Tamoxifen prevents induction of hepatic neoplasia by zeranol ... - PNAS

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 lactone (RAL) that has estrogen activity. It is synthesized by molds and is difficult to avoid in human food products. We tested the ability of this mycoestrogen to damage the liver of the Armenian hamster, a rodent that is epcialy sensitive to hepatotoxic effects of exogenous estrogens. Zeranol Induced acute hepatotoxicity and, subsequently, hepatic carcInogeneing estrge sis; both effects were blocked by tamoxifen, s receptor mediation. Because zeranol is acting alone as a primary initiator of hepatic neoplasms, this model provides an unusual opportunity to study the pathogenesis of estrogeninitiated tumorigenesis.

hamsters is known to be particularly sensitive to an exogenous estrogen, diethylstilbestrol (DES). Thus, injection of DES is followed by clinical and histological evidence of hepatocellular degeneration (24) that is blocked by concomitant injection of tamoxifen (Tam), suggesting that hepatic ER is involved in its pathogenesis (25). Furthermore, chronic exposure to DES is associated with the formation of hepatocellular carcinomas (26). Such profound hepatic toxicity and carcinogenicity induced by DES alone is unknown in other experimental animals. Therefore, it is pertinent to determine the effects of other estrogens, and zeranol is an especially appropriate compound to study because it is structurally dissimilar to DES and is a relatively weak estrogen (2-3 orders of magnitude less potent than DES or estradiol), yet is environmentally important as a (human) food contaminant (18, 19, 22). The results indicate that zeranol is a particularly suitable estrogen for induction of acute toxic and chronic neoplastic changes in the liver of the Armenian hamster. Because zeranol acts as a primary initiator of hepatic tumors, this model provides an unusual opportunity to study the pathogenesis of estrogen-induced carcinogenesis.

There is now ample evidence supporting the role of estrogens in the neoplastic process. Attention has been focused on the involvement of estrogens in the development of breast and endometrial cancer (1, 2). However, other organs such as liver also contain a functioning estrogen-receptor (ER) mediator and, accordingly, are targets for control by circulating estrogens (3-8). Indeed, estrogens taken by women for birth control purposes are implicated as a cause for development of both benign and malignant hepatic neoplasms (9-16). In addition to endogenous steroidal estrogens, a variety of compounds with estrogenic activity also are found in the environment because a number of drugs, insecticides, and natural food products representing many different structures can act like estrogens after ingestion (17). The .-resorcylic acid lactones (RALs) are a group of natural products with similar nonsteroidal structure that have estrogen activity (18, 19). RALs are important because they are stable compounds, functional after oral administration, and difficult to avoid in human food products. Zearalenone is the RAL that is found in food; it is a mycoestrogen, synthesized by molds (fusarium species) commonly contaminating grain. Another RAL, zeranol (a-zearalanol), -is found as a natural metabolic product of zearalenone and binds to the ER of uterus (20) and liver (21). The growth-promoting (anabolic) effects of zeranol have led to the commercial production and sale of this RAL for use as a growth stimulant in meat production. Therefore, human consumption of RAL compounds may be direct, by ingestion of contaminated cereal products, or indirect, by consumption of products from animals fed a mold-infected grain or injected with a RAL growth stimulant. Accordingly, RAL compounds have been tested extensively in various assays for genotoxic and neoplastic effects; to date no outstanding toxic, mutagenic, or carcinogenic changes from RAL have been observed (22), although an increased incidence of adenomas (pituitary, liver) were detected in one species after a 2-yr oral carcinogenicity study (23). The present report describes experiments in which zeranol was administered to Armenian hamsters. The liver of these

