(CANCER RESEARCH 35, 1341 1353, May 1975] Estriol Prevention of Mammary Carcinoma Induced by 7,12-Dimethylbenzanthracene and Procarbazine1 Henry M. Lemon Section of Oncology, Department of Internal Medicine, The University of Nebraska Medical Center, 42nd Street and Dewey Avenue, Omaha. Nebraska 68105 SUMMARY hexestrol, 0.60 mg/pellet, did not alter breast cancer incidence in 220 additional rats. Estrogen-treated rats The concentration of estrogenic, androgenic, progesta- usually sustained a mean 0.6 to 8.9% reduction of body tional, and adrenocortical steroid hormones in body fluids growth for the first 6 to 8 months of observation; this did of mature intact Sprague-Dawley female rats was increased not correlate with the breast carcinoma-suppressive activi by s.c. implantation of 5 to 7 mg NaCl pellets containing 1 ties of individual steroids. to 20% steroid 48 hr before administration p.o. of either Single implantation of 0.60 mg estriol 48 hr before 7,12-dimethylbenz(a)anthracene or procarbazine. The inci dimethylbenzanthracene p.o. or sustained implantation dence of rats developing one or more mammary carcinomas every 2 months of 10% estriol pellets beginning 24 hr after in each treated group was compared to that observed in si carcinogen exposure failed significantly to alter mammary multaneously treated groups receiving only the carcinogen, carcinoma development after dimethylben/anthracene ad steroid, or no treatment whatsoever, with weekly observa ministration. tion of all rats until palpably growing tumors were biopsied Inhibition of mammary carcinogenesis induced by these and proven carcinomatous or until death occurred from two dissimilar carcinogens in intact mature rats using other causes determined by autopsy. A total of 105 un sustained low dosages of estriol support previously pub treated or steroid-implanted rats followed to death (234 to lished biochemical and epidemiological data suggesting an 256 days median observation) developed no breast carci inverse relationship between the renal excretion ratio of nomas. Rats fed either of the carcinogens developed initial estriol to estrone plus 17/3-estradiol and risk of breast cancer evidence of breast carcinoma, after 136 to 156 days median in women. On a mg per kg per day basis, the calculated observation, in 51 to 57% of 318 total treated rats. Non- daily absorption of estriol from the pellets by the rats is breast carcinomas and sarcomas developed in 5 to 10% of considerably less than that contributed by the human the carcinogen-treated rats. fetoplacental unit to the mother during pregnancy (a Estriol administered as 0.15 to 0.60 mg/pellet reim- deterrent of breast cancer risk) and is within a well- planted every 2 months reduced breast carcinoma incidence tolerated p.o. dosage range in the postmenopausal human to as low as 5 to 7% of 106 rats given dimethylbenzan- female in clinical trials. thracene or procarbazine (p < 0.001) during a median observation period of 202 to 232 days. The incidence of nonbreast neoplasms was not significantly altered. Breast INTRODUCTION tumor incidence noted following implantation of 0.50 to 1.14 mg estrone and 17/3-estradiol in 80 dimethylbenzan- Multiple genetic and endocrine factors contribute to thracene-treated rats was reduced to 22 to 25%, compared to increased risk of human breast carcinogenesis, such as 45 to 47% in carcinogen controls; more significant breast female sex, Caucasian racial background, breast carcinoma cancer reduction was observed by estrone treatment, in in premenopausal Ist-degree relatives, and duration of similar doses, following procarbazine administration. 16- ovarian function; pregnancy, marked by a 1000-fold in Epiestriol also reduced breast cancer incidence to one-half crease in estriol production, is notably protective (26, 34). that observed in carcinogen-treated controls. Estrone 3-sul- Controversy exists as to the etiological significance of fate, 0.88 mg/pellet, reduced breast tumor incidence after reduced estriol excretion noted in 20% of nonpregnant procarbazine administration, but it was ineffective in a healthy Caucasians compared to 60% of patients with lower dose. D-Equilenin, 6-dehydroesterone, 16-keto-17/3- precancerous and malignant breast disease that we have estradiol, 2-hydroxy-17/3-estradiol, 2-hydroxyestriol, es- reported (27, 32). Premenopausal Asiatic women with tradiol-17«, 16,17-epiestriol, 17-epiestriol, testosterone, one-sixth the breast carcinoma risk of Caucasians com progesterone, corticosterone, dehydroepiandrosterone, and monly excrete a higher proportion of estriol relative to estrone and estradiol in their urine (28, 34). Wide variation 1Supported in part by National Cancer Institute Grant in estriol excretion by healthy Caucasian premenopausal CA 10626-01,02 from the United States Department of Health. Education, and Welfare: by the Department of Internal Medicine Memorial Cancer women may reflect several phenotypic mutant