Critical role of oxidative stress in estrogen-induced carcinogenesis Hari K. Bhat*†, Gloria Calaf‡, Tom K. Hei*‡, Theresa Loya§, and Jaydutt V. Vadgama¶ *Department of Environmental Health Sciences, Mailman School of Public Health, 60 Haven Avenue-B1, Columbia University, New York, NY 10032; ‡Center for Radiological Research, Columbia University, New York, NY 10032; and Departments of §Pathology and ¶Medicine, Charles Drew University, Los Angeles, CA 90059 Communicated by Donald C. Malins, Pacific Northwest Research Institute, Seattle, WA, December 27, 2002 (received for review August 22, 2002) Mechanisms of estrogen-induced tumorigenesis in the target quinones generates oxidative stress and potentially harmful free organ are not well understood. It has been suggested that oxida- radicals that are postulated to be required for the carcinogenic tive stress resulting from metabolic activation of carcinogenic process, and analogous to the metabolic activation of hydrocar- estrogens plays a critical role in estrogen-induced carcinogenesis. bons and other nonsteroidal estrogen carcinogens (9, 19–22). We We tested this hypothesis by using an estrogen-induced hamster have investigated the role of oxidative stress in estrogen carci- renal tumor model, a well established animal model of hormonal nogenesis by using a well established hamster renal tumor model carcinogenesis. Hamsters were implanted with 17␤-estradiol (␤E2), that shares several characteristics with human breast and uterine 17␣-estradiol (␣E2), 17␣-ethinylestradiol (␣EE), menadione, a com- cancers, pointing to a common mechanistic origin (6, 9, 23). bination of ␣E2 and ␣EE, or a combination of ␣EE and menadione Different estrogens used in the present study differ in their for 7 months. The group treated with ␤E2 developed target organ estrogenic, carcinogenic, and metabolic activation potentials specific kidney tumors. The kidneys of hamsters treated with ␣E2, (14–17). ␤E2 is a good catechol progenitor and a potent ␣EE, or menadione alone did not show any gross evidence of estrogen; its use results in 80–100% tumor incidence in the tumor. Kidneys of hamsters treated with a combination of ␣E2 and hamster kidney (6, 10, 14, 15). 17␣-estradiol (␣E2) is a nontu- ␣EE showed early signs of proliferation in the interstitial cells. morigenic, weak estrogen with a catechol-forming potential Kidneys of hamsters treated with a combination of menadione and similar to that of ␤E2 (24, 25). 17␣-Ethinylestradiol (␣EE) is a ␣EE showed foci of tumor with congested tubules and atrophic potent estrogen, but a weak catechol progenitor that is either glomeruli. ␤E2-treated tumor-bearing kidneys showed >2-fold nontumorigenic or very weakly tumorigenic in the hamster Ͼ increase in 8-iso-prostaglandin F2␣ (8-iso-PGF2␣) levels compared model with 9 months of continuous exposure required for less with untreated controls. Kidneys of hamsters treated with a than 10% tumor incidence (15, 17). Menadione (2-methyl-1,4- combination of menadione and ␣EE showed increased 8-iso-PGF2␣ napthaquinone) is used in the present study as a model com- levels compared with untreated controls, whereas no increase in pound with known oxidant stress potential to study the influence 8-iso-PGF2␣ was detected in kidneys of ␣EE-treated group. A of oxidative stress on estrogen-induced carcinogenesis (26–28). chemical known to produce oxidative stress or a potent estrogen We used a combination of an oxidant chemical menadione and with poor ability to produce oxidative stress, were nontumorigenic a noncarcinogenic estrogen ␣EE to show the induction of renal in hamsters, when given as single agents, but induced renal tumors, tumors in a rodent model of hormonal carcinogenesis. We also when given together. Thus, these data provide evidence that oxidant demonstrated increased levels of 8-iso-prostaglandin F2␣ (8-iso- ␤ stress plays a crucial role in estrogen-induced carcinogenesis. PGF2␣), a known marker of oxidant stress (29, 30), in E2- induced tumor-bearing kidneys as well as in menadione plus tumor ͉ hormonal carcinogenesis ͉ menadione ͉ prostaglandin ͉ metabolic ␣EE-treated kidneys of hamsters. Our studies suggest that activation oxidative stress plays a critical role in estrogen-induced carcinogenesis. ex hormones are implicated in the development of a variety Materials and Methods of human cancers (1–4). Estrogen administration to post- S Treatment of Animals. Male Syrian hamsters (4–6 weeks old; menopausal women is associated with an increased risk of Harlan Sprague–Dawley, Madison, WI) were housed in our endometrial and breast cancer (1–4). An increasing evidence of animal facility with Purina rodent chow and water available ad elevated breast cancer risk with increases in total lifetime libitum throughout the experiment. Hamsters were implanted exposure of women to estrogens has been presented (1–3). s.c. with 25-mg pellets of ␤E2, ␣E2, ␣EE, menadione, a com- Recently, the clinical