Introduction

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Sex hormones are implicated in the development of a variety of human cancers (1-4). Estrogen administration to postmenopausal women is associated with an increased risk of endometrial and breast cancer (1-4). An increasing evidence of elevated breast cancer risk with increases in total lifetime exposure of women to estrogens has been presented (1-3). Recently, the clinical trial of estrogen plus progestin treatment therapy was stopped because of an increased risk of breast cancer (5). Knowledge of how estrogens induce proliferation and tumorigenesis in their target organ is not well defined (6-9). The mechanism of tumor induction by estrogens is being investigated in rodent models of hormonal carcinogenesis. The natural female sex hormone 17-estradiol (E2) and the synthetic estrogen diethylstilbestrol induce tumors in rats, mice, and hamsters (10-13). It must be noted that in rodent models, different estrogens tested have not shown similar carcinogenic potential despite their similar hormonal potencies (6, 14, 15). However, carcinogenic and noncarcinogenic estrogens differ in their metabolic activation profiles (14-17). Therefore, it is postulated that estrogen metabolism may play a key role in hormonal carcinogenesis.

Estrogens can be metabolically activated into catechol estrogens by cytochrome P450 enzymes (18, 19). Metabolic redox cycling between catechol estrogens and their corresponding quinones generates oxidative stress and potentially harmful free radicals that are postulated to be required for the carcinogenic process, and analogous to the metabolic activation of hydrocarbons and other nonsteroidal estrogen carcinogens (9, 19-22). We have investigated the role of oxidative stress in estrogen carcinogenesis by using a well established hamster renal tumor model that shares several characteristics with human breast and uterine cancers, pointing to a common mechanistic origin (6, 9, 23). Different estrogens used in the present study differ in their estrogenic, carcinogenic, and metabolic activation potentials (14-17). E2 is a good catechol progenitor and a potent estrogen; its use results in 80-100% tumor incidence in the hamster kidney (6, 10, 14, 15). 17-estradiol (E2) is a nontumorigenic, weak estrogen with a catechol-forming potential similar to that of E2 (24, 25). 17-Ethinylestradiol (EE) is a potent estrogen, but a weak catechol progenitor that is either nontumorigenic or very weakly tumorigenic in the hamster model with >9 months of continuous exposure required for less than 10% tumor incidence (15, 17). Menadione (2-methyl-1,4-napthaquinone) is used in the present study as a model compound with known oxidant stress potential to study the influence of oxidative stress on estrogen-induced carcinogenesis (26-28). We used a combination of an oxidant chemical menadione and a noncarcinogenic estrogen EE to show the induction of renal tumors in a rodent model of hormonal carcinogenesis. We also demonstrated increased levels of 8-iso-prostaglandin F₂ (8-iso-PGF₂), a known marker of oxidant stress (29, 30), in E2-induced tumor-bearing kidneys as well as in menadione plus EE-treated kidneys of hamsters. Our studies suggest that oxidative stress plays a critical role in estrogen-induced carcinogenesis.

	Materials and Methods

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Treatment of Animals.

Male Syrian hamsters (4-6 weeks old; Harlan Sprague-Dawley, Madison, WI) were housed in our animal facility with Purina rodent chow and water available ad libitum throughout the experiment. Hamsters were implanted s.c. with 25-mg pellets of E2, E2, EE, menadione, a combination of E2 + EE, or a combination of EE + menadione. These hamsters received a second estrogen or menadione pellet 3 months after initial treatment. Before implantation of the drug pellets, hamsters were anesthetized with a combination of ketamine and xylazine (ketamine, 100 mg/kg body weight, i.p., and xylazine, 10 mg/kg body weight, i.p.). Estrogen and menadione pellets were prepared by using a hand press and implanted into the hamsters s.c. as described (10, 12). There were 10 hamsters in each group. A control group of 10 animals was sham operated and left untreated. Hamsters were killed after 7 months and inspected macroscopically for tumor nodules on the surface of each kidney as reported (31). Portions of liver and kidney tissue were placed on dry ice and stored at 80°C for further studies. Other portions of liver and kidney were placed in 10% buffered formalin for histopathological evaluation and immunocytochemical studies. Menadione and all estrogens used in the present study were purchased from Sigma.

