Proc. Nati. Acad. Sci. USA Vol. 89, pp. 1085-1089, February 1992 Medical Sciences prevents induction of hepatic neoplasia by , an estrogenic food contaminant JOHN E. COE*t, KAMAL G. ISHAK*, JERROLD M. WARD§, AND MARY J. Ross* *National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, Hamilton, MT 59840; tArmed Forces Institute of Pathology, Washington, DC 20306; and fLaboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick, MD 21702-1201 Communicated by Rudi Schmid, November 1, 1991

ABSTRACT Zeranol (a-zearalanol) is a P-resorcylic acid hamsters is known to be particularly sensitive to an exoge- lactone (RAL) that has activity. It is synthesized by nous estrogen, (DES). Thus, injection of molds and is difficult to avoid in human food products. We DES is followed by clinical and histological evidence of tested the ability ofthis to damage the liver ofthe hepatocellular degeneration (24) that is blocked by concom- Armenian hamster, a rodent that is epcialy sensitive to itant injection oftamoxifen (Tam), suggesting that hepatic ER hepatotoxic effects of exogenous . Zeranol Induced is involved in its pathogenesis (25). Furthermore, chronic acute hepatotoxicity and, subsequently, hepatic carcInogene- exposure to DES is associated with the formation of hepa- sis; both effects were blocked by tamoxifen, s ing estrge tocellular carcinomas (26). Such profound hepatic toxicity receptor mediation. Because zeranol is acting alone as a and carcinogenicity induced by DES alone is unknown in primary initiator of hepatic neoplasms, this model provides an other experimental animals. Therefore, it is pertinent to unusual opportunity to study the pathogenesis of estrogen- determine the effects of other estrogens, and zeranol is an initiated tumorigenesis. especially appropriate compound to study because it is structurally dissimilar to DES and is a relatively weak There is now ample evidence supporting the role ofestrogens estrogen (2-3 orders of magnitude less potent than DES or in the neoplastic process. Attention has been focused on the ), yet is environmentally important as a (human) involvement of estrogens in the development of breast and food contaminant (18, 19, 22). The results indicate that endometrial cancer (1, 2). However, other organs such as zeranol is a particularly suitable estrogen for induction of liver also contain a functioning estrogen-receptor (ER) me- acute toxic and chronic neoplastic changes in the liver of the diator and, accordingly, are targets for control by circulating Armenian hamster. Because zeranol acts as a primary initi- estrogens (3-8). Indeed, estrogens taken by women for birth ator of hepatic tumors, this model provides an unusual control purposes are implicated as a cause for development opportunity to study the pathogenesis of estrogen-induced of both benign and malignant hepatic neoplasms (9-16). In carcinogenesis. addition to endogenous steroidal estrogens, a variety of compounds with estrogenic activity also are found in the MATERIALS AND METHODS environment because a number of drugs, insecticides, and natural food products representing many different structures Animals. Armenian hamsters (Cricetulus migratorius) can act like estrogens after ingestion (17). The .-resorcylic were obtained from the Rocky Mountain Laboratories pro- acid lactones (RALs) are a group of natural products with duction unit. They were provided free access to food pellets similar structure that have estrogen activity (18, (Purina Lab Chow) and water. Hepatic synthesis of some 19). RALs are important because they are stable compounds, proteins in Armenian hamster is influenced by light (27) so all functional after oral administration, and difficult to avoid in animals were housed in a room illuminated by fluorescent human food products. is the RAL that is found lights with a 16/8-hr light/dark cycle. in food; it is a mycoestrogen, synthesized by molds (fusarium Drugs. Zeranol (a-zearalanol, 12-mg pellets, Ralgro, Pit- species) commonly contaminating grain. Another RAL, zer- man-Moore, Terra Haute, IN) (also referred to as RAL in (a-zearalanol),-is found as a natural metabolic product of this report) was surgically implanted s.c. in the anesthetized zearalenone and binds to the ER ofuterus (20) and liver (21). (Metofane, Pitman-Moore) animal. Tam (supplied by Stuart The growth-promoting (anabolic) effects of zeranol have led Pharmaceuticals, Wilmington, DE) was suspended in propy- to