Article pubs.acs.org/est
Co-occurrence of Estrogenic and Antiestrogenic Activities in Wastewater: Quantitative Evaluation of Balance by in Vitro ERα Reporter Gene Assay and Chemical Analysis Masaru Ihara,*,† Mariko O. Ihara,† Vimal Kumar,†,‡ Masanori Narumiya,† Seiya Hanamoto,† Norihide Nakada,† Naoyuki Yamashita,† Shinichi Miyagawa,§ Taisen Iguchi,§ and Hiroaki Tanaka† †
Research Center for Environmental Quality Management, Graduate School of Engineering, Kyoto University, 1-2 Yumihama, Otsu, Shiga 520-0811, Japan ‡ Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, University of South Bohemia in Ceske Budejovice, Zatisi 728/II, 389 25 Vodnany, Czech Republic § Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, and Department of Basic Biology, Faculty of Life Science, Graduate University for Advanced Studies, Sokendai, Higashiyama 5-1, Myodaiji, Okazaki, Aichi 444-8787, Japan S Supporting Information *
ABSTRACT: Endocrine-disrupting chemicals are exogenous substances that alter the function of the endocrine system, with adverse health effects on organisms or their progeny. In vitro estrogen receptor (ER) reporter gene assays have long been used to measure estrogenic activity in wastewater. Nevertheless, there is still uncertainty about their usefulness in environmental monitoring on account of a discrepancy between the estrogenic response of the in vitro assay and concentrations of estrogenic compounds determined by chemical analysis. Here, we measured estrogenic and antiestrogenic activities in wastewater by ERα reporter gene assay. All samples were simultaneously analyzed for estrone, 17β-estradiol, estriol, and 17α-ethynylestradiol, and the concentrations were used to predict estrogenic activity. All samples in which measured estrogenic activity was significantly lower than predicted showed strong antiestrogenic activity. In addition, we confirmed that the fraction that did not have antiestrogenic activity showed stronger estrogenic activity than the unfractionated wastewater extract. These results indicate that antiestrogenic compounds in wastewater suppress the activity of natural estrogens, and the reporter gene assay represents the net activity.
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bioavailable ER antagonists in wastewater.15 In vitro ER reporter gene assays have shown several compounds to have antiestrogenic activity.16−18 In vitro reporter gene assays offer a rapid, highly sensitive, cost-effective way to measure the estrogenic activity of a single compound. There is, however, uncertainty about their usefulness in environmental monitoring. Studies that compared estrogenic activity in wastewater as measured by in vitro reporter gene assay with predicted activity based on chemical analysis of EDCs have reported both good19,20 and poor21,22 correlation. Where measured estrogenic activities were lower than predicted, this discrepancy has been put down to the presence of antiestrogenic compounds.21,22 However, this speculation has not been fully verified.
INTRODUCTION Endocrine-disrupting chemicals (EDCs) are exogenous substances that alter the function of the endocrine system, with adverse health effects on organisms or their progeny.1 Effluent of wastewater treatment plants (WWTPs) is one of the major sources of EDCs in the aquatic environment.2,3 Exposure to WWTP effluent or environmental estrogens has been associated with the induction of vitellogenin in male fish and with intersex in wild fish species.4−7 The in vitro estrogen receptor-alpha (ERα) reporter gene assay is used to measure estrogenic activity in wastewater. Increasing numbers of studies using this assay have shown antiestrogenic activity in environmental samples, such as sediments from agricultural catchments,8,9 wastewater,10−12 and river water.13 Antiestrogenic chemicals have been shown to reduce the reproductive function of killifish in Newark Bay.14 Extracts of bile from fish exposed to WWTP effluents have shown antiestrogenic activity, suggesting the presence of © 2014 American Chemical Society
Received: October 2, 2013 Accepted: May 6, 2014 Published: May 6, 2014 6366
