Carcinogenic Activities of Various Steroidal and Nonsteroidal Estrogens in the Hamster Kidney: Relation to Hormonal Activity and Cell Proliferation1

[CANCER RESEARCH 55, 4347-4351, October 1, 1995] Carcinogenic Activities of Various Steroidal and Nonsteroidal Estrogens in the Hamster Kidney: Relation to Hormonal Activity and Cell Proliferation1

Jonathan J. Li,2 Sara Antonia Li, Terry D. Oberley, and Jonathan A. Parsons

Hormonal Carcinogenesis Laboratory, Division of Etiology and Prevention of Hormonal Cancers, University of Kansas Cancer Center, and Department of Pharmacology, Toxicology, and Therapeutics [J. J. L, S. A. L} and Preventive Medicine [J. J. L], University of Kansas Medical Center, Kansas City, Kansas 66160-7312; Pathology Service, William S. Middleton Memorial Veterans Administration Hospital, and Department of Pathology, University of Wisconsin Medical School. Madison, Wisconsin 53706 IT. D. O.J; and Department of Anatomy, University of Minnesota Medical School, Minneapolis, Minnesota 55417 Â¡J.A. P.}

ABSTRACT INTRODUCTION

The therapeutic use of estrogens has been associated with an increased Numerous epidemiological studies have established causal relation risk of some of the most predominant, as well as less prevalent, cancers in ships between estrogen use and increased risk for some of the pre women. The estrogen-induced renal tumor is one of the primary animal dominant cancers in women, namely, breast and endometrium (1-3). models to evaluate the carcinogenic properties of estrogens. Correlations Ingestion of estrogens has also been associated with some less prev were made with various estrogens by using parameters of estrogenicity alent cancers at hepatic, cervico-vaginal, and ovarian sites (4-6). It is end points such as competitive binding, progesterone receptor induction, estimated that at least 20 million women in the United States take and alterations in prolactin levels; in vitro renal proximal cell prolifera estrogenic hormones, largely but not solely for contraception, for the tion; and in vivo estrogen-induced carcinogenicity. The most potent estro relief of menopausa! symptoms, and to reduce the risk of osteoporosis gens were Moxestrol (MOX), diethylstilbestrol (DES), and 17ÃŸ-estradiol, and cardiovascular disease that afflicts aging women. On a worldwide followed by indenestrol B, 16a-hydroxyestrone, and llÃŸ-methoxyestra- basis, this number is most certainly multiplied many times. Menopau- diol with moderate estrogenic activities, whereas llÃŸ-methylestradiol, sal estrogens have become one of the most widely prescribed drugs, 17a-estradiol, indanestrol, and deoxoestrone were all relatively weaker. with a current usage rate of over 32% among women ages 50-70 As expected, hydrolyzed Premarin (unconjugated estrogens) was strongly years (7). Despite increasing study, there is still little understanding of estrogenic. Of the estrogens tested, MOX was the most potent carcino the cellular and molecular mechanisms(s) whereby these estrogen- genic estrogen in the hamster kidney. Both 16a-hydroxyestrone and llÃŸ- methoxyestradiol induced intermediate tumor incidences with distinctly related neoplastic events occur. The estrogen-induced renal tumor has emerged as one of the lower frequencies of renal tumor foci compared to the most potent car cinogenic estrogens. However, hamsters treated for 9.0 months with llÃŸ- primary experimental models to evaluate the carcinogenic properties methylestradiol, 17a-estradiol, deoxoestrone, and indanestrol exhibited of estrogens, whether they possess steroidal or nonsteroidal structures no tumors. In contrast, treatment with estrone, equilin plus d-equilenin, (8-10). Recent advances in estrogen carcinogenesis indicate that cell and hydrolyzed Premarin for the same time period resulted in 100% renal proliferation may play a critical early role in estrogen-induced tumor- tumor incidences and numerous tumor foci. Cell proliferation studies of igenic processes (11, 12). Interestingly, this concept has received cultured