* Cell Signaling and Cancer Group, Department of Environmental and Molecular Toxicology, North Carolina State University, Campus Box 7633, Raleigh, North Carolina 27695;
CIIT Centers for Health Research, 6 Davis Drive, Box 12137, Research Triangle Park, North Carolina 27709-2137; and
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
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ABSTRACT |
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Key Words: mirex; skin; 17ß-estradiol; tumor promotion; endocrine.
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INTRODUCTION |
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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, 2000). Additionally, mirex was listed as "reasonably anticipated to be a human carcinogen" in the 2001 Ninth Report on Carcinogens (National Toxicology Program, 2001
). The International Agency for Research on Cancer (IARC) has also classified mirex as a possible human carcinogen (Fisher, 1999
). 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- (ER-
) 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.
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MATERIALS AND METHODS |
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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- 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 105 M to 1011 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 Students t-test at p < 0.05.
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RESULTS |
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DISCUSSION |
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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- 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-
(Blair, 2000). Taken together, these results suggest that mirex and E2 interaction may involve pathways downstream from ER-
. 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 2001
), 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 or ERß and estrogen-responsive promoter reporter suggests the promotion effect of mirex is downstream of ER
/ß.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: Icagen, 4222 Emperor Blvd., Suite 350, Durham, NC 27703.
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REFERENCES |
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Binder, R. L., Gallagher, P. M., Johnson, G. R., Stockman, S. L., Smith, B. J., Sundberg, J. P., and Conti, C. J. (1997). Evidence that initiated keratinocytes clonally expand into multiple existing hair follicles during papilloma histogenesis in SENCAR mouse skin. Mol. Carcinog. 20, 151158.[ISI][Medline]
Bishop, C., Chek, A. A., Hussell, D. J. T., and Jock, K. (1995). Chlorinated hydrocarbons and mercury in sediments, red-winged blackbirds (Agelais-phoeniceus), and tree swallows (Tachyceneta bicolor) from the wetlands in the Great Lakes-St. Lawrence River basin. Environmental Toxicology and Chemistry 14, 491501.[ISI]
Blair, R. M., Fang, H., Branham, W. S., Hass, B. S., Dial, S. L., Moland, C. L., Tong, W., Shi, L., Perkins, R., and Sheehan, D. M. (2000). The estrogen receptor relative binding affinities of 188 natural and xenochemicals: Structural diversity of ligands. Toxicol. Sci. 54, 138153.
Buhl, A. E., Yuan, Y., Cornette, J. C., Frielink, R. D., Knight, K. A., Ruppel, P. A., and Kimbell, F. A. (1985). Steroid-induced urogenital tract changes and urine retention in laboratory rodents. J. Urol. 134, 12621267.[ISI][Medline]
Bulger, W. H., and Kupfer, D. (1983). Effect of xenobiotic estrogens and structurally related compounds on 2-hydroxylation of estradiol and on other monooxygenase activities in rat liver. Biochem. Pharmacol. 32, 10051010.[ISI][Medline]
Carr, B. R. (1998). Disorders of the ovaries and female reproductive tract. In Williams Textbook of Endocrinology. (J. D. Wilson, D. W. Foster, H. M. Kronenberg, and P. R. Larsen, Eds.), pp. 751818. W. B. Saunders, Philadelphia.
Chanda, S., Robinette, C. L., Couse, J. F. and Smart, R. C. (2000). 17ß-Estradiol and ICI-182780 regulate the hair follicle cycle in mice through an estrogen receptor- pathway. Am. J. Physiol. Endocrinol. Metab. 278, E202210.
Cotsarelis, G., Sun, T.-T., and Lavker, R. M. (1990). Label-retaining cells reside in the bulge area of pilosebaceous unit: Implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61, 13291337.[ISI][Medline]
Cox, N. M., Ramirez, J. L., Matamoros, I. A., Bennett, W. A., and Britt, J. H. (1987). Influence of season on estrous and luteinizing hormone responses to estradiol benzoate in ovariectomized sows. Theriogenology 27, 395405.[ISI]
Cui, W., Fowlis, D. J., Bryson, S., Duffie, E., Ireland, H., Balmain, A., and Akhurst, R. J. (1996). TGF-ß1 inhibits the formation of benign skin tumors, but enhances progression to invasive spindle carcinomas in transgenic mice. Cell 86, 531542.[ISI][Medline]
Dewailly, E., Mulvad, G., Pederson, H. S., Ayotte, P., Demers, A., Weber, J.-P., and Hansin, J. C. (1999). Concentration of organochlorines in human brain, liver, and adipose tissue autopsy samples from Greenland. Environ. Health Perspect. 107, 823828.[ISI][Medline]
Donaldson, G. M., Shutt, J. L., and Hunter, P. (1999). Organochlorine contamination in bald eagle eggs and nestlings from the Canadian Great Lakes. Arch. Environ. Contam. Toxicol. 36, 7080.[ISI][Medline]
European Commission (2001). European Commission Environment COM document; Candidate list of substances as a basis for priority setting, p. Annex 13.