the commercial production and sale ofthis RAL for use as lene glycol, and 0.1 ml (5 mg) was injected s.c. a growth stimulant in meat production. Therefore, human Experimental Design. Male and female Armenian hamsters consumption ofRAL compounds may be direct, by ingestion (in groups of 5-20) were started on experimental drug treat- ofcontaminated cereal products, or indirect, by consumption ment when 2-3 months of age. At appropriate intervals, ofproducts from animals fed a mold-infected grain or injected animals were sacrificed for histological examination. Control with a RAL growth stimulant. Accordingly, RAL compounds animals (no drug treatment) of comparable age or older than have been tested extensively in various assays for genotoxic the experimental animals were also examined to ascertain the and neoplastic effects; to date no outstanding toxic, muta- effects of aging alone. Blood samples from anesthetized genic, or carcinogenic changes from RAL have been ob- hamsters were obtained from the retroorbital area or by served (22), although an increased incidence of adenomas bleeding from the heart, and serum bilirubin was determined (pituitary, liver) were detected in one species after a 2-yr oral as before (25). At necropsy, 2- to 3-mm slices of liver were carcinogenicity study (23). carefully examined for gross pathology; presumptive nodules The present report describes experiments in which zeranol was administered to Armenian hamsters. The liver of these Abbreviations: DES, diethylstilbestrol; EGF, epidermal growth fac- tor; ER, ; RAL, resorcylic acid lactone (or zera- nol); Tam, tamoxifen; HCC, hepatocellular carcinoma(s); HCA, The publication costs of this article were defrayed in part by page charge hepatocellular adenoma(s); EMH, extramedullary hematopoiesis; payment. This article must therefore be hereby marked "advertisement" MB, Mallory body/bodies. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 1085 Downloaded by guest on September 28, 2021 1086 Medical Sciences: Coe et al. Proc. Natl. Acad. Sci. USA 89 (1992) were measured and, along with slices of normal-appearing liver, were fixed in 10o buffered formalin. After routine processing and embedding in paraffin, sections were cut at 6 A&m and stained with hematoxylin and eosin (plus other stains). The histologic diagnosis of the hepatic lesion was - 12' determined independently by two of us (K.G.I. & J.M.W.), E 10- and classification of the neoplasm was based on previously published histological criteria (28). The animal experiments a conducted for this study were performed according to the National Institutes of Health Guide for the Care and Use of 4- Laboratory Animals. 2

RESULTS 1Rai36 20 40 60 8 t 10 li2 Effect of RAL (Without and with Tam) on Serum Bilfirubin. Days after injection of RAL Armenian hamsters becamejaundiced FIG. 1. Hyperbilirubinemia induced by RAL and its prevention [three 12-mg pellets (36 mg total) on day 0]. Fig. 1 shows that by concomitant Tam administration. Serum bilirubin promptly in- increased bilirubin levels were detected in all animals within creases in Armenian hamster females (n) (n = 10) and males (A) (n 6 days, peaked at "13 mg/dl within 14 days, and then = 10) after injection of RAL pellets (36 mg) on day 0. The icterus is gradually decreased to minimal levels by 50-60 days. Male- transient, and the animals do not show such hyperbilirubinemia upon female response usually was similar in magnitude, although a repeat RAL injection (36 mg) on day 94. In addition to RAL, one female bilirubinemia usually remained elevated for a longer group of female and male hamsters (0) (n = 11) also received Tam when (5 mg twice weekly for 4 weeks and then once per week thereafter), time. Both sexes were refractory to this RAL effect which inhibited the bilirubinemia response until day 94, when serum reinjected on day 94 (Fig. 1), as a minimal bilirubin response bilirubin increased slightly (2.0-2.8 mg/dl). (Bar = +1 SEM.) was detected only after a prolonged interval (i.e., on day 112, 18 days after reimplantation). RAL pellets were very difficult Preneoplastic/neoplastic lesions grossly appeared as cir- to detect more than 2.5 months after implantation. cumscribed pale nodules. The diameter frequently correlated Another group ofmale-female hamsters implanted with 36 with the type of nodule; that is, foci were smallest (1-3 mm), mg of RAL pellets on day 0 was also injected with Tam (5 mg HCC largest (6-15 mm), and HCA intermediate in size. twice weekly for 4 weeks and then once per week thereafter). Larger nodules were usually found after longer treatment Tam inhibited the bilirubinemia response of both sexes to with RAL. No evidence of metastasis was detected. Among RAL, as no increase was detected until late (day 94) when the notable nonneoplastic changes found in liver after RAL serum bilirubin increased slightly (2.0-2.8 mg/dl) (Fig. 1). were a very marked but transient stimu- Hepatic Histolo after 36 mg of RAL. Male and female treatment (Fig. 2) Armenian hamsters injected once with 36 mg of RAL pellets lation of mitosis and the constant presence of Mallory bodies on day 0 were sacrificed for necropsy at specified intervals (MB) and extramedullary hematopoiesis (EMH). Focal ne- thereafter. The results of histological examination of the crosis and transient apoptosis were also observed. These livers are tabulated in Fig. 2 and shown as preneoplastic/ lesions were not seen in livers of control (normal) animals neoplastic or nonneoplastic changes. The first preneoplastic (not shown), but MB, EMH, and focal necrosis were found changes (foci) were detected on day 56 and by day 119 had in RAL- and Tam-treated hamsters (Fig. 2). Venous prolapse become more abundant and were joined by hepatocellular was usually found in livers examined 142 days or more after adenomas (HCA). Subsequently, hepatocellular carcinomas RAL administration (not shown) and histologically was sim- (HCC) were found in 3 of 6 female livers on day 142; at this ilar to that previously seen in livers of Armenian hamsters time 6 of 6 females had hepatic lesions versus only 1 of 3 treated with DES (24). A minimal amount of venous prolapse males. On day 236, 3 of 4 hamsters showed preneoplastic/ also was detected occasionally in livers from some old normal neoplastic change; however, no lesions were found in 5 Armenian hamsters (>1.5 years of age) (not shown). hamsters that had received RAL and also a short course of Hepatic Neopblsms Induced with Higher and Lower Doses of Tam injections (5 mg, on days 0, 4, 6, 8, 11, 14, and 28) (Fig. RAL. Various amounts of RAL (high dose, low dose) were 2). Also, no neoplastic change was found in the livers of 4 administered to determine the most effective schedule for normal animals of similar age (not shown). induction of neoplastic lesions (Fig. 3). When hamsters were Proplastic Foci 1 3 43 21 1 3 1 1 4 2 4 HCA 2 2 Neoplasic -L HICC 2 1 1 1 1

Apoptosis + +++ ||+++ ++++ t++ FocAfNerRAis + +++ ++|++ +++ + +++ ++ +++ +++ ++++ + ++++ MAB ++++|+++ ++ ++ ++ ++ ++ + ++ + + + ++ +++||++X++ + +++ + Non- EMH + + + +++ + ++ + + + ++ + ++++++++ ++ + + ++ ++ itodsi 2 1 1230 1 5 5 12 121214 S 7 2 2 7 4 3 Neoplasuic Sex FF M FF MM F F M FF F M, F FF FFFFF FFF M Day FFMMM.FMMMS ALer RAL 0 7 13 21 31 56 119 142 236 236 _ 36n PLUS TAM

FIG. 2. Kinetic analysis of histological changes in Armenian hamster liver after one injection of RAL pellets (36 mg on day 0). Female (F) and male (M) hamsters were examined at particular time intervals, and histological changes of individual hamsters are entered in vertical columns; the presence of a nonneoplastic change is noted by "+," and the presence of preneoplastic and neoplastic lesions is indicated by the number of lesions detected per liver. The mitosis number is the number of mitotic figures counted per two high-power fields. One group of 5 hamsters injected with the same RAL also was given 5 mg of Tam (on days 0, 4, 6, 8, 11, 14, and 28), which blocked chronic neoplastic lesions of RAL (day 236 necropsy). Tam also prevented the hyperbilirubinemia effect of RAL (not shown). Four normal Armenian hamsters (3 female, 1 male) 270 days old also were examined, and no pathologic changes were detected (not shown). Downloaded by guest on September 28, 2021 Medical Sciences: Coe et al. Proc. Natl. Acad. Sci. USA 89 (1992) 1087

100 100-

80- C c 0 0 'a 5) 0 60- 60-

C 40

020 0 a. 20 10' iLL 10- 12 mg (xl) RAL treatmnent 36 mg (x3) 12 mg (x3) Treatment RAL RAL RAL + TAM

