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Estrogenic and Antiprogestagenic Activities of Pyrethroid Insecticides
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
251, 855– 859 (1998)
RC989569
Estrogenic and Antiprogestagenic Activities of Pyrethroid Insecticides Joan Garey and Mary S. Wolff Department of Community and Preventive Medicine, Mount Sinai School of Medicine, New York, New York 10029
Received September 18, 1998
Many pesticides possess hormonal activity and have thus been classified as endocrine disruptors. Pyrethroids are commonly used insecticides worldwide, but little has been done to characterize their hormone agonist/antagonist potential. We tested four frequently encountered pyrethroids, fenvalerate, sumithrin, d-trans allethrin, and permethrin, for estrogen and progesterone agonist/antagonist activities using the Ishikawa Var-I human endometrial cancer cell line and the T47D human breast cancer cell line. Both cell lines produce alkaline phosphatase as an indicator of hormonal activity. Fenvalerate and sumithrin demonstrated significant estrogenicity; at concentrations of 10 mM, these compounds achieved maximal activities comparable to that of 10 nM 17a-ethynylestradiol in Ishikawa Var-I cells. None of the four compounds showed statistically significant estrogen antagonist activity or acted as progestins. However, fenvalerate and d-trans allethrin significantly antagonized the action of progesterone in T47D cells. Through these hormonal pathways, exposure to certain pyrethroids may contribute to reproductive dysfunction, developmental impairment, and cancer. © 1998 Academic Press
Estrogenic and progestagenic activities are among the hormonal responses reported for a number of pesticides, plasticizers and polychlorinated biphenyls (PCBs) (1-6). Concern exists that these synthetic environmental “endocrine disruptors” may enhance the risk of breast cancer in women (7), lower sperm counts and heighten risks of cryptorchidism, hypospadias and testicular cancer in males (8), as well as impair immunity and alter neurological development (9). Synthetic pyrethroids are among the most common pesticides in current use worldwide (10), yet little has been done to assess their potential hormonal activities. The widespread use of pyrethroids in agriculture and in the home has been encouraged by laboratory evidence suggesting that they are relatively safe to humans and wildlife (10). However, epidemiological ac-
counts, clinical reports and other laboratory studies indicate that pyrethroids can elicit a range of immunotoxic and neurotoxic effects in humans and other mammals; pyrethroid exposures have been associated with acute reproductive effects and may produce chronic and developmental impairments, as well (11-15). The mechanisms have not been firmly established in all cases, but these effects are consistent with estrogen receptor (ER) and progesterone receptor (PR) mediated actions. Here we report the first demonstration of estrogenic and antiprogestagenic activities among synthetic pyrethroid pesticides using two in vitro systems based on human cells. We tested four commonly encountered synthetic pyrethroid compounds to determine whether they act as estrogen analogs or antagonists using the Ishikawa Var-1 human endometrial andenocarcinoma cell line. This cell line, rich in estrogen receptors, produces a specific, dose-dependent increase in alkaline phosphatase (AlkP) in response to estrogens (16). In addition, we investigated the potential progesterone agonist/antagonist effects of the same four pyrethroids in T47D human breast cancer cells. In these cells, an increase in AlkP induction occurs only in response to progestins (17). Both systems have been used previously to characterize hormonal activities among a range of steroidal compounds and plant flavonoids (1820). We detected significant estrogenic activity in two widely used synthetic pyrethroids, fenvalerate and sumithrin, in the Ishikawa Var-1 AlkP assay, and we observed significant antiprogestagenic activity in the pyrethroids fenvalerate and d-trans allethrin in the T47D AlkP assay. MATERIALS AND METHODS Test compounds and standards. Four synthetic pyrethroids (dtrans allethrin [also known as bioallethrin], fenvalerate, technical permethrin and sumithrin [also known as fenothrin and d-phenothrin] (Figure 1) were a gift from McLaughlin Gormley King, Co., Minneapolis, MN. The pyrethroids obtained were of variable stated purity, with d-trans allethrin 5 96.5%; fenvalerate 5 95.9%; permethrin, 95.7%; sumithrin 5 95.4%. These values were taken into account when preparing the desired test concentrations. 17b-
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0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Vol. 251, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1.
Structures of four synthetic pyrethroid insecticides.