trial of estrogen plus progestin treatment bination of ␣E2 ϩ ␣EE, or a combination of ␣EE ϩ menadione. therapy was stopped because of an increased risk of breast cancer These hamsters received a second estrogen or menadione pellet (5). Knowledge of how estrogens induce proliferation and tu- 3 months after initial treatment. Before implantation of the drug morigenesis in their target organ is not well defined (6–9). The pellets, hamsters were anesthetized with a combination of ket- mechanism of tumor induction by estrogens is being investigated amine and xylazine (ketamine, 100 mg͞kg body weight, i.p., and in rodent models of hormonal carcinogenesis. The natural ͞ ␤ ␤ xylazine, 10 mg kg body weight, i.p.). Estrogen and menadione female sex hormone 17 -estradiol ( E2) and the synthetic pellets were prepared by using a hand press and implanted into estrogen diethylstilbestrol induce tumors in rats, mice, and the hamsters s.c. as described (10, 12). There were 10 hamsters hamsters (10–13). It must be noted that in rodent models, in each group. A control group of 10 animals was sham operated different estrogens tested have not shown similar carcinogenic and left untreated. Hamsters were killed after 7 months and potential despite their similar hormonal potencies (6, 14, 15). inspected macroscopically for tumor nodules on the surface of However, carcinogenic and noncarcinogenic estrogens differ in each kidney as reported (31). Portions of liver and kidney tissue their metabolic activation profiles (14–17). Therefore, it is were placed on dry ice and stored at Ϫ80°C for further studies. postulated that estrogen metabolism may play a key role in hormonal carcinogenesis. Estrogens can be metabolically activated into catechol estro- Abbreviations: ␤E2, 17␤-estradiol; ␣E2, 17␣-estradiol; ␣EE, 17␣-ethinylestradiol; ER, estro- gens by cytochrome P450 enzymes (18, 19). Metabolic redox gen receptor; 8-iso-PGF2␣, 8-iso-prostaglandin F2␣. BIOCHEMISTRY cycling between catechol estrogens and their corresponding †To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0437929100 PNAS ͉ April 1, 2003 ͉ vol. 100 ͉ no. 7 ͉ 3913–3918 Downloaded by guest on September 26, 2021 Other portions of liver and kidney were placed in 10% buffered wt͞vol) were prepared by using a PRO 200 homogenizer with a formalin for histopathological evaluation and immunocytochem- 5mmϫ 75 mm generator (PRO Scientific, Oxford, CT) in 2-ml ical studies. Menadione and all estrogens used in the present microfuge tubes. Homogenization was carried out by moving the study were purchased from Sigma. 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 Ϸ ␮ Tissue Preparation for Histopathology and Immunocytochemistry. 5 s. 8-iso-PGF2␣ esters in 100 l of the total kidney homoge- The formalin-fixed tissue was embedded in paraffin, and sections nate were hydrolyzed by incubation with 25 ␮lof10N NaOH at of 4- to 5-␮m thickness were cut. Paraffin-embedded sections of 45°C for 2 h. The reaction mixture was cooled on ice for 5 min the kidneys and livers were stained with hematoxylin and eosin and neutralized with 25 ␮l of 12 N HCl, and centrifuged in a for histopathological evaluation. Gross examination and histo- microcentrifuge for 5 min. The clear neutralized supernatant was logical sections were interpreted by two independent patholo- transferred into a new microfuge tube, and 50 ␮l of the neu- gists in a blinded fashion, without knowledge as to how the tralized sample was used for 8-iso-PGF2␣ assay. The samples animals were stratified. Paraffin-embedded sections were also were incubated with the 8-iso-PGF2␣ antibody for 18 h at 4°Cin used for cell-specific expression of estrogen receptor (ER)-␣ and a 96-well format. After incubation, the contents of the wells were -␤ proteins. Deparaffinized sections were incubated with the emptied and washed with wash buffer; wash buffer was removed corresponding primary antibodies: ER-␣ (Santa Cruz Biotech- from the wells, and the color was developed by incubation with nology, SC-542) and ER-␤ (Santa Cruz Biotechnology, SC-6821) 200 ␮lofp-nitrophenyl phosphate for 45 min at room temper- at dilutions suggested by the suppliers. Incubation with the ature. The reaction was stopped by the addition of 50 ␮l of stop primary antibodies was performed overnight at 4°C. Nonspecific solution, and the plate was read at 405 nm. A standard curve was sites were blocked by covering sections with solutions of 1% BSA generated by measuring the optical density of 160–100,000 ͞ (Sigma). After washing in PBS, the sections were incubated with pg ml of 8-iso-PGF2␣ standards that were processed simulta- Ј peroxidase-conjugated, affinity-purified F(ab )2 fragment of neously with unknown samples on the same plate. Protein donkey anti-rabbit IgG (Jackson ImmunoResearch) for 60 min concentrations from the neutralized homogenates were deter- at room temperature as described (12). Slides were rinsed three mined by using a Pierce protein assay kit.