Tissue Preparation for Histopathology and Immunocytochemistry.

The formalin-fixed tissue was embedded in paraffin, and sections of 4- to 5-µm thickness were cut. Paraffin-embedded sections of the kidneys and livers were stained with hematoxylin and eosin for histopathological evaluation. Gross examination and histological sections were interpreted by two independent pathologists in a blinded fashion, without knowledge as to how the animals were stratified. Paraffin-embedded sections were also used for cell-specific expression of estrogen receptor (ER)- and - proteins. Deparaffinized sections were incubated with the corresponding primary antibodies: ER- (Santa Cruz Biotechnology, SC-542) and ER- (Santa Cruz Biotechnology, SC-6821) at dilutions suggested by the suppliers. Incubation with the primary antibodies was performed overnight at 4°C. Nonspecific sites were blocked by covering sections with solutions of 1% BSA (Sigma). After washing in PBS, the sections were incubated with peroxidase-conjugated, affinity-purified F(ab')₂ fragment of donkey anti-rabbit IgG (Jackson ImmunoResearch) for 60 min at room temperature as described (12). Slides were rinsed three times in PBS, and staining was developed by incubating with 3,3'-diaminobenzidine tetrachloride/H₂O₂ (Sigma) for 2-5 min. 3,3'-Diaminobenzidine solution was prepared according to the manufacturer's recommendations. After staining, the sections were rinsed in distilled water, dehydrated in ethanol/water baths with decreasing water content, and finally rinsed in xylene before being mounted with a permanent mounting medium.

Semiquantitative Analysis of Critical Histopathological Differences.

The severity of the total kidney damage was evaluated by a scoring system that gave a semiquantitative measurement of the damage as described (32). The four types of damage for which semiquantitative analysis was performed were glomerular atrophy, glomerular congestion, tubular congestion, and nodular proliferation. One hundred nephrons (glomeruli and adjacent tubules) were examined histologically, and each nephron was scored from 0 to 4. For glomerular atrophy, glomeruli that contained >20 nuclei were graded as "0," and those glomeruli which contained <10 nuclei were scored as "4." For glomerular congestion, glomeruli that did not contain eosionophilic and pink deposits were graded as "0," and those that had 80% of glomeruli with such deposits were graded as "4." For tubular congestion, convoluted tubules that did not contain eosinophilic and pink deposits were graded as "0," and those convoluted tubules that contained  80% deposits were graded as "4." Intermediate stages were graded as 1, 2, and 3. For proliferation, sections of kidneys were graded as "4" if they contained tumor nodules, and as "0" if they did not have any tumor nodules. The sum of individual scores of 100 counts on each kidney section was used as an estimate of the severity of the kidney damage. A total score of 1,600 was given by the sum of the four descriptive damages described above (each kind of damage getting a maximum score of 400). For each type of damage, six to eight hematoxylin and eosin-stained kidney sections were examined from each treatment group and scored, and data were expressed as a mean ± SEM of the individual scores.

Analysis of 8-iso-PGF₂ Levels.