Schedule day 0 day 0,94, 183 day 0, 150,210 DAY Necropsy 174 202 202 Day necropsy 236 270 240 Number dc 4 h,4 f Number 8 S,7d' 4, 3d' S4, 2C Animals 5q ,2 7!,99d' Animals FIG. 3. Preneoplastic and neoplastic changes found in liver after FIG. 4. Preneoplastic foci (n) and neoplastic HCA (a), and HCC (n) lesions detected on days 174 and 202 after RAL treatment and maximal RAL (36 mg x 3) or minimal RAL (12 mg x 3 or 12 mg x Tam. RAL 1). The percentage of hamster livers containing foci (o), HCA (s), prevention of those lesions by concomitant injections of pellets (36 mg) were implanted on day 0 and day 94, and Tam (5 mg) and HCC (n) is shown. In the group receiving 36 mg of RAL three was injected s.c. twice per week to day 32 and then once per week times, either HCA or HCC were found in all 15 livers examined with to day 202. Results of male and female hamsters were compiled; similar incidence in males and females; however, the average number however, more HCC were found in female livers: on day 202, 6 of 7 per was greater in females range 1-4) than males of HCC liver (2.6, females (86%) and 3 of 9 males (33%) had detectable HCC. When (1.4, range 1-3). In this group, HCA were primarily basophilic in 12 of 15 livers, although 2 livers had acidophilic HCA and 4 livers Tam also was given, only one foci was found in 1 liver (male) of 8 animals examined, although MB and venous prolapse were found in contained mixed HCA; foci were basophilic in 9 of 15 livers and acidophilic in 2 livers. all livers. the acute but also blocked the given repeated high doses of RAL (36 mg of pellets on days inhibited hyperbilirubinemia chronic induced by RAL. A control group 0, 94, and 183), an impressive array of neoplastic changes neoplastic changes 2 that received the Tam occurred in all 15 livers examined on day 236; HCC were of 4 animals (2 females, males) detectable in 6 of 8 females and 6 of 7 males. Multiple doses injections only was examined histologically on day 202 (not no or lesions were detected, of RAL were well tolerated by the hamsters with little shown); preneoplastic neoplastic some MB were found in livers of the 2 females. The morbidity or mortality and resulted in more hepatic neo- although Armenian hamsters (6 female, 4 male) plasms than one dose of RAL. livers from 10 normal To evaluate the hepatic pathology induced with lower also were carefully examined for histopathological changes All these animals were more than 1.5 years of doses ofRAL, a 12-mg pellet was injected either once or three (not shown). times (Fig. 3). Livers examined from all 15 animals treated for age, and therefore, were older than any experimental hamster 240-270 days showed evidence in hepatic parenchyma of reported in this study. There was no evidence of spontaneous preneoplastic/neoplastic change or MB formation in the RAL exposure (i.e., MB and venous prolapse, not shown), livers of these old hamsters. and 1 of 3 animals showed neoplastic changes after only one 12-mg pellet. Animals receiving one 12-mg pellet three times (on days 0, 150, and 210) had greater hepatic changes, with DISCUSSION two livers neoplastic lesions found in 3 of 4 livers, and of the Zeranol (RAL) is an effective agent for induction of HCC in contained HCC. Hyperbilirubinemia was usually not de- Armenian hamsters. Such a clear-cut relationship between tected in animals given the 12-mg RAL dose. carcinogenesis and a RAL compound has not been shown Effect of Two Doses RAL ± Continuous Tam. Multiple before to our knowledge. This observation was possible injections of RAL pellets (36 mg) were a convenient regimen because of the uncommon susceptibility of the liver of the to produce maximal hepatic lesions with minimal morbidity. Armenian hamster to estrogens. However, these mycoestro- Accordingly, a larger group ofanimals (16) was evaluated 174 gens are difficult to avoid in the food ofanimals and man, and and 202 days after two injections of RAL pellets (36 mg) on their presence could represent a concern for another suscep- days 0 and 94 (Fig. 4). By day 202, neoplastic change was tible species or a susceptible individual of an otherwise detectable in all 7 females (100%o) and in 7 of 9 males (78%); resistant species. The susceptibility ofthe Armenian hamster 86% of the females (6 of 7) had at least two detectable HCC, to RAL-induced HCC is not even shared by other hamsters whereas HCC was found in only 3 of 9 males. On the other such as the closely related Chinese hamster (Cricetulus hand, the incidence of foci (females 71%, males 67%) and griseus) or by the Syrian hamster (Mesocricetus auratus) HCA (females 57%, males 56%) was similar in both sexes. (unpublished data). In fact, zeranol has little estrogenic Some of the animals (5 females, 2 males) in this experiment activity in the Syrian hamster, even when very large doses were sacrificed at