Electrochemical Evaluation of the Interaction between Endocrine

Sapporo, Hokkaido, 060-0810 Japan, Gunma University, Maebashi, Gunma 371-8510, Japan, and. Hokkaido College of Pharmacy, Otaru, Hokkaido, 047-0264 ...
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Anal. Chem. 2002, 74, 533-538

Electrochemical Evaluation of the Interaction between Endocrine Disrupter Chemicals and Estrogen Receptor Using 17β-Estradiol Labeled with Daunomycin Hideki Kuramitz,† Junpei Natsui,† Kazuharu Sugawara,‡ Shinji Itoh,§ and Shunitz Tanaka*,†

Division of Material Science, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Hokkaido, 060-0810 Japan, Gunma University, Maebashi, Gunma 371-8510, Japan, and Hokkaido College of Pharmacy, Otaru, Hokkaido, 047-0264 Japan

A new electrochemical screening method for endocrine disrupting chemicals (EDCs) was developed. To evaluate the binding capacity of EDCs to the estrogen receptor (ER), 17β-estradiol labeled with daunomycin as an electroactive compound was prepared. The electrochemical sensitivity of the prepared labeled estradiol (LE) was high, as compared with daunomycin. The interaction between LE and ER was observed by the decrease in the electrode response of LE, indicating the specific binding of LE with ER. The competitive reaction between LE and 17βestradiol for the limiting binding site on ER produced increases in the peak current of LE. The relative standard deviation at 1 × 10-8 M 17β-estradiol was about 10.0% (n ) 7). The binding affinity between EDC and ER was also evaluated by comparison with 17β-estradiol-ER interaction. Bisphenol A, p-nonylphenol and p,p′-DDT was used as a test compound. All test compounds demonstrated some ability to bind with ER. This electrochemical binding assay illustrates a new method for evaluating the binding capacity of EDCs to ER without the need for a separation procedure for the bound and free LE. A wide variety of artificial chemicals are currently being released into the environment. Some of these chemicals have the potential to cause serious effects on wildlife and human health, even when they are present at very low concentrations over a period of time. Such effects include the apparent increase in hormone-dependent cancers and disorders of the reproductive tract in wildlife and humans.1-4 Recent studies have clarified the definition of endocrine disrupting chemicals (EDCs), identified research needs,5-7 and reviewed potential EDCs screening * To whom correspondence should be addressed. Fax: +81-011-706-2219. E-mail: [email protected]. † Graduate School of Environmental Earth Science, Hokkaido University. ‡ Gunma University. § Hokkaido College of Pharmacy. (1) Davis, D. L.; Bradlow, H. L.; Wolff, M.; Woodruff, T.; Hoel, D. G.; AntonCulver, H. Environ. Health Perspect. 1993, 101, 372-377. (2) Colborn, T.; vom Saal, F. S.; Soto, A. M. Environ. Health Perspect. 1993, 101, 378-384. (3) Colborn, T. Environ. Health Perspect. 1995, 103 (suppl 7), 135-136. (4) Harrison, P. T. C.; Holmes, P.; Humfrey, C. D. N. Sci. Total Environ. 1997, 205, 97-106. 10.1021/ac010426b CCC: $22.00 Published on Web 12/22/2001