hamster kidney proximal tubule cells were carried out at varying considerable support from earlier studies using numerous experimen estrogen concentrations (0.01-100 nivi). Exposure to MOX resulted in tal models in hormonal carcinogenesis employing estrogens as well as consistently high renal cell proliferative response over a concentration other sex steroids (13-17). range of 0.1-10 n\i. Strongly carcinogenic estrogens such as estrone had a The current report presents data correlating in vivo carcinogenicity maximal renal cell proliferation response (2.4-fold above untreated con data of estrogens and in vitro cell proliferation of primary renal trol levels) between 0.1 and 10 Â»\i,DES and 17ÃŸ-estradiol responded at epithelial hamster cells in culture, grown in serum-free chemically 1.0 UM,and 4-hydroxyestradiol responded at 10 n\i. Interestingly, expo defined conditions, as well as other parameters of estrogenicity. sure to ethinylestradiol, a potent estrogen, at similar or higher doses as those used for DES and 17ÃŸ-estradiol, yielded only a 10% renal tumor incidence and induced only a 1.7-fold increase in proximal tubule cell MATERIALS AND METHODS proliferation. In contrast, 17<*-estradiol, deoxoestrone, indanestrol, and llÃŸ-methylestradiol, all weakly estrogenic and noncarcinogenic agents, Chemicals and Reagents. [2,4,6,7-3H]- 17ÃŸ-estradiol (115 Ci/mmol), [l,2,6,7-3H]-progesterone (103 Ci/mmol), and [17a-methyl-'H]-R5020 (86 had relatively little effect on tubule cell proliferation. The hydrolyzed Premarin exhibited a maximal 2.0-fold cell proliferative response at Ci/mmol) were obtained from New England Nuclear (Boston, MA). Radioinert 10 mi. Chromatographie grade estradiol, estrone, and progeslerone were purchased The present results provide clear evidence that, in the hamster kidney, from Calbiochem (Behring, Ã‡A), and all other nonlabelcd steroids were obtained either from Sigma Chemical Co. (St. Louis, MO) or Steraloids the degree of carcinogenicity of a given estrogen correlates with its ability (Wilton, NH). Deoxoestrone was prepared by Dr. Luke K. T. Lam (LKT to induce proximal tubule cell proliferation in vitro. Therefore, the ability Laboratories, Minneapolis, MN). Indenestrol B and indanestrol were gener of estrogen to enhance tubule cell proliferation is a more accurate indi ously provided by Dr. Manfred Metzler (University of Kaiserslautern, Kaiser cator of its carcinogenicity in this system than either the estrogen-respon slautern, Germany); MOX3 (llÃŸ-methoxyethinylestradiol) by Dr. J. P. sive end points used or the amount of catechol metabolites generated in Raynaud (Roussel UCLAF, Paris, France); and 16a-hydroxyestrone by Dr. this tissue as reported earlier. Jack Fishman (IVAX Corp., Miami, FL). All estrogens investigated exhibited purities of at least 95% as shown by HPLC analyses by using a Waters model 840 liquid Chromatograph equipped with a Waters 490 programmable multi- Received 4/17/95; accepted 7/27/95. wavelength detector. Estrogen samples (10 fig), dissolved in tetrahydrofuran. The costs of publication of this article were defrayed in part by the payment of page were injected and eluted on two tandem octadecyl (Cls) columns, 0.46 x 25 cm charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (IBM, Wallingford. CT), by using 29% acetonitrile in water isocratically at a 1This investigation was supported by National Cancer Institute, NIH Grants CA58030 column temperature of 35Â°Cand at a flow rate of 3 ml/min. Conjugated and CA22008 and a Kansas Masonic Oncology Research Center grant. estrogens were hydrolyzed from Premarin (Wyeth-Ayerst, Philadelphia. PA) 2 To whom requests for reprints should be addressed, at Division of Etiology and Prevention of Hormonal Cancers. University of Kansas Cancer Center, Robinson Suite 5008, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS ' The abbreviations used are: MOX, Moxestrol; PRL, prolactin; ER, estrogen receptor; 66160-7312. DES, diethylstilbestrol; PR, progesterone receptor; EE, ethinylestradiol. 4347

