Lack of Significant Estrogenic or Antiestrogenic Activity of Pyrethroid Insecticides in Three in Vitro Assays Based on Classic Estrogen Receptor {alpha}-Mediated Mechanisms

Koichi Saito1, Yoshitaka Tomigahara, Norihisa Ohe, Naohiko Isobe, Iwao Nakatsuka and Hideo Kaneko

Environmental Health Science Laboratory, Sumitomo Chemical Company, Limited., 1-98, 3-Chome, Kasugade-Naka, Konohana-Ku, Osaka 554-8558, Japan

Received February 16, 2000; accepted May 2, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogenic and antiestrogenic activity of pyrethroid insecticides (d-trans-allethrin, cypermethrin, empenthrin, fenvalerate, imiprothrin, permethrin, d-phenothrin and prallethrin) was evaluated using a suite of three in vitro assays based on classic human estrogen receptor {alpha} (hER{alpha})-mediated mechanisms. A mammalian cell-based luciferase reporter gene assay was developed for examining effects on hER{alpha}-mediated gene activation. hER{alpha}-independent effects on the gene activation were examined using control cells with constitutive luciferase activation by a herpes simplex virus thymidine kinase (HSV-TK) promoter for determining appropriate dose levels of test chemicals. Moreover, the test chemical-dependent interaction between hER{alpha} and a coactivator (transcriptional intermediary factor 2: TIF2) was analyzed by a yeast two-hybrid method, competitive binding to hER{alpha} being assayed by a fluorescence polarization method. Significant (p < 0.05) positive effects of estrogenic substances (E2/estradiol, diethylstilbestrol, and p-nonylphenol) were detected in all assays. An antiestrogen, 4-hydroxytamoxifen, significantly inhibited E2-mediated transactivation and interaction between hER{alpha} and TIF2 through hER{alpha} binding (p < 0.05). However, none of the pyrethroids tested showed significant (p < 0.05) estrogenic or antiestrogenic effects (100 nM–10 µM), indicating that they do not impact on the classic hER{alpha}-mediated activation pathway in vitro.

Key Words: estrogen receptor; pyrethroid insecticides; reporter gene assay; yeast two-hybrid assay; competitive ligand-binding assay.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several natural and synthetic chemicals that are widely distributed in the environment may have the potential to mimic estrogens or inhibit estrogen action, resulting in disruption of endocrine functions (Colborn, 1995Go; Colborn et al., 1996Go), particularly through direct interaction with the estrogen receptor (ER) (Hammond et al., 1979Go; Krishnan et al., 1993Go; White et al., 1994Go). The ER is a member of the nuclear hormone receptor (NR) superfamily, which includes receptors for steroid hormones, thyroid hormones, vitamin D3, and retinoids (Mangelsdorf et al., 1995Go). In the classic ligand-mediated activation pathway, complexes of estrogen and ER dimerize and interact with estrogen response elements (EREs) in promoter regions of estrogen-regulated genes (Truss and Beato, 1993Go; Tsai and O'Malley, 1994Go). The ER is able to stimulate transactivation of target genes through interaction with factors such as TIF2/GRIP-1/NCoA2, (Hong et al., 1996Go; Torchia et al., 1997Go; Voegel et al., 1996Go), SRC-1/NCoA1 (Oñate et al., 1995Go; Torchia et al., 1997Go) and pCIP/ACTR/AIB1 (Chen et al., 1997Go; Torchia et al., 1997Go), known as coactivators, which interact with the receptor ligand-binding domain (LBD) in the presence of estrogens.

Several in vitro assays have been developed to screen chemicals for estrogenic and antiestrogenic effects (Zacharewski, 1997Go). These include competitive ligand binding (Berthois et al., 1986Go; Korach et al., 1978Go), cell proliferation (Soto et al., 1995Go; Welshons et al., 1990Go), receptor/reporter gene (Legler et al., 1999Go; Miksicek, 1994Go), and protein expression/enzyme activity assays (Markiewicz et al., 1992Go, 1993Go). Recently, ligand-dependent association of ER with coactivators has been reported to be a useful end point for detecting estrogenic chemicals by ER-dependent mechanisms, with efficacy demonstrated for yeast two-hybrid assays (Nishikawa et al., 1999Go). The mechanisms of competitive ligand-binding, receptor/reporter gene and yeast two-hybrid assays are based on the classic ligand-mediated activation pathway, and thus are clear and specific. However, response or induction for cell proliferation and endogenous protein expression/enzyme activity are additionally regulated by ER-independent mechanisms (Dickson and Lippman, 1995Go; Zacharewski et al., 1994Go), and end points are limited to certain human carcinoma cell lines or the cell variant (Villalobos et al., 1995Go).

