K. Ohno, , Y. Azuma, K. Date, S. Nakano, T. Kobayashi, Y. Nagao and T. Yamada
Central Research Institute, Nissin Food Products Co., Ltd, 2247, Noji-Cho, Kusatsu, Shiga 525-0055, Japan
Accepted 17 June 2002. ; Available online 16 October 2002.
Abstract
Styrene dimers (SDs) and styrene trimers (STs) eluted a little from polystyrene have been suspected of having estrogenic activity in the Wingspread Declaration [Our Stolen Futures, 1996] despite the lack of scientific analysis. Therefore, we have studied and reported styrene oligomers to have no endocrine disrupting effects [J. Food Hygienic Soc. Japan 40 (1999) 36; 41 (2000) 109; Yuki Goseikagaka Kyokaishi 57 (1999) 58; Bunseki Kagaku 49 (2000) 493, 857; Food Chem. Toxicol. 39 (2001) 1233; 40 (2002) 129]. However, Ohyama et al. reported that certain styrene oligomers have estrogenic effects in E-SCREEN and estrogen receptor (ER) binding assay [Environ. Health Perspect. 109 (2001) 699]. Recently, several assay systems have been developed, and a few of them can show false positive reactions at the high concentrations to which test compounds are precipitated [J. Health Sci. 48 (2002) 83]. In order to assess the estrogenic effect of SDs and STs in more detail, we examined the accuracy of the binding assay system and tested SDs and STs by three types of ER binding assay. In one ER binding assay, the same method that Ohyama et al. performed, SDs and STs showed a little estrogenic activity at high concentration; they did not dissolve, but this assay system tended to detect false positive effects at high concentration. In contrast, in the other assay systems, SDs and STs did not show any binding affinity to ER. In addition, luciferase reporter gene assay in HeLa cells transfected with ER expression plasmid and reporter plasmid, as a newly developed standard assay, and immature rat uterotrophic assay were conducted. In these tests, styrene oligomers showed no estrogenic activity.
Author Keywords: Endocrine disruptor; Polystyrene; Styrene dimer; Styrene trimer; Estrogen receptor binding assay; Luciferase reporter gene assay; Uterotrophic assay; Anti-estrogenic activity
Abbreviations: BPA, bisphenol A; CDFBS, charcoal dextran stripped fetal bovine serum; DES, dimethylstilbestrol; DMSO, dimethyl sulfoxide; E2, 17-estradiol; [3H]E2, [2, 4, 6, 7-3H(N)] estradiol; ER, estrogen receptor; ERE, estrogen response element; HAP, hydroxylapatite; hERa, human Era; ICI, ICI 182,780; MEM, minimum essential metium; p-NP, p-nonylphenol; PS, polystyrene; SD, styrene dimer; ST, styrene trimer; Tam, tamoxifen
1. Introduction
In modern society, we live in an environment that contains an untold number of synthetic chemical substances. These substances are found in daily necessities that serve to make life easier for us. At the same time, some of these substances disrupt the endocrine system of fish, wild animals and human beings, damaging our health and the natural ecosystems. When a chemical substance comes into contact with a human being or a wild animal, it may have some detrimental effects. When a chemical substance is found to have an undesirable side-effect, it is important to demonstrate the effect scientifically and investigate whether they have undesirable effects, in other words, to carry out "Hazard Identification".
Among artificially synthesized chemical compounds are p-nonylphenol (Soto et al., 1991), bisphenol A ( Krishnan et al., 1993), phthalate esters ( Harris et al., 1997), styrene dimers (SDs) and styrene trimers (STs) ( Ohyama et al., 2001), all of which have recently received public attention as compounds used in resin products and food containers. In particular, SDs and STs are known as substances eluted in microscopic amounts from food containers made of polystyrene (PS) ( Kawamura and Yamada). They belong to a group of chemical compounds that were included in "the list of endocrine disruptors" by Colborn et al. in 1991 in their "Wingspread Declaration"( Colborn et al., 1996). They were also listed in 1996 among the "endocrine disruptors" at the end of the book, Our Stolen Future. Unlike the case with other listed compounds, we found no scientific grounds for including SDs and STs in the list of endocrine disruptors, and today it is still unclear on what grounds they come under this category.