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1995 American Association for Cancer Research. CARCINOGENIC ACTIVITY OF ESTROGENS AND CELL PROLIFERATION by using sulfatase, type H-l (Sigma), for 5 h under N2 in 0.2 N sodium acetate, [*H(-progesterone or |'H]-R502(), alone or in combination with corre pH 5.0. HPLC separation of the unconjugated estrogens in hydrolyzed Pre- sponding radioinert hormones (100-fold excess). Dcxlran-charcoal treat marin is shown in Fig. 1. The composition of the hydrolyzed Premarin ment was 10 min (20-22). Protein concentrations of the renal tumor and prepared was estrone (42%), equilin (17%), 17a-dihydroequilin (3.4%), 17a- kidney cytosols were determined by the method of Lowry el al. (24). estradiol (2.4%), 8-dihydroestrone (18%), 17ÃŸ-dihydroequilin (0.7%), equi- Hamster PRL Bioassay. To determine PRL levels in hamster serum lenin (4.3%), 17a-dihydroequilenin (10%), 17ÃŸ-estradiol (1.5%), and 17ÃŸ- samples, the Nb, node lymphoma bioassay was used as described previously dihydroequilenin (0.7%). in detail (19, 25). Serum samples were obtained at 9.0 months from each of the Animals and Treatments. Young adult castrated male Syrian golden ham estrogen-treated animals via the inferior vena cava. The Nb2 node rat lym sters, weighing 85-95 g, were purchased from HarÃanSprague-Dawley (Indi phoma cells were kindly provided by Dr. R. L. Noble and Dr. C. T. Beer. anapolis, IN). All animals were acclimated 1 week before treatment or use. (University of British Columbia, Vancouver, British Columbia, Canada). Ham Hamsters were housed in compliance with the guidelines of the United States ster serum samples were diluted 1:10 with filtered Fischer medium containing Department of Health and Human Resources (N1H). Animals were housed in 10% horse serum and added to wells in aliquots of 50 and KM)Â¡t\.Each a facility certified by the American Association for the Accreditation of hamster serum sample was bioassayed in triplicate. Laboratory Animal Care. Hamsters were maintained on a 12-h light/12-h dark Detection of Renal Tumor Foci. The occurrence of renal tumor foci cycle and fed rodent certified chow 5012 Purina diet (Ralston Purina Co., induced by the various steroidal and nonsteroidal estrogens was assessed by Richmond. IN) and tap water ail libitum. Castrated animals in the treatment procedures described earlier (19, 26, 27). Briefly, serial frozen kidney sections groups (9-12/group) were implanted s.c. with the following pellets (20 mg): ( 10-jim thick) were prepared immediately after excision and embedding whole 17ÃŸ-estradiol, MOX, l lÃŸ-methoxyestradiol, l lÃŸ-methylestradiol, 16a-hy- kidneys in ornithine carbamyl transferase (Tissue TEK, Elkhart, IN). By using droxyestrone, 17a-estradiol, deoxoestrone, DES, indenestrol B, indanestrol, or a CTF Microtome cryostat (Damon, IEC, Needham Heights, MA), maintained hydrolyzed Premarin as described previously (18, 19). To maintain estrogenic at - 18Â°C,25-30 unfixed frozen sections were taken at regular intervals from levels of each compound constant, additional pellets were implanted every 2.5 each kidney. Each frozen section was stained for nonspecific esterase activity months. The pellets were prepared by Hormone Pellet Press (Westwood. KS), by using a-naphthyl butyrate as substrate (18, 28). Tumor foci were identified and their release rates were adjusted so that mean daily absorptions were under microscopic examination by the markedly reduced esterase activity in similar (111 Â±11 jag). However, l lÃŸ-methoxyestradiol, like EE, exhibited a the tumor compared to surrounding normal kidney tissue and by morphological mean daily absorption rate of 259 Â±12 fig. For PR induction and serum PRL criteria. levels, hamsters were treated for 3.0 and 9.0 months, respectively, with various Isolation of Renal Tubules. Kidneys were removed from castrated male estrogens as reported elsewhere (19-21). hamsters immediately after decapitation. The kidneys were decapsulated, and Competitive Binding Studies. For ER. pure renal tumors were excised the cortices were