Recently, certain pyrethroid insecticides (d-trans-allethrin, fenvalerate, sumithrin and permethrin) were studied for estrogenic or antiestrogenic activity using cell proliferation and protein expression/enzyme activity assays (Garey and Wolff, 1998Go; Go et al., 1999Go). The results showed fenvalerate to significantly increase alkaline phosphatase activity in Ishikawa Var-I human endometrial cancer cells, and pS2 protein mRNA expression and cell proliferation in MCF-7 human breast cancer cells. However, the increases were not consistently inhibited by estrogen receptor antagonist ICI 164,384. Significant increases in alkaline phosphatase activity and pS2 expression were also detected with sumithrin (d-phenothrin), whereas no significant MCF-7 cell proliferation was evident. Therefore, the estrogenic potential of pyrethroids based on ER-mediated mechanisms remain equivocal.

Pyrethroids are the most common pesticides in current use worldwide for control of agriculture and indoor pests. Numerous metabolism (Roberts and Hutson, 1999Go) and toxicological studies have shown no significant toxicological concerns with regard to humans or wildlife (Miyamoto et al., 1995Go). The objective of the present study was to evaluate possible estrogenic and antiestrogenic activity of pyrethroid insecticides using three in vitro assays (cell-based luciferase reporter gene, yeast two-hybrid, and competitive ligand-binding assays) with classic ligand-mediated activation mechanisms and different end points.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Test chemicals.
17ß-Estradiol (E2) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), diethylstilbestrol (DES) from Nacalai Tesque (Kyoto, Japan), and p-nonylphenol from Kanto Chemical Co. (Tokyo, Japan). d-trans-Allethrin, cypermethrin, empenthrin, fenvalerate, imiprothrin, d-phenothrin, permethrin, prallethrin (Fig. 1Go) and 4-hydroxytamoxifen (HTM) were synthesized in Sumitomo Chemical Company (Osaka, Japan). The purity of all test chemicals used was more than 93%.



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FIG. 1. Chemical structures of the pyrethroid pesticides tested.

 
Construction of plasmids.
For the luciferase reporter gene assay, expression vector pRc/RSV-hER{alpha} was generated by insertion of an RT-PCR amplified full-length cDNA of hER{alpha}, with an efficient Kozak's translation initiator sequence (Kozak, 1984Go) from a commercial human ovary mRNA (Clontech, Palo Alto, CA), into the blunting site of a pRc/RSV vector (Invitrogen, San Diego, CA). A reporter plasmid for hER{alpha}, pGL3-TATA-EREx5, was generated by insertion of five copies of EREs from the Xenopus vitellogenin gene into a pGL3 basic vector (Promega, Madison, WI) with a mouse metallothionein minimum TATA promoter. A luciferase control plasmid, pGL3-TK was generated by insertion of a herpes simplex virus thymidine kinase (HSV-TK) promoter into a pGL3 basic vector. For the yeast two-hybrid assay, the pGBT9 cloning vector for expressing the GAL4 DNA-binding domain (DBD) fusion protein and pGAD424 for expressing the GAL4 transcriptional activation domain were purchased from Clontech. The LBD of hER{alpha} (Greene et al., 1986Go) (amino acid 249–595) was amplified from human ovary mRNA (Clontech) by RT-PCR. After subcloning into the EcoRI and SalI sites of pGBT9, cloned hER{alpha} and junctures were sequenced, and the LBD of the receptor was determined to be correctly in-frame with GAL4 DBD. The receptor interaction domain (RID) of TIF2 (transcriptional intermediary factor 2) (Voegel et al., 1996Go) (amino acid 624-1287) was amplified by RT-PCR from human brain mRNA (Clontech) using primers with EcoRI or BglII sites, and subcloned into pGAD424 digested with EcoRI-BamHI for production of a fusion protein with the GAL4 activation domain (GAL4AD). The sequence and reading frame of TIF2RID were confirmed by DNA sequencing.

