17ß-Estradiol Is a Hormonal Regulator of Mirex Tumor Promotion Sensitivity in Mice

Karen L. Porter*, Sanjay Chanda*, Hui Quin Wang*, Kevin W. Gaido{dagger}, Robert C. Smart*,1 and C. Lee Robinette{ddagger},2

* Cell Signaling and Cancer Group, Department of Environmental and Molecular Toxicology, North Carolina State University, Campus Box 7633, Raleigh, North Carolina 27695; {dagger} CIIT Centers for Health Research, 6 Davis Drive, Box 12137, Research Triangle Park, North Carolina 27709-2137; and {ddagger} Department of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27695

Received February 18, 2002; accepted May 28, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mirex, an organochlorine pesticide, is a potent non-phorbol ester tumor promoter in mouse skin. Previous studies have shown that female mice are 3 times more sensitive to mirex tumor promotion than male mice and that ovariectomized (OVX) female mice are resistant to mirex promotion, suggesting a role for ovarian hormones in mirex promotion. To determine whether the ovarian hormone 17-ß estradiol (E2) is responsible for the sensitivity of female mice to mirex promotion, female mice were initiated with DMBA; 2 weeks later groups of mice were OVX and implants, with or without E2, were surgically implanted subcutaneously. These mice were treated topically twice weekly with mirex for 26 weeks. E2 implanted OVX mice demonstrated high normal physiologic levels of serum E2 throughout the tumor promotion experiment. E2 implants restored by 80% the intact mirex-sensitive phenotype to the OVX mice. Consistent with a role for E2 and ER{alpha} and ERß, treatment of DMBA-initiated female mice with topical ICI 182,780, an estrogen-receptor antagonist, reduced mirex tumor multiplicity by 30%. However, in cells co-transfected with ER{alpha} or ERß and estrogen-responsive promoter reporter, mirex did not stimulate promoter reporter activity, suggesting that the promotion effect of mirex is downstream of ER{alpha}/ß. Finally, a tumor promotion study was conducted to determine whether E2 implants could increase the sensitivity of male mice to mirex promotion. E2 implants in male mice did increase sensitivity to mirex promotion; however, the implants did not produce the full female sensitivity to mirex tumor promotion. Collectively, these studies indicate that E2 is a major ovarian hormone responsible for mirex tumor promotion sensitivity in female mice.

Key Words: mirex; skin; 17ß-estradiol; tumor promotion; endocrine.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mirex, an organochlorine pesticide, was used as an industrial fire retardant and a control for fire ants in the southeastern United States until its cancellation by the United States Environmental Protection Agency (U.S. EPA) in 1977 (Fisher, 1999Go). Mirex continues to be used in South America and South Africa (Fisher 1999Go; National Toxicology Program, 2001Go). The United Nations environmental program has recently identified mirex as one of the 12 most important persistent organic pollutants that threaten global human and wildlife health (Fisher 1999Go; National Toxicology Program, 2001Go). Mirex has been found in Lake Ontario sediment cores at depths corresponding to the mid-1960‘s (U.S. EPA, 2000Go) and has also been found in the Great Lakes area in bald eagle eggs and nestlings, herring gulls, cormorants, mussels, and trout (Bishop, 1995; Donaldson, 1999; Fisk, 1998; Fox, 1998; Robertson, 1998Go; Ryckman, 1998). Additionally, mirex was found in plasma from fish and waterfowl consumers in Ontario, Canada in 1999 (Kearney, 1999), in human breast milk in New York state in 1996 (Madden, 1996), and in human brain, liver, and adipose tissues in Greenland in 1999 (Dewailly, 1999). In 1985, a U.S. EPA national survey of chemicals in adipose tissue estimated that 10.2% of the population in the southern United States has measurable levels of mirex in adipose tissue (Kutz, 1985). The U.S. EPA and the New York Department of Health have issued fish consumption advisories for mirex in Great Lakes fish in 2001, indicating that mirex continues to be a concern for human health (New York Department of Health, 2001Go; U.S. EPA, 2001Go).