separated from the medullas. The procedure for isolating from hamsters treated with DES for 9-11 months as described previously renal tubules has been described elsewhere (29, 30). Briefly, the kidneys were (22), homogenized in 9.0 volumes of TED buffer (10 rnw Tris-HCl, 1.5 mM minced and homogenized in an all-glass homogenizer on ice in 10 mw EDTA, l mM dithiothreitol, pH 7.4), and centrifuged at 100,000 X K. phosphate buffer and 0.133 Msodium chloride (pH 7.2). The resultant clumped Competitive binding studies of the 8S ER were carried out on filtered kidney fragments were washed through a 132-fun Nitex Nylon screen (Tetko, cytosols (22-/xm Millipore filter; Millipore) as described earlier, by using 5 nw ['H]-17ÃŸ-cstradiol and various concentrations (1â€”100-fold excess) of Elmsford. NY), allowing passage of glomeruli and broken cells. The kidney fragments were digested with protease E (Sigma) at a concentration of 0.10 either radioinert steroidal or nonsteroidal estrogens (22, 23). Prewashcd /Â¿g/mlat 37Â°Cfor 40 min, placed in Waymouth's MB 752-1 medium (GIBCO, dextran-charcoal was used to remove free hormone and some lower- affinity-binding components (I h). For PR. renal cytosol fractions from Grand Island, NY) and 10% fetal bovine serum (Hyclone, Logan, UT), and washed through a 75-jj.m Nitcx Nylon screen, thus removing the protease. The 3.0-month-old DES-treated hamsters were incubated with 5 nw of either kidney tubules were resuspended in a chemically defined serum-free medium as described later in the text. Initial light microscopy analysis of paraffin- embedded tubular preparations showed a consistency of ~97% tubules. to Culture of Primary Renal Tubules. Basement membrane-coated flasks were prepared as described previously (28). Renal tubules at a concentration of ~5(K)/flask were plated in Waymouth's MB 752-1 medium supplemented with insulin (5 jAg/ml), transferrin (5 /Â¿g/ml), triiodothyronine (7 (ig/ml). hydrocortisone (0.18 /ig/ml), and epidermal growth factor (50 ng/ml) from Sigma; selenium (5 /xg/ml) and prostaglandin E, (25 /xg/ml) from Collab orative Research (Bedford, MA); and penicillin (100 units/ml) and strep tomycin (100 /j,g/ml) from GIBCO. No serum was added to the culture medium. Fresh medium (1.0 ml) was added to each flask every other day. o Flasks were maintained at 37Â°Cin an unhumidified incubator equilibrated CO with 95% air/5% CO2. Morphology of the cultures was monitored daily by phase-contrast microscopy and periodically by transmission electron microscopy (31). Steroidal and Nonsteroidal Estrogens on Renal Tubular Proliferation. Kidney tubules (~5(K)/flask) were grown for 5 days to allow for tubular expiant attachment in 25-cirr flasks coated with PF-HR-9 basement membrane in serum-free chemically defined medium, in the absence or presence of estrogens at various concentrations. Estrogens (0.1-100 nM) were added in 10-(il aliquots containing 100% ethanol. This concentration of ethanol, applied to control cultures, had no effect on cell outgrowth. On the fifth day, the flasks were washed to remove all unattached tubules, and fresh medium with or without estrogens was added. Seven days after attachment of the renal tubule Time(rnin) cells (12 days from the initiation of the culture), the expiants and monolayers were washed with 0.1% EDTA and disaggregated to single cells with 0.25% Fig. 1. HPLC' separation of hydroly/ed Premarin (unconjugalcd estrogens) hydrolyzed trypsin and 0.1% EDTA. The cells were then counted in a hemocytometer by hy sulfatase for 5 h. Percentage composition is in descending order of quantity. Peak 10, estrone; Peak 9, K-dehydroeslronc: Peak K. equilin; Petik 5. 17a-dihydroequi!in; Peak 6, a procedure described elsewhere (30). The cell proliferation data are expressed equilenin; Peak .*, 17a-dihydroequilenin; Peak 7, I7a-estradiol; Peak l, I7ÃŸ-dihydro- as a stimulation index, the ratio of the number of cells grown in the presence equilenin: Peak 4, 17/J-estradiol: and Peak 2. 17ÃŸ-dihydroequilin. of estrogen versus the number of cells grown without estrogen. 4348

