Environ. Sci. Technol. 2005, 39, 3703-3707
Estrogenic Activity of Impurities in Industrial Grade Bisphenol A M A S A N O R I T E R A S A K I , * ,† FUJIO SHIRAISHI,‡ TOMOHIRO NISHIKAWA,‡ JOHN S. EDMONDS,‡ MASATOSHI MORITA,‡ AND MASAKAZU MAKINO† Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan, and Endocrine Disrupter Research Laboratory, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
The estrogenicities of 10 compounds found as impurities in industrial grade bisphenol A (BPA) were measured by yeast two-hybrid assays incorporating the human estrogen receptor R (hERR) or the medaka fish (Oryzias latipes) estrogen receptor R (medERR). Five impurities showed greater activity than BPA itself in an agonist assay for hERR. p-Cumylphenol, the most active of the impurities in the hERR assay, was 12 times as active as BPA. The REC10 (10% relative effective concentration: 10% of the activity of 10-8M 17β-estradiol) was 710 nM. Five impurities showed greater activity than BPA in an agonist assay for medERR: 4,4′-(1,3-dimethylbutylidene) bisphenol and 2-(4′-hydroxyphenyl)-2,4,4-trimethylchroman were nearly equipotent and 9 times as active as BPA, and the REC10 values of these compounds in the medERR assay were 280 and 320 nM, respectively. Comparison of the experimentally determined estrogenicities of mixtures of BPA and 4,4′-(1,3-dimethylbutylidene) bisphenol and those calculated by the concentrations addition (CA) method confirmed the suitability of the method for the prediction of the estrogenicities of the mixtures of BPA and its phenolic analogues. The measured estrogenicities of four samples of industrial grade BPA and laboratory grade (pure) BPA were not significantly different in either the hERR assay or the medERR assay (p > 0.05 in each case). We conclude that the impurities in industrial grade BPA, although some are of much higher estrogenic activity than BPA itself, do not significantly increase the estrogenicity of the industrial compound and therefore do not increase possible adverse health effects from such activity.
Introduction Bisphenol A (4,4′-isopropylidenediphenol, BPA; CAS Registry No. 80-05-7) (Table 1) is used as a monomer in the manufacture of polycarbonate and epoxy resins, as a stabilizer or antioxidant for many types of plastics such as poly(vinyl chloride) (PVC), and as an inhibitor of end oxidation in PVC (1). Annual production capacity of this compound in the United States is about 865 000 tons (2). As early as 1936 BPA * Corresponding author phone/fax: +81-54-264-5783; e-mail:
[email protected]. † University of Shizuoka. ‡ National Institute for Environmental Studies. 10.1021/es048932g CCC: $30.25 Published on Web 04/08/2005
2005 American Chemical Society
was shown to have estrogenic activity in vivo when administered to rats (3). The estrogenic properties of BPA have been consistently shown by in vitro assays (4), but there has been disagreement over the concentrations at which BPA exerts its estrogenic effects in vivo (5). BPA is released to the environment both accidentally and through permitted discharges (4), and its widespread distribution has been a major cause of concern to regulatory agencies and others (6). We have recently identified and quantified 15 trace impurities in samples of industrial grade BPA (7). All of these compounds, like BPA itself, possess phenolic hydroxyl groups para to other substituents and all thus might also have estrogenic properties. Although BPA has been reported to interact with the human estrogen receptor R and show weak estrogenic activity (8-10), to our knowledge, there are no studies on the activities of its impurities. The object of the study reported here was to measure the estrogenicities of 10 impurities of industrial grade BPA using yeast two-hybrid assays incorporating the human estrogen receptor R (11) or the medaka fish (Oryzias latipes) estrogen receptor R and to calculate, using the concentrations addition (CA) model, at what concentrations they might significantly raise the estrogenicity of BPA. First, we sought confirmation that the CA method was appropriate in this case by measuring the estrogenicities of mixtures of BPA and an active impurity and comparing them with predicted values. Finally, the estrogenicities of a number of samples of industrial grade BPA were determined experimentally and compared with that of the laboratory grade reagent.