Mirex has been observed to be a hepatocarcinogen in rats, producing neoplastic nodules and hepatocellular carcinomas (Timchalk, 1985; Ulland, 1977). The U.S. EPA classified mirex as a probable human carcinogen in 2000 (U.S. EPA, 2000Go). Additionally, mirex was listed as "reasonably anticipated to be a human carcinogen" in the 2001 Ninth Report on Carcinogens (National Toxicology Program, 2001Go). The International Agency for Research on Cancer (IARC) has also classified mirex as a possible human carcinogen (Fisher, 1999Go). We have found that mirex is a potent, nonphorbol ester-type tumor promoter in 7,12 dimethylbenz[a]anthracene (DMBA)-initiated mouse skin, and produces papillomas with a 90% incidence of A->T transversion in the 61st codon of Ha-ras. (Kim, 1995) However, mirex skin-tumor promotion is refractory to the classic phorbol-ester and non-phorbol-ester skin tumor promoter inhibitors, retinoic acid and the synthetic, anti-inflammatory steroid, fluocinolone acetonide (Kim, 1995). Also, unlike phorbol esters, mirex skin-tumor promotion is sexually dimorphic, promoting 3 times more tumors in female than in male mice. Additionally, ovariectomized (OVX) female mice treated with mirex exhibited about one-third the number of papillomas per mouse as intact female mice, indicating that ovarian hormones are factors likely to influence mirex-tumor promotion sensitivity (Moser, 1992, 1993).

The hair follicle contains a stem cell located in the bulge region of the follicle. Early studies have shown that when an initiating carcinogen is topically applied to shaven skin, tumor yield often depends on the phase of the hair cycle at application (Cotsarelis, 1990). Additionally, studies have shown that squamous cell, hyperplastic foci in DMBA-initiated, 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted mouse skin, histologically involves the hair follicle (Binder, 1997). Also, in H-ras transgenic TG.AC mouse skin treated with TPA, papillomas arise from hyperplastic foci of the follicular epithelium (Hansen, 1994). These data indicate that the hair follicle containing the follicular bulge stem cell may be considered the target for chemical-induced skin carcinogenesis. Recently, we have found that skin expresses estrogen receptor-{alpha} (ER-{alpha}) in the dermal papilla of the hair follicle and that topical treatment with 17-ß estradiol (E2) or ICI 182,780, an E2 antagonist, have opposing effects on the hair cycle, indicating that skin is an estrogen-responsive tissue (Chanda, 2000; Oh, 1996). Since the bulge stem cell is a putative target for chemical carcinogenesis and the hair follicle is responsive to E2, estrogen may be an ovarian hormone responsible for regulating mirex tumor promotion sensitivity. The present study was conducted to determine the role of E2 in mirex tumor promotion by examining the ability of E2 implantation and gonadectomy to alter the mirex tumor promotion.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Analytical standard (99% purity) mirex (dodecachlorooctahydro-1,3,4-methano-1H-cyclobutal[c,d] pentalene) was purchased from Radian Corporation (Dallas, TX). 17ß-estradiol (1,3,5[10]-estratriene-3, 17ß-diol), kepone, and thimersol were purchased from Sigma (St. Louis, MO). Polyethylene tubing was purchased from Becton Dickinson (Sparks, MD), and silastic tubing was obtained from VWR. Teflon beading was purchased from Cole Parmer (Vernon Hills, IL). Bloom-275 gelatin and norit-A charcoal were purchased from Fisher (Fairlawn, NJ). Sodium phosphates, monobasic and dibasic, were purchased from J.T. Baker (Phillipsburg, NJ). HPLC grade, carbonyl-free ethyl acetate was purchased from Burdick Jackson (Muskegon, MI). Rabbit anti-estradiol antibody was a kind gift from J.H. Britt (University of Tennessee, Knoxville). Estradiol-6-(O-carboxymethyl)oximino-)2-[125I]iodohistamine tracer was purchased from Amersham (Arlington Heights, IL). ICI 182,780 was a kind gift from Zeneca Pharmaceuticals (Wilmington, DE). Materials used in the HepG2 transcription assay are as previously described (29), except that the transfection reagent used was TransIT-LT1 from Pan Vera (Madison, WI).

