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-hydroxyestradiol, and 4-hydroxyestrone
1 Joint Graduate Program in Toxicology, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854 and University of Medicine and Dentistry of New Jersey – Robert Wood Johnson Medical School, USA
2 Laboratory of Cellular and Biochemical Toxicology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA
3 Laboratory for Neurotoxicology, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA
4 Hormonal Carcinogenesis Laboratory, Department of Pharmacology and Toxicology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA
5 Department of Environmental and Occupational Medicine, Environmental and Occupational Health Sciences Institute, and The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey – Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA
6 Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA
(Requests for offprints should be addressed to S Mesia-Vela, Laboratory for Cellular and Biochemical Toxicology, 41B Gordon Road, Piscataway, New Jersey 08854, USA; Email: smvela{at}yahoo.com)
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Abstract |
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-hydroxyestradiol (16-OH E2), or 4-hydoxyestrone (4-OH E1) (equimolar to 2 mg E2). Treatment with 1, 2, or 3 mg E2 resulted in the first appearance of a mammary tumor between 12 and 17 weeks, and a 50% incidence of mammary tumors was observed at 36, 19, and 18 weeks respectively. The final cumulative mammary tumor incidence in rats treated with 1, 2, or 3 mg E2 for 36 weeks was 50%, 73%, and 100% respectively. Treatment of rats with pellets containing 2-OH E2, 4-OH E2, 16-OH E2, or 4-OH E1 did not induce any detectable mammary tumors. The serum levels of E2 in rats treated with a 1 or 3 mg E2 pellet for 12 weeks was increased 2- to 6-fold above control values (~30 pg/ml). Treatment of rats with E2 enhanced the hepatic microsomal metabolism of E2 to E1, but did not influence the 2- or 4-hydroxylation of E2. In summary, we observed a dose-dependent induction of mammary tumors in female ACI rats treated continuously with E2; however, under these conditions 2-OH E2, 4-OH E2, 16-OH E2, and 4-OH E1 were inactive in inducing mammary tumors. |
Introduction |
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Numerous rodent studies have shown that administration of 17ß-estradiol (E2) is carcinogenic. E2 administration decreased the latency for the development of mammary adenocarcinoma, and increased the incidence of cervical, uterine, vaginal, and bone tumors in mice (Rudali et al. 1975, Highman et al. 1980, 1981), and increased the incidence of mammary and pituitary tumors in rats (Shull et al. 1997). Naturally occurring and synthetic estrogens are kidney carcinogens in castrated male hamsters (Li et al. 1983). Despite extensive studies, the carcinogenic mechanism(s) of estrogens remain unresolved.
Two major hypotheses have been put forth to explain the role(s) of estrogens in mammary cancer. The longest standing is the estrogen receptor agonist hypothesis (Preston-Martin et al. 1990, Henderson & Feigelson 2000, Santen 2002) which concludes that E2 and other ovarian estrogens cause proliferation of selective proliferating genes, secondary to the binding of E2 to estrogen receptor- and estrogen response elements in up-stream promoter regions. The second hypothesis is based on some hydroxylated metabolites (2-hydroxyestradiol (2-OH E2), 4-hydroxyestradiol (4-OH E2), or 4-hydroxyestrone (4-OH E1) primarily) being able to form catechol estrogen quinones that are genotoxic, inducing mutations that may lead to mammary cancer (Cavalieri et al. 2000, Liehr 2000, Yue et al. 2003).