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filtered and extracted within 6 h (samples from WWTPs A and C) or 48 h (WWTP B). The samples were stored at 4 °C (WWTPs A and C) or below 16 °C (WWTP B) before filtration. Samples for the ERα reporter gene assay were extracted by solid-phase extraction (SPE). To prevent contamination, all glassware and materials were washed before use with acetone and Milli-Q water (Millipore Corp., Billerica, MA, USA). Each 1-L sample was passed through a glass fiber filter (pore size 1 μm; GF/B, Whatman, Maidstone, UK), and the filtrate was passed through an SPE cartridge. The cartridge (Oasis HLB, 200 mg/6 cc, 30 μm particle size, Waters Corp., MA, USA) was preconditioned first with 5 mL of methanol, followed by 5 mL of Milli-Q water. The samples were loaded through the preconditioned cartridge at a constant 10 mL/min by an automated concentrator (Sep-Pak Concentrator Plus, Waters). The cartridge was dried for 2 h under gentle air pressure in a glass manifold. Then 10 mL of methanol was used to elute compounds trapped on the SPE cartridge. The eluate was evaporated to dryness under a gentle nitrogen stream at 37 °C. The residue was immediately dissolved in 1 mL of cell culture medium (described below) containing 0.2% DMSO, followed by serial dilutions with the cell culture medium. All samples were stored at −30 °C until the ERα reporter gene assay. The Milli-Q water was also treated in parallel as a blank control, which we confirmed had no estrogenic or antiestrogenic activity by human (Homo sapiens) or medaka (Oryzias latipes) ERα reporter gene assay. Recovery rates of E1, E2, E3, and EE2 during the SPE procedure for the reporter gene assay were confirmed to be comparably as high as those during the SPE procedure for the chemical analysis (SI Methods S2 and S3). Analysis of the recovery of E2 activity (SI Methods S4) confirmed the high recovery (see Results and Discussion). We thus concluded that the estrogenic activity measured by the reporter gene assays was directly comparable to the results of chemical analysis. Chemical Analysis of Natural and Synthetic Estrogens. Concentrations of E1, E2, E3, and EE2 in the wastewater samples were measured by ultraperformance liquid chromatography coupled with tandem mass spectrometry (UPLC/MS/ MS) as described25 in parallel with the ERα reporter gene assay. The procedure is described in SI Methods S2. Evaluation of Estrogenic Activity. To compare the response of ERαs from human and medaka to wastewater extracts, we chose a transient reporter gene assay system instead of a stable system such as the OECD validated T47D assay. Because HEK (human embryonic kidney) 293 cells do not express endogenous ERα, we can directly compare the response of transiently transfected ERαs from both human and medaka under identical conditions. The vectors used to express the human or medaka ERαs (pcDNA3.1-ER) have been described previously.7,26 An estrogen-regulated expression vector containing four estrogen-responsive elements and the firefly (Photinus pyralis) luciferase gene, named pGL3-4xERE, was used as the reporter vector.27 The ERα reporter gene assay was performed as described,27,28 with a slight modification. HEK 293 cells were seeded in 96-well plates (Nunc, Rochester, NY, USA) in phenol-red-free Dulbecco’s modified Eagle’s medium (SigmaAldrich Co., St. Louis, MO, USA) supplemented with 10% charcoal/dextran-treated fetal bovine serum (Hyclone, South Logan, UT, USA). After 16 h incubation, cells were transfected with 80 ng of pGL3-4xERE, 40 ng of pcDNA3.1-ER (to express
In this study, we aimed to investigate the effect of antiestrogenic activity on the measurement of estrogenic activity in wastewater by the in vitro ERα reporter gene assay. Estrogenic and antiestrogenic activities were measured by the ERα reporter gene assay and quantified as estradiol equivalent quantity (EEQ) and 4-hydroxy-tamoxifen equivalent quantity (TMXEQ), respectively. Measured concentrations of estrone (E1), 17β-estradiol (E2), estriol (E3), and 17α-ethynylestradiol (EE2) were used to predict EEQs in wastewater. We were able to extract E1, E2, E3, and EE2 at high recovery rates during the pretreatment procedure for the ERα reporter gene assay. All samples in which measured estrogenic activity was lower than predicted showed strong antiestrogenic activity. In addition, we confirmed that the fraction that did not have antiestrogenic activity showed stronger estrogenic activity than the unfractionated wastewater extract. To the best of our knowledge, this is the first study to show quantitatively that antiestrogenic activity can suppress the activity of natural estrogens in wastewater. We used both human and medaka ERαs in assays. Most current studies of estrogenic activity in wastewater use only mammalian receptor-based assays.23 However, fish ERs are likely to be more sensitive to some compounds, such as bisphenol A and alkylphenols.24 Therefore, we expected that we could detect a wider range of compounds using both.