Table 1 fc.\trogenicit\ tinti carcinogenu-it\ of various sle nmin I timi stilhene estrogens in Â¡heSvritin hamster kidne\ no. tumor % of kidney of animals ER competitive induction of PR' prolactin with tumors of tumor foci Estrogen"SteroidalI7ÃŸ-Estradiol110-Methoxy binding'55 (fmoles/mgprotein)48 (ng/ml)390 (%)10010038250001001000C'omhinedin bothkidneys17Â±422

Â±252 Â±66()Â±245 Â±76330 EE(MOX)16tt-Hydroxyestrone1 Â±448 Â±65387 Â±33Â±12Â±100019 Â±330 Â±735 Â±35389 l/3-MctrioxycslradiolllÃŸ-Mcthylestradiol17a-EstradiolDcoxocstroncStilbcncDicthylslilhcslrolIndcncstrolÂ±614 Â±618Â±26Â± Â±17149 Â±134 Â±69129 Â±514 18Â± Â±1794 Â±146 150 Â±20449

Â±446 Â±449 Â±89281 Â±411Â±30 BIndancslrolRenal Â±210Â± Â±529 Â±90103 1Hamster Â±5Serum Â±40No. " Duration of estrogen treatment was 9.0 months. Each treatment group contained 9-12 animals. Data, mean Â±SEM. Competitive binding of radioinert estrogens to ER was carried out in renal tumor cytosolic fractions obtained from DES-induced renal carcinomas. Estradiol concentration in these tumor cytosols (4 mg of protein/ml) without competitor corresponded to 0% inhibition. [3H]-estradiol at 5 nM Â±cold competitor at 1.0-fold excess. ' Determined by affinity labeling of cytosolic fractions from 4.0-month DES-treated hamsters. ['ill-progesterone at 5 nM Â±cold competitor at 100-fold excess.