© 2002 American Chemical Society

methods.8-12 The amount of screening, such as bioassays, receptor-binding assays, DNA-binding assays, and cell-based assays, that will be required is enormous, given the wide range of EDCs, metabolites, and potential environmental estrogens. The in vivo screening of EDCs is required to characterize a compound as an EDC that can cause adverse effects in any exposed whole organisms. On the other hand, in vitro assays are required to define the molecular mechanisms responsible for these effects. There is mounting evidence that many synthetic compounds mimic or block the natural estrogen activities in the living body by binding to the estrogen receptor (ER). Therefore, in vitro screening using the binding assays that evaluate the ability of a compound to bind with ER would be useful for identifying EDCs. A number of binding assays have been developed in order to evaluate the ER-ligand interaction.13-16 These binding assays are based on a competitive reaction in which a test compound displaces a labeled ligand, which is bound to the ER. Typically, a radioactive 17β-estradiol is used as a probe molecule. The use of a radioactive ligand leads to both high selectivity and the low detection limits based on the specificity of its receptor-ligand reaction. However, this technique warrants stringent regulation in handling and disposal, since isotopes are involved. Furthermore, most of these assays require a separation or wash procedure to separate free probe molecules from those bound to ER before any measurements can be made. The separation process in (5) Kavlock, R. J.; Daston, G. P.; DeRosa, C.; Fenner-Crisp, P.; Gray, L. E.; Kaattari, S.; Lucier, G.; Luster, M.; et al. Environ. Health Perspect. 1996, 104 (suppl 4), 715-740. (6) Kavlock, R. J.; Ankley, G. T. Risk Anal. 1996, 16, 731-739. (7) Ashby, J. Environ. Toxicol. Pharmacol. 1997, 3, 87-90. (8) Korach, K. S.; McLachlan, J. A. Environ. Health Perspect. 1995, 103 (suppl 7), 5-8. (9) Patlak, M. Environ. Sci. Technol. 1996, 30, 540A-544A. (10) Zacharewski, T. Environ. Sci. Technol. 1997, 31, 613-623. (11) Sadik, O. A.; Witt, D. M. Environ. Sci. Technol. 1999, 33, 368A-374A. (12) Erickson, B. Anal. Chem. 1998, 528, 8A-532A. (13) Van Aswegen, C. H.; Purdy, R. H.; Wittliff, J. L. J. Steroid Biochem. 1989, 32, 485-492. (14) Reel, J. R.; Lamb, J. C.; Neal, B. H. Fundam. Appl. Toxicol. 1996, 34, 288305. (15) Shelby, M. D.; Newbold, R. R.; Tully, D. B.; Chae, K.; Davis, V. L. Environ. Health Perspect. 1996, 104, 1296-1300. (16) Blair, R. M.; Fang, H.; Branham, W. S.; Hass, B. S.; Dial, S. L.; Moland, C. L.; Tong, W. D.; Shi, L. M.; Perkins, R.; Sheehan, D. M. Toxicol. Sci. 2000, 54, 138-153.

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Figure 1. Schematic diagram for the electrochemical assay for the evaluation of the interaction between EDC and ER using LE.

binding assays not only complicates the operation but may also causes a deviation in the reaction equilibrium between ER and ligand. Thus, binding assays that include a separation process would be useful for characterizing EDCs that have a relatively high affinity for the ER and aqueous solubility. Such procedures are not necessarily suitable for the screening of compounds that bind to the ER only weakly and have limited solubility. An excellent screening method for EDCs was developed by Bolger et al.17,18 This approach permits the evaluation of the binding between ER and EDC by monitoring changes in the fluorescence polarization of a fluorescently labeled ligand and eliminates the need to separate free from bound ligand. Electrochemical binding assays are a group of immunoanalytical techniques that have been the subject of continued research and development in recent years. Although electrochemical methods have many advantages (for example, they are relatively simple, rapid, inexpensive, and precise), a screening method of EDCs by electrochemical techniques has not yet been reported. We reported earlier on some simple electrochemical assays to detect the protein-ligand interaction, such as avidin-biotin19-22 and lectin-sugar.23,24 These methods do not require a separation procedure, since the protein-ligand interaction can be evaluated solely on the basis of changes in the electrode response of free ligand labeled with an electroactive compound. A similar attempt has been made in an immunoassay by using an antigen labeled with an electrochemically active substance for signal generation. In this case, the signal disappears or decreases on binding with an antibody.25,26 In this study, we report on the development of an electrochemical assay for the evaluation of the interaction between EDC and ER using 17β-estradiol labeled with daunomycin as an (17) Bolger, R.; Wiese, T. E.; Ervin, K.; Nestich, S.; Checovich, W. Environ. Health Perspect. 1998, 106, 551-557. (18) Parker, G. J.; Law, T. L.; Lenoch, F. J.; Bolger, R. J. Biomol. Screening 2000, 5, 77-88. (19) Sugawara, K.; Tanaka, S.; Nakamura, H. Anal. Chem. 1995, 67, 299-302. (20) Tanaka, S.; Yoshida, K.; Kuramitz, H.; Sugawara, K.; Nakamura, H. Anal. Sci. 1999, 15, 863-866. (21) Kuramitz, H.; Sugawara, K.; Nakamura, H.; Tanaka, S. J. Electroanal. Chem. 1999, 466, 117-121. (22) Kuramitz, H.; Natsui, J.; Tanaka, S.; Hasebe, K. Electroanalysis 2000, 12, 588-592.