Yeast strain.
The yeast strain Y190 (Clontech) was used in this study. Yeast cells were transformed with pGBT9-hER{alpha}LBD and pGAD424-TIF2RID using a lithium acetate method and selected with SD medium (-Trp, -Leu).

Luciferase reporter gene assay.
HeLa cells were routinely maintained in phenol red–free Eagle's modified essential medium (EMEM) containing 10% charcoal-treated fetal calf serum in 10-cm plates. Six hours before transfection, 2 x 106 cells were seeded per 10-cm plate in phenol red–free EMEM containing 10% charcoal-treated fetal calf serum. Transient transfections were performed by lipofection with Lipofectamine (Life Technologies Inc., Rockville, MD), 3.75 µg/plate of pRc/RSV-hER expression vector, and 3.25 µg /plate of pGL3-TATA-EREx5. For control assays, an amount of 7 µg/plate of pGL3-TK was used for transfections. Cells were incubated with liposome-DNA complexes at 37°C for 16 h, and then further incubated for 3 h after medium change. The cells were harvested, homogeneously mixed, and seeded into 96-well plates (2 x 104/well) containing the medium with DMSO solutions of chemicals (0.1% final DMSO concentration) (n = 6). Incubation was at 37°C for 40 h. For determining antiestrogenic activities, 100 pM of E2 was incubated with test chemicals. After incubation, cells were solubilized and luciferase activity was determined with a luminometer (Berthold MicroLumat LB96P).

Yeast two-hybrid assay.
Yeast two-hybrid assays were performed according to the method described previously (Nishikawa et al., 1999Go), with minor modification. Yeast transformants were grown overnight at 30°C with vigorous shaking in the selection medium (-Trp, -Leu) and diluted (10–15x dilution) with the medium. DMSO solutions of the test chemicals (2.5 µl) were added to 96 deep-well plates (n = 4), then mixed with 250 µl of fresh medium containing 10 µl of the diluted yeast culture. For antagonist assays, 100 pM of E2 was incubated with test chemicals. After incubation for 4 h at 30°C, ß-galactosidase induced in yeast was measured by chemiluminescent detection using a Gal-Screen (Tropix Inc., Bedford, MA) according to the manufacturer's protocols.

Competitive ligand-binding assay.
Competitive binding to hER{alpha} was assayed by a fluorescence polarization method (Bolger et al., 1998Go) using an FP Screen-for-Competitor Kit ER{alpha}, high sensitivity (PanVera, WI). Test chemical–dependent displacement of a labeling ligand, fluoromone ES1, from ER{alpha} was measured by changes in fluorescence anisotropy using a Beacon 2000 Fluorescence Polarization Instrument (Takara).

Data analysis.
Differences were tested for statistical significance using the t-test computed by Excel (Microsoft). Differences were considered statistically significant when p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estradiol Dose Response with the Three in vitro Assays
The expected dose response with the three assays was confirmed with estradiol (E2). In the luciferase reporter gene assay, dose-dependent increases in luciferase activity were observed in cells transiently transfected with hER{alpha}. In this assay, a positive response was found at concentrations of more than 0.01 nM, with maximum induction of 6.5-fold (E2:10 nM) relative to solvent controls (Fig. 2AGo). One of the difficulties of transient reporter gene assays is alteration in transfection efficiency. Therefore, we transfected plasmids into cells in large 10 cm-plates, and the transfected cells were divided as described in the Materials and Methods section. E2-dependent interaction between hER{alpha} and TIF2 was dose dependent in the yeast two-hybrid assay. In this assay, a positive response was observed at more than 0.1 nM and maximum induction was 85-fold (10 µM) relative to solvent controls (Fig. 2BGo). In the competitive ligand-binding assay, dose-dependent displacement of the fluorescence standard was found at more than 0.01 nM (Fig. 2CGo). The results showed the in vitro assays to be capable of detecting estrogenic compounds through hER{alpha}-mediated mechanisms with high sensitivity.