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MATERIALS AND METHODS Chemicals. The chemicals used in this study are described in Supporting Information [SI] Methods S1. Sampling and Sample Treatment for Biological and Chemical Analysis. Raw wastewater (influent), effluent from primary settling tanks (primary effluent), and effluent from final settling tanks after biological treatment (secondary effluent) were collected at wastewater treatment plants (WWTPs) A, B, and C in 2011 and 2012. In total, 14 samples were collected (Table 1). These WWTPs, located in different parts of Japan, all use the conventional activated sludge treatment process. All samples were collected in 1-L amber glass bottles to which 1 g/ L ascorbic acid was added as a preservative. After collection, the samples were transported to our laboratory, where they were Table 1. Sample Characteristics and Concentrations of Estrogensa concentration (ng/L)b
specifications #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14 a b
WWTP
type
date
E1
E2
E3
EE2
A A B B B B B C A A A A B C
SE SE SE SE SE SE SE SE PE PE PE PE influent influent
Jan. 2011 Mar. 2011 Feb. 2011 Dec. 2011 May 2012 May 2012 Aug. 2012 Sep. 2012 Aug. 2011 Aug. 2011 Nov. 2011 May 2012 Feb. 2011 Sep. 2012
3.3 7.8 37.0 56.6 61.7 27.3 11.1 2.1 12.8 21.4 18.1 10.1 22.1 5.5
0.8 ND ND 2.5 4.2 1.4 2.8 ND 25.3 42.1 34.4 21.9 44.6 5.90
ND ND ND ND ND ND ND ND 26.1 59.9 51.6 70.9 87.0 4.6
ND ND ND ND ND ND ND ND ND ND ND ND ND ND
SE: secondary effluent; PE: primary effluent; ND = not detected. Limits of detection (ng/L): E1, 0.3; E2, 0.5; E3, 0.5; EE2, 0.5. 6367
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concentration−response curves of fixed molar ratio mixtures of 4-OHT and E2 are described in SI Methods S7. Concentration−Inhibition Curve of Fixed Molar Ratio Mixture of 4-OHT and E2. The procedures for the analysis of concentration−inhibition curves of fixed molar ratio mixtures of 4-OHT and E2 are described in SI Methods S8. Fractionation of Wastewater Samples by SPE Cartridge. The procedures for the fractionation of wastewater samples by SPE cartridge are described in SI Methods S9.
human or medaka ERα), and 20 ng of pRL-TK using the Fugene 6 transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The pRL-TK vector containing the sea pansy (Renilla reniformis) luciferase gene (Promega) was used as an internal control to normalize variations in transfection efficiency and cell numbers seeded. After 4 h incubation, the diluted wastewater extracts were added to the medium. To avoid cell toxicity, the concentration of DMSO in serial dilutions never exceeded 0.1%. After 40 h incubation, luciferase activity was measured as follows. The exposure medium was removed, and cells were washed with phosphate-buffered saline (PBS) to prevent the interference of the compounds in the wastewater extract with the luciferase activity, and then cells were lysed in each well, and the luciferase activity in each well was measured by a chemiluminescence assay with the Dual-Luciferase Reporter Assay System (Promega). Luminescence was measured by an Infinite 200 multifunctional microplate reader (Tecan, Salzburg, Austria). The strength of transactivation was calculated as the ratio of firefly luciferase activity to sea pansy luciferase activity and expressed as a multiple of the vehicle control activity (cell culture medium including 0.1% DMSO). The cytotoxicity of each wastewater extract was analyzed under the same conditions as those for estrogenic/antiestrogenic activity measurement (SI Methods S5). ERα reporter gene assay data obtained from the dilution range of extracts in which the number of living cells was statistically lower (P < 0.05) than in vehicle were excluded from further analysis. For each assay, the activity of E2 (10−14 to 10−5 M) was analyzed in parallel as a positive control. All assays were performed at least twice, using duplicate sample points in each experiment. Estrogenic activities of E1, E3, and EE2 were also evaluated. These compounds were dissolved in DMSO and then added to the test wells at concentrations between 10−14 and 10−5 M. The concentration of DMSO in serial dilutions did not exceed 0.1% during exposure. The procedures for plasmid transfection and chemiluminescence assay were the same as above. Evaluation of Antiestrogenic Activity. 