E2 is metabolized to multiple hydroxylated metabolites, including the catechol estrogens 2-OH E2, 4-OH E2, and 4-OH E1, by rat liver microsomes (Suchar et al. 1996, Mesia-Vela et al. 2002), human liver microsomes (Lee et al. 2001), and by isolated human cytochrome P450 enzymes (Lee et al. 2003). 4-OH E2 and 16-hydroxyestradiol (16-OH E2) possess estrogenic activity and have been proposed as carcinogenic metabolites of E2. 4-OH E2, but not 2-OH E2, has strong carcinogenic activity in the Syrian hamster kidney (Liehr et al. 1986, Li & Li 1987) and in the uterus of CD-1 mice (Newbold & Liehr 2000). In addition, 4-hydroxylation of E2 was reported to be elevated in human breast tumor biopsies over that of normal breast tissue samples (Liehr & Ricci 1996, Rogan et al. 2003). Treatment with 16-OH E2 caused mammary tumors in mice (Rudali et al. 1975) and adrenal, mammary, and pituitary tumors in rats (Noble et al. 1975). Urine analyses of premenopausal women –OH showed that a higher ratio of urinary 2-OH E1 to 16 was associated with a decreased risk of developing E1 breast cancer after 5 years (Muti et al. 2000). Catechol estrogens (or the ortho quinones derived from these compounds) are believed to react with DNA and result in depurinating DNA adducts (Stack et al. 1996, Cavalieri et al. 1997, 2000, 2002) and oncogenic mutations (Chakravarti et al. 1995, 2001).
There are marked strain differences in susceptibility to estrogen-induced mammary oncogenesis in both mice and rats (Young & Hallowes 1973), and several rodent models are dependent upon treatment with 7,12-dimethyl-benz(a)anthracene, N-nitroso-N-methylurea, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine, and/or radiation to induce tumor formation (Broerse et al. 1989, Ip 1996, Snyderwine 1999). The ACI rat, a cross between the August and Copenhagen–Irish strains (Altman & Katz 1979), appears to be uniquely sensitive to estrogen-induced formation of mammary tumors (Cutts & Noble 1964, Holtzman et al. 1981, Shull et al. 1997, Spady et al. 1999, Harvell et al. 2000). Treatment of intact female ACI rats with E2 causes mammary tumors in 80–100% of the animals within a few months. In addition, the ACI strain has a very low incidence (11%) of spontaneous mammary tumors after 2 years (Segaloff & Maxfield 1971, Maekawa & Odashima 1975). After Dunning et al.(1947) first reported on the susceptibility of the ACI rat to diethylstilbestrol-induced mammary carcinogenesis, many other naturally occurring and synthetic estrogens such as E2, estrone (E1), and ethinylestradiol have also been shown to induce mammary tumors in this strain (Dunning & Curtis 1952, Stone et al. 1979, Holtzman et al. 1981). We chose to study the ACI rat model because the estrogen-induced mammary tumors closely resemble human breast tumors histologically and in many salient molecular aspects more closely than do the tumors induced in other rat strains by chemical or environmental carcinogens (Li et al. 2002a). The purpose of this study was to compare the in vivo carcinogenic potential of E2 versus the hypothesized carcinogenic metabolites.
In the present study, we have demonstrated that treatment of female ACI rats with E2 caused a dose-dependent induction of mammary tumors. We also found that administration of several metabolites of E2, including 2-OH E2, 4-OH E2, 16-OH E2, and 4-OH E1, which have been implicated as possibly playing a role in estrogen-induced mammary cancer, did not exhibit detectable oncogenic effects in the female ACI rat mammary gland.
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Materials and Methods |
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Female ACI rats were purchased from Harlan Sprague Dawley Inc. (Indianapolis, IN) at 7–8 weeks of age. The rats were housed individually in an American Association for Accreditation of Laboratory Animal Care-accredited barrier facility under controlled temperature, humidity, and lighting conditions, and provided with AIN-76A diet, a phytoestrogen-free, semi-synthetic diet (Dyets Inc., Pennsylvania, PA, USA and Research Diets, New Brunswick, NJ, USA) and water ad libitum. Treatment began 4–7 days after arrival. In one study, rats were implanted with a single 20 mg pellet containing cholesterol and 0, 1, or 3 mg E2 (n =8). In a second study, rats were implanted with a single 20 mg pellet containing cholesterol and 0 or 2 mg E2, or an equimolar dose of 2-OH E2, 4-OH E2, 16-OH E2, or 4-OH E1 (n =11–12). All pellets in the second study contained 5% ascorbic acid to stabilize the catechol estrogens. Since all of the pellets contained either 20 mg cholesterol alone (control) or with a corresponding amount of cholesterol replaced by an estrogen, we will refer to the E2 pellets by the amount of estrogen present.