Total 8-iso-PGF₂ levels in kidney tissue of hamsters treated with different chemicals were quantified by using a direct 8-iso-PGF₂ enzyme immunoassay kit from Assay Designs (Ann Arbor, MI, catalog no. 900-091) according to the supplier's recommendations. Kidney tissue (50-100 mg) was homogenized in cold PBS (pH 7.4) containing 0.005% butylated hydroxytoluene. Tissue homogenates (10% wt/vol) were prepared by using a PRO 200 homogenizer with a 5 mm × 75 mm generator (PRO Scientific, Oxford, CT) in 2-ml microfuge tubes. Homogenization was carried out by moving the motor speed dial of the homogenizer from 0 to 5 (0-30,000 rpm) back and forth five times with a total homogenization time of 5 s. 8-iso-PGF₂ esters in 100 µl of the total kidney homogenate were hydrolyzed by incubation with 25 µl of 10 N NaOH at 45°C for 2 h. The reaction mixture was cooled on ice for 5 min and neutralized with 25 µl of 12 N HCl, and centrifuged in a microcentrifuge for 5 min. The clear neutralized supernatant was transferred into a new microfuge tube, and 50 µl of the neutralized sample was used for 8-iso-PGF₂ assay. The samples were incubated with the 8-iso-PGF₂ antibody for 18 h at 4°C in a 96-well format. After incubation, the contents of the wells were emptied and washed with wash buffer; wash buffer was removed from the wells, and the color was developed by incubation with 200 µl of p-nitrophenyl phosphate for 45 min at room temperature. The reaction was stopped by the addition of 50 µl of stop solution, and the plate was read at 405 nm. A standard curve was generated by measuring the optical density of 160-100,000 pg/ml of 8-iso-PGF₂ standards that were processed simultaneously with unknown samples on the same plate. Protein concentrations from the neutralized homogenates were determined by using a Pierce protein assay kit. 8-iso-PGF₂ and protein were analyzed from 10 kidney homogenates from each group, and data were expressed as mean 8-iso-PGF₂ pg/mg protein ± SEM.

Statistical Analysis.

Statistical analysis was performed by using SPSS statistical software package (SPSS, Chicago). Unpaired t test was used to assess significance between the two different treatment groups. Tumor incidence was analyzed by Fisher's exact test.

	Results

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Tumor Incidence.

On macroscopic examination, no tumor nodules were detected in untreated hamsters and in groups of hamsters that were treated with E2, EE, menadione, or a combination of E2 and EE for 7 months (Table 1). In contrast, hamsters treated with either E2 or a combination of EE and menadione for 7 months developed renal tumors. The tumor incidence was 90% for the group treated with E2 and 30% for the group treated with a combination of EE and menadione (Table 1). The mean number of tumor nodules was higher in the group treated with E2 compared with the group treated with a combination of EE and menadione (Table 1).

Histopathological Analysis.

Sections from untreated hamster kidneys demonstrated normal convoluted tubules and glomeruli within the cortex (Fig. 1). The corticomedullary junction appeared normal, as did the renal hilum, which was lined by normal transitional urothelium.

View larger version (136K): [in this window] [in a new window]   Fig. 1.   Paraffin section of an untreated male Syrian hamster kidney stained with hematoxylin and eosin. Normal kidney architecture with normal convoluted tubules (CT) and glomerulus (G) is observed. Magnification = ×40.

Kidneys of hamsters treated with E2 for 7 months were abnormal and contained numerous tumor nodules (Fig. 2a). The nodules were composed of a combination of round hyperchromatic cells and round to spindled, irregular, hyperchromatic cells (Fig. 2b). Scattered tubules within the tumor nodules were congested (Fig. 2a). Most of the tumor nodules were in the cortical area of the kidney and, in some of the nodules, entrapped and atrophic glomeruli were seen (Fig. 2c). Sections from the kidney away from the tumor were also markedly abnormal and demonstrated large dilated congested convoluted tubules, which were lined by somewhat flattened, cuboidal epithelial cells (Fig. 2d). Many of the congested tubules were filled with pink eosinophilic deposits (Fig. 2d). A significant increase in the number of congested convoluted tubules, congested glomeruli, atrophic glomeruli, and nodular proliferation was detected in the E2-treated tumor-bearing kidneys compared with untreated controls by using the semiquantitative estimates of the total kidney damage (Fig. 3).