4-Hydroxytamoxifen (4-OHT) was used as a standard E2 antagonist. Antiestrogenic effects of samples detected by the reporter gene assay were determined as 4-OHT-equivalent (TMXEQ) activity. After plasmid transfection, serial dilutions of wastewater extracts were added to the test wells. Then, in the human ERα assay, 5.0 × 10−11 M E2, the lowest concentration that induces the maximum response of ER (called “EC100”) was added. In the medaka ERα assay, 5 × 10−9 M E2 (EC100 for medaka ERα) was added. In some wastewater extracts which showed both estrogenic and antiestrogenic activity, the maximum estrogenic response reached over 50% of maximum E2 activity (e.g., Figure 2, #11, human ERα; SI Figure S2, #1, #3−#5, #9, #12, and #13, human ERα). Because using EC50 might not detect the reduction of E2 activity by these wastewater extracts, we used EC100 in this study. After 40 h incubation, luciferase activity was measured. Other procedures were the same as for the evaluation of estrogenic activity. From the reduction of E2 activity, TMXEQ of wastewater extracts was calculated (SI Methods S6). Data Presentation. The procedures for the calculation of relative potency values, measured EEQs, predicted EEQs, and TMXEQs are described in SI Methods S6. Concentration−Response Curve of Fixed Molar Ratio Mixture of 4-OHT and E2. The procedures for the analysis of
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RESULTS AND DISCUSSION Estrogenic Activity of Single Compounds. E1, E2, E3, and EE2 gave dose-dependent responses (Figure 1A). From the
Figure 1. (A) Concentration−response curves of human and medaka ERα to E1, E2, E3, and EE2. The y-axis indicates percentage activation relative to maximum activity by E2 (100%). Dashed lines indicate 25% and 50% of the maximum response of E2. Arrows indicate EC50 and EC25 of E2. Values represent mean ± SEM (n = 8−16). (B) Concentration−inhibition curves of 4-OHT against E2-induced transactivation. Values represent mean ± SEM (n = 8). Dashed line indicates 25% reduction of the activity of EC100 of E2 (to 75%). Arrows indicate the IC25 of 4-OHT.
response curves, the average EC50 values of E2 were 9.32 × 10−12 by human ERα and 3.53 × 10−10 M by medaka ERα. The relative potency values of E1 (1.82% for human ERα, 8.74% for medaka ERα), E3 (34.0%, 5.70%), and EE2 (36.4%, 146%; SI Table S1) were used to calculate the predicted EEQs of wastewater extracts. Differences in relative potency values of E1 and E3 between human and medaka ERαs resulted in differences in the predicted EEQs of wastewater extracts (Figure 4). EC25 values of E2 (3.11 × 10−12 by human ERα, 1.23 × 10−10 M by medaka ERα) were used to calculate the measured EEQ of wastewater extracts (Figure 4). Concentration−Inhibition Curve of 4-OHT on E2Induced Transactivation. Clear concentration-dependent inhibition of E2 activity by 4-OHT (Figure 1B) shows that the reporter gene assay system can respond to the antiestrogenic compound 4-OHT. IC25 values, calculated to be 2.42 × 10−11 M by human ERα and 2.50 × 10−10 M by medaka ERα (arrows), were used to calculate the TMXEQ of 6368