Materials and Methods Test Chemicals. The following test chemicals with purities as stated by the manufacturer were purchased from Wako Pure Chemical Industries, Osaka, Japan: laboratory grade BPA (for environmental analysis 99+%), 4-hydroxyacetophenone (97+%) (1), 4,4′-(1,3-dimethylbutylidene) bisphenol (99+%) (7), and p-cumylphenol (98+%) (8). BPA (SigmaAldrich, Tokyo, Japan, Aldrich catalog no. 13,302-7, 97% purity, 3 lots (Table 1); Acros Organics, Tokyo, Japan, catalog no. 15824-1000, 97% purity) was subjected to evaluated their estrogenicity. These test chemicals were used without purification. The following compounds were synthesized and purified following procedures reported in the literature, and their purities were measured by capillary gas chromatography: 4-isopropenylphenol (12, 13), 2-isopropenylphenol (14), 4-hydroxyphenyl isobutyl methyl ketone (2) (purity, 99+%) (15, 16), 2,4′-dihydroxy-2,2-diphenylpropane (3) (99+%) (17), 2,2′-dihydroxy-2,2-diphenylpropane (5) (99+%) (17), 2,4-bis(4-hydroxycumyl)phenol (6) (98+%) (17), 2,3dihydro-3-(4′-hydroxyphenyl)-1,1,3-trimethyl-1H-inden-5ol (4) (99+%) (18), 4-(4′-hydroxyphenol)-2,2,4-trimethylchroman (9) (99+%) (19), and 2-(4′-hydroxyphenyl)-2,4,4trimethylchroman (10) (98+%) (20). Methanol-d4 was from Cambridge Isotope Laboratories, Maryland, U.S., and silica gel 60 (0.040-0.063 mm for column chromatography) from Merck, Darmstadt, Germany. Synthesis. Details of the syntheses of compounds 2-6, 9, and 10 are provided as Supporting Information. Estrogenic Agonist Activity by Yeast Two-Hybrid Assay. The agonist activities of 1-10 were measured, both with and without possible metabolic activation by rat liver S9 preparation (Kikkoman Company, Noda, Japan), with a yeast twohybrid estrogenicity assay using yeast cells (Saccharomyces cervisiae Y190) into which the human (hERR) or medaka (medERR; Oryzias latipes) estrogen receptor R and coactivator VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Compounds Found as Impurities in Samples of Industrial Grade Bisphenol A and Their Estrogenicities in Yeast Two-Hybrid Assays
a Sample 1: Aldrich Chemical Co. Lot No. JU06612EU (97%). Sample 2: Aldrich Chemical Co. Lot No. CA18206TO (97%). Sample 3: Aldrich Chemical Co. Lot No. JS03830LR (97%). Sample 4: Acros Organics Lot No. A014744401 (97%). b Yeast two-hybrid assay incorporating the human estrogen receptor R. c Yeast two-hybrid assay incorporating the medaka fish (Oryzias latipes) estrogen receptor R. d Concentration of the compound showing 10% of the activity of 10-8 M 17β-estradiol (mean, n ) 4). e Standard deviation. f Activity relative to laboratory grade BPA. g Laboratory grade bisphenol A (99+%).