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electroactive substance. Daunomycin can be detected by accumulation voltammetry sensitively, because it strongly adsorbs onto a glassy carbon electrode surface.19,23 In addition, daunomycin, which has an amino group in its sugar part, is easily labeled with a variety of commercially available reagents, and the electroactive and 17β-estradiol portions are far apart, so it is anticipated that these functions will not interfere with each other. The mechanism for this assay is illustrated in Figure 1. In the absence of a competitor, the electrode response of labeled estradiol (LE) decreases on binding with the ER. On the other hand, the presence of a large amount of competitor does not allow binding of LE to ER, and consequently, the electrode response of LE does not decrease. Therefore, EDC-ER interaction can be evaluated indirectly without a separation process. To achieve this assay, we initially investigated the electrochemical behavior of prepared LE on the glassy carbon electrode surface, and the specific binding between LE and ER or estradiol antibody was electrochemically observed. The binding affinity of ER for bisphenol A, p-nonylphenol, and p,p′-DDT as test compounds was evaluated by comparison with the 17β-estradiol-ER interaction. EXPERIMENTAL SECTION Apparatus. All voltammetric measurements were carried out by a CV-50W voltammetric analyzer (Bioanalytical Systems, Inc. (BAS)). A glassy carbon (GC) electrode (model no. 11-2012, 3.0mm diameter, BAS) was used as a working electrode. Prior to the experiments, the GC electrode was immersed in 0.1 M nitric acid for 5 min and sequentially polished with 0.3- and 0.05-µm alumina and then cleaned ultrasonically for 1 min. A platinum wire was used as a counterelectrode, and all potentials were recorded against a Ag/AgCl electrode (model no. 11-2020, BAS). The pH of buffer solutions was measured using a Horiba pH meter, F-22. Visible spectra of daunomycin and LE were measured using a V-550 UV/vis spectrophotometer (Jasco Co., Japan). (23) Sugawara, K.; Kuramitz, H.; Hoshi, S.; Akatsuka, K.; Tanaka, S.; Nakamura, H. Talanta 1998, 47, 665-671. (24) Sugawara, K.; Kuramitz, H.; Kaneko, T.; Hoshi, S.; Akatsuka, K.; Tanaka, S. Anal. Sci. 2001, 17, 21-25. (25) Heineman, W. R.; Anderson, C. W.; Halsall, H. B. Science 1979, 204, 865866. (26) Wehmeyer, K. R.; Halsall, H. B.; Heineman, W. R. Clin. Chem. 1982, 28, 1968-1972.

