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Estrogenicity of styrene oligomers and assessment of estrogen receptor binding assays. (Correspondence).

Polystyrene is frequently used in resins, and the styrene dimers and trimers eluted from polystyrene have been reported to have estrogenic activity (1). We have performed a number of in vitro and in vivo tests [i.e., estrogen receptor (ER) and androgen receptor binding assays, thyroid hormone receptor binding assays, human breast cancer cell line MCF-7 proliferation assays (E-SCREEN), uterotrophic assays in immature and ovariectomized rats, Hershberger assays, and prolactin release assays and steroidogenesis] and found no effects of styrene dimers or trimers on sex hormones in any of these assays (2-7). These results are supported by Fail et al. (8), who reported that mixtures of styrene oligomers did not show any estrogenic activity in the immature rat uterotrophic assay and the reporter gene assay. In addition, the Japan Environment Agency referred to their studies (9) and removed the styrene dimers and trimers from their list of endocrine disruptors (9). However, Ohyama et al. (10) reported that high concentrations of certain styrene dimers and trimers showed estrogenic effects in an ER binding assay and in the E-SCREEN assay. Recently, several assay systems have been used to assess endocrine-disrupting effects, but a few of these assay systems can cause false-positive reactions when test compounds are at high concentrations (11).

To assess the accuracy of the ER binding assay system and the results of Ohyama et al. (10), and to ascertain the safety of styrene dimers and trimers, we used a solubility test and three ER binding assays (12) (Table 1). The ER binding assay, which detects the direct reactivity of ligand to a receptor, is the most standardized and simple test system for the detection of specific mechanisms of estrogenic activity.

Using the radiosotope method (Method RI) as described previously (13,14), we observed that styrene dimers and trimers did not show statistically significant inhibitory action against the binding of [[sup.3]H]-17[beta]-estradiol ([E.sub.2]) to ER.

We used Method A to detect the binding affinities of test samples to human ER[alpha] (hER[alpha]). Using a fluorescence polarization Screen-for-Competitor Kit ER[alpha] (Takara, Kyoto, Japan) as described by Bolger et al. (15), we measured the difference of polarization between fluorescence-labeled [E.sub.2] (ES1) bound to ER and ES1 only. Styrene dimers and trimers did not show statistically significant inhibitory action against the binding of ESI to ER in this assay.

We also used Method B, the method used by Ohyama et al. (10), to detect the binding affinities of test samples to the human recombinant ER[alpha] coated on the microplate by competition with fluorescence-labeled [E.sub.2]; this was performed using the Estrogen Receptor ([alpha]) Competitor Screening Kit (Wako PC, Osaka, Japan). Styrene dimers and trimers showed weak inhibitory effect on the binding of fluorescein [E.sub.2] to hER[alpha] at 5 [micro] mol/L, and their binding abilities were < 30% in this assay.

To evaluate the ER binding assays themselves, we included vitamin [D.sub.3], naphthalene, 5[alpha]-dihydrotestosterone, and testosterone in each of the three ER binding assays; none of these compounds bound ER in vitro (13,16,17). A cross-reaction between estrogen and androgens cannot occur in vivo unless the androgens are metabolized. In Method RI and Method A, these nonestrogenic compounds did not show any ability to bind to the ER. However, in Method B, these compounds showed binding affinity for the recombinant hER[alpha] coated on the microplate at such high concentrations that they did not dissolve, although the binding affinity of [E.sub.2] was similar in each assay. These results suggest that Method B tends to detect false-positive effects and that it is less accurate at high concentrations because of a decline of specificity to estrogen at high concentrations at which compounds do not dissolve. The manufacturer's instructions for the Estrogen Receptor ([alpha]) Competitor Screening Kit used for Method B say to "make sure there is no precipitation in the solution." Styrene dimers and trimers are so hydrophobic that their solubility is very low in the buffer solutions used in each assay. On the basis of these results, styrene dimers and trimers have no affinity for ER in Methods RI and A. Nevertheless, styrene dimers and trimers exhibited some affinity for the recombinant hER[alpha] in the Method B study, similar to that described by Ohyama et al. (10), but at high concentrations such that the compounds were not completely dissolved. This result is not because of the difference of sensitivity between rat ER and human ER, as shown in Method A with the use of hER[alpha], but is caused by a decrease in specificity to estrogen because of the precipitation of test compounds.