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FIG. 2. Results for estradiol (E2) in the three in vitro assays with ER{alpha}-mediated mechanisms. (A) Luciferase reporter gene assay. (B) Yeast two-hybrid assay. (C) Competitive ligand-binding assay. Data presented are means ± SD (n = 3–6 test samples).

 
Analysis of Estrogenic or Antiestrogenic Activity of Positive Controls by Transient Luciferase Reporter Gene Assays
A mammalian cell–based luciferase reporter gene assay was developed to examine the effects of test chemicals on transactivation of estrogen target genes. The ER{alpha}-independent effects on the transactivation were analyzed using control cells with constitutive luciferase activation by an HSV-TK promoter. As shown in Figure 3Go, antiestrogen HTM (4-hydroxytamoxifen) inhibited E2-mediated luciferase induction significantly (p < 0.05) in a dose-dependent manner (1 nM-10 µM) without influence on control cells, showing this approach for evaluating antihormonal chemicals to be sensitive and reliable. In cells transfected with ER{alpha}, significant (p < 0.05) ligand-dependent luciferase activation was found with xenoestrogens DES and p-nonylphenol (Fig. 3Go). The lowest concentration for a response and the maximum fold induction with DES were 0.01 nM and 8.4-fold relative to solvent controls, respectively, without any effects on control cells at concentrations of 0.01 nM–10 µM. However, a marked decrease in luciferase activity was detected with exposure of test and control cells to high concentrations (50 µM) of p-nonylphenol, indicating toxic effects on luciferase transactivation. This was confirmed by decrease in cell growth (data not shown). Therefore, data from p-nonylphenol-treatment at 10 µM or less were used for assessing the estrogenic activity in this experiment. The results indicate that the present control assay is a sensitive method for detecting toxic effects on transactivation of the luciferase gene.



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FIG. 3. Analysis of estrogenic or antiestrogenic activity of estrogens (DES and p-nonylphenol) and an antiestrogen (HTM: 4-hydroxytamoxifen) by luciferase reporter gene assays. Test chemical–dependent induction or inhibition (black bars) and control activity (white bars) are presented relative to solvent control levels. For analyzing the antiestrogenic activity of HTM, E2 (100 pM) was incubated with the chemical. Data presented are means ± SD (n = 6 test samples). Asterisks indicate significant difference from the solvent control (p < 0.05) in a two-tailed Student's t-test for induction or inhibition.

 
Analysis of Estrogenic or Antiestrogenic Activity of Pyrethroid Insecticides by Luciferase Reporter Gene Assays
In control assays, significant alterations in luciferase activity were detected with high doses of d-trans-allethrin and prallethrin (data not shown). Therefore, maximum doses of these agents were set at 1 µM for the present luciferase reporter gene assay. Significantly increased turbidity was detected at 595 nm with d-trans-allethrin, cypermethrin, empenthrin, fenvalerate, imiprothrin, permethrin, and d-phenothrin in phenol red–free EMEM at 30 µM (data not shown). Therefore, maximum doses of other pyrethroid insecticides were set at 10 µM. Under these conditions, no pyrethroids showed significant (p < 0.05) estrogenic or antiestrogenic activity in the luciferase reporter gene assay (Fig. 4Go), suggesting a lack of influence on transactivation of hER{alpha}-regulated genes by classic hER{alpha}-mediated mechanisms.



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FIG. 4. Analysis of estrogenic (A) and antiestrogenic (B) activity of pyrethroid insecticides by the luciferase reporter gene assay. Dose levels for pyrethroids (1 or 10 µM) were determined by control assays or the solubility of the chemicals. For analyzing the antiestrogenic activity, E2 (100 pM) was incubated with the test chemicals. Data presented are means ± SD (n = 6 test samples). Asterisks indicate significant difference from the solvent control (p < 0.05) in a two-tailed Student's t-test for induction or inhibition.