Reagents. Daunomycin hydrochloride, 17β-estradiol (1,3,5(10)estratrien-3,17β-diol), 17β-estradiol-6-[O-carboxymethyl]oxime, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) were obtained from Sigma Chemical Co. (St. Louis, MO). Sulfo-EGS (ethylene glycol-bis(sulfosuccinimidylsuccinate)) was purchased from Pierce (Rockford, IL). Human recombinant estrogen receptor-R was supplied from PanVera Co. (Madison, WI). Polyclonal rabbit anti-estradiol antibody was from Biostride, Inc. (Redwood, CA). Bovine γ-globulin was from ICN Pharmaceuticals, Inc. (Aurora, OH). Bisphenol A (4,4′-isopropylidenediphenol) and p-nonylphenol were purchased from Kanto Chem. Co. (Tokyo, Japan). 1,1,1-Trichloro-2,2-bis-(p-chlorophenyl)ethane (p,p′-DDT) was from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Screening buffer (40 mM Tris-HCl, pH 7.5; 50 mM KCl; 5% glycerol; 20% N,Ndimethylformamide (DMF); 0.02% sodium azide) was used as a solvent for the incubation of ER and as a supporting electrolyte for the electrochemical measurements. All other reagents were of analytical grade, and water was deionized and distilled twice. Preparation of Estradiol Labeled with Daunomycin. According to the procedure of Tiefenauer et al.,27 3,17β-dihydroxy1,3,5(10)-estratriene-6R-amine was synthesized by acetylation of the starting material, 17β-estradiol, with acetic anhydride in pyridine, followed successively by carbonylation of the 6-position with chromium (VI) oxide, reductive amination of the carbonyl group using a combination of ammonium acetate and sodium cyanoborohydride, and finally by base-catalyzed hydrolysis of the protective acetyl groups. The structure of 3,17β-dihydroxy-1,3,5(10)-estratriene-6R-amine was determined by IR, 1H NMR and SI-MS. IR: 3500-3200, 2924, 2870, 1611 cm-1. 1H NMR (DMSO-d6): δ 9.04 (1H, bs, 3-OH), 7.00 (1H, d, J ) 8.3 Hz, 1-H), 7.00 (1H, d, J ) 2.7 Hz, 4-H), 6.52 (1H, dd, J ) 8.3, 2.7 Hz, 2-H), 4.45 (2H, bs, 6RNH2), 3.72 (1H, dd, J ) 11, 6 Hz, 6β-H), 3.51 (1H, t, J ) 8.3 Hz, 17R-H), 0.65 (3H, s, 18-H3). SI-MS (positive, m/z): 288 (MH+), 271 (MH+ - 17(HH3)). Two LEs having spacers of different lengths were prepared. LE-L was prepared by mixing 2 mM daunomycin, 1 mM 3,17βdihydroxy-1,3,5(10)-estratriene-6R-amine, and 1.5 mM sulfo-EGS in 0.1 M phosphate buffer (pH 8.5)-DMF solution (7:3, v/v%) and incubating for 3 days at 4 °C. LE-S was prepared by mixing 2 mM daunomycin, 2 mM 17β-estradiol-6-[O-carboxymethyl]oxime and 5 mM EDAC in 0.1 M phosphate buffer (pH 8.5)-DMF solution (7:3, v/v%) and incubating for 1 day at 4 °C. The product was separated from unlabeled daunomycin and other byproducts by thin-layer chromatography (silica gel 60 F254 alumina sheet, Merck). Chloroform plus methanol (8:2, v/v%) was used as a developing solvent. After developing, the portion of the silica gel to which the LE was absorbed was stripped from the sheet and collected. The LE was dissolved in ethanol, and the solution was them centrifuged to remove the silica gel. From the simplicity of the reaction for labeling and the difference in the Rf value on the thin-layer chromatography, structures of the product are suggested as shown in Figure 2. The concentration of LE was determined by its absorbance at 475 nm. To examine the stability of LEs, the cyclic voltammogram and UV spectra of LE after 1 month were measured. (27) Tiefenauer, L. X.; Bodmer, D. M.; Frei, W.; Andres, R. Y. J. Steroid Biochem. 1989, 32, 251-257.

Figure 2. Structure of 17β-estradiol labeled with daunomycin (LE).