Estradiol (E2), 17a-ethynylestradiol (EE) and progesterone (P), obtained from Sigma Chemical Co., St. Louis, MO, were used as positive controls. The antiestrogen ICI 164,384, a gift from Dr. A. Wakeling of Imperial Chemical Industries (Macclesfield, UK), was used to verify estrogenicity of the test compounds. All compounds were prepared in ethanol to achieve the desired concentrations. Cell lines. The methods described here for both cell lines are based on those of Markiewicz et al. (21). The Ishikawa Variant-I was originally described by Holinka et al. (16). Cells were maintained in an estrogen-free basal medium (BM) comprised of a 1:1 mixture of Dulbecco’s Modified Eagle’s medium and Ham’s Nutrient Mix F12 without phenol red (Sigma Chemical Co., St. Louis, MO); the medium was supplemented with an antibiotic/antimycotic mixture, HEPES, sodium bicarbonate (all supplements from Sigma) and 5% calf serum (Atlanta Biologicals, Atlanta, GA) stripped of endogenous estrogens with dextran-coated charcoal (Sigma). The de novo induction of AlkP in T47D cells by progestins was originally described by DiLorenzo et al. (17). T47D cells were maintained in Dulbecco’s Modified Eagle’s Medium containing phenol red (DMEM, purchased from Sigma) and supplemented with penicillin/ streptomycin (Sigma), sodium bicarbonate and 10% fetal bovine serum (FBS; purchased from Atlanta Biologicals). The cell lines were cultured in 100 mm culture dishes and maintained at 37°C in a humidified atmosphere of 5% CO2-95% air. Cells were used in the assay when they reached confluence. Ishikawa and T47D AlkP assays. Cells were harvested with 0.25% trypsin (Atlanta Biologicals) and seeded for experiments in 96-well flat-bottom Microtest-III tissue culture plates (Becton Dickinson, Franklin Lakes, NJ). The Ishikawa cells were plated at a density of between 15,000-20,000 cells/well in 100 ml aliquots; the T47D cells were plated at a density of 50,000 cells/well in 100 ml aliquots. Test compounds and standards were dissolved in ethanol and diluted in BM with 5% stripped calves serum to appropriate concentrations (no greater than 0.2% final solvent concentration). Each dilution (50 ml) was added to eight wells per plate after a 24 hour incubation period and returned to the incubator. After a 48 hour exposure period, the plates were washed in 2 L PBS (0.15M NaCl, 10 mM sodium phosphate, pH 7.4) and drained. (For the T47D assays only, plates were then placed in a 272°C freezer for 15 min and then thawed at room temperature). An icecold solution of 5 mM p-nitrophenyl phosphate, 0.24 mM MgCl and 1 M diethanolamine (all reagents purchased from Sigma), pH 9.8, was added to each well. Formation of p-nitrophenol at room temperature
was detected at 405 nm in a Titertek Multiscan Plus MKII (type 314) plate reader. Plates were considered fully developed when the wells containing E2 at 1028 M read approximately 1.0-1.4 optical density (OD) units. At this time, the control level was between 0.1-0.3 OD units. The development time for the Ishikawa bioassay was 5-30 minutes and 30-60 min for the T47D bioassay. The mean of eight wells on one plate constituted a single experiment, and all results represent at least three independent experiments. Data analysis. Differences were tested for statistical significance using the t-test (two-tailed, equal variance) computed by Excel (Microsoft) or SAS PC (Cary, NC) supported by the City University of New York Academic Computing Facility. Differences were considered statistically significant when p , 0.05. Data are presented as the mean and, where shown, the standard deviation, of at least three experiments. Dose-response curves were prepared through the use of p-Fit software (Biosoft, Ferguson, MO).
RESULTS AND DISCUSSION Fenvalerate and sumithrin demonstrated estrogenic activity at 30 mM as evidenced by a significant increase of AlkP activity in Ishikawa Var-I endometrial cancer cells. Permethrin and d-trans allethrin were not significantly different from the control (Figure 2). To confirm that fenvalerate and sumithrin acted through the ER, the antiestrogen ICI 164,384 was added at 1 mM in a separate experiment, causing a total reversal of the AlkP response to basal levels (data not shown). The dose-response curves for fenvalerate and sumithrin in the same assay system exhibited maximal activities comparable to that of EE, when using approximately 104-105 fold higher concentrations (Figure 3). The EC50s of fenvalerate and sumithrin were approximately 1 and 4 mM, respectively. In addition, we tested the two compounds which did not demonstrate estrogenic activity, d-trans allethrin and permethrin, in combination with 10 pM and 100 pM E2 (Figure 3). While permethrin did not reduce E2 activity, d-trans allethrin showed a trend toward re-
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FIG. 2. Estrogen-induced AlkP production (OD units) of four pyrethroid insecticides in Ishikawa Var-I cells. d-trans Allethrin (A), fenvalerate (F), permethrin (PM), and sumithrin (S) were tested at 30 mM. 17a-Ethynylestradiol (EE) at 10 nM was used as a positive control; C represents the vehicle control. For each compound, n 5 3. Both fenvalerate and sumithrin demonstrated levels of AlkP activity significantly higher than the vehicle control, as indicated by * for fenvalerate, p , 0.01; for sumithrin, ** indicates p 5 0.001.