MATERIALS AND METHODS Animals. Armenian hamsters (Cricetulus migratorius) were obtained from the Rocky Mountain Laboratories production unit. They were provided free access to food pellets (Purina Lab Chow) and water. Hepatic synthesis of some proteins in Armenian hamster is influenced by light (27) so all animals were housed in a room illuminated by fluorescent lights with a 16/8-hr light/dark cycle. Drugs. Zeranol (a-zearalanol, 12-mg pellets, Ralgro, Pitman-Moore, Terra Haute, IN) (also referred to as RAL in this report) was surgically implanted s.c. in the anesthetized (Metofane, Pitman-Moore) animal. Tam (supplied by Stuart Pharmaceuticals, Wilmington, DE) was suspended in propylene glycol, and 0.1 ml (5 mg) was injected s.c. Experimental Design. Male and female Armenian hamsters (in groups of 5-20) were started on experimental drug treatment when 2-3 months of age. At appropriate intervals, animals were sacrificed for histological examination. Control animals (no drug treatment) of comparable age or older than the experimental animals were also examined to ascertain the effects of aging alone. Blood samples from anesthetized hamsters were obtained from the retroorbital area or by bleeding from the heart, and serum bilirubin was determined as before (25). At necropsy, 2- to 3-mm slices of liver were carefully examined for gross pathology; presumptive nodules Abbreviations: DES, diethylstilbestrol; EGF, epidermal growth factor; ER, estrogen receptor; RAL, resorcylic acid lactone (or zeranol); Tam, tamoxifen; HCC, hepatocellular carcinoma(s); HCA, hepatocellular adenoma(s); EMH, extramedullary hematopoiesis; MB, Mallory body/bodies. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

1085

1086

Proc. Natl. Acad. Sci. USA 89 (1992)

Medical Sciences: Coe et al.

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 determined independently by two of us (K.G.I. & J.M.W.), and classification of the neoplasm was based on previously published histological criteria (28). The animal experiments conducted for this study were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

12' E 10-

-

a

42

RESULTS

1Rai36

+++

EMH

itodsi Sex

Day ALer RAL _

36n

li2

t 10

8

FIG. 1. Hyperbilirubinemia induced by RAL and its prevention by concomitant Tam administration. Serum bilirubin promptly increases in Armenian hamster females (n) (n = 10) and males (A) (n = 10) after injection of RAL pellets (36 mg) on day 0. The icterus is transient, and the animals do not show such hyperbilirubinemia upon a repeat RAL injection (36 mg) on day 94. In addition to RAL, one group of female and male hamsters (0) (n = 11) also received Tam (5 mg twice weekly for 4 weeks and then once per week thereafter), which inhibited the bilirubinemia response until day 94, when serum bilirubin increased slightly (2.0-2.8 mg/dl). (Bar = +1 SEM.)

Preneoplastic/neoplastic lesions grossly appeared as circumscribed pale nodules. The diameter frequently correlated with the type of nodule; that is, foci were smallest (1-3 mm), HCC largest (6-15 mm), and HCA intermediate in size. Larger nodules were usually found after longer treatment with RAL. No evidence of metastasis was detected. Among the notable nonneoplastic changes found in liver after RAL treatment (Fig. 2) were a very marked but transient stimulation of mitosis and the constant presence of Mallory bodies (MB) and extramedullary hematopoiesis (EMH). Focal necrosis and transient apoptosis were also observed. These lesions were not seen in livers of control (normal) animals (not shown), but MB, EMH, and focal necrosis were found in RAL- and Tam-treated hamsters (Fig. 2). Venous prolapse was usually found in livers examined 142 days or more after RAL administration (not shown) and histologically was similar to that previously seen in livers of Armenian hamsters treated with DES (24). A minimal amount of venous prolapse also was detected occasionally in livers from some old normal Armenian hamsters (>1.5 years of age) (not shown). Hepatic Neopblsms Induced with Higher and Lower Doses of RAL. Various amounts of RAL (high dose, low dose) were administered to determine the most effective schedule for induction of neoplastic lesions (Fig. 3). When hamsters were 1

3 43 2

21 1 3 1 1 2 1 2 1

424 1

1

++++ t++ +++ ||+++ + + +++ ++|++ +++ +++ +++ ++++ + ++++ + +++ ++ + +++ + + ++ + + ++ + + ++ ++ ++ ++ ++ +||++X+ ++++|++ ++ +