Procedure of Electrochemical Binding Assay for EDC. Prior to the measurements, a screening buffer solution was deaerated for 15 min with nitrogen gas. LE and ER were mixed in 500 µL of the screening buffer under stirring over 1 h at room temperature to reach equilibrium. To evaluate the binding capacity of an EDC to ER, 5 × 10-8 M ER was added to the screening buffer solution that contained 4 × 10-8 M LE and various concentrations of EDC, and the solution was incubated at the same conditions. Following the incubation step, the voltammetric measurement was carried out. A potential at -300 mV was applied to the cleaned electrode for 5 min with stirring to accumulate LE on the electrode surface. After a rest period of 15 s, the electrode response for reduction of LE was recorded by scanning the potential in the range from -300 to -800 mV by Osteryoung square wave voltammetry (OSWV). RESULTS AND DISCUSSION Electrochemical Behavior of 17β-Estradiol Labeled with Daunomycin on the Glassy Carbon Electrode. The cyclic voltammogram of 5 × 10-5 M daunomycin in a screening buffer solution (pH 7.5) at the GC electrode is shown in Figure 3A. Two pairs of redox peaks were observed at the different potentials. The more positive pair of peaks (an anodic peak potential at 465 mV and a cathodic peak potential at 270 mV) is due to two hydroxyl groups, and other pair of peaks (an anodic potential of -670 mV and a cathodic potential of -690 mV) is a redox based on the quinone portion of the molecule. In addition, 17β-estradiol exhibited a typical irreversible oxidation electrode response of a phenolic compound over 800 mV. The cyclic voltammograms of 1 × 10-5 M LEs were identical to that of daunomycin itself. Therefore, it was confirmed that the electrode reaction of daunomycin portion in LE was not affected by the labeling. In addition, the cyclic voltammogram and UV spectra of LEs after 1 month were the same as those at the time of preparation. In this electrochemical binding assay for EDC, a cathodic peak at -690 mV of LEs was used to evaluate the binding of LE to ER considering the sensitivity and the interference by the oxidation of the 17β-estradiol portion in LE. Analytical Chemistry, Vol. 74, No. 3, February 1, 2002

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Figure 3. (A) Cyclic voltammogram of 5 × 10-5 M daunomycin in screening buffer solution (pH 7.5); scan rate, 50 mV/s. (B) The relationship between the peak current and the concentration of (a) daunomycin, (b) LE-S, and (c) LE-L in screening buffer solution (pH 7.5). The peak currents were obtained by OSWV. Ea, -300 mV; accumulation time, 5 min.

Figure 4. (A) Osteryoung square wave voltammograms of 4 × 10-8 M LE-L in screening buffer solution (pH 7.5) containing (a) 0, (b) 1 × 10-8, (c) 2 × 10-8, (d) 5 × 10-8 M ER, and (e) background solution. (B) Relationship between the peak current of 4 × 10-8 M (a) LE-S and (b) LE-L and the concentration of ER. The peak currents were obtained by OSWV.

Figure 3B shows the relationship between the peak current and the concentration of daunomycin, LE-S, and LE-L in a screening buffer solution (pH 7.5). The peak current for the reduction of LEs was larger than that for daunomycin. This indicates that the LEs adsorbed onto the glassy carbon electrode surface more efficiently than daunomycin. This strong adsorption property of LEs can be attributed to the increase in hydrophobicity caused by labeling with 17β-estradiol. That is, the labeling converted the ionic amino group of daunomycin into a hydrophobic moiety. Furthermore, the adsorption property of LE-L on the electrode surface was stronger than that of LE-S because of the presence of a spacer between the 17β-estradiol and daunomycin portions. The peak current of LEs was linear in the range of about 1 × 10-8 to 4 × 10-8 M. The relative standard deviation (RSD) at 4 × 10-8 M daunomycin, LE-S, and LE-L was 13.2, 6.2, and 9.0% (n ) 5), respectively. The sensitivity for the electrochemical detection improved by about 1.5-2.0-fold as the result of labeling with 17β-estradiol. Electrochemical Evaluation of the Interaction between Labeled Estradiol and the Estrogen Receptor. Figure 4A shows the Osteryoung square wave voltammograms in a screening buffer solution (pH 7.5) containing 4 × 10-8 M LE-L and the several concentration of ER. The peak shape for the reduction of LE-L remained unchanged in the presence of ER, but the peak current decreased significantly. Figure 4B shows the binding curve 536 Analytical Chemistry, Vol. 74, No. 3, February 1, 2002

