You are on page 1of 9

Environmental Toxicology and Pharmacology 12 (2002) 27 /35 www.elsevier.


Identication of estrogenic activity of chlorinated bisphenol A using a GFP expression system

Ryoko Kuruto-Niwa a,*, Yoshiyasu Terao b, Ryushi Nozawa a

Laboratory of Microbiology and Host Defenses, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan b Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan Received 10 October 2001; received in revised form 15 February 2002; accepted 18 February 2002

Abstract A green fluorescent protein (GFP)-reporter vector regulated by an estrogen response element (ERE) was constructed and transfected into human breast carcinoma MCF7 cells. Stable transfectants were selected and their GFP fluorescence intensity was measured using a quantitative fluorescent imaging system. 17b-estradiol (E2) and bisphenol A (BPA) induced a dose-dependent increase in GFP intensity in the cells, reaching maximum response at 5 )/10 (10 and 10 (5 M, respectively. Using this GFP expression system, we examined the estrogenicity of mono-, di-, tri-, and tetra-chlorinated BPAs, which were detected in wastewater from waste-paper recycling plants using sodium hypochlorite as a bleaching agent. 3-ClBPA and 3,3?-diClBPA showed similar estrogenicities, effective at lower concentrations than parent BPA. On the other hand, the maximum activities of BPA and 3,3?,5triClBPA, whose EC50 were similar, were higher than other chlorinated BPAs. This is the first demonstration of the estrogenicity of chlorinated BPAs. Since polychlorinated BPAs were not easily biodegraded, chlorinated BPAs might be more severe endocrine disruptors than BPA. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: Green uorescent protein; Estrogen response element; MCF-7 cells; Estrogen; Bisphenol A; Chlorinated bisphenol A

1. Introduction It is known that certain chemicals in the environment such as bisphenol A (BPA) and 4-nonylphenol (NP) are estrogenic (Krishnan et al., 1993; Soto et al., 1995). These chemicals may modulate the endocrine system contributing to adverse health, reproduction, and developmental effects in humans and wildlife (McLachlan et al., 1980; Guillette et al., 1994), and, therefore, are commonly referred to as endocrine disruptors. Recently, analyses of estrogenic hormones and chemicals in municipal wastewater effluent and surface water have been performed in various areas of the world (Korner et al., 2000; Huang and Sedlak, 2001; Khim et al., 2001). BPA is a common raw material used in the polymer and plastics industries, and is also used to
* Corresponding author. Tel.: '/81-54-264-5554; fax: '/81-54-2645553. E-mail address: (R. KurutoNiwa).

produce paper, such as thermal paper and carbonless copy paper. Therefore, BPA might be released into wastewater from recycling plants that process such paper products. On the other hand, BPA readily reacts with sodium hypochlorite, which is used as a bleaching agent in paper factories, to produce chlorinated BPAs. Recently, chlorinated BPAs have been detected in the final effluent from waste-paper recycling plants using sodium hypochlorite (Fukazawa et al., 2001). Staples et al. (1998) reported that BPA was not persistent based on its rapid biodegradation in acclimated wastewater treatment plants and receiving waters (half-lives 2.5 /4 days). In contrast, a biodegradation test using activated sludge as the supernatant revealed that polychlorinated BPAs were not found to be easily biodegraded (Fukazawa et al., 2001). Therefore, it is important to evaluate the properties, such as estrogenic activity and toxicity, of the chlorinated BPAs. To determine the estrogenicity of chemicals, various methods such as the cell proliferation assay, receptor binding assay, reporter gene assay, and uterotrophic

1382-6689/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 2 - 6 6 8 9 ( 0 2 ) 0 0 0 1 1 - X


R. Kuruto-Niwa et al. / Environmental Toxicology and Pharmacology 12 (2002) 27 /35

assay have been performed (Lan and Katzenellenbogen, 1976; Pons et al., 1990; Soto et al., 1995; Klinge et al., 1997; Fielden et al., 1997). However, the merits and limitations of these assays have been discussed (Zacharewski, 1998; Legler et al., 1999). A cell proliferation assay based on MCF7 cells, whose growth is estrogen dependent, has been proposed as a possible assay of estrogenicity (Soto et al., 1995). However, the assay generally takes several days to assess the estrogenicity and is quite time consuming. In addition, it is complicated by the cytotoxicity of some chemicals. Among a number of in vitro assays, the reporter gene assay based on stably transfected cell line seems to be one of the most sensitive and simple assays. Several reporter gene systems have been already developed (Pons et al., 1990; Legler et al., 1999). In this report, we tried to establish an assay using green fluorescent protein (GFP) as a reporter gene. GFP was first cloned and sequenced from the jellyfish Aequorea victoria (Prasher et al., 1992). Wild-type GFP emits green fluorescence when excited with blue or UV light without any additional substrates or cofactors. Therefore, GFP has emerged as a reporter for gene expression (Chalfie et al., 1994; Gerdes and Kaether, 1996; Plautz et al., 1996). EGFP encodes a redshifted variant of wild-type GFP that has been optimized for brighter fluorescence and higher expression in mammalian cells. We constructed an estrogen response element (ERE)-regulated GFP reporter vector containing a neomycin-resistant gene. The vector was transfected into human breast carcinoma MCF7 cells, which are estrogen-responsive. Stably transfected MCF-7 cells expressed GFP in an estrogen-dependent manner. We then devised a simple GFP expression system to detect estrogenic chemicals using a quantitative fluorescent imaging system. In this report, the estrogenic activity of halogenated BPAs was investigated using the established GFP expression system.