FocAfNerRAis MAB

NonNeoplasuic

60

Days

Effect of RAL (Without and with Tam) on Serum Bilfirubin. Armenian hamsters became jaundiced after injection of RAL [three 12-mg pellets (36 mg total) on day 0]. Fig. 1 shows that increased bilirubin levels were detected in all animals within 6 days, peaked at "13 mg/dl within 14 days, and then gradually decreased to minimal levels by 50-60 days. Malefemale response usually was similar in magnitude, although female bilirubinemia usually remained elevated for a longer time. Both sexes were refractory to this RAL effect when reinjected on day 94 (Fig. 1), as a minimal bilirubin response was detected only after a prolonged interval (i.e., on day 112, 18 days after reimplantation). RAL pellets were very difficult to detect more than 2.5 months after implantation. Another group of male-female hamsters implanted with 36 mg of RAL pellets on day 0 was also injected with Tam (5 mg twice weekly for 4 weeks and then once per week thereafter). Tam inhibited the bilirubinemia response of both sexes to RAL, as no increase was detected until late (day 94) when serum bilirubin increased slightly (2.0-2.8 mg/dl) (Fig. 1). Hepatic Histolo after 36 mg of RAL. Male and female Armenian hamsters injected once with 36 mg of RAL pellets on day 0 were sacrificed for necropsy at specified intervals thereafter. The results of histological examination of the livers are tabulated in Fig. 2 and shown as preneoplastic/ neoplastic or nonneoplastic changes. The first preneoplastic changes (foci) were detected on day 56 and by day 119 had become more abundant and were joined by hepatocellular adenomas (HCA). Subsequently, hepatocellular carcinomas (HCC) were found in 3 of 6 female livers on day 142; at this time 6 of 6 females had hepatic lesions versus only 1 of 3 males. On day 236, 3 of 4 hamsters showed preneoplastic/ neoplastic change; however, no lesions were found in 5 hamsters that had received RAL and also a short course of Tam injections (5 mg, on days 0, 4, 6, 8, 11, 14, and 28) (Fig. 2). Also, no neoplastic change was found in the livers of 4 normal animals of similar age (not shown). Foci Proplastic HCA Neoplasic -L HICC

Apoptosis

40

20

+ + + + + + + + + ++++++++ + +

+ ++

2 1 1230 1 5 5 12 121214 S 7 2 2

FF M 0

F F MM F F M 13 7

F F F M, 21

31

7

FF F 56 119

4

+ + ++ ++

3

F FF FFMMM.FMMMS

FFF F F 142

236

M

236 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).

Medical Sciences: Coe

et


al.

100

1087

100-

C 0

c

80-

0

'a

5)

0

60-

60-

C

020

40

0

10' RAL treatmnent

Schedule Day necropsy Number Animals

iLL 12 mg (xl)

36 mg (x3)

12 mg (x3)

day 0, 94, 183

day 0, 150,210

day 0

236

270

8 S, 7d'

4, 3d'

240 S4, 2C

a.

20 10-

Treatment

RAL

RAL

RAL + TAM

DAY Necropsy

174

202

202

5q ,2 dc

7!,99d'

4 h,4 f

Number Animals

FIG. 3. Preneoplastic and neoplastic changes found in liver after maximal RAL (36 mg x 3) or minimal RAL (12 mg x 3 or 12 mg x 1). The percentage of hamster livers containing foci (o), HCA (s), and HCC (n) is shown. In the group receiving 36 mg of RAL three times, either HCA or HCC were found in all 15 livers examined with similar incidence in males and females; however, the average number of HCC per liver was greater in females (2.6, range 1-4) than males (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 contained mixed HCA; foci were basophilic in 9 of 15 livers and acidophilic in 2 livers.

FIG. 4. Preneoplastic foci (n) and neoplastic HCA (a), and HCC (n) lesions detected on days 174 and 202 after RAL treatment and prevention of those lesions by concomitant injections of Tam. RAL pellets (36 mg) were implanted on day 0 and day 94, and Tam (5 mg) was injected s.c. twice per week to day 32 and then once per week to day 202. Results of male and female hamsters were compiled; however, more HCC were found in female livers: on day 202, 6 of 7 females (86%) and 3 of 9 males (33%) had detectable HCC. When 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 all livers.