As part of our research effort to ascertain the safety of PS-made cup noodle containers, especially with respect to SDs and STs––chemical substances that are eluted from the cup noodle containers in trace amounts (Yamada et al., 2000b)––we have carried out a number of tests and studies both in vitro and in vivo, including estrogen and androgen receptor binding assays, thyroid hormone receptor binding assays, human breast cancer cell line MCF-7 proliferation assays (E-SCREEN), immature rat and ovariectomized rat uterotrophic assays, Hershberger assays, and prolactin release assays and steroidogenesis. We then presented a report summarizing the test results, stating that in any of these assays SDs or STs have no effects on sex hormones ( Nobuhara; Yamada; Azuma; Yamada; Ohno and Date).
Recently, Ohyama et al. reported that certain SDs and STs in high concentrations showed estrogenic effects in an the ER binding assay and E-SCREEN (Ohyama et al., 2001), pointing out the fear that they may disrupt the endocrine system. In the present study, we performed three different ER binding assays. One method was the ER binding assay using radiolabeled E2. The others were fluorescent-labeled E2, as used in Ohyama et al., and a polarization detection method. We performed the above assays with a view to ascertaining the accuracy of the ER binding assay system used by Ohyama et al. (2001). and to determine the limits of use in this method, and also the propriety of using E-SCREEN.
Furthermore, we evaluated all SDs and STs by reporter gene assay, a new method using HeLa cells, which are useful for the first screening method of large numbers of synthetic chemical substances. We also carried out an immature rat uterotrophic assay, in vivo, which is seen as the subsequent assay method following various others developed using cells and ER binding assay. Thus, in the present report a series of estrogenic effects were investigated in great detail, not only at the genetic level but also at the animal level, and we carried out "Hazard Identification" of SDs and STs on endocrine disrupting effects.
2. Materials and methods
2.1. Test compounds
All SDs and STs were synthesized by the Central Research Institute of Nissin Food Products Co., Ltd (Table 1). A quality test and eluting test of the cup noodle container made of polystyrene showed the presence of three SDs (NSD-01, 08 and 09), and seven STs (NST-01, 03–1, 2, 3, 4 and 12–1, 2) (Yamada and Yamada) ( Fig. 1). NST-01 was racemic mixture, and NST-03 and NST-12 has diastereomers. These were isolated and their chemical structures were investigated by nuclear magnetic resonance, mass spectrometry and element analysis. Their purity was determined as 98.5% or higher by gas chromatography–flame ionization detector (GC–FID). Bisphenol A, naphthalene, p-nonylphenol and testosterone were purchased from Katayama Chemical Inc. (Osaka, Japan). 5a-Dihydrotestosterone, 17b-estradiol (E2), diethylstilbestrol (DES), tamoxifen and vitamin D3 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). ICI 182,780 was purchased from Tocris Cookson Ltd. (Bristol, UK), and [2, 4, 6, 7-3H(N)] estradiol ([3H] E2) was purchased from NEN(TM) Life Science Products, Inc. (Boston, MA, USA). All other reagents used were of reagent grade.
Table 1. Solubility of tested compounds in buffer solution contained 5% DMSO
Solubility Compounds (µmmol/l) Estrogenic compounds 17b-Estradiol >10 Bisphenol A >10 Styrene dimers NSD-01 (2,4-diphenyl-1-butene) 1.3 NSD-08 (cis-1,2-diphenylcyclobutane) 9.4 NSD-09 (trans-1,2-diphenylcyclobutane) 4.0 Styrene trimers NST-01 (2,4,6-triphenyl-1-hexene) <0.16a NST-03–1 (1e-phenyl-4e-(1-phenylethyl) tetralin) <0.16a NST-03–2 (1a-phenyl-4e-(1-phenylethyl) tetralin) <0.16a NST-03–3 (1a-phenyl-4a-(1-phenylethyl) tetralin) 0.17 NST-03–4 (1e-phenyl-4a-(1-phenylethyl) tetralin) 0.16 NST-12–1 (1e-phenyl-4a-(2-phenylethyl) tetralin) <0.16a NST-12–2 (1a-phenyl-4a-(2-phenylethyl) tetralin) <0.16a Androgens Testosterone <10b 5a-Dihydrotestosterone <10b Non-estrogenic compounds Vitamin D3 0.19 Naphthalene 100 a These compounds could not dissolve at 0.16 µmmol/l. b Solubility of these compounds was in the ranging from 1 to 10 µmmol/l.