an earlier time (day 174) (Fig. 4). Hepatic are used (29). pathology was less severe than on day 202, and only one HCC As measured by other parameters, the estrogenic activity was detected in 1 female hamster liver. Another group of of RAL should be 2-3 orders of magnitude less than that of animals was given the same multiple-dose RAL treatment DES (18, 22). However, the effect of RAL on Armenian and also injected weekly with Tam (5 mg s.c.) until sacrificed hamster liver appeared to be much greater than predicted. on day 202 (Fig. 4). Eight animals were examined, and only For example, the acute hepatitic effect (hyperbilirubinemia) one acidophilic focus was detected in 1 liver (male); however, produced by 36 mg of RAL pellets (Fig. 1) was similar to that all hamsters (RAL + Tam) had MB and venous prolapse in detected after a 6-mg DES pellet (unpublished observation). nonneoplastic portions of liver. Therefore, Tam not only Also, the incidence of chronic hepatic lesions was more Downloaded by guest on September 28, 2021 1088 Medical Sciences: Coe et al. Proc. Natl. Acad. Sci. USA 89 (1992) consistent after injection of 36 mg of RAL pellets than after effectiveness of Tam in blocking estrogen-induced hamster administration of a 6-mg DES pellet (unpublished observa- hepatotoxicity and carcinogenesis suggests that ER is in- tions). Differences in pellet characteristic (absorption, etc.) volved in the pathogenesis, although Tam can also affect complicate a direct comparison between the RAL and DES other cell constituents as calmodulin (44) and protein kinase effect on Armenian hamster liver, but a smaller-than- C (45). This experimental model may provide a new percep- expected difference in activity (i.e., 10-25 fold) appears tion about the mechanism by which Tam controls breast likely. cancer in women and experimental animals (41, 46). For induction of hepatic tumors, administration of RAL Although estrogens are clearly crucial for development of has distinct advantages when compared with DES because some human neoplasms, such as breast cancer, it is apparent DES is frequently lethal, whereas little mortality/morbidity that in addition to estrogen, other factors (genetic, dietary, is observed with RAL. Unlike natural estrogens, DES is a etc.) are also necessary (1). Similarly, the results of experi- known carcinogen (30) and after injection into Armenian ments in animals suggest that natural estrogens usually do not hamsters, HCC developed much more rapidly with DES than play a primary role in tumorigenesis because they rarely RAL; that is, HCC were common (five of nine hamsters) 84 cause cancer when injected alone; however, estrogens will days after DES administration (26), whereas, at least 142 days enhance (promote) the development of neoplasia when given were required after RAL injection before the first HCC was in concert with a known carcinogen (initiator) (47, 48). detected in two of seven hamsters. This protracted HCC Accordingly, from clinical and experimental experience, latent period with RAL permitted observation of a variety of estrogens have been assigned the oncological role of a preneoplastic hepatic lesions that were seen frequently after promoter agent, enhancing the susceptibility of certain or- 56 days of RAL treatment but were rarely seen in livers after to hamster thus DES treatment (26). Progression offoci/HCA to HCC could gans initiator carcinogens. The Armenian is not be defined in the present study, although areas of HCC an unusual animal because estrogen (as zeranol) alone is developing at the center of an HCA were observed to occur acting as the apparent initiator of HCC. The oncogenic as has been reported before (28). Similar to other kinetic mechanism in this experimental animal may relate to the studies of liver tumorigenesis (31-33), we also observed that susceptibility of some women to estrogen-induced carcino- foci and HCA appeared at an earlier time than HCC. We do genesis. not know if any of these hepatic lesions are reversible; Other organs in the Armenian hamster contain ER; how- however, foci were observed many months after complete ever, only the liver is affected adversely by estrogen. This absorption of only one RAL dose (=90 days), suggesting a organ tropism may be a function of preferential liver expo- certain permanence of this change and also indicating that it sure to estrogen (via concentration or prolonged half life) or was not dependent upon