Fig. 1. Chemical structure of BPA and its derivatives.

chased from Aldrich (USA). The structures of BPA derivatives are shown in Fig. 1.

2.2. Cells and culture Human breast cancer MCF7 cells were maintained in Dulbeccos modified Eagle medium (DMEM) (Nissui, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma) and 100 mg/ml each of sodium ampicillin and kanamycin sulfate at 37 8C under humidified 5% CO2/95% air.

2. Materials and methods 2.1. Materials 17b-estradiol (E2), tamoxifen (Tam), dexamethasone (Dex), progesterone, and testosterone were purchased from Sigma (St. Louis, MO). Dihydrotestosterone (DHT) was purchased from Wako Pure Chemical Industries (Osaka, Japan). BPA was purchased from Kanto-Chemical (Tokyo, Japan). 4-NP was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Genistein and daidzein were kindly provided by Dr. A. Hirota, University of Shizuoka (Hirota et al., 2000). Chlorinated BPAs, 3-ClBPA, 3,3?-diClBPA, 3,5diClBPA, 3,3?,5-triClBPA, and 3,3?,5,5?-tetraClBPA were synthesized and purified as described previously (Fukazawa et al., 2001). 3,3?,5,5?-tetraBrBPA was pur-

2.3. Construction of ERE-regulated GFP vector pGL3-pro-3(EREc38), which contains three tandem copies of EREc38 (a 38-bp ERE consensus sequence) at the upstream of SV40 promoter linked to the firefly luciferase reporter gene (Klinge et al., 1997), was digested with Not I and Nco I. A fragment containing 3(EREc38) and SV40 promoter were inserted into pEGFP-C1 (Clontech, Palo Alto, CA) instead of cytomegalovirus (CMV) promoter. We designated it pEGFP-3(EREc38), which is an ERE-regulated GFP reporter vector containing a neomycin resistance gene.

R. Kuruto-Niwa et al. / Environmental Toxicology and Pharmacology 12 (2002) 27 /35


2.4. Transfection and selection of GFP expressing-MCF7 cells MCF7 cells (1 )/106) were plated in a 60 mm tissue culture dish in DMEM supplemented with 10% FBS. After 24 h, the cells were transfected with 1.66 mg of pEGFP-3(EREc38). For the transfection, DNA was preincubated with cationic lipid Tfx-20 transfection reagents (Promega, Madison, WI) at a ratio of 1 mg DNA/6 nmol Tfx-20 in DMEM for 15 min at room temperature (Schenborn et al., 1996; Ishimi et al., 1998; Kuruto-Niwa et al., 2000). MCF7 cell medium was removed and replaced with 2 ml of preincubated Tfx-20/ DNA mixture, and cells were incubated at 37 8C for 60 min in a CO2 incubator. At the end of the incubation period, 4 ml of fresh medium was added to the mixture. After 48 h of transfection, antibiotic G418 (Gibco BRL, NY, USA, final 800 mg/ml) was added to the medium to select the stable transfectants (Southern and Berg, 1982). The medium was renewed every 3 /4 days. After 2 /3 weeks, colonies were isolated and transferred to the assay medium [phenol red-free DMEM (Cosmo Bio, Tokyo, Japan) supplemented with 10% heat-inactivated charcoal/dextran treated FBS (Hyclone, Logan, UT)] with 10(10 M E2 and further cultured. The cells that expressed GFP in the presence of E2 were selected and designated as ERE-GFP-MCF7. 2.5. Cell monitoring by microscope Cells were observed under a fluorescence microscope (IMT-2, Olympus). Green fluorescent images and transmission images were monitored with 3CCD High Gain Color Camera (HCC-1200, FLOVEL) and analyzed using the Macintosh image analysis program Mac SCOPE. 2.6. Cell proliferation assay and image analysis of estrogenicity ERE-GFP-MCF7 cells (1 or 2)/104) were plated in each well of a 96-well plate (Costar, Corning, NY) in the assay medium as described above. After 3 h, indicated amounts of estrogen and/or the test materials or an equal volume of ethanol or DMSO (solvent) as controls were added, and cells were further incubated. Cell proliferation was measured using Cell Counting Kit-8 (Dojin, Kumamoto, Japan), where a water-soluble tetrazolium salt WST-8, namely 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt, is used as a substrate (Ishiyama et al., 1997; Tominaga et al., 1999). The counting solution containing WST-8 and 1-methoxyphenazinium salt (0.5 mM and 20 mM, respectively, as the final concentration) was added to each well, and cells were incubated at 37 8C for 2 h in a CO2