The pellet implantation procedure, performed in a sterile environment, was adapted from a previously described method (Li et al. 1968). An incision was made in a shaved area in the middle of the back in rats anesthetized with an i.p. injection of 80 mg/kg ketamine and 12 mg/kg xylazine. Pellets were implanted subcutaneously between the scapulas through the incision site, which was closed with a single wound clip. Rats were monitored daily for a week after which the wound clip was removed. Pellets with or without estrogens (Sigma Chemical Co., St Louis, MO, USA) were purchased from Hormone Pellet Press (Shawnee Mission, KS, USA).
The rats were palpated for tumors twice weekly and weighed every 2 weeks for the duration of the study. The dates of appearance and location of tumors were noted. Rats were terminated when a mammary tumor reached approximately 3 cm2 in size or at the end of experimental periods (36 weeks). Additional rats were implanted with 0, 1, or 3 mg E2, and killed for E2 metabolism studies and serum E2 measurements after 6 or 12 weeks of treatment, which was prior to the development of mammary tumors (n=6).
Blood and tissue collection
Blood was collected by cardiac puncture during anesthesia immediately prior to necropsy. The blood was allowed to clot at 4°C for 6 h, and centrifuged. The serum was collected and stored at –80°C. Serum levels of E2 were determined by RIA, using Coat-A-Count estradiol RIA kits, according to the manufacturer’s instructions (Diagnostic Products Corporation, Los Angeles, CA, USA).
At necropsy, all the rats were subjected to gross pathologic examination, and the presence of mammary tumors was recorded. The mammary glands and the mammary tumors were quickly removed, portions were fixed in Carnoy’s solution for 4 h and processed for paraffin embedding, while unfixed portions of tissues were immediately frozen in liquid nitrogen and stored at –80°C. Pituitary gland, thymus, liver, uterus, kidneys, and adrenals were removed and weighed. Portions of pituitary gland and liver were fixed in Carnoy’s solution. For morphological examination, tissue sections (6 µm) were prepared and stained with hematoxylin and eosin.
Hepatic microsomal metabolism of E2
Hepatic microsomes were prepared by differential centrifugation as described previously (Thomas et al. 1983) and stored at –80°C until analyzed. Protein concentrations were determined by the BCA protein assay kit (Pierce, Rockford, IL, USA) according to the supplier’s instructions, using bovine serum albumin as a standard.
NADPH-dependent oxidation of E2 was carried out as previously described (Suchar et al. 1995). Hepatic micro-somes were incubated with 5 mM ascorbic acid, 3 mM magnesium chloride, 50 mM sodium-phosphate buffer (pH 7.4), and 50 µM [3H]E2 (0.5 µCi) for 20 min at 37°C. The enzyme reaction was initiated by the addition of 2 mM NADPH and terminated by the addition of 5 ml spectral grade ethyl acetate and shaken to extract E2 and its metabolites. The ethyl acetate extracts were evaporated to dryness under nitrogen. The residues were resolubil-ized in methanol and analyzed for estrogen metabolite formation by HPLC as previously described (Suchar et al. 1995).
Quantification of E2 and its metabolites in cholesterol pellets
Pellets from rats treated with 2 mg E2, an equimolar amount of 2-OH E2, 4-OH E2, 16-OH E2, or 4-OH E1, and corresponding controls were removed at necropsy and kept frozen at –20°C until HPLC analysis. The pellets to be analyzed were those removed from rats treated with 2 mg E2 for 35–36 weeks (n =6), and those removed from the other treatment groups after 36 weeks (n=11–12). After removal of adherent tissue from each pellet, they were individually dissolved in 1 ml of a mixture consisting of methanol:chloroform (1:2). Twenty microliters of this solution were diluted in 1 ml methanol for HPLC analysis.
