transiently transfected with 1.9 µg of p(ERE)3-SV40-luc and 0.1 µg 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 µl 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 (56 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 x g for 20 min. The S9 fraction was further centrifuged at 105,000 x 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 µl ethanol, an NADPH-generating system (0.5 mM NADP+, 5 mM glucose-6-phosphate, 5 mM MgCl2), 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 µl ethanol, then 10 µl 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 µl 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 x 150 mm, 5 µm; Supelco, Bellefonte, PA). The separation of metabolites was performed with a linear gradient of 070% 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 NADPH-generating 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 µl acetonitrile was applied to a preparative reversed-phase column (Supelcosil ABZ+ Plus; 10 x 250 mm, 5 µm; Supelco, Bellefonte, PA). The separation of metabolites was performed with a linear gradient of 070% 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 µl acetonitrile; 10 µl of each sample solution was applied for the yeast estrogenicity assay and the remainder for GC/MS or LC/MS analysis.
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RESULTS |
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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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NOTES |
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