Fig. 1. Chemical structures of styrene dimers (SDs) and styrene trimers (STs).
2.2. Cell culture
HeLa cells derived from ATCC were obtained from Dainippon Pharmaceutical Co., Ltd (Osaka, Japan). Cells were maintained in phenol red-free Earle's minimum essential medium (MEM; GIBCO, Invitrogen Corp., Carlsbad, CA, USA) supplemented with 10% (v/v) charcoal dextran-stripped fetal bovine serum (FBS; GIBCO), 1% non-essential amino acid (GIBCO), at 37 °C in a saturated atmosphere containing 5% CO2.
2.3. Animals
Female Sprague-Dawley (SD) rats with body weights of 180–210 g and 20 days old were obtained from Charles River Japan Inc. (Tokyo, Japan). The animals were kept in an SPF facility maintained in a 12-h light/dark period (8.00–20.00) at a controlled humidity (55±10%) and temperature (23.5±2 °C).
2.4. Estrogen receptor binding assay (radiolabeled E2 method; Method RI)
A classical estrogen receptor (ER) binding assay using radioisotope was performed according to the method described previously (EORTC; Blair and Ohno) and EDSTAC ( US EPA, 1998). Uteri of rats were homogenized and centrifuged and the supernatant yielded was used as a cytoplasm fraction (ER-rich fraction). A reaction mixture consisting of 25 µl of cytoplasm fraction, 2 nmol/l of [3H] E2 and 100 µl of TEDG buffer E containing test compounds or E2 was incubated in glass tubes at 4 °C for 18 h. Test compounds and E2 were dissolved in dimethyl sulfoxide (DMSO, final concentration 0.2%) before they were added to the reaction mixture. After the incubation period, hydroxylapatite (HAP) slurry was added to each tube to separate the bound [3H]E2 from the free ligand. The resulting HAP pellet was washed and extracted the radiolabeled E2 with ice-cold ethanol and decanted into vials containing 10 ml of scintillation cocktail. Radioactivity was measured on liquid scintillation counter (Tri-Carb 2700TR, Packard Instrumental Co., Meriden, CT, USA). Non-specific binding was assessed by the addition of 100 molar excesses of non-labeled E2. Data were plotted as percent of [3H]E2 bound vs molar concentration of test samples.
2.5. Estrogen receptor binding assay (fluorescence E2 method; Method A)
ER binding assay detecting with fluorescence-labeled E2 was carried out with a Beacon(TM) 2000 (Takara, Kyoto, Japan) and an FP Screen-for-Competitor Kit ER (Takara) according to the manufacturer's protocol and previous report (Bolger et al., 1998). Binding affinity for human ER (hER) was measured by changes in polarization of the fluorescence-labeled E2 (ES1) in this assay. Estrogen receptors bound to ES1 and form a chemical complex, showing a high degree of fluorescent polarization. In the presence of test compounds, the degree of polarization is subject to change due to a competitive reaction between the test sample and ES1. The value obtained from the degree of change is evaluated as the test compound's affinity for ER. Data were plotted as percent of ES1 bound vs molar concentration of test samples.
2.6. Estrogen receptor binding assay (fluorescence E2 method; Method B)
ER binding assay using fluorescein-labeled E2 and recombinant hERa coated on the microplate was carried out with Estrogen Receptor (a) Competitor Screening Kit (Wako PC, Osaka, Japan) according to the manufacturer's protocol. This assay kit measured binding ability of test samples to recombinant hERa coated on the microplate by using fluorescence-labeled E2 as the competitor with competitive format. This assay system is heterogeneous. Data were plotted as percent of fluorescence-labeled E2 bound vs molar concentration of test samples.