View larger version (127K): [in this window] [in a new window]   Fig. 2.   Paraffin section of a tumor-bearing kidney stained with hematoxylin and eosin. The tumors were induced by treatment of male Syrian hamsters with E2 for 7 months. (a) An abnormal kidney architecture with tumor nodules (T) and congestion of scattered convoluted tubules (arrow) within the tumor nodules can be observed. (b and c) The tumor nodules are composed of a combination of round to spindled hyperchromatic cells (b, arrows), and in some of the tumor nodules (T), entrapped and atrophic glomeruli (G) are present (c). Many of the congested tubules (Tb) are filled with pink eosinophilic deposits (arrow in c, arrowhead in d) and are lined by somewhat flattened epithelial cells (d, arrow). Magnification: a = ×4; b = ×40; c = ×10; d = ×40.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Semiquantitative estimate of the total kidney damage in hamsters treated with 25-mg pellets of E2, E2, EE, menadione, a combination of E2 + EE, or a combination of EE + menadione for 7 months as s.c. implants. The control group was sham operated and received no estrogen implants. Hematoxylin and eosin-stained sections of paraffin-embedded tissues were analyzed microscopically for the extent of renal damage as described in Materials and Methods. Increased congestion of the convoluted tubules (c-ct) and glomeruli (c-g) was observed in kidneys of hamsters treated with E2, menadione, a combination of E2 + EE, or a combination of EE + menadione. Glomerular atrophy (a-g) and proliferation (p) is observed in kidney tissue of the groups treated with E2 or a combination of EE + menadione for 7 months. 1, untreated control; 2, E2; 3, EE; 4, E2 + EE; 5, menadione; 6, menadione + EE; 7, E2. a, P < 0.05 for both c-g and c-ct when compared with untreated, E2-treated, or EE-treated hamster kidney by an unpaired t test. b and c, P < 0.05 for a-g and p, respectively, when compared with any other group by an unpaired t test. For each type of damage, six to eight hematoxylin and eosin-stained kidney sections were examined from each treatment group and scored, and data are expressed as a mean of the individual scores. The SEM for all of the four types of kidney damage (c-ct, c-g, a-g, and p) in all of the seven different treatment groups varied from 0% to 22% except for c-ct for the group that was treated with E2, where the % error was 64. This group showed very little kidney damage.

Microscopic evidence of tumor nodules was not seen in any of the kidneys of hamsters treated with E2 for 7 months. Kidney sections showed normal glomeruli and convoluted tubules within the renal cortex. No abnormal proliferations were seen. The medulla and hilum were microscopically normal (data not shown).

Atypical congested convoluted tubules were seen in kidneys of hamsters treated with EE for 7 months (Fig. 3). In some areas, the convoluted tubules were lined by crowded hyperchromatic cuboidal cells, which had decreased cytoplasm, and the kidney sections also showed some congested glomeruli (see Fig. 7, which is published as supporting information on the PNAS web site, www.pnas.org). No nodular proliferation was detected in the kidney sections of hamsters treated with EE for 7 months (Fig. 3).

Kidney sections of hamsters treated with menadione for 7 months demonstrated renal convoluted tubules with evidence of vascular as well as glomerular congestion (Fig. 3) and the presence of an altered epithelial layer of convoluted tubules (see Fig. 8, which is published as supporting information on the PNAS web site). No tumor nodules were identified, and there was no evidence of atrophic glomeruli (Fig. 3).

On microscopic examination, vascular congestion of glomeruli and of convoluted tubules was observed in the kidneys of hamsters treated with a combination of E2 plus EE for 7 months (Figs. 3 and 4a). Kidneys contained foci where there were atypical collections of both interstitial cells and convoluted tubules (Fig. 4b). In these areas, some of the tubules contained cuboidal cells, which had scant cytoplasm and irregular, slightly pleomorphic, hyperchromatic nuclei (Fig. 4c). Some slightly crowded hyperchromatic renal collecting tubules were observed (Fig. 4d).