The HPLC system consisted of a Shimadzu SCL-6B system controller, Shimadzu CR4-A integrator (Shimadzu Scientific Instruments, Columbia, MD, USA), Luna Phenomenex, Torrance, CA, USA 250x2.0 mm C-18 column maintained at 25°C, and a u.v. detector set at 280 nm. Mobile phase A consisted of 0.1% acetic acid, and mobile phase B (B) consisted of methanol:acetonitrile (20:80) containing 0.1% acetic acid. Aliquots (10 µl) of the pellet dilutions were injected into the HPLC. Fractions from the solutions of control and E2-containing pellets were eluted at a constant flow rate of 0.25 ml/min with a 20 min linear curve from 35 to 90% mobile phase B, 2 min 90% B, 2 min linear curve from 90 to 35% B, and re-equilibrated for 12 min at 35% B. Similarly, fractions from the solutions of pellets containing 2-OH E2, 4-OH E2, 16-OH E2, or 4-OH E1 were eluted at a constant flow rate of 0.25 ml/min with a 2 min linear curve from 35 to 70% B, 18 min convex gradient (SCL-6B time/ program menu –10) to 90% B, 2 min 90% B, 2 min linear curve from 90 to 35% B, and re-equilibrated for12 min at 35% B. The amount of estrogen present in each pellet was calculated by fitting the data to standard curves that were created from serial dilutions and HPLC analysis of authentic steroids (Steraloids, Wilton, NH, USA).
Statistical analysis
The data presented represent the means±S.E.M. Differ-ences between means were assessed using two-way ANOVA followed by Bonferroni post hoc test using GraphPad Prism software (San Diego, CA, USA). P values <0.05 were considered significant.
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Results |
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In the first study, we compared the formation of mammary tumors in female ACI rats treated with 1 or 3 mg E2 (Fig. 1
) while, in the second study, we examined the effect of a 2 mg E2 pellet (Fig. 2). A 50% incidence of palpable mammary tumors was observed after 18 weeks for 3 mg E2, 19 weeks for 2 mg E2, and 36 weeks for 1 mg E2. At termination after 36 weeks, the incidence of palpable mammary tumors was 100% for 3 mg E2, 73% for 2 mg E2, and 50% for 1 mg E2 (Figs 1 and 2). A 100% incidence of mammary tumors was observed after 24 weeks of treatment with 3 mg E2.
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Body and organ weight responses
Treatment of ACI rats with 1 or 3 mg for 6 or 12 E2 weeks had no effect on body weight, uterine weight, or adrenal weights, while the weights of the liver and pituitary gland were significantly increased and those of the thymus were significantly decreased (Table 1). Kidney weights were significantly increased in rats at all time-periods of 3 mg E2 treatment. Rats treated with 1 or 3 mg E2 for 24–36 weeks exhibited significant decreases in body weights, and significant increases in liver and pituitary weights.
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Treatment of ACI rats with 2 mg E2 had similar effects as were observed after treatment with 1 or 3 mg E2 for 24–36 weeks. Liver and pituitary weights were signifi-cantly increased with 2 mg treatment, while body E2 weight was significantly reduced compared with control rats (Table 2). The hepatic morphologic changes observed after treatment with 2 mg E2 were similar to those described above for rats treated with 1 and 3 mg E2. Treatment with 2-OH E2, 4-OH E2, 16-OH E2, or4-OH E1 did not affect liver histology or body, uterus/ovary, liver, or pituitary weights.