incubator. Cells are not damaged by WST-8 counting solution. The absorbance of each well was measured at 450 nm with a reference wavelength at 630 nm. The number of cells was calculated by standard curve. After the counting assay, the medium in the 96-well plate was removed. Then the intensity of green fluorescence in the cells was scanned using a FluorImager SI (Amersham pharmacia biotech). The cell counting solution in the kit did not affect the measurement of fluorescence (data not shown). Fluorescence intensity was estimated with the ImageQuant program (Amersham pharmacia biotech). Each experiment was performed in triplicate, and the assay was repeated two or three times. 2.7. Dose-response curves and statistics In all data, mean9/S.E.M. is calculated. To determine the EC50, dose response curves were fitted (sigmoid fit) using GraphPad Prism 3.0 (GraphPad Software, Inc., CA, USA). The EC50 was calculated by determining the concentration at which 50% of the maximum fluorescence intensity was reached using the sigmoidal fit equation. Statistical comparisons among groups were determined using one-way analysis of variance (ANOVA). Individual comparisons were performed using Fishers Protected LSD test. Statistical significance was ascribed to the datum when it was significant at P B/0.05.

3. Results 3.1. Establishment of an ERE-regulated GFP expression system Stable transfectants, ERE-GFP-MCF7 cells, were established by transfection of MCF7 cells with the plasmid pEGFP-3(EREc38), and by then selecting resistant clones in the presence of G418. The expression of GFP in ERE-GFP-MCF7 cells was dependent on the presence of E2 as shown in Fig. 2A and B. Green fluorescence of the cells under the microscope was also induced by BPA (Fig. 2C). On the other hand, Tam, which is an estrogen antagonist, inhibited E2-dependent induction of GFP (Fig. 2D). Also, fluorescence intensity of GFP in ERE-GFPMCF7 cells was measured using a FluorImager with the ImageQuant program. The clearest results were obtained when measured after 2 /3 days of incubation with test chemicals (data not shown). We calculated the GFP intensity per cell in the combination of the cell proliferation assay. As shown in Fig. 3, ERE-GFP-MCF7 cells expressed GFP in response to E2 in a dosedependent manner. The lowest effect of E2 was observed at 10(12 M (P B/0.05 vs. control). The maximum


R. Kuruto-Niwa et al. / Environmental Toxicology and Pharmacology 12 (2002) 27 /35

3.2. Specicity of the estrogenic response To determine the specificity of the ERE-regulated GFP expression system, we tested the estrogenic chemicals and various hormones. 4-NP, genistein, and daidzein as well as BPA are known to exhibit estrogenic activity. BPA induced a dose-dependent increase up to 10(5 M in GFP intensity in the cells (Fig. 3). The maximum GFP intensity of BPA though, exceeded the maximum activity induced by E2, at a 50 000-fold higher concentration than E2. The EC50 of BPA was 1.1 )/10(6 M (Table 1). 4-NP, genistein, and daidzein also showed estrogenic activity. These EC50 values were 2.3 )/10(7, 5.8 )/10(8, and 3.2 )/10(7 M, respectively (Table 1). Among them, genistein was the most potent estrogenic chemicals, having the lowest EC50 and the higher maximum fluorescence intensity. We then examined the responsiveness to other hormones, glucocorticoid Dex, progesterone, testosterone, and DHT, which is an active form of testosterone. These hormones did not induce GFP expression when tested in concentrations ranging from 10 (11 to 10(5 M for progesterone and Dex, 10 (11 to 10(7 M for testosterone and DHT. Thus, GFP expression in our system is specific for estrogenic chemicals. This GFP expression system was useful for the detection of endocrine disruptors that had estrogenic effects. 3.3. Effects of chlorinated bisphenol A on cell proliferation To evaluate the properties of the chlorinated BPAs, the effects of chlorinated BPAs on cell proliferation were examined. The ERE-GFP-MCF7 cells were plated in 96well microplates at a density of 1 )/104 cells per well, and treated with a range of chemical concentrations from 10(10 to 10(4 M. Fig. 4 shows the growth of cells treated with BPA and three chlorinated BPAs, 3-ClBPA, 3,3?-diClBPA, and 3,3?,5-triClBPA. At 10 (6 to 10 (4 M, BPA greatly stimulated the growth of ERE-GFP-MCF7 cells (Fig. 4A). However, 10(4 M BPA was less estrogenic than 10(5 M BPA as calculated using the GFP expression system (data not shown). Similar growth stimulatory effect was observed in 10 (6 to 10(5 M treatments of chlorinated BPAs (Fig. 4B /D). At 10(6 M, 3,3?,5-triClBPA had a lower effect on cell proliferation than other chemicals. TetraClBPA at 10 (5 M stimulated cell growth to a lesser degree, and tetraBrBPA had no effect on cell growth (data not shown). On the other hand, 10 (4 M of chlorinated BPAs was cytotoxic to cells in a manner different to that of BPA. Fig. 5 shows typical images. Cells were round and dead at 10(4 M of 3,3?-diClBPA as well as 3-ClBPA (Fig. 5C), and were expanded at 10 (4 M of 3,3?,5-triClBPA (Fig. 5D). 3,5-diClBPA, tetraClBPA, and tetraBrBPA-

Fig. 2. Fluorescence microscopy showing GFP expression in EREGFP-MCF7 cells. ERE-GFP-MCF7 cells were left untreated (A), treated with 10 (10 M E2 (B), 10 (6 M BPA (C), or 10 (11 M E2 plus 5)/10 (7 M Tam (D) for 3 days. Bar0/50 mm.