variants for Research Funds of the University of Nebraska Medical Center in Omaha: the endogenous production of estriol (28), which appear and by research grants from Carnrick Laboratories, Cedar Knolls, N. J. susceptible to genotypic quantitation by leukocyte incuba Received September 27, 1973: accepted February 3, 1975. tion with tritiated precursors of estriol in vitro (29). MAY 1975 1341 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1975 American Association for Cancer Research. H. M. Lemon The present investigation was planned to explore the Table 1 possible carcinogenic or anticarcinogenic function not only Compounds tested of estriol but also of other metabolites of estradiol hydrox- ylated at C-2, C-6. C-16 and of nonestrogenic steroids. Trivia] name Chemical name Some estrogen metabolites resemble estriol in altering 17«-Estradiol Estra-l.3.5(10)-triene-3,17«-diol physiological response to estradiol and are classified among 17rf-Estradiol Estra-1.3.5(10)-triene-3,l7/3-diol "impeded" estrogens (16, 27, 51). Commencing in 1967, we Estrone 3-Hydroxyestra-1,3,5( 10)-trien-17-one Estriol Estra-1.3.5( 10)-triene-3.16«. 17/3-triol adapted the model system of rat mammary carcinogenesis 16-Epiestriol Estra-1,3,5( IO)-triene-3,16/3.170-lriol developed by Dao (5) and Muggins el al. (14, 15) to our 17-Epiestriol Estra-1,3,5( IO)-triene-3,16a, 17a-triol needs, by superimposing upon the normal ovarian estradiol 16,17-Epiestriol Estra-1,3,5( 10)-triene-3,16/3,17a-triol secretory activity (44), required for DM BA2 induction of 16-Ketoestradiol 3.17j3-Dihydroxyestra-1.3.5(10)-trien-16-one mammary carcinogenesis, periodic s.c. implantation of 2-Hvdroxyestradiol Estra-1,3,5( IO)-triene-2,3,17/3-triol 0.06- to 1.3-mg doses of the test steroid (Table 1). Mam 2-Hydroxyestriol Estra-1,3,5( 10)-2,3,16a. 17/i-tetrol D-Equilenin 3-Hydroxyestra-1,3.5( 10),6,8-pentaen-17-one mary carcinogenesis induced in the female Sprague-Dawley 6-Dehydroestrone 3-Hydroxyestra-1.3.5( I0).6-tetraen-17-one rat by this agent has many similarities to human mammary Corticosterone 1li3.21-Dihydroxypregn-4-ene-3.20-dione carcinogenesis, especially relating to the near identity of Dehydroepiandros- 3d-H \droxyandrost-5-en-17-one estrogen receptor proteins and the response of these tumors terone Testosterone 17-Hydroxyandrost-4-en-3-one to various types of endocrine therapy (Table 2). Further Progesterone Pregn-4-ene-3.20-dione more, the rat offered an acceptable model for determining Estrone 3-sulfate 17-Oxoestra-l,3,5(IO)-trien-3-yl sulfate the potential chronic toxicity and tolerance of various doses Hexestrol p.p'-(1.2-Diethylethylene)diphenol of steroid metabolites, which might have significant inhibi tory effect upon mammary carcinogenesis, and also be suitable for human clinical trials. PC was later substituted with gross examination of the heart, lungs, and abdomen. In for DMBA as the carcinogenic agent to determine whether sacrificed animals, the uterus was dissected free and its the inhibition of breast carcinogenesis by estriol was related weight was recovered as an index of estrogenic response. to the chemical structure or possible endocrine activity of Steroid Incorporation into Pellets. The pellets were con either carcinogen (13). structed in a Forbes pellet press (9), from 1 to 20% mixtures of the authentic crystalline steroid with crystalline NaCl. They were prepared a few days before use, weighed, and MATERIALS AND METHODS kept in a dessicator until implantation. Their weight usually varied between 5 and 7 mg and they could be identified as RaCs and Their Maintenance. Throughout the study, long as 2 to 3 weeks after implantation, at necropsy. The intact female Sprague-Dawley rats were obtained from a dynamics of steroid release from such pellets has been single source (Sasco, Inc., Omaha, Nebr.) at 50 to 55 days described (10) and, in the case of 100% estrogen pellets, of age. They were randomized upon receipt and housed hormone release continues for at least 90 to 100 days in the individually in suspended cages. They were fed Wayne Lab rat. Pellet reimplantation was carried out every 2 months Blox (South Omaha Terminal Co., Omaha, Nebr.) and for the 2.5 to 20% pellets and monthly for the 1% pellets. municipal water, with temperature maintained within 22-23° and 40 to 50% relative humidity. Prophylactic The percentage composition of nonestrogenic steroids or synthetic nonsteroidal estrogens in pellets was adjusted to hormone therapy was achieved by implanting s.c. in the provide equimolar activity compared to estriol. Steroids posterior nuchal region 5- to 7-mg pellets once every 1 or 2 were obtained from commercial sources. Estrogens were months under light ether anesthesia. The 1st pellet implan screened for identity and purity by thin-layer silica gel tation was 48 hr before administration by gavage of the chromatography using 5% ethanol, 47.5% ethyl acetate, carcinogen, again under light ether anesthesia.