given repeated high doses of RAL (36 mg of pellets on days 0, 94, and 183), an impressive array of neoplastic changes occurred in all 15 livers examined on day 236; HCC were detectable in 6 of 8 females and 6 of 7 males. Multiple doses of RAL were well tolerated by the hamsters with little morbidity or mortality and resulted in more hepatic neoplasms than one dose of RAL. To evaluate the hepatic pathology induced with lower doses of RAL, a 12-mg pellet was injected either once or three times (Fig. 3). Livers examined from all 15 animals treated for 240-270 days showed evidence in hepatic parenchyma of RAL exposure (i.e., MB and venous prolapse, not shown), 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 neoplastic lesions found in 3 of 4 livers, and two of the livers contained HCC. Hyperbilirubinemia was usually not detected in animals given the 12-mg RAL dose. Effect of Two Doses RAL ± Continuous Tam. Multiple injections of RAL pellets (36 mg) were a convenient regimen to produce maximal hepatic lesions with minimal morbidity. Accordingly, a larger group of animals (16) was evaluated 174 and 202 days after two injections of RAL pellets (36 mg) on days 0 and 94 (Fig. 4). By day 202, neoplastic change was detectable in all 7 females (100%o) and in 7 of 9 males (78%); 86% of the females (6 of 7) had at least two detectable HCC, whereas HCC was found in only 3 of 9 males. On the other hand, the incidence of foci (females 71%, males 67%) and HCA (females 57%, males 56%) was similar in both sexes. Some of the animals (5 females, 2 males) in this experiment were sacrificed at an earlier time (day 174) (Fig. 4). Hepatic pathology was less severe than on day 202, and only one HCC was detected in 1 female hamster liver. Another group of animals was given the same multiple-dose RAL treatment and also injected weekly with Tam (5 mg s.c.) until sacrificed on day 202 (Fig. 4). Eight animals were examined, and only one acidophilic focus was detected in 1 liver (male); however, all hamsters (RAL + Tam) had MB and venous prolapse in nonneoplastic portions of liver. Therefore, Tam not only

inhibited the acute hyperbilirubinemia but also blocked the chronic neoplastic changes induced by RAL. A control group of 4 animals (2 females, 2 males) that received the Tam injections only was examined histologically on day 202 (not shown); no preneoplastic or neoplastic lesions were detected, although some MB were found in livers of the 2 females. The livers from 10 normal Armenian hamsters (6 female, 4 male) also were carefully examined for histopathological changes (not shown). All these animals were more than 1.5 years of age, and therefore, were older than any experimental hamster reported in this study. There was no evidence of spontaneous preneoplastic/neoplastic change or MB formation in the livers of these old hamsters.

DISCUSSION Zeranol (RAL) is an effective agent for induction of HCC in Armenian hamsters. Such a clear-cut relationship between carcinogenesis and a RAL compound has not been shown before to our knowledge. This observation was possible because of the uncommon susceptibility of the liver of the Armenian hamster to estrogens. However, these mycoestrogens are difficult to avoid in the food of animals and man, and their presence could represent a concern for another susceptible species or a susceptible individual of an otherwise resistant species. The susceptibility of the Armenian hamster to RAL-induced HCC is not even shared by other hamsters such as the closely related Chinese hamster (Cricetulus griseus) or by the Syrian hamster (Mesocricetus auratus) (unpublished data). In fact, zeranol has little estrogenic activity in the Syrian hamster, even when very large doses are used (29). As measured by other parameters, the estrogenic activity of RAL should be 2-3 orders of magnitude less than that of DES (18, 22). However, the effect of RAL on Armenian hamster liver appeared to be much greater than predicted. For example, the acute hepatitic effect (hyperbilirubinemia) produced by 36 mg of RAL pellets (Fig. 1) was similar to that detected after a 6-mg DES pellet (unpublished observation). Also, the incidence of chronic hepatic lesions was more

1088

Medical Sciences: Coe et al.