2.7. Solubility of test compounds in buffer solution
SDs and STs are so hydrophobic that their solubility in buffer solution was very low. A precipitation of insoluble compounds causes incorrect result in vitro assays, especially in a ligand binding assay. Thus solubility in buffer solution used in ER binding assay was measured according to the standard method described in the Japanese pharmacopoeia (Japanese Pharmacopoeia, 13th edition). 0.5 ml of each test compound (10 mmol/l; solvent was DMSO) was added to 9.5 ml of TEDG buffer and shaken at 250 rpm for 30 min at 30 °C. Each solution was filtered thorough a 0.2 mm membrane filter and 100 ml of 1,4-bis (2-methylstyryl) benzene, an internal control, was added to 100 ml of filtrate, then 50 ml was injected into the HPLC system. HPLC was conducted under the following conditions: Column, µ-BONDASPHERE 5µ C18 (3.9 × 150 mm, Waters, MA, USA); mobile phase, 80% acetonitrile; flow rate, 0.7 ml/min; column temperature, 40 °C; detector, ultraviolet (vitamin D3: 265 nm, others: 215 nm).
2.8. Construction of plasmids
For luciferase reporter gene assay, estrogen receptor expression plasmid and reporter plasmid were constructed according to the methods described previously (Pons; Saito and Sumida). Standard protocols were used for DNA recombination. pRc /RSV-hERa, human estrogen receptor expression plasmid, was constructed by inserting human estrogen receptor sequence prepared from human liver total cDNA (Clontech Laboratories, Palo Alto, CA, USA) by PCR into the pRc/RSV plasmid (Invitrogen). ptk-Luc plasmid, encoding firefly-derived luciferase gene downstream of thymidine kinase, was constructed by inserting thymidine kinase promoter sequence into the PGV-P2 (Promega, Madison, WI, USA). The pERE3-tk-Luc reporter plasmid was constructed by inserting the three tandem repeat synthetic oligonucleotide for the estrogen responsive element (ERE) derived from the Xenopus vitellogenin gene into the ptk-Luc plasmid. The pRL-SV40 control plasmid encoding the seapansy-drived luciferase gene downstream of the SV40 enhancer/immediate early promoter site was purchased from Promega.
2.9. Transfection conditions
Transient transfection with the commercially available TransFast(TM) (Promega) transfection reagent containing a synthetic cationic lipid was carried out according to our previous report (Ohno et al., 2001). 18 h before transfection, HeLa cells were plated at initial concentration of 50,000 cells/well in 24-well plates (FALCON, Flanklin Lakes, NJ, USA) using phenol red-free MEM supplemented with 10% charcoal dextran-stripped FBS (CDFBS). The cells in each dish were co-transfected with 0.2 µg of pERE3-tk-Luc plasmid, 0.1 µg of pRc/RSV-hERa plasmid and 0.5 ng of pRL-SV40 plasmid with TransFast(TM) transfection reagent, and incubated at 37 °C and 5% CO2 for 1 h.
2.10. Luciferase reporter gene assay in HeLa cells
Luciferase reporter gene assay was carried out according to the methods of Saito et al. (2000). At 24 h after transient transfection, test compounds or E2 (final concentration of solvent was 0.1% DMSO) was added and incubated for 18 h at 37 °C and 5% CO2. The cells were washed with phosphate buffered saline three times and lysed by adding 100 µl/well of Picagene lysis buffer solution (TOYO INK MFG, Tokyo, Japan). 20 µl of cell lysate was transferred to the 96-well plate for luminoassay (EG & Berthold, Wildbad, Germany) and, firefly luciferase activity and seapansy luciferase activity were measured with Picagene Dual Seapansy Luciferase Kit (TOYO INK MFG) using luminometer (Micro Lumat P96V; EG & Berthold). Firefly luciferase activity was normalized with seapansy luciferase activity as an internal standard and expressed by the percentage to that of control vehicle treated cells.
2.11. Immature rat uterotrophic assay
Immature rat uterotrophic assay was carried out according to the methods described previously (Odum; US and Laws). Prepubertal 21-day-old female rats were dosed once per day for 3 days by subcutaneous injections with vehicle (corn oil). The dosing volume was 5 ml/kg body weight. Prepubertals were anesthetized using ether and euthanized by exsanguination 24 h after the last dose. Uteri were removed and adhering fat was trimmed away. The body of the uterus was cut just above its junction with vagina and bladder, and cut at the junction of the uterine horns with the ovaries. The uterus was then weighed.