Ohyama et al. (10) reported that high concentrations of styrene dimers and trimers showed proliferative activity in the E-SCREEN assay. Cell proliferation can be induced by other growth factors, although proliferation of MCF-7 cell is basically [E.sub.2] dependent (18-20), and the response to [E.sub.2] in MCF-7 cells varies because of the various mutation of ER (21). Therefore, a false-positive response might only be shown in tests using proliferation as a target. The luciferase reporter gene assay, which indicates direct gene expression reactivity through the receptor, has been considered to be a more suitable assay for evaluating estrogenicity at the cellular level because of specificity to [E.sub.2] response (22,23). Styrene dimers and trimers did not show any estrogenic effect in the E-SCREEN assay and the reporter gene assay in our previous study (6). In addition, at high concentrations at which test compounds were precipitated, cells indicated an abnormal response in the luciferase activity of control plasmids and in morphology (data not shown). To construct a stable assay system, we used HeLa cells transfected with an hER[alpha] expression plasmid derived from normal human liver ER[alpha]. In this assay system, styrene dimers and trimers did not show any increase in [E.sub.2]-dependent luciferase transcription activity. These results agreed with the result of the ER binding assay. We presume that styrene dimers and trimers had no binding affinity to ER and they did not affect [E.sub.2]-dependent transcription.

As a result, in our comparison of three ER binding assays using estrogenic and nonestrogenic compounds, it appeared that Method RI and Method A were useful for evaluating binding affinity for the ER, but Method B, similar to the method of Ohyama et al. (10), tended to indicate false-positives in high concentrations in which test chemicals were insoluble; this reduced the specificity of ER to [E.sub.2]. Based on our present results and previous reports (2-7), we found no endocrine-disrupting activities in styrene dimers and trimers eluted from polystyrene-containing instant noodle containers.
Table 1. Solubility and binding affinity for ER of tested compounds.

 Binding
 affinity
 for ER
 ([ED.sub.30])
 [micro]
 mol/L)

Compounds ([micro] mol/L) Method RI
Estrogenic compounds
 17[beta]-Estradiol > 10 0.0012 ***
 Bisphenol A > 10 5.0 ***
Styrene dimers
 2,4-Diphenyl-1-butene 1.3 NC
 cis-1,2-Diphenylcyclobutane 9.4 NC
 trans-1,2-Diphenylcyclobutane 4.0 NC
Styrene trimers
 2,4,6-Triphenyl-1-hexene < 0.16 NC
 1e-Phenyl-4e-(1-phenylethyl) tetralin < 0.16 NC
 1a-Phenyl-4e-(1-phenylethyl) tetralin < 0.16 NC
 la-Phenyl-4a-(1-phenylethyl) tetralin 0.17 NC
 le-Phenyl-4a-(1-phenylethyl) tetralin 0.16 NC
 1e-Phenyl-4a-(2-phenylethyl) tetralin < 0.16 NC
 1a-Phenyl-4a-(2-phenylethyl) tetralin < 0.16 NC
Androgens
 Testosterone < 10 NC
 5[alpha]-Dihydrotestosterone < 10 NC
Nonestrogenic compounds
 Vitamin [D.sub.3] 0.19 NC
 Naphthalene 100 NC

 Binding affinity for ER
 ([ED.sub.30]) ([micro] mol/L)