of LE-S and LE-L to the ER obtained from the peak current for the reduction of LEs plotted as a function of increasing ER concentration. The electrochemical behaviors of LE-S and LE-L in the solution containing ER were similar. The peak current of 4 × 10-8 M LEs decreased with increasing concentrations of ER present in the solution and reached a constant value over 5 × 10-8 M ER. The same measurements were performed using daunomycin instead of LE. In this case, the peak current of daunomycin remained unchanged, even if a sufficient amount of ER was added to the solution. Therefore, the decrease in the peak current of LEs is not due to nonspecific binding between the daunomycin portion in LE and ER. To investigate nonspecific interactions between LE and a protein, bovine γ-globulin and estradiol antibody were used in place of ER. Figure 5 shows plots of the peak current for LE-S vs the concentrations of bovine γ-globulin, estradiol antibody, and ER (already shown in Figure 4B). With the addition of estradiol antibody, the peak current of LE-S was decreased and rapidly reached a constant value. On the other hand, the addition of bovine γ-globulin did not cause a significant decrease in the peak current of LE. These results indicate that LE has a specific affinity with estradiol antibody and ER. The specific interaction between LE and the protein is responsible for the decrease in the electrode response of LE, but not for the interference in the electrode reaction, such as nonspecific interaction with the protein or the

Figure 5. Relationship between the peak current of 4 × 10-8 M LE-S and the concentration of (a) ER, (b) estradiol antibody, and (c) bovine γ-globulin. The peak currents were obtained by OSWV.

Figure 6. Competitive binding curves obtained using a competitive reaction between 17β-estradiol and LE for ER. The peak currents of 4 × 10-8 M (a) LE-S and (b) LE-L in screening buffer solution (pH 7.5) including 5 × 10-8 M ER and a various concentration of 17βestradiol were obtained by OSWV.

adsorption of protein on the electrode surface. Possible reasons for the decrease in the peak current of LE are (1) the binding of LE with ER or estradiol antibody may sequester the electroactive daunomycin portion from the electrochemical reaction by burying them in the binding site, or (2) the diffusion coefficient of LE decreases on binding with ER or estradiol antibody. The diffusion coefficient of globular proteins (Mr 150 000-160 000) in aqueous media are reportedly in the 10-7 cm2/s range,28 whereas those of small biomolecules, such as estradiol, are in the 10-5 cm2/s range.29 This principle for the detection of the interaction between protein and ligand is called “sequestration electrochemistry”.26 Electrochemical Evaluation of the Interaction between Endocrine Disrupting Chemicals and the Estrogen Receptor Using Labeled Estradiol. The competitive binding curves obtained with the competitive reaction between 17β-estradiol and LE-S or LE-L against ER are shown in Figure 6. The measurements for the reduction in peak current of LE were carried out over a range of 10-12-10-4 M 17β-estradiol in the screening buffer (28) CRC Handbook of Biochemistry: Selected Data for Molecular Biology, 2nd ed.; The Chemical Rubber Co.: Cleveland, OH, 1970; p C-39. (29) Miller, T. A.; Lamb, B.; Prater, K.; et al. Anal. Chem. 1964, 36, 418-420. (30) Waller, C. L.; Oprea, T. I.; Chae, K.; Park, H.-K.; Korach, K. S.; Laws, S. C.; Wiese, T. E.; Kelce, W. R.; Gray, L. E. Chem. Res. Toxicol. 1996, 9, 12401248.

Figure 7. The competitive binding curves obtained from competitive reaction between (a) p-nonylphenol, (b) bisphenol A, (c) p,p′-DDT, (d) 17β-estradiol, and LE-S for ER. The peak currents of 4 × 10-8 M LE-S in screening buffer solution (pH 7.5), including 5 × 10-8 M ER and various concentrations of the test compounds, were obtained by OSWV.