Fisher, B. E. (1999). Most unwanted persistent organic pollutants. Environ. Health Perspect. 107, A1823.[ISI][Medline]
Fisk, N. R., Cymbalisty C. D., and Muir D. C. G. (1998). Dietary accumulation and depuration of hydrophobic organochlorines: Bioaccumulation parameters and their relationship with the octanol/water partition coefficient. Environ. Toxicol. Chem. 17, 951961.[ISI]
Fox, G., Won, H., and Grasman, K. A. (1998). Monitoring the elimination of persistent toxic substances from the Great Lakes: Chemical and physiological evidence from adult herring gulls. Environmental Monitoring and Assessment 53, 147168.[ISI]
Gaido, K., Maness, S. C., McDonnell, D. P., Dehal, S. S., Kupfer, D., and Safe, S. (2000). Interaction of methoxychlor and related compounds with estrogen receptor alpha and beta and androgen receptor: Structure-activity studies. Mol. Pharmacol. 58, 852858.
Hansen, L. A., and Tennant, R. W. (1994). Follicular origin of epidermal papillomas in v-Ha-ras transgenic TG. AC mouse skin. Proc. Natl. Acad. Sci. U.S.A. 91, 78227826.[Abstract]
Kaminuma, T., and Ohtake, C. (1997). Illinois EPA Endocrine Disruptors Strategy. Illinois EPA.
Kearney, J. P., Cole, D. C., Ferron, L. A., and Weber, J. (1999). Blood PCB, p,p-DDE, and mirex levels in Great Lakes fish and waterfowl consumers in two Ontario communities. Environ. Res. 80, S138149.[ISI][Medline]
Kim, T.-W., and Smart, R. C. (1995). Lack of effect of retinoic acid and fluocinolone acetonide on mirex tumor promotion indicates a novel mirex mechanism. Carcinogenesis 16, 21992204.[Abstract]
Kutz, F. W., Strassman, S. C., Stroup, C. R., Carra, J. S., Leininger, C. C., Watts, D. L., and Sparacino, C. M. (1985). The human body burden of mirex in the southeastern United States. J. Toxicol. Environ. Health 15, 385394.[ISI][Medline]
Lu, D., and Giguere, V. (2001). Requirement of Ras-dependent pathways for activation of the transforming growth factor beta3 promoter by estradiol. Endocrinology 142, 751759.
Madden, A., and Makarewicz, J. C. (1996). Salmonine consumption as a source of mirex in human breast milk near Rochester, New York. Journal of Great Lakes Research 22, 810817.[ISI]
Moser, G. J., Meyer, S. A., and Smart, R. C. (1992). The chlorinated pesticide mirex is a novel nonphorbol ester-type tumor promoter in mouse skin. Cancer Res. 52, 631636.[Abstract]
Moser, G. J., Robinette, C. L., and Smart, R. C. (1993). Characterization of skin tumor promotion by mirex: Structure-activity relationships, sexual dimorphism, and presence of Ha-ras mutation. Carcinogenesis 14, 11551160.[Abstract]
National Toxicology Program. (2001). The Ninth Report on Carcinogens. National Toxicology Program, Washington, DC.
New York Department of Health. (2001). Health AdvisoriesChemicals in Sport Fish and Game. New York Department of Health, Albany, NY.
Oh, H.-S., and Smart, R. C. (1996). An estrogen-receptor pathway regulates the telogen-anagen hair follicle transition and influences epidermal cell proliferation. Proc. Natl. Acad. Sci. U.S.A. 93, 1252512530.
Robertson, A. (1998). Distribution of chlorinated organic contaminants in dreissenid mussels along the southern shores of the Great Lakes. Journal of Great Lakes Research 24, 608619.[ISI]
Ryckman, D., Hamr, P., Fox, G. A., Collins, B., Edwins, P. J., and Norstrom, R. J. (1998). Spacial and temporal trends in organochlorine contamination and bill deformities in double-crested cormorants (Phalacrocorax auritus) from the Canadian Great Lakes. Environmental Monitoring and Assessment 53, 169195.[ISI]
Teo, S., and Vore, M. (1990). Mirex exposure inhibits the uptake of estradiol-17ß (ß-D-glucuronide), taurocholate, and L-alanine into isolated rat hepatocytes. Toxicol. Appl. Pharmacol. 104, 411420.[ISI][Medline]
Timchalk, C., Charles, A. K., and Abraham, R. (1985). Comparative changes in rat liver cytosolic proteins by mirex, diethylnitrosamine, and dimethylnitrosamine exposure. Proc. Soc. Exp. Biol. Med. 180, 214218.[Abstract]
Ulland, B. M., Page, N. P., Squire, R. A., Weisburger, E. K., and Cypher, R. L. (1977). A carcinogenicity assay of Mirex in Charles River CD rats. J. Natl. Cancer Inst. 58, 13340.[ISI][Medline]
U.S. EPA (2000). Great Lakes Pesticide Report, pp. 4.14.45. U.S. EPA, Washington, DC.
U.S. EPA. (2000). Integrated Risk Information System, Vol. 2000. U.S. EPA, Washington, DC.
U.S. EPA. (2001). Fish Consumption Advisory 2001, pp. 4.14.45. U.S. EPA, Washington, DC.
van Steenbrugge, G. J., Groen, M., van Kreuningen, A., de Jong, F. H., Gallee, M. P., and Schroder, F. H. (1988). Transplantable human prostatic carcinoma (PC-82) in athymic nude mice: III. Effects of estrogens on the growth of the tumor tissue. Prostate 12, 157171.[ISI][Medline]
Walker, S. E., McMurray, R. W., Besch-Williford, C. L., and Keisler, D. H. (1992). Premature death with bladder outlet obstruction and hyperprolactinemia in New Zealand black x New Zealand white mice treated with ethinyl estradiol and 17ß-estradiol. Arthritis Rheum. 35, 13871392.[ISI][Medline]