ducing the AlkP activity of E2, although the differences were not statistically significant. The same four compounds were also tested for progestagenic/antiprogestagenic activities in T47D cells. At 30 mM, none of the compounds induced an AlkP response signficantly different from the control (Figure 4). However, when combined with 5 nM P, both d-trans allethrin and fenvalerate demonstrated significant antagonist effects, with d-trans allethrin being the most potent antagonist, reducing the AlkP activity of 5 nM P by an average of 68% (Figure 5). Endocrine disruption research has primarily focused on identifying exogenous sources of estrogen as well as estrogen antagonists (9). However, the action of progestins may enhance or inhibit the action of estrogen, depending upon their concentration relative to estrogen (22). Moreover, recent research has demonstrated a role for neuronally-produced progesterone in neurological development and repair of damaged nerve cells (23,24,25). Screening efforts have therefore been expanded to include the analysis of environmental compounds for their potential to produce PR-mediated effects (2,3,4,5,6). Many environmental substances demonstrating ERand/or PR-mediated activites have been banned from use in the United States (e.g., the organochlorine insecticides aldrin, dieldrin, DDT, heptachlor and chlordane, as well as PCBs) (4,26), although these compounds persist in the environment and are used in developing countries (27). Other organochlorine insecticides still in use in the United States and elsewhere exhibit estrogenic or antiprogestagenic activity in some assays, e.g., endosulfan and toxaphene (2,28), as does the current use acetanilide herbicide, Alachlor (3,29).
FIG. 3. Estrogenic response (OD units) of fenvalerate, sumithrin and 17a-ethynylestradiol (EE). F 5 EE, Œ 5 fenvalerate ■ 5 sumithrin. Average half-maximal doses (EC50s) were: fenvalerate 5 1100 6 75 nM; sumithrin 5 4500 6 1100 nM; EE 5 40 6 10 pM. Each concentration data point on the curve represents the average of three or four separate experiments.
Antiestrogenic activity has been reported for certain current use chloro-S-triazine and carbamate pesticides (3), while other carbamates are weakly estrogenic and progestagenic, as well as antiprogestagenic (3). Pyrethroids are synthetic derivatives of pyrethrins, the active ingredients of the natural insecticidal chrysanthemum extract known as pyrethrum (10). The pyrethrins are highly lipophilic but unstable in light and heat and, therefore, have been limited to indoor use (10). The pyrethroids are also highly lipophilic, possess enhanced environmental stability and have been widely marketed as alternatives to organochlorine and organophosphate pesticides for both agricultural and
FIG. 4. Inhibition of estrogen-induced AlkP production by two pyrethroid insecticides in Ishikawa Var-I cells. d-trans Allethrin (A) and permethrin (PM) were tested at 30 mM alone and in combination with 10 pm E2 (E10) and 100 pM E2 (E100). C represents the vehicle control. For each condition tested, n 5 3. The AlkP activity is expressed as optical density (OD) units. E2 activity was not significantly suppressed by PM or A, although A 1 E10 approached statistical significance (t 5 2.6, p 5 0.06).
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FIG. 5. The inhibition of progesterone-induced AlkP production by four pyrethroid insecticides in T47D cells. Cells were incubated for 48 hr with vehicle (C) or 5 nM progesterone (P), in the presence or absence of d-trans allethrin (A), fenvalerate (F), permethrin (PM), and sumithrin (S), all at 30 mM. The resulting AlkP activity is expressed as optical density (OD) units. The * indicates activity significantly lower than 5 nM P, p , 0.01.
