Proc. Nati. Acad. Sci. USA Vol. 89, pp. 1085-1089, February 1992 Medical Sciences Tamoxifen prevents induction of hepatic neoplasia by zeranol, 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 estrogen activity. It is synthesized by nous estrogen, diethylstilbestrol (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 mycoestrogen 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 estrogens. 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 estradiol), 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 nonsteroidal 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. Zearalenone 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 anol (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, estrogen receptor; 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.