RESULTS steroidal estrogens at varying concentrations (0.01-100 nM) under serum-free chemically defined conditions. Estrone attained a maximal Estrogenicity Studies. To assess the relative estrogenicity of the 2.4-fold increase in renal cell proliferation between 0.1 and 10 nM; various steroidal and nonsteroidal estrogens studied in the hamster, 4-hydroxyestradiol exhibited a similar maximum proliferative re competitive binding to kidney tumor cytosolic ER, induction of PR in sponse at 10 nM. Additionally, 4-hydroxyestrone exposure resulted in hamster kidneys, and changes in serum PRL levels were studied. a maximal 2.1-fold rise in tubule cell proliferation at 1.0 nM. It is Untreated normal levels of PR and PRL in castrated male hamsters noted that 2-hydroxylated metabolites of 17ÃŸ-estradioland estrone were 3.3 Â±0.2 fmol/mg protein and 34 Â±5 pg/ml, respectively. exhibited lower renal cell proliferative activity compared to their Generally, there was a good correlation among these parameters corresponding 4-hydroxylated counterparts (data not shown). Inter concerning the relative estrogenic potency of the steroidal and non estingly, renal proximal tubule cell proliferation increased only a steroidal compounds studied (Table 1). maximal of 1.7-fold after exposure to EE. At 100 nM, all of the above Utilizing the hormonal end points described herein, it is evident that indicated estrogens exhibited a decline in tubular cell proliferation. MOX, 17ÃŸ-estradiol, and DES were the most potent estrogens, Exposure of the proximal tubule cell cultures to MOX resulted in a followed closely by indenestrol B, 16a-hydroxyestrone, and llÃŸ- consistently high cell proliferation response over a concentration methoxyestradiol with moderate estrogenic potencies, whereas range of 0.1-10 nM (Fig. 3/4). Moreover, 17ÃŸ-estradiolresulted in a llÃŸ-methylestradiol, 17a-estradiol, indanestrol, and deoxoestrone similar 2.4-fold rise in tubule cell proliferation at 1.0 nM. At 1 nM exhibited distinctly lower estrogenic activities (Table 1). The expected concentration, however, exposure of the tubule cells to either 16a- strong estrogenicity of hydrolyzed Premarin (unconjugated estro hydroxyestrone or l lÃŸ-methoxyestradiol elicited a lower 1.4- and gens) was confirmed by its competitive binding to renal tumor ER 1.3-fold increase in cell proliferative response, respectively. The hy (1.0-fold excess) at 42 Â±3%, its induction of hamster kidney PR to drolyzed Premarin exhibited a 2.0-fold increase in tubule cell prolif 53 Â±2 fmol/mg protein, and its elevation of serum PRL levels to eration with a maximum occurring at 10 nM. On the other hand, 339 Â±93 ng/ml. llÃŸ-methylestradiol exhibited little ability to effect renal tubule cell Carcinogenic Activity of Various Estrogens. As reported previ proliferation (Fig. 3/4). In contrast, DES exhibited a strong cell ously (19, 26), 17ÃŸ-estradioland DES were equally carcinogenic in proliferative response, between 1.0 and 10 nM, when exposed simi the hamster kidney. These potent carcinogenic estrogens served as larly to proximal tubule cells in culture (Fig. 3ÃŸ).Indenestrol B positive references for the rest of the steroidal and nonsteroidal exposure also showed a strong, 2.2-fold, cell proliferative response at estrogens studied. MOX was found to be the most potent carcinogenic 1.0 nMconcentration. However, exposure of hamster renal tubule cells estrogen in the hamster kidney (Table 1), effecting a 100% tumor to weak estrogens, such as 17a-estradiol, indanestrol (Fig. 3/4 and incidence and more numerous individual tumor foci even at shorter 3ÃŸ),and deoxoestrone (data not shown), did not result in significant treatment periods (6.0-9.0 months) compared to either DES or 17ÃŸ- renal tubule cell proliferative responses in these cultures. estradiol (data not shown). Both 16a-hydroxyestrone- and llÃŸ-methoxyestradiol-treated hamsters exhibited moderate tumor incidences and distinctly lower frequencies of renal tumor foci compared to DISCUSSION MOX, DES, or 17ÃŸ-estradiol.In contrast, in hamsters treated for 9.0 Previous studies from our laboratory (19, 26, 27) have concluded that months with either 17a-estradiol, deoxoestrone, llÃŸ-methylestradiol, a good correlation exists between hormonal activity and carcinogenicity or indanestrol exhibited no tumors. These evidently noncarcinogenic estrogens did not induce renal tumors even when the dose was increased to 40 mg (2 pellets) and implanted every 2.5 months for 9.0 Table 2 Carciiuwtucity of itm'onjtt^titeil estru months (data not shown). of animals no. Interestingly, hamsters treated for 9.0 months with estrone, equilin with lumors(%)100 of tumor foci Estrogen"Estrone plus d-equilenin, or hydrolyzed Premarin exhibited 100% renal tumor in bothkidneys15 incidences and abundant tumor foci for the same treatment period Â±3 Equilin + d-Equilcnin 100100Combined 18Â± 1 (Table 2). PremarinNo. 16 Â±2 Renal Tubule Cell Proliferation. Fig. 2 depicts the cell prolifer " Duration of estrogen treatment was 9.0 months. Each group contained 6-8 animals. ation of cultured hamster renal proximal tubule cells with several Data, mean Â±SEM. 4349