home use (10). They are effective against a variety of insects, including fleas, roaches, ticks, mosquitoes and lice (10,30,31). They are used to protect livestock against ectoparasites and are also used to protect a variety of crops (32,33) Human exposures may occur occupationally, during application, or through pyrethroid residues such as those detected in cow’s milk, bread, fruits, vegetables and carpeting (11,32-34). Previous reports indicate that pyrethrum is not estrogenic (1) and that pyrethrins, as well as several synthetic pyrethroids, including fenvalerate, permethrin and fenothrin (sumithrin), are androgen antagonists (35-37). An androgen receptor (AR) mechanism was proposed to explain an “epidemic” of gynecomastia seen among men in one community in Haiti where extensive pyrethroid spraying had been conducted (36). However, gynecomastia is ordinarily associated with estrogen exposure (38). Our data suggest that pyrethroids may have produced the symptoms of gynecomastia among Haitian men in 1981 via estrogenicity, as well. Both p,p9-DDE, the main mammalian metabolite of DDT, and Vinclozolin, have been found to be antagonists of AR binding (39-40), while p,p9-DDE has also demonstrated an ability to bind the ER and produce ER-mediated transcription of the lacZ reporter gene in an in vitro yeast assay system (41). Therefore, several classes of pesticides are now known to be able to target multiple hormone receptors. Our results are the first to suggest that some pyrethroids may act as estrogen analogs and progestin antagonists. These findings add to the growing evidence over the past decade that pesticides of several chemical classes have ER and PR agonist and antagonist effects (1-6). Moreover, estrogenicity by two pyrethroids but antiprogestagenicity by another illustrates the complexity of endocrine effects and the need for further research on structure-activity relationships.
Exposures to a variety of contaminants, including pharmaceuticals, plant-derived hormone agonists and environmental toxicants, may modulate endogenous endocrine signals in ways that can influence human health. The discovery of estrogenic and antiprogestagenic activities in some synthetic pyrethroids, a large and widely used family of pesticides, suggests the urgent need for further investigation of these current use chemicals and thus provides added evidence that pyrethroids belong on the list of “endocrine disruptors” (9). ACKNOWLEDGMENT This research was supported by NIEHS Superfund Basic Research Grant P42 ES07384.
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10. Naumann, K. (1990) Synthetic Pyrethroid Insecticides: Structures and Properties. Chemistry of Plant Protection, Vol. 4, Springer-Verlag, NY. 11. Landrigan, P. J., Claudio, L., and McConnell, R. (1998) in Environmental Toxicants (Lippman, M., Ed.), VanRhinehold, NY. 12. Miyamoto, J., Kaneko, H., Tsuiji, R., and Okuno, Y. (1995) Tox. Lett. 82/83, 933–940. 13. Tanenbaum, D. M., Wang, Y., Williams, S. P., and Sigler, P. B. (1998) Proc. Natl. Acad. Sci. USA 95, 5998 – 6003. 14. He, F., Wang, S., Liu, L., Chen, S., Zhang, Z., and Sun, J. (1996) Arch Toxicol. 15, 389 –390. 15. He, F. (1994) Toxicology 91, 43– 49. 16. Holinka, C. F., Hata, H., Kuramoto, H., and Gurpide, E. (1986) Cancer Res. 46, 2771–2774. 17. DiLorenzo, D., Albertini, A., and Zava, D. (1991) Cancer Res. 51, 4470 – 4475. 18. Littlefield, B. A., Gurpide, E., Markiewicz, L., McKinley, B., and Hochberg, R. B. (1990) Endocrinology 127, 2757–2762. 19. Markiewicz, L., Hochberg, R. B., and Gurpide, E. (1992) J. Steroid Biochem. Mol. Biol. 41, 53–58. 20. Markiewicz, L., Garey, J., Adlercreutz, H., and Gurpide, E. (1993) J. Steroid Biochem. Mol. Biol. 45, 399 – 405. 21. Markiewicz, L. and Gurpide, E. (1994) in The Human Endometrium. Annals of the New York Academy of Sciences, Vol. 734 (Buletti, C., Gurpide, E., and Flamigni, C., Eds.), pp. 285–297, New York Academy of Sciences, NY. 22. Kraus, W. L., Weis, K. E., and Katzenellenbogen, B. S. (1995) Mol. Cell Biol. 15, 1847–1857. 23. Koenig, H.L., Schumacher, M., Ferzaz, B., Do Thi, A. N., Ressouches, A., Guennoun, R., Jung-Testas, I., Robel, P., Akwa, Y., and Baulieu, E.-E. (1995) Science 268, 1500 –1503. 24. Schumacher, M. and Baulieu, E.-E. (1995) in Non-Reproductive Actions of Sex Steroids (Bock, G. R., and Goode, J. A., Eds.), pp. 90 –111, Wiley, NY. 25. Guennoun, R., Schumacher, M., Robert, F., Dilespierre, B., Gouezou, M., Eychenne, B., Akwa, Y., Robel, P., and Baulieu, E. E. (1997) Eur. J. Neurosci. 9, 2236 –2247.