solution, including 4 × 10-8 M LE and 5 × 10-8 M ER. In the absence of 17β-estradiol, the peak current of LE decreases as a result of the specific binding with ER. However, the presence of a large amount of 17β-estradiol in the solution does not allow binding of LE to ER because 17β-estradiol occupies the limited binding site on the ER; consequently, the peak current of LE increases. The IC50 values (competitor concentration needed to achieve a 50% inhibition of ER-ligand binding) for 17β-estradiol that competes with LE-L and LE-S were 4.1 × 10-8 M and 3.6 × 10-8 M, respectively. This result indicates that LE-L has a strong affinity to ER, as compared with that of LE-S. This strong affinity can be attributed to the long-chain spacer (ca. 16 Å) between the 17β-estradiol and daunomycin portion in LE-L. That is, LE-L is superior to LE-S for preventing steric interference. The RSD at 1 × 10-8 M 17β-estradiol in the dose curve obtained from LEs was about 10.0% (n ) 7). The binding affinity to ER for three EDCs was evaluated using this electrochemical assay. Bisphenol A, p-nonylphenol, and p,p′DDT were selected as test compounds. It is known that these compounds bind with ER and exert estrogenic action. On the other hand, it is well-known that phenolic compounds show a typical irreversible electrochemical oxidation, and their electrochemical oxidation causes the fouling of the electrode surface by the electropolymerization. However, the oxidation potential of bisphenol A and p-nonylphenol is over 600 mV; hence, the presence of these compounds causes no interference. Figure 7 shows that the competitive binding curves for bisphenol A, p-nonylphenol, and p,p′-DDT that competes with LE-S against ER. The y axis is reported as the inhibition for the binding of LE-S to ER. Although these test compounds have a very weak binding affinity to the ER, compared with 17β-estradiol, all compounds demonstrated some ability to bind to the ER. To more easily compare the binding affinity to ER for test compounds to each other, the IC50 and the RBA (relative binding affinity) values for each test compound are shown in Table 1. The RBA values were calculated from (IC50 of 17β-estradiol/IC50 of competitor) × 100. The calculated RBA values (31) Kuiper, G. G. J. M.; Carlsson, B.; Grandien, K.; Enmark, E.; Haggblad, J.; Nilsson, S.; Gustafsson, J. A. Endocrinology 1997, 138, 863-870.

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Table 1. IC50 and RBA Values and of Tested Compound compd 17β-estradiol p-nonylphenol bisphenol A p,p′-DDT a

RBA

RBA values from literaturea

ref

100 1.14 0.07 0.0009-0.0022

0.3 h, 0.313 m, 0.05 t 0.04 h, 0.05 h, 0.018 m 0.03-0.09 h, 0.00026 m

16, 30, 15 16, 30, 31 16, 30

IC50 10-8

M 3.6 × 3.1 × 10-6 M 5.2 × 10-5 M >1.0 × 10-4 M

The letters after the RBA value indicate the source of ER in the assay: h, human; m, mouse; t, trout.

in this study were relatively high, as compared with that from other literature values. These results indicate that the monitoring of binding between EDC and ER can be achieved sensitively by this electrochemical assay. CONCLUSIONS The electrochemical binding assay was achieved on the basis of monitoring the changes in the electrode response of 17βestradiol labeled with daunomycin by the interaction between the 17β-estradiol portion in LE and ER. The electrode response of LE decreased as a result of the specific binding with the ER. The screening test for bisphenol A, p-nonylphenol, and p,p′-DDT was performed by a competitive reaction with LE and a test compound for the ER. Although EDCs have very weak binding affinity to the ER, the competitive reaction between LE and 17β-estradiol or EDCs was able to evaluate the binding capacity of these compounds for ER. This electrochemical binding assay can be

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run at physiological temperature and does not involve the use of radioactivity. Furthermore, it does not require a separation or wash procedure for free LE from the molecules bound to the ER before measurements. Therefore, a deviation of the reaction equilibrium between ER and EDC may be not caused. In addition, another possible interference source is not introduced to the assay, unlike other binding assays which use immobilized ER. The approach described in this research can be applied to other hormone receptor assay systems, as well. ACKNOWLEDGMENT This work was supported by the Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (no. 07021). Received for review April 16, 2001. Accepted November 7, 2001. AC010426B