2.12. Statistical analysis
Values were expressed as the mean±S.D. The statistical significance of differences between multiple groups, the Dunnett's multiple comparison test was applied. Student's t-test was also used in the comparison between the two groups. A difference was considered to be statistically significant when the P-value was less than 0.05.
3. Results
3.1. Solubility in buffer solution
In order to perform the in vitro assays more correctly, a solubility test was carried out. Estrogenic compounds, such as p-nonylphenol and bisphenol A, were soluble in a buffer well at the concentration 200 µmol/l. A non-estrogenic hydrocarbon, naphthalene, was insoluble at >100 µmol/l. The solubility of vitamin D3 was very low and maximum solubility was 0.19 µmol/l. Androgens, such as testosterone and 5-dihydrotestosterone, were insoluble >10 mol/l. The solubility of SDs and STs were very low and they did not dissolved at > 9.4 mol/l and >0.17 µmol/l, respectively (Table 1).
3.2. Estrogen receptor binding assay
An estrogen receptor (ER) binding assay was carried out to evaluate the direct interaction between test compounds and a receptor. Recently, several ER binding assay systems were used and three assay systems were performed in this study to achieve a more accurate result (Fig. 2). In the ER binding assay (method RI), the SDs, and STs did not show statistically significant inhibitory action against the binding of [3H]E2 to ER at concentrations from 5 nmol/l to 10 µmol/l, and they did not bind to the ER (Ohno et al., 2001).
Fig. 2. Binding ability of xenoestrogens, SDs and STs to estrogen receptor (Method RI, Method A and Method B). Method RI was classical binding assay using radiolabeled E2. Results of Method RI were the same as our previous reports (Ohno et al., 2001). Method A was performed with FP Screen-for-Competitor Kit ERa (Takara). Method B using fluorescence labeled E2 was performed with estrogen receptor (a) Competitor Screening Kit (Wako). Each value represents the mean±S.D. of triplicate assays. *P<0.05, **P<0.01, ***P<0.001 (vs control vehicle, Dunnett's test).
Methods A and B were newly performed in this study. In Method A, the binding affinities of test samples to hERa were measured by detecting the difference of polarization between fluorescence labeled E2 (ES1) bound to ER and ES1 only. E2 and DES inhibited the binding of ES1 to hERa in a concentration-dependent manner and exhibited great affinity for the ER with an IC50 value of 14.2 and 5.4 nmol/l, respectively. p-Nonylphenol and bisphenol A inhibited ES1 binding to hERa in a concentration-dependent manner. This data were in agreement with the previous report (Bolger et al., 1998). In contrast, SDs, and STs did not show statistically significant inhibitory action against the binding of ESI to ER at concentrations from 10 nmol/l to 10 µmol/, and they did not bind to the hERa in this assay. In Method B, the binding affinities of test samples to the human recombinant ER coated on the microplate were measured. Fluorescence labeled-E2 was used as a competitor. E2 inhibited the binding of fluorescence labeled-E2 to hERa coated on the microplate in a concentration-dependent manner and exhibited great affinity for ER with IC50 values of 2.1 and 3.2 nmol/l, respectively. SDs, and STs showed weak inhibitory effect on the binding of fluorescein E2 to hERa at 5 µmol/l, and their binding abilities were below 30% in this assay.
3.3. Comparison of three types of ER binding assay
In order to assess the specificity of binding ability of E2 to ER, three types of ER binding assay were compared using non-estrogenic compounds, vitamin D3, naphthalene, 5a-dihydrotestosterone and testosterone (Fig. 3). All three methods responded well to E2, although there was little difference in sensitivity between them. Vitamin D3, naphthalene, 5a-dihydrotestosterone and testosterone, that were reported non-estrogenic in the binding assay, did not show any binding ability to ER in Method RI and Method A (Blair; Nishihara and Swami); nevertheless, in Method B, they exhibited binding ability to human recombinant ER at high concentrations at which these compounds did not dissolve.