Compounds Method A Method B
Estrogenic compounds
 17[beta]-Estradiol 0.005 *** 0.001 ***
 Bisphenol A 1.7 *** 2.0 **
Styrene dimers
 2,4-Diphenyl-1-butene NC > 10.0
 cis-1,2-Diphenylcyclobutane NC 10.0 **
 trans-1,2-Diphenylcyclobutane NC > 10.0
Styrene trimers
 2,4,6-Triphenyl-1-hexene NC > 10.0
 1e-Phenyl-4e-(1-phenylethyl) tetralin NC > 10.0
 1a-Phenyl-4e-(1-phenylethyl) tetralin NC > 10.0
 la-Phenyl-4a-(1-phenylethyl) tetralin NC > 10.0
 le-Phenyl-4a-(1-phenylethyl) tetralin NC 5.2 **
 1e-Phenyl-4a-(2-phenylethyl) tetralin NC > 10.0
 1a-Phenyl-4a-(2-phenylethyl) tetralin NC > 10.0
Androgens
 Testosterone NC 105.0 ***
 5[alpha]-Dihydrotestosterone NC 45.0 ***
Nonestrogenic compounds
 Vitamin [D.sub.3] NC 100.0 ***
 Naphthalene NC 1010.0 ***

Abbreviations: [ED.sub.30], concentration equivalent to 30% activity
of 100 nmol/L [E.sub.2]; NC, no competition for binding of labeled
[E.sub.2]. Each value represents the mean of triplicate assays..

(a) Concentration at which test compounds are saturated.

** p < 0.01,

*** p < 0.001 (vs. control, Dunnett test).


REFERENCES AND NOTES

(1.) Colborn T, Dumanoski D, Myers JP. Our Stolen Future. New York:Dutton, 1996.

(2.) Yamada T. Synthesis, analysis and biological evaluation of styrene oligomers. Yuki Goseikagaku Kyokaishi 57:58-64 (1999).

(3.) Nobuhara Y, Hirano S, Azuma Y, Date K, Ohno K, Tanaka K, Matsushiro S, Sakurai T, Shiozawa S, Chiba M, et al. Biological evaluation of styrene oligomers for endocrine-disrupting effects. J Food Hyg Soc Japan 40:36-45 (1999).

(4.) Yamada T, Hirano S, Kobayashi K, Sakurai T, Takagi K, Tanaka M, Nagao Y, Azuma Y, Date K, Ohno K, et al. Identification, determination and biological evaluation of novel styrene trimer in polystyrene container. Bunseki Kagaku 49:493-501 (2000).

(5.) Azuma Y, Nobuhara Y, Date K, Ohno K, Tanaka K, Hirano S, Kobayashi K, Sakurai T, Chiba M, Yamada T. Biological evaluation of styrene oligomers for endocrine-disrupting effects (II). J Food Hyg Soc Japan 41:109-115 (2000).

(6.) Ohno K, Azuma Y, Nakano T, Kobayashi S, Hirano T, Nobuhara Y, Yamada T. Assessment of styrene oligomers eluted from polystyrene-made food container for estrogenic effects in vitro assays. Food Chem Toxicol 39:1233-1241 (2001).

(7.) Date K, Ohno K, Azuma Y, Hirano S, Kobayashi K, Sakurai T, Nobuhara Y, Yamada T. Endocrine-disrupting effects of styrene oligomers that migrated from polystyrene containers into food. Food Chem Toxicol 40:129-139 (2001).

(8.) Fail PA, Hines JW, Zacharewski T, Wu ZF, Borodinsky L. Assessment of polystyrene extract for estrogenic activity in the rat uterotrophic model and an in vitro recombinant receptor reporter gene assay. Drug Chem Toxicol 21(suppl 1):101-121 (1998).

(9.) JEA. Strategic Programs on Environmental Endocrine Disruptors '98. Available: http://www.env.go.jp/en/pol/ speed98/sp98.pdf [cited 30 April 2002].