26. Dich, J., Hoar Zahm, S., Hanberg, A., and Adami, H.-O. (1997) Cancer Causes Control 8, 420 – 443. 27. World Health Organization (1992) Our Planet, Our Health. WHO, Geneva. 28. Soto, A. M., Lin, T.-M., Justicia, H., Silvia, R. M., and Sonnenschein, C. (1992) in Chemically Induced Alterations in Sexual Development: The Wildlife/Human Connection (Colborn, T., and Clement, C., Eds.), pp. 295–309, Princeton, NJ. 29. Huntley, S. L., Bonnevie, N. L., Wenn, R. J., and Bedbury, H. (1993) Bull. Environ. Contam. Toxicol. 51, 865– 872. 30. Rowland, M., Hewitt, S., Durrani, N., Bano, N., and Wirtz, R. (1997) Trans. R. Soc. Trop. Med. Hyg. 91, 252–255. 31. Bissacot, D. Z., and Vassilieff, I. (1997) Vet. Hum. Toxicol. 39, 6 – 8. 32. WHO (1990) Fenvalerate. Environmental Health Criteria 95, WHO, Geneva. 33. FAO/WHO (1985) FAO Plant Production and Protection Paper 67, FAO/WHO, Rome. 34. Boge, K. P., Brokof, H., Prohl, A., and Alsen-Hinrichs, C. (1996) Gesundheitswesen 58, 673– 681. 35. Eil, C. and Nisula, B. C. (1990) J. Steroid Biochem. Molec. Biol. 35, 409 – 414. 36. Brody, S. A., and Winterer (1983) Program and Abstracts of the 65th Annual Meeting of the Endocrine Society, San Antonio, TX, p. 261. 37. Parker, C., Patterson, D. R., Van Gelder, G. A., Gordon, E. B., Valerio, M. G., and Hall, W. C. (1984) J. Toxicol. Environ. Health 13, 83–97. 38. Wilson, J. D., Aiman, J., and MacDonald, P. C. (1980) A. Rev. Med. 36, 1–32. 39. Kelce, W. R., Stone, C. R., Laws, S. C., Gray, L. E., Kemppalnen, J. A., and Wilson, E. M. (1995) Nature 375, 581–585. 40. Kelce, W. R., Monosson, E., Gamcsik, M. P., Laws, S. C., and Gray, Jr. (1994) Toxicol. Appl. Pharmacol. 126, 276 –285. 41. Chen, C. W., Hurd, C., Vorojeikina, D. P., Arnold, S. F., and Notides, A.C. (1997) Biochem. Pharmacol. 53, 1161–1172.
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251, 855– 859 (1998)
RC989569
Estrogenic and Antiprogestagenic Activities of Pyrethroid Insecticides Joan Garey and Mary S. Wolff Department of Community and Preventive Medicine, Mount Sinai School of Medicine, New York, New York 10029
Received September 18, 1998
Many pesticides possess hormonal activity and have thus been classified as endocrine disruptors. Pyrethroids are commonly used insecticides worldwide, but little has been done to characterize their hormone agonist/antagonist potential. We tested four frequently encountered pyrethroids, fenvalerate, sumithrin, d-trans allethrin, and permethrin, for estrogen and progesterone agonist/antagonist activities using the Ishikawa Var-I human endometrial cancer cell line and the T47D human breast cancer cell line. Both cell lines produce alkaline phosphatase as an indicator of hormonal activity. Fenvalerate and sumithrin demonstrated significant estrogenicity; at concentrations of 10 mM, these compounds achieved maximal activities comparable to that of 10 nM 17a-ethynylestradiol in Ishikawa Var-I cells. None of the four compounds showed statistically significant estrogen antagonist activity or acted as progestins. However, fenvalerate and d-trans allethrin significantly antagonized the action of progesterone in T47D cells. Through these hormonal pathways, exposure to certain pyrethroids may contribute to reproductive dysfunction, developmental impairment, and cancer. © 1998 Academic Press
Estrogenic and progestagenic activities are among the hormonal responses reported for a number of pesticides, plasticizers and polychlorinated biphenyls (PCBs) (1-6). Concern exists that these synthetic environmental “endocrine disruptors” may enhance the risk of breast cancer in women (7), lower sperm counts and heighten risks of cryptorchidism, hypospadias and testicular cancer in males (8), as well as impair immunity and alter neurological development (9). Synthetic pyrethroids are among the most common pesticides in current use worldwide (10), yet little has been done to assess their potential hormonal activities. The widespread use of pyrethroids in agriculture and in the home has been encouraged by laboratory evidence suggesting that they are relatively safe to humans and wildlife (10). However, epidemiological ac-