Fig. 3. Analysis of the induction of GFP expression by estrogenic chemicals and various hormones in ERE-GFP-MCF7 cells. EREGFP-MCF7 cells were treated with the indicated concentrations of E2, BPA, NP, genistein, daidzein, progesterone, testosterone, DHT, and Dex for 3 days. Fluorescence intensity was assessed using a FluorImager. The values are relative to the maximum uorescence intensity per cell obtained with E2 (50 pM). Points are means (n 0/6)9/S.E.M. for each concentration. Two replicates were taken from each treatment, which was performed in triplicate. The controls were treated with ethanol or DMSO at a nal concentration of 0.1%.

response is reached at 5)/10(11 M E2. The EC50 value, a concentration at which 50% of the maximum fluorescence intensity is reached, was calculated by a doseresponse curve to 5.9 )/10 (12 M. Similar EC50 and maximum effect concentration were calculated from the data per well without the combination of cell counting (data not shown). Estrogenicity of the chemicals can then be simply assessed by monitoring the GFP intensity per well. However, fluorescence intensity of the data in this report was calculated per cell.

R. Kuruto-Niwa et al. / Environmental Toxicology and Pharmacology 12 (2002) 27 /35 Table 1 Potency of estrogenic chemicals relative to estradiol in the GFP expression system Chemicals Natural hormone Estradiol EC50 (M)a Relative potency (EC50)b Maximum effect concentration (M)c Maximum fluorescence intensityd



1 5.19E-6 2.54E-5 1.02E-4 1.86E-5

5.0E-11 1.0E-5 1.0E-6 1.0E-6 1.0E-5

100 1199/5.4 84.59/3.4 1419/8.3 1449/4.7

Industrial chemicals BPA 1.14E-6 4-NP 2.34E-7 Phytoestrogen Genistein Daidzein

a b c d

5.83E-8 3.20E-7

Concentration at which 50% of the maximum fluorescence intensity is reached. Ratio between EC50 of chemicals and EC50 of estradiol. Concentration at which maximum estrogenic effect is detected. Maximum fluorescence intensity 9/S.E.M. relative to the intensity obtained with E2 (50 pM).

treated cells were similar (data not shown). The inhibition of cell growth in 10(8 to 10(10 M of 3-ClBPA and 3,3?-diClBPA might be due to appearance of cytotoxicity rather than to estrogenicity (Fig. 4B and C). 3.4. Estrogenicity of chlorinated bisphenol A measured using a GFP expression system Using the established GFP expression system, we evaluated the estrogenic activity of chlorinated BPAs. Fig. 6 shows the results of GFP fluorescence intensity per cell at chemical concentrations ranging from 10 (9 to 10(5 M. 3-ClBPA, 3,3?-diClBPA, 3,5-diClBPA, and 3,3?,5-triClBPA all exhibited estrogenic activity. The ranking of EC50 was 3-ClBPA (1.5 )/10(7 M), 3,3?diClBPA (2.3 )/10 (7 M) /3,5-diClBPA (9.5 )/10 (7 M), BPA (1.1 )/10(6 M), 3,3?,5-triClBPA (2.0 )/10 (6 M) (Table 2). On the other hand, the ranking of maximum activity was BPA /3,3?,5-triClBPA /3,5diClBPA /3-ClBPA /3,3?-diClBPA. The maximum activity of 3,5-diClBPA, 3-ClBPA, and 3,3-diClBPA was significantly different from that of BPA at P B/0.05. 3-ClBPA and 3,3?-diClBPA showed similar estrogenic potency, and were more effective at lower concentrations than parent BPA. 3,3?,5,5?-tetraClBPA exhibited lower estrogenic activity compared with other chlorinated BPAs. 3,3?,5,5?-tetra BrBPA had a much weaker estrogenic effect.