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the wastewater extracts (Figure 4). We confirmed that 4-OHT blocked E2 activity competitively (SI Figure S1), shifting the E2 curve to a higher dose. Concentration−Response Curve of Estrogenic Activity and Concentration−Inhibition Curve of Antiestrogenic Activity. In the influents and primary effluents, E1, E2, and E3 were detected at high concentrations (samples #9−#14; Table 1). In secondary effluents, on the other hand, mainly E1 was detected (#1−#8). EE2 was not detected in any samples, as the contraceptive pill is not a popular method of contraception in Japan.29 From these concentrations, the predicted EEQs were calculated (Figure 4). To compare the measured EEQs obtained by the ERα reporter gene assays with the predicted EEQs based on chemical analysis, we evaluated the recovery rates of E1, E2, E3, and EE2 during the SPE procedure used for the ERα reporter gene assay. Chemical analysis (SI Methods S3) confirmed high recovery rates of 85% (E1 in primary effluent) to 101% (E2 in primary effluent) (SI Table S2). The recovery of E2 activity (SI Methods S4) confirmed the high recovery: recovery rates ± relative SD (reproducibility) were 85% ± 12% (in Milli-Q water) and 88% ± 18% (in primary effluent). Thus, we conclude that estrogenic activity measured by the reporter gene assay is directly comparable to results of chemical analysis. Therefore, if a measured EEQ is higher than predicted from the concentrations of E1, E2, E3, and EE2, there are other estrogenic compounds in samples beside E1, E2, E3, and EE2. In contrast, if a measured EEQ is lower than predicted, antiestrogenic compounds are present. As we simultaneously measured antiestrogenic activity in all samples, we could state whether or not antiestrogenic activity existed in these samples. Concentration−response curves of estrogenic activity and concentration−inhibition curves of antiestrogenic activity were obtained from the results of ERα reporter gene assays (Figure 2, SI Figure S2). The inhibition was not due to cytotoxicity of samples (top). The human ERα assay of all wastewater extracts showed a decrease of estrogenic response induced by E2 with increasing relative enrichment factor (REF, which is the ratio of the enrichment factor from the SPE step to the dilution factor of wastewater extracts in reporter gene assay) of the extract (Figure 2, SI Figure S2, middle, blue), indicating antiestrogenic activity. The curve shapes of samples #6, #11, and #14 were typical of secondary effluents, primary effluents, and influents, respectively, that showed antiestrogenic activity to human ERα (Figure 2A−C, middle). The estrogenic activity (red) initially increased but then decreased at higher concentrations (it showed a turning point) as the antiestrogenic activity became evident. The maximum activity was suppressed to below the maximum E2 activity. Other wastewater extracts that were antiestrogenic to human ERα gave similar curves (SI Figure S2, #1−#5, #7−#10, #12). EC25 was determined from the concentration−response curve in its continuously increasing range (red arrows). The medaka ERα assay of samples #2 and #4−#8 (secondary effluent) indicated antiestrogenic activity. The curve shape of sample #6 was typical of secondary effluent that was antiestrogenic to medaka ERα (Figure 2A, bottom). The strength of estrogenic activity increased to 30% of the maximum E2 activity (red). Samples #5 and #7 also showed a moderate increase of estrogenic activity (SI Figure S2). The curve shapes of samples #11 and #14 were typical of primary effluents and influents that were not antiestrogenic to medaka ERα (Figure 2B, C, bottom). In contrast to sample #6, the
Figure 2. Concentration−response curves of estrogenic activity (red) and concentration−inhibition curves of antiestrogenic activity (blue) by human ERα assay (middle) and medaka ERα assay (bottom). Top: Results of cytotoxicity tests. (A) Sample #6, secondary effluent; (B) sample #11, primary effluent; (C) sample #14, influent. Dashed lines indicate either 25% of the maximum response of E2 or 25% reduction of the activity of E2 (to 75%). Red arrows, EC25; blue arrows, IC25. Values represent mean ± SEM (n = 4−6). Asterisk indicate that the number of living cells at this REF was statistically lower than the control (P < 0.05); activity data of this REF were excluded from further analysis.