3.0 or serum PRL levels in this animal. Previous studies in our laboratory have demonstrated that 2-hydroxyestrogen (estradici and estrone) metabolites were devoid of carcinogenic activity in the hamster kid ney (27). In contrast, 4-hydroxyestrogen metabolites, particularly estradiol, exhibited significant carcinogenic activity at this organ site. In regard to PR induction in the hamster kidney, 2-hydroxyestradiol was shown to be a poor inducer when compared to 4-hydroxyestradiol (data not shown). This observation is consistent with the relatively stronger estrogenic activity reported for 4-hydroxyestradiol in other target tissues when compared to 2-hydroxyestradiol (39, 40). The relative abilities of estrone, 4-hydroxyestradiol, 4-hydroxy estrone, and EE to elicit tubule cell proliferation essentially coincides with the abilities of these estrogens to induce renal tumors in the hamster (27). Similarly, the ability of DES and indenestrol B to effect tubule cell proliferative responses also correlates well with the ability of these nonsteroidal estrogens to induce kidney tumors. It should be 0.5 0.01 0.1 1.0 10 100 noted that the relative estrogenicity of these stilbene estrogens re Concentration ported herein is similar to those reported elsewhere using uterotropic activity studies in the mouse (41). Fig. 2. Effect of various sleroidal estrogens (0.1-100 nM) on proliferative outgrowth of kidney tubule cells grown on PF-HR9 basement membrane in serum-free chemically Although determination of hormonal activity described in these defined medium. Hamster cortical expiants (~5(H)/flask) were plated in separate 25 cm2 studies is a useful marker of estrogenicity, and the relative amounts of flasks. Twelve days after initiation of the culture after Â±estrogen exposure, all tubules catechol estrogen metabolites generated by various estrogens on the were washed and treated with 0.1% EDTA. Both parent expiants and monolayer cells were removed and disaggregated into single cells. The cells were then counted in a hamster kidney have been assessed (42, 43), neither of these param hemocytometer. Points, mean of six counts from three individual flasks; bars, eters has adequately predicted the carcinogenic potential of a given mean Â±SEM. The data are expressed as a stimulation index, the ratio of the number of cells grown in the presence of estrogen versus the number of cells grown in the absence estrogen. A more accurate indicator of carcinogenicity for an estrogen of estrogen. The steroidal estrogens shown are estrone (T), 4-hydroxyestradiol (â€¢), in the hamster kidney is the ability of the hormonal agent to enhance 4-hydroxyestrone (V). cthinylestradiol (ED),and 17a-cstradiol (â€¢). cell proliferation in the proximal tubule cells. For example, neither 16a-hydroxyestrone nor l lÃŸ-methoxyestradiol (data not shown) elic ited strong proliferative responses in these cells, consistent with their among estrogens tested in the hamster kidney. However, EE was a observed lower carcinogenic activity. Although exposure of these notable exception because it exerted poor carcinogenic activity at this renal tubule cells to highly potent carcinogenic estrogens (MOX, organ site (10% tumor incidence), and yet is considered to be at least five estrone, 17ÃŸ-estradiol,DES, and indenestrol B) in the hamster kidney times more estrogenic than 17ÃŸ-estradiolby standard uterus assay (32) resulted in significant renal tubule cell proliferative responses, estro and is also evidently a strong estrogen in the hamster by most of the gens that were either poorly carcinogenic or exhibited no tumor estrogenic criteria used herein (19, 27). It is clear, however, from the development showed markedly less renal cell proliferation activity present results and those reported elsewhere (33) that various estrogens (llÃŸ-methylestradiol, indanestrol, deoxoestrone, and 17a-estradiol). can have profoundly different effects in the same target tissue. For In contrast to our finding that 2-methylestradiol is devoid of carcino example, compared to other potent estrogens, hamsters treated with EE genic activity in the hamster kidney, it has been reported by others that exhibited a very different series of morphological changes in the kidney. this estrogen induced renal tumors in 2 of 10 hamsters after 7.0 After only brief treatment (sl.O month), consistent unusual hyperplasia months of treatment, but no renal neoplasms in 0 of 10 hamsters after was seen in a subset of proximal tubule cells located deep in the cortex 8 months of treatment (42). Because estrone comprised >40% of adjacent to the renal pelvis (33). This unique hyperplasia observed in hydrolyzed Premarin, it was not surprising that this preparation was EE-treated hamsters was only uncommonly observed in either DES or highly carcinogenic in the hamster renal tumor model and also sig 17ÃŸ-estradioltreated animals. This finding may in part help explain the nificantly affected renal tubule cell proliferation. It should be noted low carcinogenic activity of EE at this organ site. On the other hand, in the rat and human, MOX has an estrogenic potency at least five times greater than EE and about ten times that of 17ÃŸ-estradioland its metab olites (2- and 4-hydroxylation; Refs. 34, 35) by virtue of its lower affinity to plasma proteins, slower dissociation from ER, and markedly reduced metabolism, particularly to catechol intermediates, in both human liver and hamster kidney (35, 36). As a result of these properties, it was not surprising that MOX exhibited strong estrogenic activity in the hamster based on the hormonal end points studied, but is also the most potent T T\l carcinogenic estrogen tested thus far in the hamster kidney. iâ€”i-=tr5 Although a good association exists between estrogenicity and carcinogenicity by using the hormonal end points described herein, the correlation is apparently limited in some respects. For example, 16a- 0.01 0.1 1.0 10 100 nM 0.01 0.1 1.0 10 Concentration hydroxyestrone exhibited appreciable estrogenicity by these end Fig. 3. Effect of different steroidal estrogens (A ) and nonsteroidal stilbene estrogens points and yet yielded only a moderate, 38% tumor incidence. It (B) at varying concentrations (0.01-100 nM) on proliferative outgrowth of kidney tubule should be noted that 16a-hydroxyestrone, unlike in humans, is not cells grown on PF-HR9 basement membrane in serum-free chemically defined medium. formed from either 17ÃŸ-estradiolor estrone in the hamster kidney or All culture conditions are the same as described in Fig. 2. A, stimulation by steroidal estrogens 17ÃŸ-estradiol(â€¢),MOX (T), hydrolyzed Premarin (V). 16a-hydroxyestrone liver (12, 37, 38). Moreover, the significant binding of 17a-estradiol (D), and 11/3-methylestradiol (â€¢).B. stimulation by nonsteroidal stilbene estrogens DES to renal ER in the hamster did not result in substantial elevation in PR (O), indenestrol B (â€¢),and indanestrol (V). 4350