Fig. 3. Comparison of three types of ER binding assay to investigate the specificity to E2. Non-estrogenic compounds, naphthalene, vitamin D3, testosterone, and 5-dihydroxytestosterone, showed no affinity to ER in Method RI and Method A. In contrast, they showed affinity to ER in Method B at high concentrations in which they were insoluble. Each value represents the mean±S.D. of triplicate assays. *P<0.05, **P<0.01, ***P<0.001 (vs control vehicle, Dunnett's test).
3.4. Luciferase reporter gene assay in HeLa cells
Transcriptional activity assays can directly show receptor-mediated responses to gene expression. In this assay, reporter plasmid (pERE3-tk-Luc) was constructed to insert three tandem ERE into the ptk-Luc plasmid, and human ER expression plasmid was constructed to insert ERa sequence derived from human liver into pRc/RSV plasmid. The estrogen-dependent transcriptional activity of styrene oligomers and xenoestrogens were investigated. The luciferase activity of the HeLa cells transfected with pERE3-tk-Luc plasmid and pRc/RSV-hERa responded to E2, about 4.5-fold higher than control vehicle. E2 induced significant luciferase activity at concentration from over 1 pmol/l. Xenoestrogens, that is p-nonylphenol and bisphenol A, significantly increased luciferase activity, three- to four-fold higher from control, but their estrogenic activity were weaker than that of E2. In contrast, SDs and STs did not induce statistically significant luciferase activity (Fig. 4). This assay can detect anti-estrogenic activity with simultaneous addition of E2 (100 pmol/l) and test samples (10 µmol/l) (Fig. 5). ER antagonist, ICI 182,780 (ICI, 10 nmol/l) (Wakeling and Bowler, 1992) and tamoxifen (Tam, 100 nmol/l) ( Roos et al., 1982), abolished E2-dependent luciferase activity to the control level, but SDs and STs did not inhibit statistically significant luciferase activity. Hence it was concluded that they did not affect estrogen-dependent transcription activity in these assays.
Fig. 4. Effects of E2, bisphenol A (BPA), p-nonylphenol (p-NP), SDs and STs on E2-dependent luciferase transcription activity in HeLa cells transfected with pERE3-tk-Luc and pRc/RSV-hERa plasmids. HeLa cells transfected with pRc/RSV-hERa and pERE3-tk-Luc plasmid were treated with test compounds for 18 h as described in Materials and Methods. Each value represents the mean±S.D. of triplicate assays. *P<0.05, **P<0.01, ***P<0.001 (vs control, Dunnett's test).
Fig. 5. Effects of ER antagonist, SDs and STs on E2-induced luciferase transcription activity in HeLa cells transfected with pERE3-tk-Luc and pRc/RSV-hERa plasmids. HeLa cells transfected with pRc/RSV-hERa and pERE3-tk-Luc plasmid were treated with simultaneous addition of E2 (100 pmol/l) and test compounds (ICI 182,780, 10 nmol/l: tamoxifen, 100 nmol/l; SDs and STs, 10 µmol/l) for 18 h, as described in Materials and Methods. Each value represents the mean±S.D. of triplicate assays. **P<0.01, ***P<0.001 (vs control, Student's t-test).
3.5. Immature rat uterotrophic assay
In immature prepubertal rats, E2 (0.04 mg/kg) induced significant increase in uterine weight as compared with control (Fig. 6). p-Nonylphenol and bisphenol A (200 mg/kg) also induced significant increases in the uterine weight (about 230% of control). In contrast, SDs and STs (0.2, 2, 20 and 200 mg/kg) did not show any increase in uterine weight. The plates shows that the uterus treated with E2 0.04 mg/kg were significantly enlarged; in contrast, those of NSD-08 or NST-03–4 treated were not enlarged.
Fig. 6. Effects of bisphenol A (BPA), p-nonylphenol (p-NP) and SDs and STs on uterus weight in immature rat uterotrophic assay. Rats were dosed once per day for 3 days by subcutaneous injections with vehicle (corn oil) as described in Materials and Methods. Uterus weight of E2 treated group was 220 mg. Each value represents the mean±S.D. n=5. *P<0.05, **P<0.01, ***P<0.001(vs control, Dunnett's test). Photographs show the uterus treated with NST-08 or NST03–4 was not enlarged.