strength of estrogenic activity of samples #11 and #14 increased sharply (red). Neither cytotoxicity nor nonreceptor-mediated pathways was the reason for the decrease of the estrogenic response (SI Discussion S1). Concentration−Response Curves of Fixed Molar Ratio Mixtures of 4-OHT and E2. As described above, the concentration−response curves of wastewater extracts that had antiestrogenic activity showed a turning point and a reduced maximum estrogenic activity. We investigated whether this pattern could be explained by the coexistence of estrogenic and antiestrogenic compounds by analyzing the concentration− response curves of 4-OHT and E2 in fixed molar ratios (SI Methods S7, Figure 3). As expected, these curves also showed a turning point (arrowheads) and a reduced maximum estrogenic activity in both human and medaka ERα assays. The greater the proportion of 4-OHT in the mixture, the more the maximum response was suppressed. The EC25 values of mixture became larger than those of E2 alone (arrows). These results indicate that the antiestrogenic activity could interfere with the estrogenic activity, and, as a result, the shape of the estrogenic response curve was changed. Therefore, the estrogenic activity of wastewater extracts measured in our reporter gene assay was lower than predicted from the concentration of estrogenic compounds because of the presence of antiestrogenic compounds. We also investigated the concentration−inhibition curve of antiestrogenic activity of fixed molar ratio mixtures of 4-OHT and E2 by the human ERα assay (SI Methods S8) and confirmed that 4-OHT acted as an antagonist in the presence of E2 (SI Figure S3). IC25 and EC25 values show that 6369
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#9−#12, #14). By medaka ERα, in contrast, measured EEQs in many extracts were higher than predicted (Figure 4B, #1, #3, #5; Figure 4D #9−#14). E1, E2, and E3 additively affected both ERαs following the concentration addition model (data not shown). These results indicate the presence of other estrogenic compounds specific to medaka ERα. The limits of detection of samples #10, #11, and #13 (antiestrogenic activity) by medaka ERα were not comparable to those of other samples. However, in contrast to human ERα, medaka ERα detected antiestrogenic activities only in extracts of secondary effluents (Figure 4B, #2, #4−#8). In the human ERα assay, there was not always a perfect match between the strength of antiestrogenic activity and the gap between measured and predicted EEQs. For example, sample #12 showed a very low measured EEQ compared with predicted, but at the same time the lowest TMXEQ of the influents and primary effluents. However, by both human and medaka ERα assays, all extracts in which measured EEQs were lower than predicted showed antiestrogenic activity (Figure 4A, #2, #5, #7; Figure 4B, #2, #4, #6−#8; Figure 4C, #9−#12, #14). In contrast, most extracts in which measured EEQ was higher than predicted did not show antiestrogenic activity (Figure 4B, #1, #3; Figure 4D). As described above, E1, E2, and E3 additively affected both ERαs. It seems that there is a tendency for antiestrogenic compounds in wastewater to suppress the activity of natural estrogens. If so, estrogenic activity should be increased by the separation of antiestrogenic and estrogenic compounds. To test this hypothesis, we fractionated the substances adsorbed on the SPE cartridge by a series of elution steps in which the volume of methanol in methanol/water eluent was increased from 20% to 100% (SI Methods S9). Figure 5 shows the concentration− response curves of estrogenic activity and the concentration− inhibition curves of antiestrogenic activity for fractionated and unfractionated extracts of sample #14. By human ERα assay, IC25 values indicated that most of the antiestrogenic activity was separated into the 80% methanol fraction (B, antiestrogenic, red arrow). On the other hand, the EC25 value indicated that most of the estrogenic activity was separated into the 60%
Figure 3. Concentration−response curves of fixed molar ratio mixtures of 4-OHT and E2 by (A) human and (B) medaka ERα assays. 4-OHT and E2 were mixed in fixed molar ratios from 1:1 to 100:1 for human ERα and from 1:1 to 10:1 for medaka ERα. The maximum response decreased as the proportion of 4-OHT increased. EC25 values of mixtures (arrows) became larger than those of E2 alone. The response induced by E2 increased as the concentration of E2 increased but eventually decreased at the turning point concentration (arrowheads). Values represent mean ± SEM (n = 6−10).