counts, clinical reports and other laboratory studies indicate that pyrethroids can elicit a range of immunotoxic and neurotoxic effects in humans and other mammals; pyrethroid exposures have been associated with acute reproductive effects and may produce chronic and developmental impairments, as well (11-15). The mechanisms have not been firmly established in all cases, but these effects are consistent with estrogen receptor (ER) and progesterone receptor (PR) mediated actions. Here we report the first demonstration of estrogenic and antiprogestagenic activities among synthetic pyrethroid pesticides using two in vitro systems based on human cells. We tested four commonly encountered synthetic pyrethroid compounds to determine whether they act as estrogen analogs or antagonists using the Ishikawa Var-1 human endometrial andenocarcinoma cell line. This cell line, rich in estrogen receptors, produces a specific, dose-dependent increase in alkaline phosphatase (AlkP) in response to estrogens (16). In addition, we investigated the potential progesterone agonist/antagonist effects of the same four pyrethroids in T47D human breast cancer cells. In these cells, an increase in AlkP induction occurs only in response to progestins (17). Both systems have been used previously to characterize hormonal activities among a range of steroidal compounds and plant flavonoids (1820). We detected significant estrogenic activity in two widely used synthetic pyrethroids, fenvalerate and sumithrin, in the Ishikawa Var-1 AlkP assay, and we observed significant antiprogestagenic activity in the pyrethroids fenvalerate and d-trans allethrin in the T47D AlkP assay. MATERIALS AND METHODS Test compounds and standards. Four synthetic pyrethroids (dtrans allethrin [also known as bioallethrin], fenvalerate, technical permethrin and sumithrin [also known as fenothrin and d-phenothrin] (Figure 1) were a gift from McLaughlin Gormley King, Co., Minneapolis, MN. The pyrethroids obtained were of variable stated purity, with d-trans allethrin 5 96.5%; fenvalerate 5 95.9%; permethrin, 95.7%; sumithrin 5 95.4%. These values were taken into account when preparing the desired test concentrations. 17b-
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0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Vol. 251, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1.
Structures of four synthetic pyrethroid insecticides.
Estradiol (E2), 17a-ethynylestradiol (EE) and progesterone (P), obtained from Sigma Chemical Co., St. Louis, MO, were used as positive controls. The antiestrogen ICI 164,384, a gift from Dr. A. Wakeling of Imperial Chemical Industries (Macclesfield, UK), was used to verify estrogenicity of the test compounds. All compounds were prepared in ethanol to achieve the desired concentrations. Cell lines. The methods described here for both cell lines are based on those of Markiewicz et al. (21). The Ishikawa Variant-I was originally described by Holinka et al. (16). Cells were maintained in an estrogen-free basal medium (BM) comprised of a 1:1 mixture of Dulbecco’s Modified Eagle’s medium and Ham’s Nutrient Mix F12 without phenol red (Sigma Chemical Co., St. Louis, MO); the medium was supplemented with an antibiotic/antimycotic mixture, HEPES, sodium bicarbonate (all supplements from Sigma) and 5% calf serum (Atlanta Biologicals, Atlanta, GA) stripped of endogenous estrogens with dextran-coated charcoal (Sigma). The de novo induction of AlkP in T47D cells by progestins was originally described by DiLorenzo et al. (17). T47D cells were maintained in Dulbecco’s Modified Eagle’s Medium containing phenol red (DMEM, purchased from Sigma) and supplemented with penicillin/ streptomycin (Sigma), sodium bicarbonate and 10% fetal bovine serum (FBS; purchased from Atlanta Biologicals). The cell lines were cultured in 100 mm culture dishes and maintained at 37°C in a humidified atmosphere of 5% CO2-95% air. Cells were used in the assay when they reached confluence. Ishikawa and T47D AlkP assays. Cells were harvested with 0.25% trypsin (Atlanta Biologicals) and seeded for experiments in 96-well flat-bottom Microtest-III tissue culture plates (Becton Dickinson, Franklin Lakes, NJ). The Ishikawa cells were plated at a density of between 15,000-20,000 cells/well in 100 ml aliquots; the T47D cells were plated at a density of 50,000 cells/well in 100 ml aliquots. Test compounds and standards were dissolved in ethanol and diluted in BM with 5% stripped calves serum to appropriate concentrations (no greater than 0.2% final solvent concentration). Each dilution (50 ml) was added to eight wells per plate after a 24 hour incubation period and returned to the incubator. After a 48 hour exposure period, the plates were washed in 2 L PBS (0.15M NaCl, 10 mM sodium phosphate, pH 7.4) and drained. (For the T47D assays only, plates were then placed in a 272°C freezer for 15 min and then thawed at room temperature). An icecold solution of 5 mM p-nitrophenyl phosphate, 0.24 mM MgCl and 1 M diethanolamine (all reagents purchased from Sigma), pH 9.8, was added to each well. Formation of p-nitrophenol at room temperature