4. Discussion A number of in vitro assays have been developed to identify and assess the estrogenicity of chemicals (Zacharewski, 1998). Among the assays, an ERE-regulated reporter gene assay seems to be the most commonly used. Several systems have been established (Pons et al., 1990; Klinge et al., 1997; Legler et al., 1999). Recently, using GFP as a new reporter, Miller et al. (2000)

reported the development of a rapid and sensitive method using the MCF7-ERE cells that expressed GFP in the presence of E2 by fluorescence microscopy. The advantages of GFP compared with the luciferase are the lack of a requirement for additional expensive reagent and the ease of measurement (read directly in the living cells without cell disruption). We independently constructed ERE-GFP-MCF7 cells and devised an estrogenic assay to measure GFP fluorescence in the cells treated with certain chemicals in a 96-well plate using a FLUORIMAGER. In this system, it was easier to assess the estrogenic activity of a large number of chemicals at once compared with the methods of Miller et al. (2000). Our GFP expression system requires a longer incubation time with test chemicals than other reporter gene assays such as the ER-CALUX assay reported by Legler et al. (1999). However, no additional work such as medium renewal and cell lysis is required after plating in our GFP expression system. Also, GFP expression is observed in the living cells in real-time. It can be applied to further experiments following a time course. As a first screening in vitro, our system is a useful method because it is a simple and inexpensive assay. It has also been reported that estrogen-induced GFP expression systems have been developed in transgenic plants and animals (Carvan et al., 2000; Zuo et al., 2000; Thackray et al., 2000). We demonstrated the specificity of the GFP expression system by testing several estrogenic chemicals and various hormones (Fig. 3). The GFP expression system appears to be specific for estrogenic chemicals, since other various hormones tested, such as progesterone, testosterone, DHT, and Dex, did not induce GFP expression at tested concentrations. The responses of estrogenic chemicals in our GFP expression system were compared with the data obtained with other reporter gene assays and the proliferation assay. Legler et al. (1999) reported that the ER-CALUX assay was the most sensitive and responsive among reporter gene


R. Kuruto-Niwa et al. / Environmental Toxicology and Pharmacology 12 (2002) 27 /35

Fig. 5. Light microscopy showing ERE-GFP-MCF7 cells untreated (A), treated with 10 (4 M of BPA (B), 3,3?-diClBPA (C), and 3,3?,5triClBPA (D) for 2 days. Bar0/50 mm.

Fig. 6. The estrogenic activity of chlorinated BPAs, 3-ClBPA, 3,3?diClBPA, 3,5-diClBPA, 3,3?,5-triClBPA, 3,3?,5,5?-tetraClBPA, and 3,3?,5,5?-tetraBrBPA using a GFP expression system. ERE-GFPMCF7 cells were treated with the indicated concentrations of chemicals for 3 days. The control was treated with DMSO at a nal concentration of 0.1%. Fluorescence intensity was assessed using a FluorImager. The values are relative to the maximum uorescence intensity per cell obtained with E2 (50 pM). Points are means (n0/6)9/ S.E.M. for each concentration. Two replicates were taken from each treatment, which was performed in triplicate. Fig. 4. Effects of chlorinated BPAs on cell growth. ERE-GFP-MCF7 cells were left untreated (control), treated with the indicated concentrations of (A) BPA, (B) 3-ClBPA, (C) 3,3?-diClBPA, or (D) 3,3?,5triClBPA. The illustrated results (means9/S.E.M.) are from a representative experiment that was repeated three times independently.

assays using mammalian cells. A comparison of the potency of maximum effect concentration relative to E2 indicates that our system had almost the same potency as that of the ER-CALUX and E-screen assay, and lower potency than that of others (Table 3). This was due to the differences of the maximum effect concentra-

tions of E2. The luciferase maximal expression in our GFP expression system occurred at 5 )/10(11 M E2, comparable to the ER-CALUX assay (3 )/10(11 M E2, Legler et al., 1999). In addition, the EC50 of 5.9 )/10 (12 M in our system was same as that in the ER-CALUX assay. On the other hand, the ERE-PGK-GFP maximal expression was reached at 3)/10 (10 M E2 (Miller et al., 2000). In the other reporter gene assays, the maximum effect concentrations of E2 were 10 (9 M (Shelby et al., 1996) and 10(8 M (White et al., 1994), respectively. In the E-SCREEN assay, i.e. the MCF7 cell proliferation

R. Kuruto-Niwa et al. / Environmental Toxicology and Pharmacology 12 (2002) 27 /35 Table 2 Estrogenic activity of BPA derivatives relative to estradiol in the GFP expression system Chemicals BPA 3-ClBPA 3,3?-diClBPA 3,5-diClBPA 3,3?,5-triClBPA 3,3?,5,5?-tetraClBPA 3,3?,5,5?-tetraBrBPA
a b c d


EC50 (M)a 1.14E-6 1.54E-7 2.31E-7 9.54E-7 1.95E-6 /1.0E-5 /

Relative potency (EC50)b 5.19E-6 3.87E-5 2.57E-5 6.23E-6 3.05E-6 B/1.0E-6 /

Maximum effect concentration (M)c 1.0E-5 5.0E-6 1.0E-6 5.0E-6 1.0E-5 1.0E-5 5.0E-6

Maximum fluorescence intensityd 1199/5.4 92.79/5.8 88.49/4.4 99.49/6.7 1069/2.3 53.99/7.7 40.59/4.7