consistent after injection of 36 mg of RAL pellets than after administration of a 6-mg DES pellet (unpublished observations). Differences in pellet characteristic (absorption, etc.) complicate a direct comparison between the RAL and DES effect on Armenian hamster liver, but a smaller-thanexpected difference in activity (i.e., 10-25 fold) appears likely. For induction of hepatic tumors, administration of RAL has distinct advantages when compared with DES because DES is frequently lethal, whereas little mortality/morbidity is observed with RAL. Unlike natural estrogens, DES is a known carcinogen (30) and after injection into Armenian hamsters, HCC developed much more rapidly with DES than RAL; that is, HCC were common (five of nine hamsters) 84 days after DES administration (26), whereas, at least 142 days were required after RAL injection before the first HCC was detected in two of seven hamsters. This protracted HCC latent period with RAL permitted observation of a variety of preneoplastic hepatic lesions that were seen frequently after 56 days of RAL treatment but were rarely seen in livers after DES treatment (26). Progression of foci/HCA to HCC could not be defined in the present study, although areas of HCC developing at the center of an HCA were observed to occur as has been reported before (28). Similar to other kinetic studies of liver tumorigenesis (31-33), we also observed that foci and HCA appeared at an earlier time than HCC. We do not know if any of these hepatic lesions are reversible; however, foci were observed many months after complete absorption of only one RAL dose (=90 days), suggesting a certain permanence of this change and also indicating that it was not dependent upon continued presence of RAL. As is the case with other experimental models of hepatocarcinogenesis, the dose and duration of zeranol exposure correlated directly with the number of lesions detected (3133). Multiple doses of RAL were well tolerated, and after the first injection, the hamsters acquired a resistance to acute RAL hepatotoxicity (at least as measured by bilirubinemia), similar to results with DES (26). In the Armenian hamster, the actual relationship between acute hepatic toxicity and subsequent neoplastic change is unknown. However, a number of acute pathologic changes were observed that may be important. For example, the marked hepatic mitotic activity during the first month after RAL treatment could be a critical element in subsequent neoplastic development; this may represent an acute restorative-type hyperplasia or a direct anabolic effect of RAL that is necessary for genotoxic change and/or for its amplification. The anabolic effect of RAL could reflect an enhanced expression of epidermal growth factor (EGF), a factor that has been shown to mediate estrogen effects (34), or could represent overexpression of an EGF receptor, such as Her2/ neu (35), an oncogene product that is frequently up-regulated in development of human breast cancer (36). The rapid appearance of MB in the liver of Armenian hamsters after estrogen treatment is extraordinary, as a prolonged induction period is necessary to develop hepatocyte MB in other experimental models (37, 38). These cytoplasmic inclusions are a morphologic feature often seen in neoplastic hepatocytes (39, 40). In the liver of Armenian hamster, MB may be a very sensitive indicator of estrogen effect, since they are present in hamsters treated with Tam and RAL where Tam had blocked other effects of RAL-i.e., hyperbilirubinemia and neoplastic lesions. Mallory bodies also are detected in livers of some animals after treatment with Tam alone, indicating that Tam may have both antagonistic and agonistic effects in Armenian hamster liver similar to findings in other experimental animals (41). Injections of Tam alone did not induce neoplastic changes in Armenian hamster; however, Tam did promote preneoplastic lesions in livers of mice and rats when used with a carcinogenic agent (42, 43). The


effectiveness of Tam in blocking estrogen-induced hamster hepatotoxicity and carcinogenesis suggests that ER is involved in the pathogenesis, although Tam can also affect other cell constituents as calmodulin (44) and protein kinase C (45). This experimental model may provide a new perception about the mechanism by which Tam controls breast cancer in women and experimental animals (41, 46). Although estrogens are clearly crucial for development of some human neoplasms, such as breast cancer, it is apparent that in addition to estrogen, other factors (genetic, dietary, etc.) are also necessary (1). Similarly, the results of experiments in animals suggest that natural estrogens usually do not play a primary role in tumorigenesis because they rarely cause cancer when injected alone; however, estrogens will enhance (promote) the development of neoplasia when given in concert with a known carcinogen (initiator) (47, 48). Accordingly, from clinical and experimental experience, estrogens have been assigned the oncological role of a promoter agent, enhancing the susceptibility of certain organs to initiator carcinogens. The Armenian hamster is thus an unusual animal because estrogen (as zeranol) alone is acting as the apparent initiator of HCC. The oncogenic mechanism in this experimental animal may relate to the susceptibility of some women to estrogen-induced carcino-

genesis.