View larger version (123K): [in this window] [in a new window]   Fig. 4.   Paraffin section of a male hamster kidney stained with hematoxylin and eosin. Male Syrian hamsters were treated with a combination of EE and E2 for 7 months. (a) Vascular congestion of glomeruli (G) and convoluted tubules (CT) is observed. (b) Kidneys contain foci where there are atypical collections of both interstitial cells (arrow) and CT. (c) Some of the tubules contain cuboidal cells that have scant cytoplasm and irregular, slightly pleomorphic hyperchromatic nuclei (arrowheads). (d) Some slightly crowded renal collecting tubules (arrows) are also observed in kidneys of hamsters treated with a combination of EE and E2 for 7 months. Magnification: a-c = ×40; d = ×10.

Kidney sections of hamsters treated with a combination of EE plus menadione demonstrated foci of tumor that contained congested tubules (Fig. 5a). The tumor nodules were characterized by hyperchromatic, spindled to rounded cells with marked nuclear crowding and overlapping (Fig. 5b). This group showed significant congestion of both renal convoluted tubules and glomeruli when compared with E2 and EE treated groups (Fig. 3). Glomerular atrophy and nodular proliferation were detected in the kidneys of hamsters treated either with a combination of EE plus menadione, or with E2 for 7 months (Figs. 3 and 5b).

View larger version (135K): [in this window] [in a new window]   Fig. 5.   Paraffin section of a tumor-bearing kidney stained with hematoxylin and eosin. The tumors were induced by treatment of male Syrian hamsters with a combination of EE and menadione for 7 months. (a) Sections of the kidney demonstrate foci of tumor (T) with congested renal tubules (Tb and arrowheads). (b) The tumor is comprised of hyperchromatic cells with nuclear crowding (arrows). Congested renal tubules (arrowheads) and atrophied glomeruli (G) are also seen entrapped within the tumor nodule. Magnification: a = ×10; b = ×40.

Microscopic examination of tissue sections from livers of hamsters treated with E2, E2, EE, menadione, E2 + EE, or menadione + EE did not show any evidence of tumor or dysplasia (data not shown).

Immunocytochemical Analysis.

Renal tumors induced by a carcinogenic estrogen or by a combination of a noncarcinogenic estrogen and a chemical known to produce oxidative stress were characterized immunocytochemically. Cell-specific expression of ER- and - was examined in the kidneys of hamsters treated with different estrogens, either alone or in combination with menadione. ER- protein expression was not detected either in control or in tumor-bearing kidneys (data not shown). A very poor immunoreactivity for ER- protein was observed in the tubules of untreated control kidneys (see Fig. 9, which is published as supporting information on the PNAS web site). Intense nuclear staining for ER- protein was found in E2-induced renal tumors (see Fig. 9). ER- protein expression was not detected in the glomeruli or kidney tubules of menadione-treated animals (data not shown). Moderate to strong immunoreactivity for ER- protein was observed in the renal tumor area of EE plus menadione-treated hamsters and some of the tubules within the tumor mass also expressed high levels of ER- protein (see Fig. 9).

8-iso-PGF₂ Analysis.