Concentration at which 50% of the maximum fluorescence intensity is reached. Ratio between EC50 of chemicals and EC50 of estradiol. Concentration at which maximum estrogenic effect is detected. Maximum fluorescence intensity 9/S.E.M. relative to the intensity obtained with E2 (50 pM, set at 100).

assay, the maximum effect concentration of E2 was 1/ 3 )/10(11 M (Soto et al., 1995). Of interest is that BPA, genistein, and daidzein at high concentrations induced GFP expression higher than that of the maximum level by E2 (Fig. 3, Table 1). Legler et al. (1999) also reported that a number of chemicals including BPA and genistein showed maximum luciferase activity exceeding that of E2 in ER-CALUX assay. The mechanism behind this is unclear but it may be due to stimulation of the receptor and coactivator renewal by estrogenic chemicals (Legler et al., 1999). In recent years, endocrine disruption has been observed in wild fish and in fish caged in rivers that receive significant inputs of wastewater effluents. Balaguer et al. (1999) detected estrogenic activity in wastewater sewage treatment effluents by reporter gene assays in MCF7 cells. In fact, a large number of endocrine disruptors such as BPA, 4-NP, octylphenol, and polychlorinated biphenyls were detected in wastewater effluents in various area of the world (Staples et al., 1998; Korner et al., 2000; Khim et al., 2001). Recently, chlorinated BPAs as well as BPA have been detected in wastewater from paper manufacturing plants using sodium hypochlorite as a bleaching agent (Fukazawa et al., 2001).

The activities of the chlorinated BPAs have not been examined yet, although BPA is known to exhibit estrogenic activity. We then evaluated the estrogenic activity of chlorinated BPAs using this GFP expression system. Of tested chemicals, 3-ClBPA, 3,3?-diClBPA, 3,5diClBPA, and 3,3?,5-triClBPA exhibited estrogenic activities (Fig. 6). Recently, Meerts et al. (2001) reported the estrogenic activity of brominated BPAs. It showed that 3-BrBPA and 3,3?-diBrBPA exhibited estrogenicity with similar EC50 values. This was consistent with our results of 3-ClBPA and 3,3?-diClBPA. It was also reported that an increasing number of bromo-substitutions decreased estrogenic activity of the brominated BPAs (Meerts et al., 2001; Samuelsen et al., 2001). In chlorine substituent, 3-ClBPA and 3,3?-diClBPA were more effective at lower concentration than parent BPA. This may be due to the difference of structures of chlorine and bromine atoms. Perez et al. (1998) reported that the longer the alkyl substituent at the bridge carbon, the lower the concentration needed for maximal estrogenicity in BPA derivatives. The representatives, 3,3-bis(4-hydroxyphenyl)pentane and 4,4-bis(4-hydroxyphenyl)heptane, were shown to be more active than

Table 3 A comparison of potency of estrogenic chemicals relative to estradiol in different in vitro assays for estrogenicity Chemicals BPA Assay ER-CALUX E-screen This assay ER-CALUX E-screen ERE-PGK-GFP Transient transfection in HeLa Transient transfection in MCF7 This assay ER-CALUX E-screen ERE-PGK-GFP This assay Relative potency (maximum effect concentration)a 3E-6 1E-6 5E-6 3E-5 3E-5 1E-4 2E-4 1E-3 5E-5 3E-5 1E-4 1E-4 5E-5 References Legler et al., 1999 Perez et al., 1998 Legler et al., 1999 Soto et al., 1995 Miller et al., 2000 Shelby et al., 1996 White et al., 1994 Legler et al., 1999 Mayr et al., 1992 Miller et al., 2000



Ratio between maximum effect concentration of chemicals and that of estradiol.