Other organs in the Armenian hamster contain ER; however, only the liver is affected adversely by estrogen. This organ tropism may be a function of preferential liver exposure to estrogen (via concentration or prolonged half life) or could result from a unique hepatic metabolism. Perhaps this hamster's liver somehow has been endogenously initiated. If so, no evidence for spontaneous occurrence of neoplastic lesions has been seen in normal animals, even after 2 years of age. Also preliminary experiments with nitrosodimethylamine as an initiating agent did not suggest a peculiar susceptibility of the Armenian hamster liver to neoplastic change at least by this agent (unpublished results). On the other hand, the Armenian hamster may have an endogenous hepatic infection by a retrovirus (49) or a virus similar to human hepatitis B virus, an agent important in human HCC induction (50), but histopathological evidence of hepadnavirus infection is lacking. Studies of human colorectal tumors indicate that increasing chromosomal damage correlates with the progression from early adenoma to carcinoma; the additive effects ofmutations progressively enhance the opportunities for clonal expansion of cells that are over expressing oncogenes (growth factors) and/or have lost tumor-suppressor genes (51). A similar accumulation of structural changes may occur during the multistage progression of hepatic carcinogenesis (foci -) HCA -) HCC) (31-33). The zeranol-induced hepatic tumor model in the Armenian hamster provides a series of easily identified and isolated premalignant-to-malignant lesions; these lesions are probably of monoclonal origin, and characterization of genetic damage should be feasible at the molecular level. Definition of the genetic change initiated by estrogen in this model may provide new insight into the pathogenesis of hormone-induced carcinogenesis.

Lippman, M. E. & Dickson, R. B. (1989) Yale J. Biol. Med. 62, 459-480. 2. Voigt, L. F. & Weiss, N. S. (1989) in Endometrial Cancer, eds. Surwitt, E. & Alberts, D. (Kluwer, Boston), pp. 1-27. 3. Eisenfeld, A. J. (1974) J. Steroid Biochem. 5, 328-329. 4. Chamness, G. C., Costlow, M. E. & McGuire, W. L. (1975) Steroids 26, 363-371. 5. Powell-Jones, W., Davies, W. P. & Griffiths, K. (1976) Endocrinology 69, 167-171. 6. Beers, P. C. & Rosner, W. (1977) J. Steroid Biochem. 8, 1.

251-258.

Medical Sciences: Coe et al. 7. Aten, R. F., Weinberger, M. J. & Eisenfeld, A. J. (1978) Endocrinology 102, 433-442. 8. Powell-Jones, W., Nayfeh, S. N. & Lucier, G. W. (1978) Biochem. Biophys. Res. Commun. 85, 167-173. 9. Baum, J. K., Holtz, F., Bookstein, J. J. & Klein, E. W. (1973) Lancet U, 926-929. 10. Mahboubi, E. & Shubik, P. (1976) Cancer Lett. 1, 331-338. 11. Mays, E. T. & Christopherson, W. M. (1984) Sem. Liver Dis. 4, 147-157. 12. Rooks, J. B., Ory, H. W., Ishak, K. G., Strauss, L. T., Greenspan, J. R., Hill, A. P. & Tyler, C. W., Jr. (1979) J. Am. Med. Assoc. 242, 644-648. 13. Edmondson, H. A., Henderson, B. & Benton, B. (1976) N. Engl. J. Med. 29. 470-472. 14. Neuberger, J., Forman, D., Doll, R. & Williams, R. (1986) Br. Med. J. 292, 1355-1357. 15. Forman, D., Vincent, T. J. & Doll, R. (1986) Br. Med. J. 292,