 
Yeast Two-Hybrid Assays
A coactivator, TIF2, can associate with estrogen-ER{alpha} complexes (Nishikawa et al., 1999Go). Therefore, ligand-dependent interaction between hER{alpha} and TIF2 was analyzed by a yeast two-hybrid method with induction of ß-galactosidase activity as an indicator. As shown in Figure 5Go, significant interaction was detected for DES (10 µM) and p-nonylphenol (10 µM) (p < 0.05) (Fig. 5AGo), whereas HTM (1 µM) inhibited the E2-mediated interaction (p < 0.05) (Fig. 5BGo). For this assay, maximum dose levels of pyrethroids (10 µM) were determined on the basis of the chemical solubility. Significant changes (p < 0.05) in ß-galactosidase activity induced by ligand-dependent interaction between hER{alpha} and TIF2 were not observed for any pyrethroid tested (Fig. 5AGo). Moreover, no pyrethroids showed significant (p < 0.05) antiestrogenic activity in the assay (Fig. 5BGo).



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FIG. 5. Analysis of estrogenic (A) and antiestrogenic (B) activity of pyrethroid insecticides by the yeast two-hybrid assay. Dose levels for pyrethroids (10 µM) were determined by the solubility of the chemicals. For analyzing the antiestrogenic activity, E2 (100 pM) was incubated with the test chemicals. Data presented are means ± SD (n = 4 test samples). Asterisks indicate significant difference from the solvent control (p < 0.05) in a two-tailed Student's t-test for induction or inhibition.

 
Competitive Ligand-Binding Assays
In the competitive ligand-binding assay (Fig. 6Go), dose-dependent displacement of fluoromone ES1 from hER{alpha} was detected with DES, p-nonylphenol, and HTM (10 nM–10 µM), indicating that these chemicals can bind to the hER{alpha} and exert estrogenic or antiestrogenic effects. However, no apparent effects were observed with the pyrethroids tested, suggesting that they have no potential for receptor binding under the experimental conditions employed.



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FIG. 6. Analysis of hER{alpha}-binding capacity of pyrethroid insecticides by competitive ligand-binding assays with the fluorescence polarization method. Data presented are means ± SD (n = 3 test samples).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we first developed a suite of three assays with the luciferase reporter gene, yeast two-hybrid, and competitive ligand-binding approaches, which were dependent on established ligand-mediated activation mechanisms of hER{alpha}. Neither significant estrogenic nor antiestrogenic activity was observed for the pyrethroid insecticides tested, whereas the positive controls showed (anti)estrogenic activities.

In previous studies, however, some pyrethroid insecticides were suggested to have estrogenic potency (Garey and Wolff, 1998Go; Go et al., 1999Go). Garey and Wolff reported that fenvalerate and sumithrin (d-phenothrin) could increase alkaline phosphatase activity significantly (p < 0.05) in Ishikawa Var-I endometrial cancer cells at 30 µM; this activity is inhibited by cotreatment with 1 µM of a pure antiestrogen, ICI 164,384. However, no significant antiestrogenic activities were found with d-trans-allethrin and permethrin, consistent with the present study. Go and co-workers (1999) estimated the estrogenic and antiestrogenic activity of the above four pyrethroids using mRNA expression for pS2 protein, fenvalerate, and d-phenothrin, increasing pS2 expression significantly (p < 0.05) at 30 µM. The increase with d-phenothrin was inhibited by cotreatment with ICI 164,384. Unexpectedly, no inhibition was evident with fenvalerate. No significant alteration in pS2 expression was observed with d-trans-allethrin or permethrin. Assays of endogenous protein expression/enzyme activity such as alkaline phosphatase and pS2 induction have low specificity, and induction mechanisms are unclear, as such endogenous genes are additionally regulated by other cellular mechanisms (Zacharewski et al., 1994Go). Therefore, discrepancy of response to ICI 164,384 suggests ER-independent effects that are linked to specific events in certain human carcinoma cells. In the MCF-7 human breast cancer cell proliferation assay, fenvalerate (10–100 µM), d-trans-allethrin (10 µM), and permethrin (100 µM) but not d-phenothrin (1 nM-100 µM) were reported to cause a significant increase (p < 0.05) (Go et al., 1999Go). Therefore, the previous reports suggest that estrogenic effects of pyrethroids depend on the assays or cells used. In our preliminary experiment, significantly increased turbidity at 595 nm was detected with d-trans-allethrin, cypermethrin, empenthrin, fenvalerate, imiprothrin, permethrin, and d-phenothrin in phenol red–free EMEM at 30 µM (data not shown), indicative of insolubilization because of high lipophilicity (Log Kow = 3.0–7.4) (Roberts and Hutson, 1999Go). The results indicate that data obtained with high concentrations of pyrethroids should be interpreted carefully. However, the rationale for dose levels has not attracted sufficient attention in previous studies (Garey and Wolff, 1998Go; Go et al., 1999Go). Needless to say, determination of appropriate dose levels for test chemicals is indispensable for accurate evaluation and to keep the validity of assays not only in vivo but also in vitro. In the present study, we used data from control assays in the case of the luciferase reporter gene assay as well as solubility of test chemicals as the rationale for dose levels. The control assay demonstrated toxicity for p-nonylphenol. Inhibition of E2-mediated pS2 expression was reported with d-trans-allethrin at 5 µM (Go et al., 1999Go). In the present study, an influence on control transactivation by d-trans-allethrin and prallethrin was detected at 10 µM (data not shown). Although we did not investigate the effects of d-trans-allethrin on control transactivation at 5 µM in the present study, the results suggested inhibition of pS2 transactivation by ER{alpha}-independent mechanisms.