R. Kuruto-Niwa et al. / Environmental Toxicology and Pharmacology 12 (2002) 27 /35 Carvan, M.J., Dalton, T.P., Stuart, G.W., Nebert, D.W. 2000. Transgenic zebrash as sentinels for aquatic pollution. Ann. NY Acad. Sci. 919, 133. Chale, M., Tu, Y., Euskirchen, G., Ward, W.W., Prasher, D.C. 1994. Green uorescent protein as a marker for gene expression. Science 263, 802. Dorn, P.B., Chou, C.-S., Gentempo, J.J. 1987. Degradation of bisphenol A in natural waters. Chemosphere 16, 1501. Elsby, R., Maggs, J.L., Ashby, J., Park, B.K. 2001. Comparison of the modulatory effects of human and rat liver microsomal metabolism on the estrogenicity of bisphenol A: implications for extrapolation to humans. J. Pharmacol. Exp. Ther. 297, 103. Fielden, M.R., Chen, I., Chittim, B., Safe, S.H., Zacharewski, T.R. 1997. Examination of the estrogenicity of 2,4,6,2?,6?-pentachlorobiphenyl (PCB 104), its hydroxylated metabolite 2,4,6,2?,6?pentachloro-4-biphenylol (HO-PCB 104), and a further chlorinated derivative, 2,4,6,2?,4?,6?-hexachlorobiphenyl (PCB 155). Environ. Health Perspect. 105, 1238. Fukazawa, H., Hoshino, K., Shiozawa, T., Matsushita, H., Terao, Y. 2001. Identication and quantication of chlorinated bisphenol A in wastewater from wastepaper recycling plants. Chemosphere 44, 973. Gerdes, H.H., Kaether, C. 1996. Green uorescent protein: applications in cell biology. FEBS Lett. 389, 44. Guillette, L.J, Jr, Gross, T.S., Mason, G.R., Matter, J.M., Percival, H.F., Woodward, A.R. 1994. Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida. Environ. Health Perspect. 102, 680. Hirota, A., Taki, S., Kawaii, S., Yano, M., Abe, N. 2000. 1,1Diphenyl-2-picrylhydrazyl radical-scavenging compounds from soybean miso and antiproliferative activity of isoavones from soybean miso toward the cancer cell lines. Biosci. Biotechnol. Biochem. 64, 1038. Huang, C.H., Sedlak, D.L. 2001. Analysis of estrogenic hormones in municipal wastewater efuent and surface water using enzymelinked immunosorbent assay and gas chromatography/tandem mass spectrometry. Environ. Toxicol. Chem. 20, 133. Ishimi, Y., Komamura, Y., You, Z., Kimura, H. 1998. Biochemical function of mouse minichromosome maintenance 2 protein. J. Biol. Chem. 273, 8369. Ishiyama, M., Miyazono, Y., Sasamoto, K., Ohkura, Y., Ueno, K. 1997. A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability. Talanta 44, 1299. Khim, J.S., Lee, K.T., Kannan, K., Villeneuve, D.L., Giesy, J.P., Koh, C.H. 2001. Trace organic contaminants in sediment and water from Ulsan Bay and its vicinity, Korea. Arch. Environ. Contam. Toxicol. 40, 141. Klinge, C.M., Silver, B.F., Driscoll, M.D., Sathya, G., Bambara, R.A., Hilf, R. 1997. Chicken ovalbumin upstream promoter-transcription factor interacts with estrogen receptor, binds to estrogen response elements and half-sites, and inhibits estrogen-induced gene expression. J. Biol. Chem. 272, 31465. Korner, W., Bolz, U., Sussmuth, W., Hiller, G., Schuller, W., Hanf, V., Hagenmaier, H. 2000. Input/output balance of estrogenic active compounds in a major municipal sewage plant in Germany. Chemosphere 40, 1131. Krishnan, A.V., Stathis, P., Permuth, S.F., Tokes, L., Feldman, D. 1993. Bisphenol-A: an estrogenic substance is released from polycarbonate asks during autoclaving. Endocrinology 132, 2279. Kuruto-Niwa, R., Inoue, S., Ogawa, S., Muramatsu, M., Nozawa, R. 2000. Effects of Tea Catechins on the ERE-regulated estrogenic activity. J. Agric. Food Chem. 48, 6355. Lan, N.C., Katzenellenbogen, B.S. 1976. Temporal relationships between hormone receptor binding and biological responses in

BPA and were suitable for appropriate hydrogen bonding to the acceptor site of estrogen receptor (Perez et al., 1998). The estrogenic activity of 3,5-diClBPA was also different from that of 3,3?-diClBPA. The structureactivity relationship of these chemicals is now under investigation. BPA is reportedly biodegradable in river water (Dorn et al., 1987 Staples et al., 1998). Similarly, 3-ClBPA was biodegraded rapidly, but the polychlorinated derivatives, 3,3?,5-triClBPA and 3,3?,5,5?-tetraClBPA, were scarcely biodegraded under the same conditions using the supernatant of activated sludge in waste-paper recycling plants (Fukazawa et al., 2001). The dichlorinated BPA, 3,3?-diClBPA, underwent biodegradation very slowly (Fukazawa et al., 2001). These findings suggested that polychlorinated BPAs, if produced, preferentially contaminate the final effluent without degradation. Therefore, chlorinated BPAs can not be ignored as endocrine disruptors even though they were present in smaller amounts than BPA in wastewater effluents (Fukazawa et al., 2001). In addition, chlorinated BPAs were more cytotoxic than BPA (Fig. 4B and C). In view of these results, it might be suggested that alternatives to sodium hypochlorite bleaching should be used as bleaching agents in waste-paper recycling plants. The metabolism of BPA and chlorinated BPAs in vivo and the estrogenicity of the metabolites should also be studied in detail. BPA was found to conjugate highly with glucuronic acid in male rat liver microsomes studied in vitro (Yokota et al., 1999). Elsby et al. (2001) reported that incubations of BPA with pooled human or rat liver microsomes, in the presence of NADPH, resulted in the formation of 5-hydroxybisphenol A, which was 10-fold less potent than BPA in the yeast estrogenicity assay. Considering the environments, synergistic effects of many chemicals should be examined. Further studies are needed.

Acknowledgements We thank H. Hagenmaier for providing the MCF7 cells, C.M. Klinge for providing pGL3-pro-3(EREc38), A. Hirota and Y.-C. Chen for providing genistein and daidzein, and N. Oku for the use of the Olympus uorescence microscope with 3CCD High Gain Color Camera. We also thank A. Hills for her careful reading and editing of the manuscript.