was detected at 405 nm in a Titertek Multiscan Plus MKII (type 314) plate reader. Plates were considered fully developed when the wells containing E2 at 1028 M read approximately 1.0-1.4 optical density (OD) units. At this time, the control level was between 0.1-0.3 OD units. The development time for the Ishikawa bioassay was 5-30 minutes and 30-60 min for the T47D bioassay. The mean of eight wells on one plate constituted a single experiment, and all results represent at least three independent experiments. Data analysis. Differences were tested for statistical significance using the t-test (two-tailed, equal variance) computed by Excel (Microsoft) or SAS PC (Cary, NC) supported by the City University of New York Academic Computing Facility. Differences were considered statistically significant when p , 0.05. Data are presented as the mean and, where shown, the standard deviation, of at least three experiments. Dose-response curves were prepared through the use of p-Fit software (Biosoft, Ferguson, MO).
RESULTS AND DISCUSSION Fenvalerate and sumithrin demonstrated estrogenic activity at 30 mM as evidenced by a significant increase of AlkP activity in Ishikawa Var-I endometrial cancer cells. Permethrin and d-trans allethrin were not significantly different from the control (Figure 2). To confirm that fenvalerate and sumithrin acted through the ER, the antiestrogen ICI 164,384 was added at 1 mM in a separate experiment, causing a total reversal of the AlkP response to basal levels (data not shown). The dose-response curves for fenvalerate and sumithrin in the same assay system exhibited maximal activities comparable to that of EE, when using approximately 104-105 fold higher concentrations (Figure 3). The EC50s of fenvalerate and sumithrin were approximately 1 and 4 mM, respectively. In addition, we tested the two compounds which did not demonstrate estrogenic activity, d-trans allethrin and permethrin, in combination with 10 pM and 100 pM E2 (Figure 3). While permethrin did not reduce E2 activity, d-trans allethrin showed a trend toward re-
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FIG. 2. Estrogen-induced AlkP production (OD units) of four pyrethroid insecticides in Ishikawa Var-I cells. d-trans Allethrin (A), fenvalerate (F), permethrin (PM), and sumithrin (S) were tested at 30 mM. 17a-Ethynylestradiol (EE) at 10 nM was used as a positive control; C represents the vehicle control. For each compound, n 5 3. Both fenvalerate and sumithrin demonstrated levels of AlkP activity significantly higher than the vehicle control, as indicated by * for fenvalerate, p , 0.01; for sumithrin, ** indicates p 5 0.001.
ducing the AlkP activity of E2, although the differences were not statistically significant. The same four compounds were also tested for progestagenic/antiprogestagenic activities in T47D cells. At 30 mM, none of the compounds induced an AlkP response signficantly different from the control (Figure 4). However, when combined with 5 nM P, both d-trans allethrin and fenvalerate demonstrated significant antagonist effects, with d-trans allethrin being the most potent antagonist, reducing the AlkP activity of 5 nM P by an average of 68% (Figure 5). Endocrine disruption research has primarily focused on identifying exogenous sources of estrogen as well as estrogen antagonists (9). However, the action of progestins may enhance or inhibit the action of estrogen, depending upon their concentration relative to estrogen (22). Moreover, recent research has demonstrated a role for neuronally-produced progesterone in neurological development and repair of damaged nerve cells (23,24,25). Screening efforts have therefore been expanded to include the analysis of environmental compounds for their potential to produce PR-mediated effects (2,3,4,5,6). Many environmental substances demonstrating ERand/or PR-mediated activites have been banned from use in the United States (e.g., the organochlorine insecticides aldrin, dieldrin, DDT, heptachlor and chlordane, as well as PCBs) (4,26), although these compounds persist in the environment and are used in developing countries (27). Other organochlorine insecticides still in use in the United States and elsewhere exhibit estrogenic or antiprogestagenic activity in some assays, e.g., endosulfan and toxaphene (2,28), as does the current use acetanilide herbicide, Alachlor (3,29).