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TIF2 had been introduced. Work in this laboratory following the amplification of the gene for medaka estrogen receptor R and its introduction to a yeast recombinant plasmid for use in the yeast two-hybrid assay has shown that, in general, the relative estrogenic activities to xenoestrogens of the medaka estrogen receptor R is higher than those of the human estrogen receptor R (Nishikawa et al., unpublished data). Expression plasmids for each hormone receptor ligand binding domain and pGAAD424-TIF-2 were introduced into yeast cells that carried out the β-galactosidase reporter gene (21). Both assays were adapted to a chemiluminescent reporter gene (for β-galactosidase) method employing a 96well culture plate (11). Further details of the assay have been published (22). Agonist activity was recorded as the REC10 (10% relative effective concentration)sthat is, the concentration of the test compound showing 10% of the activity of 10-8 M 17β-estradiol. Estrogenic Activity of Mixtures and of Industrial Grade BPA. The agonist activities of mixtures of BPA and 7 (the most active impurity in the assay using medERR and the second most active in the hERR assay) were measured with both the human (hERR) and medaka (medERR) assays. Mixtures containing 95:5, 90:10, 80:20, and 70:30% BPA:7, on a molar basis, were assayed. The estrogenicities (measured as the REC10 values) of the mixtures of BPA and 7 were also predicted from the measured values for the individual components, assuming that the effects of the components were additive, by the CA method (23, 24):
REC10mix ) [
∑p /REC i
FIGURE 1. Agonist activity of compounds found as impurities in BPA and BPA itself in a yeast two-hybrid assay for hERr. Compound numbers correspond to Table 1. Each point represents the mean of four replicates.
-1 10i]
where REC10mix is the REC10 of the mixture, pi is the concentration of the compound i relative to the total mixture concentration, and REC10i is the REC10 calculated from the observed response for each individual compound i. The CA method was also used to predict the estrogenicities (measured as the REC10 values) of binary mixtures of BPA and 8 (the most active impurity in the human hERR assay) and for binary mixtures of BPA and 3 (the most abundant impurity in the industrial samples). The plots of REC10 values of the mixtures against percentage composition were then used to show the theoretical concentration of each impurity necessary to yield an estrogenicity twice that of pure (laboratory grade) BPA. The estrogenicities of four samples of industrial grade BPA were measured (and recorded as the REC10 values) by both the human (hERR) and medaka (medERR) assays. The predicted estrogenicities of these samples were calculated by the CA method based on their composition as revealed by liquid chromatographic analysis (7). The components with unknown estrogenicities were assumed to be equipotent with BPA for the sake of the calculations. The measured estrogenicities of the samples of industrial grade BPA were compared with that of laboratory grade (pure) BPA and the significance of differences estimated with two-tailed Student’s t tests.
Results and Discussion Synthesis. Seven compounds found as impurities in samples of industrial grade BPA were synthesized and characterized (1H and 13C NMR spectroscopy, high-resolution mass spectrometry). Estrogenic Agonist Activity in Yeast Two-Hybrid Assay. The estrogenicities of 1-10 and BPA, recorded as their REC10 values and measured by assays incorporating either hERR or medERR are presented in Table 1. The yeast two-hybrid assay incorporating the hERR showed that the estrogenicities of five impurities were more potent than BPA itself. The doseresponse relationships for the active compounds are shown
FIGURE 2. Agonist activity of compounds found as impurities in BPA and BPA itself in a yeast two-hybrid assay for medERr. Compound number correspond to Table 1. Each point represents the mean of four replicates. in Figure 1. p-Cumylphenol (8) was the most active compound tested and was 12 times as active as BPA. 4,4′-(1,3Dimethylbutylidene) bisphenol (7) and the catechin derivative 10 both showed 9-fold greater estrogenicity than BPA. Weak estrogenic activities were also recorded for 3 and 9. Five impurities showed greater activity than BPA itself in the yeast two-hybrid assay incorporating the medERR. Doseresponse relationships are shown in Figure 2. Compounds 7-10 showed activities from 7- to 9-fold of that of BPA whereas 3 showed weaker activity. Potential metabolic transformation of each compound by exposure to rat liver S9 preparation reduced or eliminated estrogenicities in each case in assays using both hERR and medERR. In this regard their behavior was analogous to that of 17β-estradiol itself. The assay using hERR showed greater sensitivity to 17βestradiol than did that using medERR. In contrast, the medERR assay was more sensitive for all chemicals examined in this study, including BPA itself, than was the hERR assay. These results indicated that an assay system incorporating medERR is likely to be the more effective for monitoring xenoestrogens. Estrogenicity of Mixtures and of Industrial Grade BPA: Observation and Prediction. Predicted estrogenicities (REC10 values) of binary mixtures of BPA and 7 in the hERR and medERR assays are shown in Figure 3, panels a and b, respectively, as plots of estrogenicity against percentage mixture composition. The measured estrogenicities of the mixtures are superimposed as red lines in each case. There was close agreement between the experimentally measured and predicted values, particularly in the case of the medERR assay. We therefore concluded that the estrogenic effects of BPA and 7 were additive and that the CA approach to predicting the estrogenicities of mixtures of BPA and its VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Estrogenic activity (REC10 values) vs % BPA in mixtures of BPA and 4,4′-(1,3-dimethylbutylidene) bisphenol (7). The black line represents calculated values, and the red line represents experimentally determined values. The vertical bars show the standard deviations (n ) 4). Activities were measured by yeast two-hybrid assays incorporating (a) the human estrogen receptor r and (b) the medaka estrogen receptor r. REC10 values (10% relative effective concentration) are the concentrations of the test compound showing 10% of the activity of 10-8 M 17β-estradiol.