Balaguer, P., Francois, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.C., Casellas, C. 1999. Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci. Total Environ. 233, 47.

R. Kuruto-Niwa et al. / Environmental Toxicology and Pharmacology 12 (2002) 27 /35 the uterus: studies with short- and long-acting derivatives of estriol. Endocrinology 98, 220. Legler, J., van den Brink, C.E., Brouwer, A., Murk, A.J., van der Saag, P.T., Vethaak, A.D., van der Burg, B. 1999. Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line. Toxicol. Sci. 48, 55. Mayr, U., Butsch, A., Schneider, S., 1992. Validation of two in vitro test systems for estrogenic activities with zearalenone, phytoestrogens and cereal extracts. Toxicology 74, 135. McLachlan, J.A., Newbold, R.R., Bullock, B.C. 1980. Long-term effects on the female mouse genital tract associated with prenatal exposure to diethylstilbestrol. Cancer Res. 40, 3988. Meerts, I.A.T.M., Letcher, R.J., Hoving, S., Marsh, G., Bergman, A., Lemmen, J.G., van der Burg, B., Brouwer, A. 2001. In vitro estrogenicity of polybrominated diphenyl ethers, hydroxylated PBDEs, and polybrominated bisphenol A compounds. Environ. Health Perspect. 109, 399. Miller, S., Kennedy, D., Thomson, J., Han, F., Smith, R., Ing, N., Piedrahita, J., Busbee, D.A. 2000. Rapid and sensitive reporter gene that uses green uorescent protein expression to detect chemicals with estrogenic activity. Toxicol. Sci. 55, 69. Perez, P., Pulgar, R., Olea-Serrano, F., Villalobos, M., Rivas, A., Metzler, M., Pedraza, V., Olea, N. 1998. The estrogenicity of bisphenol A-related diphenylalkanes with various substituents at the central carbon and hydroxy groups. Environ. Health Perspect. 106, 167. Plautz, J.D., Day, R.N., Dailey, G.M., Welsh, S.B., Hall, J.C., Halpain, S., Kay, S.A. 1996. Green uorescent protein and its derivatives as versatile markers for gene expression in living Drosophila melanogaster , plant and mammalian cells. Gene 173, 83. Pons, M., Gagne, D., Nicolas, J.C., Mehtali, M. 1990. A new cellular model of response to estrogens: a bioluminescent test to characterize (anti)estrogen molecules. Biotechniques 9, 450. Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergrast, F.G., Cormier, M.J. 1992. Primary structure of the Aequorea victoria green uorescent protein. Gene 111, 229. Samuelsen, M., Olsen, C., Holme, J.A., Meussen-Elholm, E., Bergmann, A., Hongslo, J.K. 2001. Estrogen-like properties of bromi-


nated analogs of bisphenol A in the MCF-7 human breast cancer cell line. Cell Biol. Toxicol. 17, 139. Schenborn, E., Oler, J., Goiffon, V. 1996. A trio of TfxTM transfection reagents for eukaryotic cells. Promega Notes 59, 24. Shelby, M.D., Newbold, R.R., Tully, D.B., Chae, K., Davis, V.L., 1996. Assessing environmental chemicals for estrogenicity using a combination of in vitro and in vivo assays. Environ. Health Perspect. 104, 1296. Soto, A.M., Sonnenschein, C., Chung, K.L., Fernandez, M.F., Olea, N., Serrano, F.O. 1995. The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ. Health Perspect. 103, 113. Southern, P.J., Berg, P. 1982. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1, 327. Staples, C.A., Dorn, P.B., Klecka, G.M., OBlock, S.T., Harris, L.R. 1998. A review of the environmental fate, effects, and exposures of bisphenol A. Chemosphere 36, 2149. Thackray, V.G., Young, R.H., Hooper, J.E., Nordeen, S.K. 2000. Estrogen agonist and antagonist action on the human estrogen receptor in Drosophila. Endocrinology 141, 3912. Tominaga, H., Ishiyama, M., Ohseto, F., Sasamoto, K., Hamamoto, T., Suzuki, K., Watanabe, M. 1999. A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Anal. Commun. 36, 47. White, R., Jobling, S., Hoare, S.A., Sumpter, J.P., Parker, M.G., 1994. Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology 135, 175. Yokota, H., Iwano, H., Endo, M., Kobayashi, T., Inoue, H., Ikushiro, S., Yuasa, A. 1999. Glucuronidation of the environmental oestrogen bisphenol A by an isoform of UDP-glucuronosyltransferase, UGT2B1, in the rat liver. Biochem. J. 340, 405. Zacharewski, T. 1998. Identication and assessment of endocrine disruptors: limitations of in vivo and in vitro assays. Environ. Health Perspect. 106, 577. Zuo, J., Niu, Q.W., Chua, N.H. 2000. Technical advance: an estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24, 265.