FIG. 3. Estrogenic response (OD units) of fenvalerate, sumithrin and 17a-ethynylestradiol (EE). F 5 EE, Œ 5 fenvalerate ■ 5 sumithrin. Average half-maximal doses (EC50s) were: fenvalerate 5 1100 6 75 nM; sumithrin 5 4500 6 1100 nM; EE 5 40 6 10 pM. Each concentration data point on the curve represents the average of three or four separate experiments.
Antiestrogenic activity has been reported for certain current use chloro-S-triazine and carbamate pesticides (3), while other carbamates are weakly estrogenic and progestagenic, as well as antiprogestagenic (3). Pyrethroids are synthetic derivatives of pyrethrins, the active ingredients of the natural insecticidal chrysanthemum extract known as pyrethrum (10). The pyrethrins are highly lipophilic but unstable in light and heat and, therefore, have been limited to indoor use (10). The pyrethroids are also highly lipophilic, possess enhanced environmental stability and have been widely marketed as alternatives to organochlorine and organophosphate pesticides for both agricultural and
FIG. 4. Inhibition of estrogen-induced AlkP production by two pyrethroid insecticides in Ishikawa Var-I cells. d-trans Allethrin (A) and permethrin (PM) were tested at 30 mM alone and in combination with 10 pm E2 (E10) and 100 pM E2 (E100). C represents the vehicle control. For each condition tested, n 5 3. The AlkP activity is expressed as optical density (OD) units. E2 activity was not significantly suppressed by PM or A, although A 1 E10 approached statistical significance (t 5 2.6, p 5 0.06).
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FIG. 5. The inhibition of progesterone-induced AlkP production by four pyrethroid insecticides in T47D cells. Cells were incubated for 48 hr with vehicle (C) or 5 nM progesterone (P), in the presence or absence of d-trans allethrin (A), fenvalerate (F), permethrin (PM), and sumithrin (S), all at 30 mM. The resulting AlkP activity is expressed as optical density (OD) units. The * indicates activity significantly lower than 5 nM P, p , 0.01.
home use (10). They are effective against a variety of insects, including fleas, roaches, ticks, mosquitoes and lice (10,30,31). They are used to protect livestock against ectoparasites and are also used to protect a variety of crops (32,33) Human exposures may occur occupationally, during application, or through pyrethroid residues such as those detected in cow’s milk, bread, fruits, vegetables and carpeting (11,32-34). Previous reports indicate that pyrethrum is not estrogenic (1) and that pyrethrins, as well as several synthetic pyrethroids, including fenvalerate, permethrin and fenothrin (sumithrin), are androgen antagonists (35-37). An androgen receptor (AR) mechanism was proposed to explain an “epidemic” of gynecomastia seen among men in one community in Haiti where extensive pyrethroid spraying had been conducted (36). However, gynecomastia is ordinarily associated with estrogen exposure (38). Our data suggest that pyrethroids may have produced the symptoms of gynecomastia among Haitian men in 1981 via estrogenicity, as well. Both p,p9-DDE, the main mammalian metabolite of DDT, and Vinclozolin, have been found to be antagonists of AR binding (39-40), while p,p9-DDE has also demonstrated an ability to bind the ER and produce ER-mediated transcription of the lacZ reporter gene in an in vitro yeast assay system (41). Therefore, several classes of pesticides are now known to be able to target multiple hormone receptors. Our results are the first to suggest that some pyrethroids may act as estrogen analogs and progestin antagonists. These findings add to the growing evidence over the past decade that pesticides of several chemical classes have ER and PR agonist and antagonist effects (1-6). Moreover, estrogenicity by two pyrethroids but antiprogestagenicity by another illustrates the complexity of endocrine effects and the need for further research on structure-activity relationships.
Exposures to a variety of contaminants, including pharmaceuticals, plant-derived hormone agonists and environmental toxicants, may modulate endogenous endocrine signals in ways that can influence human health. The discovery of estrogenic and antiprogestagenic activities in some synthetic pyrethroids, a large and widely used family of pesticides, suggests the urgent need for further investigation of these current use chemicals and thus provides added evidence that pyrethroids belong on the list of “endocrine disruptors” (9). ACKNOWLEDGMENT This research was supported by NIEHS Superfund Basic Research Grant P42 ES07384.
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