When chemicals exert estrogenic activity, at least three pathways can be considered with ER as the key point of convergence. One is the classic ER{alpha}-mediated activation pathway. Others are ERß-mediated activation and ligand-independent signaling pathways (Saunders, 1998Go). However, the predominant estrogen receptor isoform is reported to be ER{alpha} in almost all human breast cancer cells such as the MCF-7, BT474, and MDA-MB-468 cell lines (Vladusic et al., 2000Go). The effects of estrogenic compounds on cell proliferation and pS2 mRNA induction in MCF-7 are considered to be related to ER{alpha}. In the absence of exogenous ligands, it has been shown that ER can be activated by dopamine receptor agonists of the D1 subtype (Smith et al., 1993Go) and agents that elevate intracellular cAMP levels (Aronica and Katzenellenbogen, 1993Go). Although the action of pyrethroid insecticides is reported to be related to modification of ion channels such as the Na+ channel, GABA and glutamate receptor-channel complex, and voltage-activated Ca2+ channel (Narahashi, 1992Go), the relationship between ligand-independent signaling pathways and pyrethroid insecticide actions have not yet been clarified. However, many endocrine-disrupting chemicals (EDCs) are considered to modify natural endocrine function. In addition EDCs can disrupt physiological processes involved in differentiation, homeostasis, and reproductive functions, particularly through direct association with nuclear hormone receptors (NRs) such as steroid and thyroid hormone receptors (Colborn et al., 1996Go). Therefore, to our knowledge, evaluation of the classic ER-mediated activation pathways are the first priority in understanding biological actions of environmental estrogens and antiestrogens.

The present three in vitro ER{alpha}-mediated assays are dependent on established ligand-mediated activation mechanisms. Moreover, one of the benefits of our suite of assays is involvement of the appropriate dose setting. We conducted each assay more than twice; reproducibility in each assay was confirmed. It is clear that our assays do not completely reflect the (anti)estrogenic effects of chemicals in vivo because it cannot evaluate pharmacokinetic parameters such as absorption, distribution, and biotransformation. We believe that the present assay allows reliable evaluation of (anti)estrogenic activities of chemicals in vitro.

The lack of significant effects at 100 nM–10 µM in the present study, therefore, strongly suggests that the pyrethroid insecticides tested are not estrogenic or antiestrogenic by classic ER-mediated pathways in vitro.


    ACKNOWLEDGMENTS
 
We thank Drs. Tsutomu Nishihara and Jun-ichi Nishikawa, both at Osaka University, for advice on yeast two-hybrid and competitive ligand-binding assays, and Norihisa Yamashita and Akane Hayashida for expert technical assistance.


    NOTES
 
1 To whom correspondence should be addressed. Fax: 81-6-6466-5442. E-mail: saitok5{at}sc.sumitomo-chem.co.jp. Back


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