Total 8-iso-PGF₂ levels in kidney tissue of hamsters treated with different chemicals were quantified. A >2-fold increase in 8-iso-PGF₂ was detected in tumor-bearing kidney homogenates of hamsters treated with E2 for 7 months compared with untreated controls (Fig. 6). Kidney homogenates of hamsters treated with EE for 7 months did not show any increase in 8-iso-PGF₂ levels compared with untreated controls (Fig. 6). The fold increases in 8-iso-PGF₂ were 1.38, 1.50, 1.52, and 1.55, respectively, for kidneys of hamsters treated with E2, menadione, E2 + EE, and menadione + EE compared with untreated controls (Fig. 6). There were no significant differences in 8-iso-PGF₂ levels between E2-, menadione-, E2 + EE-, and menadione + EE-treated groups.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   8-iso-PGF2 levels in kidney homogenates of hamsters treated with E2, E2, EE, menadione, E2 + EE, or menadione + EE for 7 months. 8-iso-PGF2 was quantified by using a commercially available kit from Assay Designs as described in Materials and Methods. A >2-fold increase in 8-iso-PGF2 is detected in E2-treated tumor-bearing kidneys of hamsters. The fold increases in 8-iso-PGF2 are 1.38, 1.50, 1.52, and 1.55, respectively, for kidneys of hamsters treated with E2, menadione, E2 + EE, and menadione + EE compared with untreated controls. 8-iso-PGF2 and protein were analyzed from 10 kidney homogenates from each group, and data are expressed as mean 8-iso-PGF2 pg/mg protein ± SEM. *, P < 0.05 compared with untreated controls by an unpaired t test; **, P < 0.05 compared with EE treated group by an unpaired t test.

	Discussion

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Several potent estrogens, such as EE, 2-fluoroestradiol, and 11-methylestradiol, have previously been identified, which are weakly or not at all carcinogenic despite their high hormonal potencies as measured by receptor binding, uterine wet weight increase, or other assays of estrogenic activity (6, 14, 15, 24, 33, 34). A number of studies of estrogen-induced carcinogenesis indicate that tumor induction depends on estrogen metabolism, and oxidative stress as a result of the redox cycling of estrogen metabolites (6, 16, 20, 21, 35). It has been shown that E2 in target organs of estrogen-induced cancer is metabolized to 2- and 4-hydroxyestradiol, commonly known as catechol estrogens (16, 36, 37). Elevated estradiol-4-hydroxylase activity has also been shown in organs that are prone to estradiol-induced hyperplasia or cancer in rodents, in humans, and in human breast cancer cell lines (16, 18, 36-40). Catechol estrogens are orthohydroquinones that are capable of undergoing metabolic redox cycling (16, 19, 20). Metabolic redox cycling between catechol estrogens and their corresponding quinones generates oxidative stress and potentially harmful free radicals (21, 22). Therefore, redox cycling and free radical generation represent a potential mechanism, whereby relatively low concentrations of active metabolites might generate major cell damage. Reactive oxygen species and metabolic activation have been known to modulate gene expression and several studies support the role of oxidative stress in tumor formation (21, 22, 41-45).

