[CANCER RESEARCH 44, 3862-3869, September 1984] Enhancement of Hepatocarcinogenesis in Female Rats by Ethinyl Estradici and Mestranol but not Estradici1 James D. Yager,2 Harold A. Campbell, Daniel S. Longnecker, B. D. Roebuck, and Mary C. Benoit Departments ölAnatomy [J. D. Y.¡,Pathology [D. S. L], Pharmacology and Toxicology [B. D. R.¡,and Medicine [M. C. B.], Dartmouth Medical School, Hanover, New Hampshire 03756, and McArdle Laboratory tor Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706 [H. A. C.] ABSTRACT In experimental studies in animals, results have been obtained which suggest that synthetic estrogen treatment following car The effect of dietary exposure to synthetic estrogens on cinogen exposure (initiation) can enhance hepatic neoplasia. hepatocarcinogenesis was evaluated. Diethylnitrosamine-initi- Taper (32) was the first investigator to demonstrate that 2 ated and 0.85% NaCI solution-treated noninitiated female synthetic steroids used in oral contraceptive preparations in Sprague-Dawley rats were transferred to semisynthetic diets Europe, estradiol 17-phenylpropionate and estradiol benzoate, containing mestranol (0, 0.1, or 0.5 ppm), ethinyl estradiol (0.5 enhanced hepatocarcinogenesis in castrated female rats previ ppm), estradiol (0.6 ppm), or mestranol plus 0-methasone (0.5 ously initiated with A/-nitrosomorpholine In the United States, 2 and 0.2 ppm, respectively). 7-Glutamyl transferase (GGT>posi- synthetic estrogens, mestranol (170-ethinyl estradiol 3-methyl tive transections and hematoxylin and eosin-detectable nodules ether) and EE, are widely used in oral contraceptive preparations. and carcinomas were scored at 9 and 12 months. Quantitative We reported that, in intact, DEN-initiatedfemale Sprague-Dawley stereological calculations were performed to determine GGT rats, mestranol alone and together with norethynodrel enhanced lesion number and size. At 9 months, in diethylnitrosamine- the appearance of GGT-positive, presumptive preneoplastic foci initiated rats, ethinyl estradiol and mestranol caused 3.5- and (39, 40, 43). The data of Vesselinovitch and Mihailovich (33) 4.4-fold increases, respectively, in the number of GGT lesions demonstrated that mestranol enhanced the incidence and mul per liver and an increased incidence of hepatocellular carcinomas tiplicity of benign liver tumors in DEN-initiated female C57BL/6 while estradiol had no enhancing effect. Addition of /S-methasone x C3H Fi mice. Cameron et al. (3, 4) reported that EE enhanced to the mestranol-containing diet caused a significant decrease in the appearance of GGT foci and hyperplastic nodules in DEN- GGT lesion number but not carcinoma incidence compared to initiated male Fischer 344 rats. Wanless and Mediine (34) also mestranol alone. At 12 months, in diethylnitrosamine-initiated reported that EE enhanced the appearance of grossly visible rats, mestranol caused a dose-dependent increase in GGT lesion hyperplastic nodules and microscopic hyperplastic foci in male number. The hepatocellular carcinoma incidence was signifi Fischer 344 rats. They indicated that some of the grossly visible cantly increased at the high mestranol dose. Small increases in nodules were well-differentiated HCs but did not report the the numbers of larger GGT lesions were also observed in non- incidence of these neoplasms. initiated animals treated with mestranol and ethinyl estradiol and In 5 (3, 4, 32-34) of the studies mentioned above, initiation are most probably due to promotion of spontaneously initiated preceded treatment with the synthetic estrogens by a period hepatocytes. These results indicated that the synthetic estro ranging from 1 to 4 weeks. This suggests that the enhancement gens cause dramatic increases in the number of presumptive of hepatocarcinogenesis caused by the synthetic estrogens is preneoplastic GGT lesions. Carcinoma incidence is also en due to promotion (Refs. 34 and 38; see "Discussion"). However, hanced. Thus, these results confirm and extend our previous to date, for mestranol and EE, no studies have been reported studies which together with the results of others have shown where their enhancing activities have been directly compared, that synthetic estrogens can act as promoters of hepatocarci where dose-response effects were analyzed, and where HC nogenesis. incidence was definitively determined. The present report de scribes the results of studies designed to address these issues. INTRODUCTION In addition, since corticosteroids are growth inhibitory for liver (9, 18), the effect of addition of ¿3-methasonetoa mestranol- In humans,the evidencethat useof oralcontraceptivesteroids containing diet was also determined. Two- and 3-dimensional is associated with an increased incidence of benign liver neo analyses of GGT lesion number and size were performed as well plasms is strong (see Refs. 36, 39, 40, and 43). While the results as the histopathological evaluation of advanced lesions. The of several studies have also suggested an increased incidence of HC3 in women following prolonged use of these agents (11, results clearly show the strong enhancing ability of EE and 2 doses of mestranol but not estradiol on GGT lesion number and 15, 19), the strength of this association remains controversial HC incidence. (11,14) and deserves continued study. ' Supported by USPHS Grant CA32916/CA36701 from the National Cancer MATERIALS AND METHODS Institute. 