Animals.
Male and female CD-1 mice (6 weeks old) were purchased from Charles River Laboratories (Raleigh, NC) and kept in our animal facility for one week prior to use. The mice were fed rodent chow ad libitum, kept on corncob bedding, and placed on a 12-h light/dark cycle. Male or female CD-1 mice (7 weeks old) were shaved on their dorsal surface with electric clippers. One week later, the mice that did not show hair regrowth were treated with a single topical application of 200 nmol DMBA in 200 ml acetone for males and 50 nmol DMBA in 200 ml acetone for females. Two weeks after initiation, the mice were put under halothane anesthesia and either castrated or ovariectomized (OVX), or sham operated, and given subcutaneous empty or 17ß-estradiol (E2) containing silastic tubing implants (female) or polyethylene tubing implants (male) over the scapula. Beginning 2 weeks later, the mice were treated topically with 200 nmol mirex in 200 µl acetone twice weekly for 27 weeks. DMBA-treated OVX female mice given E2 implants were treated topically with acetone, and acetone sham-initiated OVX female mice that were given E2 implants were treated topically with mirex, serving as the control groups. A separate group of mice were DMBA-initiated, given silastic E2 implants, and promoted with mirex for various periods of time up to 31.5 weeks to provide timed interval determination of serum estradiol levels. Blood was drawn at the start of mirex promotion, which was 2 weeks post-implantation, at 18 weeks of promotion (20 weeks post-implantation) and at 29.5 weeks of promotion (31.5 weeks post-implantation) by cardiac puncture under halothane anesthesia, prior to sacrifice. The blood was allowed to clot, centrifuged for 10 min, and serum was separated from cells and stored at –20° until analysis. At the end of each tumor-promotion study, blood was drawn, and serum was separated and stored at –20° until analysis. In the ICI 182,780 study, female mice were treated with a single topical application of 50 nmol DMBA at 8 weeks of age, then treated topically twice weekly with 200 nmol mirex in 200 ml acetone 2 weeks later for 25 weeks. The mice were treated with 10 nmol ICI 182,780 in 200 ml acetone or acetone alone 30 min prior to each mirex treatment.

Implants.
Implants for male mice were made from 12-mm lengths of polyethylene tubing (1.77 mm I.D., 2.80 mm O.D.) filled with an 8-mm crystalline E2 column, which was plugged with Teflon beading and heat-sealed. Implants for female mice were made from1-cm lengths of silastic tubing, (1.57 mm I.D., 3.12 mm O.D), filled with 2.95 mg crystalline E2 (2.5 mm length), and plugged with Teflon. This tube was inserted into a second 1.2-cm length of silastic tubing, (2.64 mm I.D., 4.88 mm O.D.). This outer tube was then sealed at both ends with silicone medical adhesive and cured overnight. Male mice were given 4 polyethylene implants each, and female mice were given 1 silastic implant. All implants were conditioned overnight in carrier mice.

Estradiol radioimmunoassay (RIA).
Serum estradiol was assayed by a method previously described (Chanda 2000; Cox 1987). Briefly, 100 ml of serum was freeze extracted with 2 ml ethyl acetate, evaporated at 37°C under nitrogen, reconstituted with PBS-gel buffer (0.01 M PBS, 0.1% gelatin, pH 7.0), and incubated overnight with 200 ml antibody (diluted 1:1,500,000 with PBS-gel). The next day, 100 ml tracer diluted with PBS-gel (approximately 8000 cpm) were added and incubated at 4°C for 6 h. Dextran-coated charcoal (500 ml, 0.05% dextran, 0.5% charcoal in PBS-gel) was added, vortexed, incubated for 45 min at 4°C, then centrifuged at 1550 x g for 15 min. The supernatant was decanted and counted using a gamma counter (1272 Clinigamma, Wallac Instruments, Gaitherburg, MD). Estradiol levels were determined from a standard curve. Recovery was measured using a pooled serum sample spiked with tracer.