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Histopathological examination of mammary glands from rats treated with cholesterol for 6, 12, or 36 weeks showed complete absence of mammary lesions. Rats treated for 36 weeks with 1 mg exhibited an increased number of E2 mammary gland ducts and proliferation of the terminal lobular units; however, only 50% of the rats presented palpable mammary tumors within that time-period. The terminal duct units were dilated and filled with epithelial and myoepithelial cells and displayed intraductal epithelial hyperplasia. The mammary glands of ACI rats treated with for as little as 6 weeks presented atypical epithelial E2 hyperplasia. The rats treated with 1 and 3 mg that E2 developed palpable mammary tumors showed classical morphological features of mammary ductal adenocarcino-mas with a variety of histological patterns. Most of the mammary tumors displayed solid phase histology, with masses of closely packed neoplastic cells with large zones of necrosis. Other mammary tumors showed a classical cribiform phenotype or a ‘ribbon’ pattern consisting of long rows of cells, often three to five deep, aligned in undulating strips. Generally, most mammary tumors appeared encapsulated by bands of connective tissue, while others showed compression of adjacent stroma and micro-glandular invasion. Considerable nuclear pleomorphism was present and mitotic figures were abundant within the tumors. There was no evidence of distant metastasis.
At necropsy, the pituitaries of the E2-treated rats appeared tan-brown to frankly hemorrhagic and displayed modest to massive enlargement. By 12 weeks, the pituitaries of rats treated with 3 mg E2 were increased by as much as 6-fold in weight, by which time the masses had begun to erode the sella turcica and compress the overlying cerebrum. Similar features were observed in 2 mg rats after 24–36 weeks. Morphologically, E2-treated the pituitaries consisted predominantly of hyperplastic lactotrophic pituicytes, often interspersed with large zones of hemorrhage and necrosis. Mitotic figures were frequent in many visual fields.
Serum E2 levels in rats treated with E2
Circulating serum E2 levels were determined at 6, 12, 27, and 36 weeks in rats treated with 1 and 3 mg E2 (Fig. 3). In control rats, serum E2 levels remained relatively constant at 30±5 pg/ml. After 12 weeks of treatment with 1 or 3 mg E2, the serum E2 level increased 2.2- or 6.4-fold above control levels respectively. No further changes in serum E2 levels were observed at later time-intervals.
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NADPH-dependent oxidation of E2 by hepatic microsomes produced several metabolites (Table 3). The major products of E2 oxidation in hepatic microsomes from control rats were 2-OH E2 and E1 as previously reported (Mesia-Vela et al. 2002), and small amounts (~5%) of 4-OH E2 and 2-OH E1 were also detected. Treatment of rats with 1 or 3 mg pelleted E2 for 6 or 12 weeks resulted in a 50–70% increase in the hepatic microsomal formation of E1 (Table 3); however, the formation of 2- and 4-hydroxylated estrogens was not affected.
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-OH E2, and 4-OH E1 as mammary carcinogens
In the second study, we compared the oncogenic effects of E2, 2-OH E2, 4-OH E2, 16-OH E2, and 4-OH E1 in the mammary gland of female ACI rats. Since the data from our first study indicated that a 3 mg dose of E2 resulted in a 100% mammary tumor incidence while a 1 mg dose of E2 induced a 50% incidence of mammary tumors, we selected an intermediate dose, 2 mg E2, for this comparative study. ACI rats treated with 2 mg E2 exhibited a 73% incidence of mammary tumors after 36 weeks of treatment. No mammary tumors were detected in any of the rats treated with equimolar doses of 2-OH E2, 4-OH E2, 16-OH E2, or 4-OH E1 after the same time-period (Fig. 2
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Quantification of E2 and its metabolites in cholesterol pellets
To ensure that the catechol estrogens present in the pellets were released in an amount similar to that of E2, the percentage of estrogen remaining in the pellets containing 2 mg E2 or equimolar amounts of 2-OH E2, 4-OH E2, 16 -OH E2, or 4-OH E1 was determined at necropsy. The HPLC retention times observed for each of the individual estrogens were the same as those for their authentic respective standards, indicating that the remaining estrogen present in the pellets was not altered from its original form. Estrogens were not detected in any of the 20 mg cholesterol pellets (control). In the estrogen-containing pellets, significantly less 4-OH E1 remained in the pellet at necropsy than was left in pellets from E2, 2-OH E2, 4-OH E2, or 16-OH E2, and significantly less 4-OH E2 remained compared with E2 (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-OH E2, and 4-OH E1, administered in cholesterol pellets in the estrogen-sensitive female ACI rat. The data presented herein indicated that only low amounts of exogenous are required to consistently E2 induce mammary tumors in female ACI rats. The incidence and the latency periods for mammary tumors observed in these studies are similar to those induced by diethylstilbestrol, using a similar pellet delivery system in ACI rats (Stone et al. 1979). We observed highly mitotic solid, cribiform, and papillary patterns in some mammary tumor samples, but not the carcinomas with invasive and metastatic features that have been reported elsewhere (Meites 1972, Harvell et al. 2000).