(10.) Ohyama K, Nagai F, Tsuchiya Y. Certain styrene oligomers have proliferative activity on MCF-7 human breast tumor cells and binding affinity for human estrogen receptor [alpha]. Environ Health Perspect 109:699-703 (2001).

(11.) Nakano S, Nagao Y, Kobayashi T, Tanaka M, Hirano S, Nobuhara Y, Yamada T. Problems with methods used to screen estrogenic chemicals by yeast two-hybrid assays. J Environ Health 48:83-88 (2002).

(12.) The Japanese Pharmacopoeia. 13th ed. Tokyo:Society of Japanese Pharmacopoeia, 1997.

(13.) Blair RM, Fang B, Braham WS, Hass BS, Dial SL, Moland C-L, Tong W, Shi L, Perkins R, Sheehan DM. The estrogen receptor binding affinities of 188 natural and xenochemicals: Structural diversity of ligands. Toxicol Sci 54:138-153 (2000).

(14.) Laws SC, Carey SA, Kelce WR, Cooper RL, Gray LE. Vinclozolin does not alter progesterone receptor (PR) function in vivo despite inhibition of PR binding by its metabolites in vitro. Toxicology 110:1-11 (1996).

(15.) Bolger R, Weise TE, Evin K, Nestich S, Checovich W. Rapid screening of environmental chemicals for estrogen receptor binding capacity. Environ Health Perspect 106:551-557 (1998).

(16.) Swami S, Krishnan AV, Feldman D. 1[alpha], 25-dihydroxyvitamin [D.sub.3] down-regulates estrogen receptor abundance and suppresses estrogen actions in MCF-7 human breast cancer cells. Clin Cancer Res 6:3371-3379 (2000).

(17.) Nishihara T, Nishikawa J, Kanayama T, Dakeyama F, Saito K, Imagawa M, Takatori S, Kitagawa Y, Hori S, Utsumi H. Estrogenic activity of 517 chemicals by yeast two-hybrid assay. J Health Sci 46:282-298 (2000).

(18.) Karey KP, Sirbasku DA. Differential responsiveness of human breast cancer cell lines MCF-7 and T47D to growth factors and 17[beta]-estradiol. Cancer Res 48:4083-4092 (1988).

(19.) Soto AM, Sonnenschein C. The role of estrogens on the proliferation of human breast tumor cells (MCF-7). J Steroid Biochem 23:87-94 (1985).

(20.) Soto AM, Sonnenschein C, Chung KL, Fernandez MF, Olea N, Serrano FO. The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ Health Perspect 103(suppl 7):113-122 (1995).

(21.) Pink JJ, Fritsch M, Bilimoria MM, Assikis VJ, Jordan VC. Cloning and characterization of a 77-kDa oestrogen receptor isolated from a human breast cancer cell line. Br J Cancer 75:17-27 (1997).

(22.) Pons M, Gagne D, Nicolas JC, Mehtali M. A new cellular model of response to estrogens: a bioluminescent test to characterize (anti)estrogen molecules. Biotechniques 9:450-459 (1990).

(23.) Saito K, Tomigahara Y, Ohe N, Isobe N, Nakatsuka I, Kaneko H. Lack of significant estrogenic or antiestrogenic activity of pyrethroid insecticides in three in vitro assays based on classic estrogen receptor [alpha]-mediated mechanisms, Toxicol Sci 57:54-60 (2000).
Katsutoshi Ohno
Yukimasa Azuma
Katsuhiro Date
Shigeru Nakano
Toru Kobayashi
Yasuhiro Nagao
Toshihiro Yamada
Central Research Institute
Nissin Food Products Co., Ltd.
Shiga, Japan
E-mail: k-ono@mb1.nissinfoods.co.jp
COPYRIGHT 2002 National Institute of Environmental Health Sciences
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Author:Yamada, Toshihiro
Publication:Environmental Health Perspectives
Date:Jul 1, 2002
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