4. Discussion
In our previous reports (Nobuhara; Yamada; Azuma; Yamada; Ohno and Date), we assessed the endocrine-disrupting activity of SDs and STs, and reported that they (ST as a mixture of diastereomers) were negative for endocrine-disrupting activity by all of the tests in vitro and in vivo. These reports were supported by Fail et al. reported that mixtures of styrene oligomers, extracted from PS, did not show any estrogenic activity in immature rat uterotrophic assay and the reporter gene assay ( Fail et al., 1998). In addition, the Japan Environment Agency (JEA) referred to their studies in JPA and our reports, and deleted the SDs and STs from the list of endocrine disruptors in SPEED' 98 (2000 Edition) ( JEA, 2000). In order to assess estrogenic effects of SDs and STs in more detail, we investigated the estrogenic activity by three types of ER binding assay, luciferase reporter gene assay and immature rat uterotrophic assay. SDs and STs did not have a hydrophilic base, unlike alkyl phenol. SDs and STs are so hydrophobic that their solubility in buffer solution used in each assay is very low. In fact, the solubility of SDs and STs in buffer solution was very low. In vitro assays to investigate the estrogenicity of SDs and STs were carried out ranging from 10 nmol/l to 10 µmol/l, but the concentration of SDs and STs are saturated over 10 µmol and 1 µmol/l in buffer solution, respectively. The ER binding assay, a test to detect the direct reactivity of ligand to a receptor, is the most standardized and simple test system for the first step in the detection of specific mechanisms of estrogenic activity. Three types of ER binding assay systems (Method RI, Method A and Method B) were carried out for the purpose of evaluating the true binding affinities of SDs and STs to the ER and assay method itself. Method RI is classical ligand binding assay with radio labeled E2 as a competitor. Methods A and B are newly developed assay systems using fluorescence labeled E2 as a competitor, although the method of detection, and hERa used in each assay and the part of labeling fluorescence are different each other. In order to assess the accuracy of ER binding assay itself, vitamin D3, naphthalene, 5a-dihydrotestosterone and testosterone were assayed in three types of ER binding assay. All these compounds were reported not to have estrogenic activity in receptor and transcription level ( Blair; Nishihara and Swami). Vitamin D3, 5a-dihydrotestosterone and testosterone are known to bind the vitamin D3 receptor and the AR ( Keenan and Swami), respectively, and both belong to the group of nucleus receptors such as the ER. Vitamin D3 shows an anti-estrogenic effect, but this reaction is not through the ER pathway ( Swami et al., 2000). Testosterone acts like estrogen at the cellular level, because the endogenous aromatase changes testosterone into estrogen. Therefore, none could exhibit a direct effect on binding affinity for ER in the ligand binding assay. It cannot be considered that cross-reaction between estrogen and androgens occurred in a living body unless androgens are metabolized. In Method RI and Method A, these non-estrogenic compounds and styrene oligomers showed no binding ability to the ER. However, in Method B, they showed binding affinity for the recombinant hERa coated on the microplate at high concentration that they did not dissolve, although the binding affinity of E2 was similar in each assay. These results suggest that Method B tends to detect false positive effects and to lose accuracy in high concentration because of the decline of specificity to estrogen at high concentration that compounds do not dissolve. In the manufacture's protocol for Method B, it is states "make sure there is no precipitation in the solution" to prevent precipitate of compounds and fluorescence E2 from interacting. On the basis of these results, SDs and STs have no affinity for ER in Method RI and in Method A. One reason why hydrocarbons such as styrene oligomer fail to show binding ability to the ER activity is that, unlike alkylphenols, they lack the phenolic OH-base, which has an important role in ER binding ( Anstead and Blair). Nevertheless, in Method B, the same method as Ohyama et al. (2001) conducted, SDs and STs exhibited little affinity for the recombinant hERa at high concentration such that SDs and STs did not dissolve. Our result for Method B and Ohyama's report are not due to the difference of sensitivity between rat ER and human ER, but to a decrease in specificity to estrogen because of the precipitation of test compounds.
