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
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ABSTRACT |
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Key Words: estrogen receptor; pyrethroid insecticides; reporter gene assay; yeast two-hybrid assay; competitive ligand-binding assay.
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INTRODUCTION |
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Several in vitro assays have been developed to screen chemicals for estrogenic and antiestrogenic effects (Zacharewski, 1997). These include competitive ligand binding (Berthois et al., 1986
; Korach et al., 1978
), cell proliferation (Soto et al., 1995
; Welshons et al., 1990
), receptor/reporter gene (Legler et al., 1999
; Miksicek, 1994
), and protein expression/enzyme activity assays (Markiewicz et al., 1992
, 1993
). 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., 1999
). 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, 1995
; Zacharewski et al., 1994
), and end points are limited to certain human carcinoma cell lines or the cell variant (Villalobos et al., 1995
).
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, 1998; Go et al., 1999
). 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, 1999) and toxicological studies have shown no significant toxicological concerns with regard to humans or wildlife (Miyamoto et al., 1995
). 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.
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MATERIALS AND METHODS |
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Yeast strain.
The yeast strain Y190 (Clontech) was used in this study. Yeast cells were transformed with pGBT9-hERLBD 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 redfree 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 redfree 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., 1999), with minor modification. Yeast transformants were grown overnight at 30°C with vigorous shaking in the selection medium (-Trp, -Leu) and diluted (1015x 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 was assayed by a fluorescence polarization method (Bolger et al., 1998
) using an FP Screen-for-Competitor Kit ER
, high sensitivity (PanVera, WI). Test chemicaldependent displacement of a labeling ligand, fluoromone ES1, from ER
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.
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RESULTS |
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DISCUSSION |
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In previous studies, however, some pyrethroid insecticides were suggested to have estrogenic potency (Garey and Wolff, 1998; Go et al., 1999
). 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., 1994
). 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 (10100 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., 1999
). 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 redfree EMEM at 30 µM (data not shown), indicative of insolubilization because of high lipophilicity (Log Kow = 3.07.4) (Roberts and Hutson, 1999
). 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, 1998
; Go et al., 1999
). 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., 1999
). 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
-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-mediated activation pathway. Others are ERß-mediated activation and ligand-independent signaling pathways (Saunders, 1998
). However, the predominant estrogen receptor isoform is reported to be ER
in almost all human breast cancer cells such as the MCF-7, BT474, and MDA-MB-468 cell lines (Vladusic et al., 2000
). The effects of estrogenic compounds on cell proliferation and pS2 mRNA induction in MCF-7 are considered to be related to ER
. 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., 1993
) and agents that elevate intracellular cAMP levels (Aronica and Katzenellenbogen, 1993
). 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, 1992
), 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., 1996
). 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-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 nM10 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.
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ACKNOWLEDGMENTS |
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NOTES |
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