HepG2 cell luciferase assay for estrogenicity.
Briefly, HepG2 cells were grown in complete, phenol red-free MEM with stripped fetal bovine serum, and transiently transfected with plasmids containing ß-galactosidase, ER-{alpha} or ER-ß, and an ERE-containing luciferase reporter gene as previously described (Gaido 2000) except that TransIT transfection reagent (2 µl/µg of plasmid DNA) was used. After transfection, cells were incubated for 3 h, then media were removed and the plates washed with PBS. Mirex, kepone, and E2 were serially diluted in DMSO to yield final concentrations in culture media from 10–5 M to 10–11 M, after adding 1 µl of the DMSO solution to l ml of media. Each chemical solution was added to the plates, 3 wells per chemical per dilution, and incubated for 24 h. Luciferase activity was corrected for ß-galactosidase activity.

Statistics.
Tumor multiplicity data was analyzed using the nonparametric Mann-Whitney test at p < 0.05. Serum E2 data was analyzed using Student’s t-test at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of subcutaneous 17ß-estradiol-containing implants on mirex tumor promotion in ovariectomized female mice.
In order to determine whether E2 is the ovarian hormone responsible for the sensitivity of female mice to mirex tumor promotion, we placed E2-containing subcutaneous implants in OVX female mice in an attempt to restore sensitivity to mirex tumor promotion. Female mice were treated topically with a single dose of 50 nmol DMBA, then 2 weeks later, groups of mice were OVX or sham OVX, and silastic implants, with or without E2, were surgically implanted subcutaneously. Two weeks later, the mice were treated topically with mirex, twice weekly, for 26 weeks. As shown in Figure 1Go, intact female mice developed 7.5 papillomas/mouse and 100% of the mice developed papillomas, while OVX female mice with sham implants developed only 3 papillomas/mouse and 65% tumor incidence. In contrast, OVX mice with E2-containing implants developed 6 papillomas/mouse and 90% of the mice developed papillomas. Mann-Whitney non-parametric statistical analysis showed that there is significant difference in tumor multiplicity (p < 0.05) between OVX and intact mice, and OVX- and OVX-E2-implanted mice during weeks 23–26, the period of maximal plateau. A preliminary experiment performed using the same protocol also showed similar results. As shown in Figure 1Go, control DMBA-treated OVX mice with E2-containing implants, treated topically with acetone, and control, acetone-treated OVX mice with E2-containing implants, treated topically with mirex, did not develop papillomas. Collectively, these results demonstrate that chronic systemic administration of E2 can increase the sensitivity to mirex tumor promotion in OVX female mice to nearly that observed in intact female mice.



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FIG. 1. Subcutaneous estradiol implants nearly restore intact female mouse mirex tumor-promotion sensitivity to OVX mice. Seven-week-old female mice were initiated with 50 nmol DMBA; 2 weeks later they were sham OVX and given sham implants, or OVX and given subcutaneous 17ß-estradiol implants, then treated topically 2 weeks later with 200 nmol mirex twice weekly for 26 weeks. Intact (square), OVX (diamond), and OVX with subcutaneous estradiol implants (circle) female mice; n = 25 mice per group. Control groups of female mice were initiated with 50 nmol DMBA (triangle) or acetone (X), given subcutaneous 17ß-estradiol implants, then promoted 2 weeks later with 200 nmol acetone (triangle) or mirex (X), respectively, twice weekly for 26 weeks; n = 15.