FIGURE 4. Calculated estrogenic activities (REC10 values) in the yeast two-hybrid assay incorporating hERr vs % BPA in mixtures of (a) BPA and p-cumylphenol (8), the most active impurity compound in the assay incorporating hERr, and (b) BPA and 2,4′-dihydroxy-2,2diphenolpropane (3), the most abundant impurity in the samples of industrial grade BPA. The horizontal and vertical straight lines illustrate the composition of the mixtures that would give estrogenicities two times (i.e., REC10 values half, i.e., 4350) that of 100% BPA. In panel a, the mixture would contain 91% BPA; in panel b, it would contain 50% BPA. REC10 values (10% relative effective concentration) are the concentrations of the test compound showing 10% of the activity of 10-8 M 17β-estradiol. impurities was justified (i.e., the suitability of the CA method for the prediction of the estrogenicities of the mixtures of BPA and phenolic analogues was confirmed). Figure 4a,b shows plots of the predicted estrogenicities (as REC10 values based on the hERR assay) of binary mixtures of BPA and 8 (the most active compound in the hERR assay) and of BPA and 3 (the most abundant impurity in the mixtures). As an illustration of the concentrations at which the impurities would have to be present to markedly increase the estrogenicity of BPA, we have indicated in the figures the level of each impurity required to produce an REC10 value two times that of BPA alone. In the case of 3, 50% would be necessary, but in the case of 8 only 9% would be required to yield an REC10 value double that of pure BPA. Finally, the estrogenicities (REC10 values) of four samples of industrial grade BPA, determined experimentally by both human and medaka assays, and their predicted estrogenicities based on the CA method are shown in Table 2. The predicted estrogenicities differ little from the experimentally measured estrogenicity of BPA itself and lie within the standard deviations of the measured values of BPA. In 3706
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TABLE 2. Experimentally Determined and Calculated Estrogenicities of Samples of Industrial Grade Bisphenol A
sample 1a sample 2a sample 3a sample 4a BPAf a-e
hERr assayb REC10d SDe
calcd REC10
medERr assayc REC10d SDe
calcd REC10
7200 8600 7500 7800 8700
8156 8817 8390 8877
2300 2700 2400 2500 2500
2454 2567 2503 2561
1000 1400 600 370 1300
160 420 270 350 270
See footnotes to Table 1. f Laboratory grade bisphenol A (99+%).
addition there was no significant differences between the measured estrogenicities of the samples of industrial grade BPA and of laboratory grade (pure) BPA (p > 0.05) in the Student’s t tests. We conclude that the impurities in industrial grade BPA, although some are of much higher estrogenicity than BPA itself, do not significantly increase the estrogenic activity of the industrial compound and therefore do not increase possible adverse health effects from such activity.
Acknowledgments We thank M. Katsu (National Institute for Environmental Studies) for assistance.
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Supporting Information Available
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Experimental details of syntheses and characterization of 4-isopropenylphenol, 2-isopropenylphenol, and compounds 2-6, 9, and 10. This material is available free of charge via the Internet at http://pubs.acs.org.
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Received for review July 11, 2004. Revised manuscript received March 16, 2005. Accepted March 17, 2005. ES048932G
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