antiestrogenic activity could be measured at higher REFs than estrogenic activity in the human ERα assay (Figure 2, SI Figure S2: #1, #3−#5, #7, #9−#13, arrows). This gap between IC25 and EC25 values could be explained by the competition between estrogenic and antiestrogenic compounds (SI Figure S3). Comparison of Measured EEQ, Predicted EEQ, and TMXEQ in Wastewater. From the concentration−response curves of estrogenic activity and the concentration−inhibition curves of antiestrogenic activity, we calculated the measured EEQ and TMXEQ values (Figure 4). From the concentrations of E1, E2, and E3 (Table 1) and their relative potency values (SI Table S1), we calculated the predicted EEQ values (Figure 4). By human ERα assay, all wastewater extracts showed antiestrogenic activity (Figure 4A, C). In particular, primary effluent and influent in which measured EEQs were lower than predicted showed antiestrogenic activity (Figure 4C, samples
Figure 4. Comparison of measured EEQ, predicted EEQ, and TMXEQ by (A, C) human and (B, D) medaka ERαs. Values represent mean ± SEM (n = 4−8). Lines indicate the limits of detection (LOD) of activity, which differed between samples depending on the highest value of the sample extract concentration factor in the assay (1−10× for influent or primary effluent, 10−100× for secondary effluent). 6370
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have antiestrogenic activity showed estrogenic activity despite a lack of activity in the unfractionated extract (SI Figure S4, medaka ERα). The results for sample #7 also indicate competition between estrogenic and antiestrogenic compounds (SI Figure S5). Taken together, these results (Figures 3, 5; SI Figures S3− S5) indicate that estrogenic and antiestrogenic compounds compete with each other in the wastewater extracts, and the estrogenic response measured by our reporter gene assay represents the net estrogenic activity in the balance of estrogenic and antiestrogenic compounds. The patterns of antiestrogenic activities differed between human and medaka ERαs. Stronger antiestrogenic activities were detected in influents and primary effluents by human ERα but in secondary effluents by medaka ERα (Figure 4). There are three possible explanations for this difference. First, the human and medaka ERαs detect different antiestrogenic compounds; second, they respond differently to the same antiestrogenic compounds; and third, estrogenic compounds suppress the antiestrogenic activity in influents and primary effluents in the medaka ERα assay (Figure 5C). EEQs of influents and primary effluents measured by medaka ERα were higher than those predicted by medaka ERα and measured by human ERα (Figure 4C, D, #9−#12, #14). There are two possible explanations for the differences. First, there were other estrogenic compounds specific to medaka ERα that might have synergistic effects. Second, there were other estrogenic compounds common to both human and medaka ERαs, but strong antiestrogenic activity specific to human ERα suppressed the estrogenic activity detected by human ERα (Figure 5B). A study comparing fish and mammalian ER properties in the reporter gene assay concluded that ER transactivation in one vertebrate species could be extrapolated to another species in chemical screening.30 Our results, however, show different responses of human and medaka ERαs to estrogenic and antiestrogenic activities in wastewater. Some previous studies determined antiestrogenic activity in wastewater by the analysis of how the E2 control curve changed; that is, how the antiestrogenic activity suppressed the upper limb of the curve or shifted the curve to a higher dose.11−13 We found similar changes to the E2 curve in both human and medaka ERα assays (SI Methods S10; SI Figure S6). Causative Antagonists. Competitive antagonists reversibly bind to receptors at the same binding site as the endogenous ligand but without activating the receptor. 4-OHT, a typical competitive ERα antagonist, has been shown to shift the E2 curve to a higher E2 dose (SI Figure S1; Martel et al.31). Therefore, such shifts caused by the addition of wastewater extracts (SI Figure S6, arrows) indicate that competitive antagonists were responsible for the antiestrogenic activity we detected. As higher doses of E2 mitigated the antiestrogenic activities of the wastewater extracts (SI Figure S7), this result confirms the presence of competitive antagonists. On the other hand, the suppression of the upper limb of the E2 control curve (SI Figure S6, arrowheads) indicated the presence of noncompetitive antagonists also (SI Discussion S2). Several compounds show antiestrogenic effects: e.g., chlorinated 4-NP,16 PCB metabolites,17 di-n-butyl phthalate,18 polycyclic musks,32 organic ultraviolet-absorbents,33 PFOS and PFOA,34 textile dyes,35 and disinfection byproducts of tryptophan and phenylalanine formed by chlorination.36,37 As all of our samples were collected before chlorination, these
Figure 5. Concentration−response curves of estrogenic activity and concentration−inhibition curves of antiestrogenic activity of fractionated and unfractionated sample #14 by (B) human ERα assay and (C) medaka ERα assay; (A) results of cytotoxicity tests. Arrowheads indicate the maximum estrogenic response. Arrows indicate EC25 or IC25. Data of unfractionated extract are the same as sample #14 in Figure 2C. Asterisk indicates that the number of living cells at this REF was statistically lower than the control (P < 0.05). Data of 40% and 20% methanol fractions are not shown because there was no estrogenic or antiestrogenic response. Values represent mean ± SEM (n = 4).