 
Since silastic E2-containing implants were capable of restoring the sensitivity of OVX female mice to mirex promotion, it was important to determine the serum concentration of E2 in OVX E2-implanted mice and to compare these values to the serum E2 concentration of E2 in intact and OVX mice. As shown in Figure 2Go, serum E2 levels in intact female mice ranged between 5.8 and 79.6 pg/ml, while E2 levels in OVX mice were low and ranged between 3.6 and 9.4 pg/ml. The broad range of E2 values measured in the intact female mice is due to the mice being in different stages of the estrous cycle. In E2-implanted OVX mice, E2 levels were measured at various time points after implantation. At 2 weeks post-implantation, which represents the start of tumor promotion, serum E2 ranged from 82 to 125 pg/ml with an average of 106 ± 16 pg/ml at 20 weeks implantation, which represents 18 weeks of promotion; serum E2 levels range from 40 to 85 pg/ml with an average of 60 ± 13 pg/ml, and at 31.5 weeks post-implantation, which represents 29.5 weeks of promotion, serum E2 levels ranged from 50 to 75 pg/ml, with an average of 62 ± 9.7 pg/ml. There was a significant difference in E2 levels of intact versus OVX mice and between OVX-E2 and OVX mice (Student’s t-test, p < 0.05). These data indicate that silastic E2-containing implants can raise OVX female mouse serum E2 levels to high physiologically intact female mouse levels and sustain those levels throughout the tumor promotion study.



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FIG. 2. Subcutaneous estradiol implants maintain high physiological serum 17ß-estradiol levels. OVX (square), n = 22, and intact (triangle) n = 34 female mice, sacrificed at 26 weeks of promotion, and E2 implanted OVX female mice sacrificed at 2 weeks (open circle), n = 6, 20 weeks (dark circle) n = 9, and 31.5 weeks (X) n = 7, post-implantation.

 
Effect of topical E2 antagonist treatment on mirex tumor promotion in female mouse skin.
To determie if an ER-mediated pathway modulates the sensitivity of female mice to mirex tumor promotion, we initiated intact female mice with a single topical dose of 50 nmol DMBA, then 2 weeks later, they were treated topically with 200 nmol mirex and 10 nmol ICI 182,780, an ER antagonist, or mirex and acetone vehicle twice weekly, for 25 weeks. A 10-nmol dose of ICI 182,780 was selected for topical application, because 10 nmol ICI 182,780 has been shown to have a profound effect on the hair follicle cycle in mice. (Oh, 1996) As shown in Figure 3Go, mice treated with mirex alone develop 11.8 papillomas/mouse, while mice treated with mirex and ICI 182,780 develop 8.2 papillomas/mouse, a 30% decrease in mirex-induced tumor multiplicity. Additionally, there was a 2-week delay in tumor formation in the ICI 182,780-treated group. However, Mann-Whitney non-parametric statistical analysis showed that there is no significant difference in tumor multiplicity (p < 0.05) between ICI 182,780-treated and mirex/acetone-treated mice. These results suggest that treatment with an ER antagonist can inhibit mirex tumor promotion, further supporting the idea that an ER or an ER pathway is important in mirex tumor promotion.



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FIG. 3. ICI 182,780 treatment reduces mirex-promoted tumor multiplicity. Seven-week-old female mice were initiated with 50 nmol DMBA, then promoted 2 weeks later with 200 nmol mirex twice weekly and treated topically with acetone or 10 nmol ICI 182,780 twice weekly for 25 weeks. Acetone-treated (square) and ICI 182,780-treated (diamond) intact female mice; n = 25 mice per group.

 
HepG2 cell luciferase reporter assay for mirex and kepone estrogenicity.
To determine whether mirex can induce ER-{alpha} or ER-ß transactivation in mammalian cells, we transiently transfected HepG2 cells with ER-{alpha} or ER-ß, ß-galactosidase, and an estrogen-response element (ERE) containing luciferase reporter gene construct. The transfected HepG2 cells were then treated with increasing concentrations of mirex or kepone, an organochlorine pesticide structurally similar to mirex. As shown in the top panel of Figure 4Go, mirex, at concentrations up to 1 x 10–5 M, did not induce luciferase activity, but kepone at 1.5 x 10–7 M did induce luciferase activity in the ER-{alpha} system. However, as shown in the bottom panel of Figure 4Go, neither mirex nor kepone induced luciferase activity in the ER-ß system. We were unable to test concentrations of mirex higher than 10–5 M since mirex is a lipophilic compound with low solubility in aqueous solutions. These data show that mirex does not increase ER-{alpha} or ER-ß transactivation activity in mammalian cells and suggests that the promotional effects of mirex are downstream of ER{alpha}/ERß.