The luciferase reporter gene assay was carried out to directly detect the gene expression reactivity through the receptor. This method has been described in the EDSTAC Tier 1 Screening (US EPA, 1998). MCF-7, the human breast cancer cell line, is more popular in reporter gene assay because of having endogenous ER. However, MCF-7 has various mutations in ERa and the responsibilities to E2 are different in each laboratory ( Pink et al., 1997). In contrast, HeLa cells, as used in this test, did not have endogenous ER cascade. To construct a stable assay system, we used HeLa cells transfected with hERa expression plasmid derived from normal human liver ERa. In this assay system, SDs and STs did not show any increase in E2-dependent luciferase transcription activity. In the anti-estrogenic test, they did not show any inhibition of E2-dependent luciferase transcription activity. This result agreed with that of the ER binding assay and luciferase reporter gene assay in MCF-7 cells ( Ohno et al., 2001). It can be presumed that SDs and STs had no binding ability to ER and they did not affect E2-dependent transcription. Ohyama et al. reported that SDs and STs showed proliferation activity in E-SCREEN at high concentration ( Ohyama et al., 2001). Proliferation of MCF-7 cell is basically E2 dependent; however, it has been known that the cells can be proliferated by other growth factors ( Soto; Karey and Soto), and responsibility to E2 in MCF-7 cells is various in each laboratory because of variety of ER ( Pink et al., 1997). In the high concentration that test compounds were precipitated, the cells did not indicate normal response in the luciferase activity of control plasmid and morphology (data not shown). Therefore, false positive response might be shown only by the tests applying proliferation as the target. At the cellular level, the luciferase reporter gene assay in HeLa cells transfected with hERa expression plasmid was thought to be a more suitable assay for evaluation of estrogenicity at the cellular level.
Following these tests, we carried out an immature rat uterotrophic assay to confirm the results of the ER binding assay and reporter gene assay at the in vivo level, newly used optimal isomers of STs. The uterotrophic assay was the most popular and simple assay system to test the estrogenicity in vivo, and EDSTAC was also adopted this assay in Tier 1 (US EPA, 1998). A sc injection was adopted in order to examine by higher sensitivity in this study ( Odum et al., 1997), and sensitivity to E2 in the prepubertal rat was similar to that of the ovariectomized rat ( Date et al., 2002). The immature rat uterotrophic assay as a simpler test system, using newly isolated isomers of NST-03 and NST-12, was carried out in more detail than in the previous report ( Date et al., 2002). As a result, SDs and STs did not induce a statistically significant increase in uterus weight, although doses of isomers of NST-03 and NST-12 series were higher than previously reported ( Date et al., 2002). This result reflected the results of the ER binding assay, luciferase reporter gene assay and our previous reports.
The mechanisms of sex hormones are very complicated and it is thought that in vitro assays exhibit only one of many ways to cause estrogenic activity and may not to be a substitute for whole-animal data. In order to judge the sex hormone-like effects more correctly, it would be necessary to combine several tests containing not only in vitro but also in vivo assay systems.
As a result, we compared three types of ER binding assay using estrogenic and non-estrogenic compounds and it appeared that Method RI and Method A were useful for evaluating binding affinity for the ER, but Method B tended to indicate false positives in the range of high concentration, in which test compounds were insoluble because of a reduction of specificity of ER to E2. Estrogenic activity of three SDs (NSD-01, 08, 09) and seven STs (NST-01, 03–1, 03–2, 03–4, 12–1, 12–2), small amounts of substances eluted from PS-made instant noodle containers, were evaluated by ER binding assay, luciferase reporter gene assay and immature rat uterotrophic assay adopted to EDSTAC Tier 1 screening (US EPA, 1998). Consequently, on the basis of the "Hazard Identification" containing the present results and previous reports ( Nobuhara; Yamada; Azuma; Ohno; Yamada and Date), it can be concluded that the SDs and STs eluted from PS-made cup noodle containers have no endocrine-disrupting activities.
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Corresponding author. Tel.: +81-77-561-9120; fax: +81-77-561-9199; email: k-ono@mb1.nissinfoods.co.jp
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