We hypothesized that if we use a combination of a potent estrogen with poor ability to form catechol estrogens such as EE (15, 17) and a chemical that is known to produce oxidative stress such as menadione (26-28), we should be able to mimic the effect of E2, a tumorigenic potent estrogen that is also a strong catechol progenitor (10, 12, 16, 19, 24). A similar effect may be observed if hamsters are treated with a combination of a noncarcinogenic estrogen of poor hormonal potency but with good catechol forming capability such as E2 (24, 25) and a potent estrogen such as EE (15, 17, 24). As expected, the group of hamsters treated with E2 showed 90% tumor incidence, which is in agreement with the previously reported results (10, 12). This group also showed a >2-fold increase in 8-iso-PGF₂ levels compared with untreated controls, suggesting an increased state of oxidative stress. Chronic treatment of hamsters with E2 for 9 days has previously been shown to result in increased lipid hydroperoxide levels in kidney, the target organ of estrogen-induced carcinogenesis in hamster, but not in liver, a nontarget organ (44). Kidneys of hamsters treated with a combination of E2 and EE showed signs of early neoplastic changes with proliferation of interstitial cells. Tumors were clearly seen in kidneys of hamsters treated with a combination of EE and menadione. This treatment group also showed a significant increase in 8-iso-PGF₂ levels compared with EE-treated group and compared with untreated controls. Menadione is reduced to a semiquinone radical through one electron reduction catalyzed by cellular reductases (26). Redox cycling of menadione has been shown to generate free radicals/reactive oxygen species, and has been widely used to investigate chemical-induced oxidative stress (26-28). Metabolic activation of E2 and redox cycling of estrogen-quinone metabolites generates free radicals by a mechanism similar to redox cycling of menadione and its semiquinone (20, 21, 26, 41). Moreover, tumors induced by E2, or by a combination of EE plus menadione, showed similar histological and immunocytochemical characteristics. Although early signs of proliferation in the renal interstitium, as demonstrated by foci with atypical collection of interstitial cells and increased 8-iso-PGF₂ levels, were seen in the kidneys of hamsters treated with a combination of E2 and EE, tumors were not clearly visible. It may take >7 months for tumors to develop with this treatment regimen. It has been shown earlier that tumor foci and early neoplastic cells arise in the renal interstitium (12, 46, 47). It is also possible that EE, which is known to inhibit cytochrome P450 enzyme activity, may reduce the catechol forming potential of both E2 and EE (48, 49). E2 forms 8-iso-PGF₂ at reduced levels compared with E2. This observation may indicate that E2 catechols may be methylated faster than E2 catechols, thus making them available at a reduced level for catechol-quinone redox cycling. Cotreatment with menadione and EE resulted in tumor development in the hamster kidney. Menadione may induce cytochrome P450 activity, or it may antagonize the inhibitory effect on P450 by EE. The reduced ability of EE to form catechol estrogens has been suggested to be responsible for the poor carcinogenic potential of EE (17). Menadione may also inhibit the catechol-O-methyl transferase-mediated conversion of 2- and 4-hydroxylated EE to methoxyestrogens, thus leading to an increased state of oxidant stress as a result of metabolic redox cycling of estrogen catechols and quinones. No evidence of hemosiderin was observed in kidney sections of hamsters treated with menadione or menadione plus EE, suggesting that the histopathological effects of menadione are not through its effects on iron homeostasis (50). Although subchronic treatment of hamsters with menadione or E2 also increased 8-iso-PGF2 levels compared with untreated controls, tumors were not detected in these two treatment groups. These chemicals either lack or have weak estrogenic activities (24). It appears that both estrogenic potential and oxidative stress as a result of metabolic redox cycling of estrogen metabolites are essential for estrogen-induced carcinogenesis, because, if estrogen is withdrawn, tumors will regress (10). The hormonal effects of estrogens may promote the development of tumors.

Our results demonstrate that tumors can be induced in a rodent model of hormonal carcinogenesis by subchronic treatment with a combination of a noncarcinogenic estrogen and a chemical known to produce oxidative stress. Our results are in agreement with the role of oxidant stress in estrogen-induced carcinogenesis. It has been recently shown that hydroxylated and O-methylated estrogens account for nearly 95% of the total estrogen in normal human breast and in human breast tumor tissue, with no difference in O-methylated estrogens between the tumor and control group (51). Both 2- and 4-hydroxy-E2 levels have been shown to be significantly increased in the human breast tumor compared with normal tissue, with 4-hydroxy-E2 showing an 20-fold increase in breast tumor tissue compared with normal tissue (51). The elevated expression of estrogen-4-hydroxylase activity has been demonstrated in organs of rodents that develop estrogen-induced tumors, in MCF-7 human breast cancer cells, in human uterine myoma, and in human breast cancer, but not in livers of these species (16, 18, 36-40). As suggested by several investigators, 4-hydroxy-E2 may undergo metabolic redox cycling between its catechol and quinone metabolites and potentially generate harmful free radicals and oxidative stress (19-22). Therefore, cancer may develop only in those organs that synthesize hydroxyl metabolites of estrogen at the target site of carcinogenesis, which may elicit biological activities distinct from E2; most notably, an oxidant stress response induced by free radicals generated by metabolic redox cycling reactions. In summary, our data support the concept that oxidative stress plays a critical role in estrogen-induced carcinogenesis.