Estrogens have been proposed to trigger breast cancer through oxidative stress induced by catechol estrogens formed by NADPH-dependent oxidation of E2 (Liehr 2000). We studied alterations in catechol estrogen formation by isolated microsomes from livers of rats treated with E2. In our studies, no alterations in the amount of 2- or 4-hydroxylation of E2 by hepatic microsomes from ACI rats treated with were found at any of the doses or E2 time-periods studied. This suggests that E2 treatment did not significantly alter the expression of hepatic cytochrome P450 1A, 2B, or 3A isoforms responsible for the NADPH-dependent oxidation of E2 to catechol estrogens in rats. Strain differences in estrogen metabolite formation can be ruled out, as we have shown previously that the liver of the ACI rat generates similar levels of catechol estrogens as the Sprague–Dawley rat (Mesia-Vela et al. 2002), which is less sensitive to mammary tumors induced by E2 treatment.
Additionally, the possible role of 2-OH E2, 4-OH E2, 16-OH E2, or 4-OH E1 in mammary carcinogenesis was studied by investigating their effects on tumor incidence when these metabolites were administered exogenously. No mammary tumors were induced by 2-OH E2, 4-OH E2, 16-OH E2 or 4-OH E1 when administered in a manner similar to E2 and at doses equimolar to 2 mg E2. The absence of mammary tumors with these metabolites of E2 was not due to poor release of these metabolites from the cholesterol pellets. The amount of metabolites recovered from the pellet at necropsy was similar to or less than the amount of E2 recovered (Fig. 4). This indicates that the total exposure to each of these E2 metabolites was either greater (4-OH E2 and 4-OH E1) than that of E2 or was comparable with that of E2. Since all of these metabolites are more water soluble than E2, they could be cleared faster. It may be critical for these metabolites to be formed in or near the mammary tissues to be effective as initiators of carcinogenesis. Although unlikely, one or more of the metabolites could have initiated mammary carcinogenesis in a few cells but the circulating levels of endogenous estrogens was insuffcient to promote those cells to a stage that was palpable or visible at the time of necropsy. It is of interest that El-Bayoumy et al. (1996) injected E1-3,4-quinone under the nipples of 30 day old female CD rats but, detected no tumors 44 weeks post treatment. All of the metabolites in our study are markedly less effec-tive estrogen receptor agonists compared with itself E2 (Martucci & Fishman 1979, Kuiper et al. 1997). The low doses of E2, but not any of the metabolites, were sufficient to affect various estrogen-responsive organs such as liver, uterus, and pituitary gland.
In summary, the results have indicated that female ACI rats treated with cholesterol pellets containing low doses of E2 are a useful animal model for evaluating the mechanisms of estrogen-induced breast cancer. This is the first report to our knowledge that shows a dose dependency for E2-induction of mammary tumors using an amount of E2 that approximates physiological concentrations. Collectively, the lack of increased formation of reactive catechol estrogens by hepatic microsomes during continuous E2 administration together with the failure of these hydroxylated estrogen metabolites administered via cholesterol implants to exhibit any oncogenic potential suggests that the E2 induction of mammary cancer in the ACI rat occurs as a consequence of molecular events arising primarily from E2 or from E2 metabolites generated within or in close proximity to oncogenic targets in the mammary gland.
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Acknowledgements |
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Received 24 June 2004
Accepted 13 July 2004
Made available online as an Accepted Preprint 23 July 2004
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)), 1 mg E2 (
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) or an equimolar dose of 2-OH E2, 4-OH E2, 16