continued presence of RAL. could result from a unique hepatic metabolism. Perhaps this As is the case with other experimental models of hepato- hamster's liver somehow has been endogenously initiated. If carcinogenesis, the dose and duration of zeranol exposure so, no evidence for spontaneous occurrence of neoplastic correlated directly with the number of lesions detected (31- lesions has been seen in normal animals, even after 2 years 33). Multiple doses ofRAL were well tolerated, and after the of age. Also preliminary experiments with nitrosodimethyl- first injection, the hamsters acquired a resistance to acute amine as an initiating agent did not suggest a peculiar RAL hepatotoxicity (at least as measured by bilirubinemia), susceptibility of the Armenian hamster liver to neoplastic similar to results with DES (26). change at least by this agent (unpublished results). On the In the Armenian hamster, the actual relationship between other hand, the Armenian hamster may have an endogenous acute hepatic toxicity and subsequent neoplastic change is hepatic infection by a retrovirus (49) or a virus similar to unknown. However, a number of acute pathologic changes human hepatitis B virus, an agent important in human HCC were observed that may be important. For example, the induction (50), but histopathological evidence of hepadnavi- marked hepatic mitotic activity during the first month after rus infection is lacking. RAL treatment could be a critical element in subsequent Studies ofhuman colorectal tumors indicate that increasing neoplastic development; this may represent an acute restor- chromosomal damage correlates with the progression from ative-type hyperplasia or a direct anabolic effect of RAL that early adenomato carcinoma; the additive effects ofmutations is necessary for genotoxic change and/or for its amplifica- progressively enhance the opportunities for clonal expansion tion. The anabolic effect of RAL could reflect an enhanced of cells that are over expressing oncogenes (growth factors) expression of epidermal growth factor (EGF), a factor that and/or have lost tumor-suppressor genes (51). A similar has been shown to mediate estrogen effects (34), or could accumulation of structural changes may occur during the represent overexpression of an EGF receptor, such as Her2/ multistage progression of hepatic carcinogenesis (foci -) neu (35), an oncogene product that is frequently up-regulated HCA -) HCC) (31-33). The zeranol-induced hepatic tumor in development of human breast cancer (36). The rapid model in the Armenian hamster provides a series of easily appearance of MB in the liver of Armenian hamsters after identified and isolated premalignant-to-malignant lesions; estrogen treatment is extraordinary, as a prolonged induction these lesions are probably of monoclonal origin, and char- period is necessary to develop hepatocyte MB in other acterization of genetic damage should be feasible at the experimental models (37, 38). These cytoplasmic inclusions molecular level. Definition of the genetic change initiated by are a morphologic feature often seen in neoplastic hepato- estrogen in this model may provide new insight into the cytes (39, 40). In the liver of Armenian hamster, MB may be pathogenesis of hormone-induced carcinogenesis. a very sensitive indicator of estrogen effect, since they are present in hamsters treated with Tam and RAL where Tam 1. Lippman, M. E. & Dickson, R. B. (1989) Yale J. Biol. Med. 62, had blocked other effects of RAL-i.e., hyperbilirubinemia 459-480. and neoplastic lesions. Mallory bodies also are detected in 2. Voigt, L. F. & Weiss, N. S. (1989) in Endometrial Cancer, eds. livers of some animals after treatment with Tam Surwitt, E. & Alberts, D. (Kluwer, Boston), pp. 1-27. alone, 3. Eisenfeld, A. J. (1974) J. Biochem. 5, 328-329. indicating that Tam may have both antagonistic and agonistic 4. Chamness, G. C., Costlow, M. E. & McGuire, W. L. (1975) effects in Armenian hamster liver similar to findings in other 26, 363-371. experimental animals (41). Injections of Tam alone did not 5. Powell-Jones, W., Davies, W. P. & Griffiths, K. (1976) Endo- induce neoplastic changes in Armenian hamster; however, crinology 69, 167-171. Tam did promote preneoplastic lesions in livers of mice and 6. Beers, P. C. & Rosner, W. (1977) J. Steroid Biochem. 8, rats when used with a carcinogenic agent (42, 43). The 251-258. 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