methanol fraction (B, estrogenic, blue arrow). The estrogenic activity of the 60% fraction (calculated to be 5.8 ng-E2/L) was higher than that of the unfractionated extract (4.1 ng-E2/L). The cytotoxicity of samples was not related to the decrease of estrogenic response (A). These results indicate that antiestrogenic compounds in the 80% fraction suppress the activity of estrogenic compounds in the 60% fraction. In addition, the maximum estrogenic response of the 60% fraction was higher than that of the unfractionated extract (B, estrogenic, blue and gray arrowheads, respectively). These results indicate that antiestrogenic compounds suppress the maximum response of estrogenic compounds in the unfractionated extract and agree well with the shape of concentration−response curve of fixed molar ratio mixtures of 4-OHT and E2, in which increasing the proportion of 4-OHT increased the suppression of the maximum response (Figure 3). By medaka ERα assay (Figure 5C), the 80% methanol fraction (red) showed antiestrogenic activity despite a lack of activity in the unfractionated extract (black). On the other hand, most of the estrogenic activity was separated into the 100% fraction (C, estrogenic). These results indicate that estrogenic compounds in the 100% fraction likewise suppress the activity of antiestrogenic compounds in the 80% fraction. The results for sample #8 show that the fraction that did not 6371
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latter two are unlikely to be present. Adsorption of E2 by dissolved organic matter might also decrease estrogenic activity38,39 and might explain part of the antiestrogenic activity detected here. Effects of Antiestrogenic Activity on the Measurement of Estrogenic Activity. We confirmed that the fraction that did not have antiestrogenic activity showed stronger estrogenic activity than the unfractionated wastewater extract. Overall, our results show that the estrogenic response measured by the ERα reporter gene assay represents the net estrogenic activity in the balance of estrogenic and antiestrogenic compounds. Thus, the absence of measured EEQs detected by the reporter gene assay does not prove the absence of estrogenic compounds. Consequently, when analysts use the ERα reporter gene assay to investigate exposure to estrogenic compounds in complex environmental samples, they should also determine the presence or absence of antiestrogenic activity to avoid underestimation of the exposure level. The health effects of mixtures of endocrine disruptors on humans and wildlife must be examined, and considerable progress has been made in assessing the effects of mixtures with components with the same effect (e.g., estrogenic or antiandrogenic).40,41 The net effects of mixtures of compounds with opposite effects such as estrogenic and antiestrogenic also need to be assessed, especially since these compounds occur together, as shown here.
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ASSOCIATED CONTENT
S Supporting Information *
EC50s and relative potency values of estrogens, recovery rates of estrogens by SPE, competitive inhibition by 4-OHT on the E2 curve, concentration−response curves of estrogenic activity and concentration−inhibition curves of antiestrogenic activity of wastewater extracts, concentration−inhibition curves of antiestrogenic activity of mixtures of 4-OHT and E2, estrogenic and antiestrogenic activity of fractionated samples, the effects of wastewater extracts on E2 control curves, the relationship between E2 dose and observed antiestrogenic activity of wastewater extracts, methods for other experiments, discussion about the cytotoxicity of samples and nonreceptor mediated pathways, discussion about causative antagonists. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81-77-527-6220. Fax: +81-77-527-9869. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Ms. K. Nakano for technical assistance. This work was supported by a UK−Japan cooperative project of the Ministry of the Environment, Japan; the Japan Science and Technology Agency’s Core Research for Evolutional Science and Technology (CREST) research promotion program; and grants from the Ministry of Education, Culture, Sport, Science and Technology.
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