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FIG. 4. Mirex is not estrogenic in an HEPG2 mammalian cell bioassay. HEPG2 cells were transiently transfected with ER{alpha} (top panel) or ERß (bottom panel) constructs and luciferase promoter/reporter constructs under the transcriptional control of promoter and containing ERE. Cells were treated with 17ß-estradiol (square), mirex (circle), or kepone (triangle).

 
Effect of subcutaneous 17ß-estradiol-containing implants on mirex tumor promotion in male mice.
In order to determine if E2 is responsible for decreased sensitivity to mirex tumor promotion in male mice and to attempt to induce intact female mouse mirex tumor promotion sensitivity in male mice, we placed polyethylene E2-containing subcutaneous implants in castrate male mice. Male mice are more sensitive to E2 toxicity than female mice, so polyethylene implants, which produce lower E2 levels than silastic implants, were used in the male mice, who were then treated topically with a single 50-nmol dose of DMBA. Two weeks later, groups of mice were castrated or sham castrated and polyethylene implants, with or without E2, were surgically implanted subcutaneously. Two weeks later, the mice were treated topically with mirex, twice weekly for 27 weeks. As shown in Figure 5Go, intact male mice with sham implants developed 2.8 papillomas/mouse and 55% of the mice developed papillomas, while castrate mice with sham implants developed ~1.0 papillomas/mouse and 25% of the mice developed papillomas. In contrast, castrate E2-implanted mice developed 3.4 papillomas/mouse and 60% of the mice developed papillomas. Mann-Whitney non-parametric statistical analysis showed that there is significant difference in tumor multiplicity (p < 0.05) between castrate and intact mice, and between castrate and castrate E2-implanted mice during weeks 24 through 27, the period of maximal plateau. These results demonstrate that castration decreases mirex promotion sensitivity and that chronic systemic administration of E2 can increase the sensitivity to mirex tumor promotion in castrate male mice to that observed in intact male mice, but not to that observed in intact female mice.



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FIG. 5. Subcutaneous estradiol implants restore intact male mouse mirex tumor-promotion sensitivity to castrated mice. Seven-week-old male mice were initiated with 200 nmol DMBA; 2 weeks later they were sham castrated and given empty implants, castrated and given empty implants, or castrated and given subcutaneous 17ß-estradiol implants, then treated topically 2 weeks later with 200 nmol mirex twice weekly for 27 weeks. Intact (square), castrate (diamond), and castrate with subcutaneous estradiol-implanted (circle) male mice; n = 25 mice per group.

 
Since polyethylene implants are capable of restoring the sensitivity of castrate male mice to mirex promotion, we assayed serum E2 levels in intact male mice, castrate male mice, and castrate E2-implanted males. Serum E2 levels in intact male mice ranged from 6.5 to 13.7 pg/ml, with an average of 10.1 ± 3.6 pg/ml, and serum E2 levels in castrate male mice ranged from 6.0 to 12.2 pg/ml, with an average of 9.1 ± 3.1. However, serum E2 levels in E2-implanted mice ranged between 9.8 and 21.2 pg/ml, with an average of 15.4 ± 5.9 at 29 weeks after implantation and were 55% higher and significantly different, using Student’s t-test (p < 0.05), than E2 levels observed in intact male mice. E2 levels in the E2-implanted male mice were within the low-normal serum E2 range observed in intact female mice. Mortality due to urinary retention and hydronephrosis was observed in 3 of 25 E2-implanted mice early in the experiment.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we observed that subcutaneous E2 implants were able to restore 80% of the intact female mouse mirex-promotion sensitivity to OVX female mice, indicating that E2 is a primary ovarian hormone responsible for regulating mirex tumor-promotion sensitivity. The E2 implants produced continuous serum E2 levels that were within the upper range of intact cycling female mice. However, intact female mice cycle from high E2 levels in proestrus, decreasing E2 levels in estrus, and low E2 levels in diestrus during the 4–5 day estrus cycle (Carr, 1998Go). This difference between cycling and non-cycling E2 levels may explain why the E2 implants did not completely restore the full intact female response to mirex tumor promotion. In addition, other hormones may also be important, since the ovary is the primary source of testosterone in females, and LH, FSH, and progesterone levels also rise and fall during the estrus cycle (Carr 1998Go). Serum prolactin levels also increase in female mice given silastic implants, 6 mg E2 per implant (Walker, 1992).