1357-1361. 16. Henderson, B. S., Preston-Martin, S., Edmondson, H. A., Peters, R. L. & Pike, M. C. (1983) Br. J. Cancer 48, 437-440. 17. McLachlan, J. A., ed. (1985) in Proceedings ofthe Symposium, Estrogens in the Environment (Elsevier, New York). 18. Lindsay, D. G. (1985) Food Chem. Toxicol. 23, 767-774. 19. Schoental, R. (1985) Adv. Cancer Res. 45, 217-290. 20. Katzenellenbogen, B. S., Katzenellenbogen, J. A. & Mordecai, D. (1979) Endocrinology 105, 33-40. 21. Powell-Jones, W., Raeford, S. & Lucier, G. W. (1981) Mol. Pharmacol. 20, 35-42. 22. Kuiper-Goodman, T., Scott, P. M. & Watanabe, H. (1987) Regul. Toxicol. Pharmacol. 7, 253-306. 23. National Toxicology Program (1982) National Toxicology Program Technical Report 81-54 (Natl. Inst. Health, Bethesda, MD), NIH Publ. No. 83-1791 7. 24. Coe, J. E., Ishak, K. G. & Ross, M. J. (1983) Hepatology 3, 489-496. 25. Coe, J. E. & Ross, M. J. (1988) Endocrinology 122, 137-144. 26. Coe, J. E., Ishak, K. G. & Ross, M. J. (1990) Hepatology 11, 570-577. 27. Coe, J. E. & Ross, M. J. (1990) Am. J. Physiol. 259, R341R349. 28. Frith, C. H. & Ward, J. M. (1979) J. Environ. Pathol. Toxicol. 3, 329-351. 29. Coe, J. E. & Ross, M. J. (1990) J. Exp. Med. 171, 1257-1267.

Proc. Nati. Acad. Sci. USA 89 (1992)

1089

30. Herbst, A. L., Ultelder, H. & Poskanzer, D. C. (1971) N. Engl. J. Med. 248, 878-881. 31. Farber, E. & Sarma, D. S. R. (1987) Lab. Invest. 56, 4-22. 32. Pitot, H. C. (1988) in The Liver: 2nd Ed. Biology and Pathobiology, eds. Arias, J. M., Jakoby, W. B., Popper, H., Schachter, D. & Shafritz, D. A. (Raven, New York), pp. 1125-1146. 33. Bannasch, P. (1990) J. Gastroenterol. Hepatol. S, 149-159. 34. Nelson, K. G., Takahashi, T., Bossert, N. L., Walmer, D. K. & McLachlan, J. A. (1991) Proc. Natl. Acad. Sci. USA 88, 21-25. 35. Schechter, A. L., Stem, D. F., Vaidyanathan, L., Decker, S. J., Drebin, J. A., Greene, M. I. & Weinberg, R. A. (1984) Nature (London) 312, 513-516. 36. Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J., Ullrich, A. & Press, M. F. (1989) Science 24, 707-712. 37. Denk, H., Gschnait, F. & Wolff, K. (1975) Lab. Invest. 32, 773-776. 38. Yokoo, H., Harwood, T. R., Racker, D. & Arak, S. (1982) Gastroenterol. 83, 109-113. 39. Cadrin, M., Kawahara, H., Ohta, M., Katsuma, Y., Marceau, N. & French, S. W. (1990) in Mouse Liver Carcinogenesis: Mechanisms and Species Comparisons (Liss, New York), pp. 231-248. 40. Hoso, M. & Nakanuma, Y. (1990) Liver 10, 264-268. 41. Furr, B. J. A. & Jordan, V. C. (1984) Pharmacol. Ther. 25, 127-205. 42. Yager, J. D. & Shi, Y. E. (1991) Prev. Med. 20, 27-37. 43. Dragan, P. D., Xu, Y.-D. & Pitot, H. C. (1991) Prev. Med. 20, 15-26. 44. Lam, H.-Y. (1984) Biochem. Biophys. Res. Commun. 118, 27-32. 45. O'Brian, C. A., Liskamp, R. M., Solomon, D. H. & Weinstein, I. B. (1986) J. Natl. Cancer Inst. 76, 1243-1246. 46. Jordan, V. C., Lababidi, M. K. & Langon-Fahey, S. (1991) J. Natd. Cancer Inst. 83, 492-496. 47. Hertz, R. (1978) Pediatrics 62, 1138-1142. 48. Lipsett, M. B. (1983) Cancer 51, 2426-2429. 49. Weinstein, I. B. (1988) Cancer Res. 48, 4135-4143. 50. Popper, H., Shafritz, D. A. & Hoofnagle, J. H. (1987) Hepatology 7, 764-772. 51. Fearon, E. R. & Vogelstein, B. (1990) Cell 61, 759-767.