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1995 American Association for Cancer Research. CARCINOGENIC ACTIVITY OF ESTROGENS AND CELL PROLIFERATION that in human and animal studies, it is the unconjugated or "free" form carcinogenic activity of various synthetic and natural estrogens in the hamster kidney. Cancer Res., 4j: 5200-5204, 1983. of Premarin that is the hormonally active form because the conjugated 20. Li, S. A., and Li, J. J. Estrogen-induced progesterone receptor in the Syrian form has been found to be hydrolyzed by target-tissue sulfatases hamster kidney. I. Modulation by antiestrogcns and androgens. Endocrinology, (44, 45). 103: 2119-2128, 1978. 21. Li, J. J., and Li, S. A. Estrogen-induced progesterone receptor in the Syrian hamster Therefore, it is possible that the disparity observed among some kidney. II. Modulation by synthetic progestins. Endocrinology, 103: 1751-1756, estrogens in the hamster kidney, as it concerns hormonal activity and 1978. the response to estrogen-dependent cell proliferation, may be due to 22. Li. J. J., Li, S. A., and Cuthbertson. T. L. Nuclear retention of all steroid hormone receptor classes in the hamster renal carcinoma. Cancer Res., 39: 2647-2651, 1979. the fact that elevation of serum PRL and renal PR (46) requires the 23. Li, J. J., Talley, D. J., Li, S. A., and Villee, C. A. Receptor characteristics of specific expression of only a few genes, which may be more readily expressed estrogen binding in the renal adenocarcinoma of the golden hamster. Cancer Res., 36: by virtually all estrogens to varying degrees. However, cell prolifer 1127-1132, 1976. 24. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall. R. J. Protein measurements ation elicited by most potent estrogens likely requires the expression with the Polin phenol reagent. J. Biol. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1995 American Association for Cancer Research. Carcinogenic Activities of Various Steroidal and Nonsteroidal Estrogens in the Hamster Kidney: Relation to Hormonal Activity and Cell Proliferation

Jonathan J. Li, Sara Antonia Li, Terry D. Oberley, et al.

Cancer Res 1995;55:4347-4351.

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