2 To whom requests for reprints should be addressed. Animal Treatment. Female Sprague-Dawley rats (Charles River 'The abbreviations used are: HC, hepatocellular carcinoma; DEN, diethylnitro- Breeding Laboratories, Inc., Wilmington, MA) obtained in 2 groups were samine; GGT, -»-glutamyltransferase; M(L), mestranol, tow dose; M(H), mestranol, housed individually in wire-bottomed cages under controlled conditions high dose; EE, 17ß-ethinylestradiol; H & E, hematoxylin and eosin; PB, phénobar of temperature, humidity, and lighting. The 12-month experiment was bital; TCDD, 2,3,7,8-tetrachtorodibenzo-p-dioxin; SAL animals, animals given 0.85% NaCI solution. started 2 weeks prior to the 9-month experiment. Food and distilled Received March 8, 1984; accepted June 12, 1984. water were available ad libitum. Upon arrival, the animals were fed a 3862 CANCER RESEARCH VOL. 44 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1984 American Association for Cancer Research. Promotion by Synthetic Estrogens defined semisynthetic basal control diet (43) obtained from Teklad (Mad Campbell er al. (5). The smallest size class has been designated Size ison, Wl). At approximately 7 weeks of age, the rats were subjected to Class 1, and the smallest lesion transectton reproducibly definable within a surgical two-thirds partial hepatectomy; 24 hr later, they were given this size class had a diameter of 50 /im. The size classes are defined by i.p. injections of either 0.85% NaCI solution (noninitiated, -DEN groups) a tog scale of maximum diameters u/m) by the series: Size Class 1, or DEN at 20 mg/kg body weight (initiated, +DEN groups). Twenty-four antilog 1.8; Size Class 2, antitog 1.9; Size Class 3, antilog 2.0 ... Size hr later, the animals were transferred (indicated by -»)to the various Class 20, antilog 3.7, as originally described by Campbell er al. (5). In treatment groups shown in Table 1. this size classification system, lesions in each subsequent size class The estrogens (Sigma Chemical Co., St. Louis, MO) were added to have a volume 2 times greater than those in the previous size class, the basal diet as described previously (43), and the diet was subsequently thus reflecting the increased tumor burden to the rat. The stereological fed in powder form. Mestranei was fed at 2 dose levels with the low calculations were not corrected for potential artifacts due to stretching dose [M(L)] being 0.1 ppm and the high dose [M(H)J being 0.5 ppm and shrinkage. The GGT lesion transections were essentially circular, which represents approximately 3 and 15 times the human dose, re thus representing essentially spherical growths. There was no evidence spectively. On the tow-dose diet, rats ingested approximately 6 ng of distortion in an elliptical fashion as a result of the histotogical proce mestranol/kg/day; at the high dose, they ingested 30 j/g mestranol/kg/ dure. day. The concentration of ethinyl estradici was 0.5 ppm, and that of Statistical Analyses of the Data. The data were subjected to a one estradici was 0.6 ppm. In one treatment group, animals were fed diet way analysis of variance with multiple comparisons among groups per containing M(H) plus ..i-methasone. Initially, this synthetic corticosteroid formed using the Neuman-Keuls procedure (2). Results reported as was also present at 0.5 ppm, but the animals failed to gain weight and significant using this procedure are less than the 5% level of significance. suffered hair toss. After 2 weeks, the dose of .-i-methasone was reduced In addition, in selected instances, 2 groups were compared using a 2- to 0.2 ppm, and it remained at that level for the duration of the experi tailed r test. Nodule and HC incidence data were analyzed using the \- ment. Food consumption and body weight data were obtained through test. out the course of the experiment. Histochemical and Histopathological Analyses. At the time of kill, RESULTS the livers were removed and weighed, and a liver sample from 2 of the Effects of Treatment on Animal Growth. Chart 1 showsthe remaining 4 lobes was obtained by the use of a 15-mm diameter cork borer. This procedure4 provides samples of similar size from each tobe body weight curves for the various groups of animals. In the 9- and each rat. The liver samples were frozen on dry ice and processed for GGT histochemistry as described previously (43) to reveal the pres ence of putative preneoplastic and neoplastic lesions. The GGT lesions —Bral•oc»-» RIMaS* detected consisted of foci and more advanced nodules and carcinomas. »- — ADEN—M No attempt was made to histopathologically identify and evaluate the 500400300200100öS«osti—E* DEN •EvSH— GGT lesions in H & E-stained serial sections.