Although castrate male mice develop one-third the tumors of intact male mice, intact and castrate male mice had statistically the same levels of serum E2. Studies in nude mice also show that intact and castrate male mice have similar serum E2 levels (van Steenbrugge, 1988). These data indicate that altered mirex tumor promotion sensitivity in castrate male mice does not require changes in the endogenous levels of circulating E2, but exogenous E2 at levels above physiological levels for male mice is sufficient to overcome the effects of castration and restore intact male mouse response. Since castration did produce a significant decrease in the number of mirex-induced papillomas but not in serum E2 levels, this suggests that factors other than E2 can modulate mirex tumor promotion. However, it is possible that the decrease in circulating levels of testosterone in mice results in a decrease in the conversion of testosterone to E2 in skin. This results in a decrease in cutaneous E2 and decreased sensitivity to mirex, and this can be overcome by the superphysiological serum E2 levels seen in the E2-implanted mice. We observed mortality in E2-implanted male mice from E2 toxicity and no mortality in E2-implanted female mice from E2 toxicity. This observation is consistent with the findings of earlier studies, where male mice develop urinary retention and hydronephrosis resulting in increased mortality after receiving silastic E2 implants (Buhl, 1985).

ICI 182,780, a pure estrogen antagonist, reduced tumor multiplicity by 30% at the dose used. However, we observed that mirex does not stimulate ER-{alpha} or ER-ß transactivation in a HepG2 mammalian cell reporter assay. Our transactivation results are in agreement with in vitro competitive binding assays, which have shown that mirex has no binding affinity for ER-{alpha} (Blair, 2000). Taken together, these results suggest that mirex and E2 interaction may involve pathways downstream from ER-{alpha}. It is possible that mirex interacts with ER target genes to produce alterations in growth, apoptosis, and/or differentiation. Mirex has also been shown to inhibit E2 uptake in cultured rat hepatocytes (Teo, 1990) and to increase 2-hydroxylation of estradiol in rat liver (Bulger, 1983). Furthermore, the European Commission lists mirex as having evidence for endocrine disruption in wildlife and humans (European Commission 2001Go), and the Illinois State EPA lists mirex as a probable chemical to cause endocrine disruption (Kaminuma, 1997).

TGF-ß3 and TGF-ß1 have been shown to participate in skin carcinogenesis (Cui, 1996). Recent studies have shown that E2 can induce the TGF-ß3 promoter via a novel activated Ras-dependent pathway (Lu, 2001). Since our laboratory has determined that greater than 90% of mirex-promoted tumors express mutant Ha-ras (Moser, 1993); it is possible that E2 activation of TGF-ß3 can occur in initiated mutant Ha-ras-containing keratinocytes. Additionally, E2 has been demonstrated to increase levels of TGF-ß1 in dermal fibroblasts (Ashcroft 1997), which may indicate an additional role for E2 in skin carcinogenesis.

In summary, our results indicate that E2 is a primary hormone responsible for modulating mirex skin tumor-promotion sensitivity in female mice. Since mirex did not activate the estrogen-responsive promoter in cells co-transfected with ER{alpha} or ERß and estrogen-responsive promoter reporter suggests the promotion effect of mirex is downstream of ER{alpha}/ß.


    ACKNOWLEDGMENTS
 
This research was supported by Grant ES08127 (to R.C.S. and C.L.R.) from the National Institute of Environmental Health Sciences and Grant CA-46637 (to R.C.S.) from the National Cancer Institute.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (919) 515-7169. E-mail: rcsmart{at}unity.ncsu.edu. Back

2 Present address: Icagen, 4222 Emperor Blvd., Suite 350, Durham, NC 27703. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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