Estriol Prevention of Mammary Carcinoma Induced by 7,12-Dimethylbenzanthracene and Procarbazine1

Home , Equilenin, Nonsteroidal estrogen

(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-17Â«, 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

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 17Â«-Estradiol Estra-l.3.5(10)-triene-3,17Â«-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-3.16Â«. 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 22-23Â° 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. The rats 47.5% cyclohexane liquid phase, and UV spotting to were all examined for tumors and weighed every 1 to 2 measure RF values. weeks. After 1 or more tumors were identified in the Carcinogens. DMBA was obtained from Eastman Kodak mammary areas and their size(s) were measured, they were Co., Rochester, N. Y., and PC was from the National reexamined after 1 to 2 weeks to confirm the continued Cancer Institute, Bethesda, Md. Both were administered by growth of the tumor(s). Firm enlarging masses were biop- flexible gavage tubes under light ether anesthesia. DMBA sied and the animals were sacrificed upon pathological was given in a 20-mg dose in 1.0 ml sesame oil for most of verification of mammary carcinomas, as defined by Murad the experiments, producing a 10 to 40% mortality from and von Haam (43). Animals with nonbreast tumors were adrenocortical necrosis (17). Increasing the sesame oil observed until near death, when they were sacrificed and vehicle to 2.0 ml without changing the DMBA dose necropsied. Non-tumor-bearing rats were reimplanted with abolished any mortality consequent upon carcinogen ad steroids according to protocol, until death, when they were ministration within the 1st 2 to 3 weeks of observation and necropsied. At this time, gross examination was conducted this volume was adopted. PC was kept in crystalline form of the dissected axillary and inguinal mammary pads, along until just before use at 4Â°,when it was made up to 50- 70-mg/ml individual rat doses in distilled H2O, prepared in 2The abbreviations used are: DMBA. 7.12-dimethvlbenz(a)an- 5.0-ml batches immediately before administration. No thracene: PC, procarbazine. mortality resulted from PC administration at any time, but

1342 CANCER RESEARCH VOL. 35

Table 2 Comparison of induced breast carcinomas in rodents with human mammary carcinoma

Rat Human

Sex incidence Rare in males (1.4. 5, 7. 8) 1:IOOM:F(34) Effect of prior castration on car- Reduced tumor incidence by 80 90% (5. 7. 8. 14, 19) Reduces cancer risk by 60 70<Ã¯dependingupon age cinogenesis (26. 34)

Estrogen replacement therapy in Restored carcinogenic activity in young ovariecto- Little, if any. influence on cancer risk in postmeno- castrate mized rats (7. 33) pause women (26. 34)

Pregnancy Pregnancy soon after DMBA reduces risk (7, 15) Reduces cancer risk (26. 34)

Role of pituitary in carcinogene- Required (5. 7. 40. 42. 45, 50) Required (26. 34) sis

Evidence of viral cocarcinogene- Not proven Not proven sis

Tumor histogenesis Origin from both myoepithelial cells and duct epithe- Duct epithelial components predominate (43) lium (43)

Pathology Adenoid cystic carcinoma, adenocarcinoma. medul- Varies from well-differentiated adenocarcinomas to lary. papillary, follicular. and myxoid carcinoma; undifferentiated carcinoma rare in situ ductular carcinoma resembling human (43)

Multiple primary tumors In 40 80%(4. 7,43) In 10 20n;

Metastasis Local invasion Common (1.4. 5, 14. 15.43) Common Distant Very rare (I. 4, 5. 14, 15,43) In 50 70%

Spontaneous regression Up to 20% tumors (1, 4. 5, 7. 8. 14, 40) Rare but documented

Estrophile proteins In 50-70% of tumors (21. 23, 38. 41) similar to those In 30 50% of neoplasms, chiefly postmenopausal (20, isolated in human (37. 48) bind to estriol as well as 21, 25) 17/3-estradiol (30)

Tumor response to castration or Tumors with estrophile proteins regress (23. 40. 41) Some but not all tumors with estrophile proteins adrenalectomy regress (20. 21. 48)

Hypophysectomy Induces regression (5, 7. 40. 45) Induces remission in 20 30%

Prolactin Restores tumor growth after castration-induced Possibly sensitive to prolactin regression (39. 40. 44) the interval required for mammary carcinogenesis was tration. The median duration in days of observation of all considerably longer than that required for DMBA activity. rats in each group was also determined: premature mortality Experimental Design. Initial experimental protocols in from infectious disease was the major factor reducing tumor cluded groups of 4 to 12 rats assigned as untreated controls, incidence in carcinogen-treated groups, owing to the 120- to steroid-implanted controls, carcinogen-treated rats, and 150-day median latent period of carcinogenesis. All breast steroid-implanted rats followed by carcinogen feeding. As carcinomas were diagnosed between the 40th and 370th day experience developed, and no breast carcinomas were noted after administration of either carcinogen. The mean of in over 100 rats observed during their natural life-span in the mean weights of steroid in pellet implants every 1 to 2 1st 2 control groups, these were no longer included in the months has also been included in the tables when appropri routine design of experiments. Most of the later experiments ate, to indicate the actual dosage administered to each involved comparison of 2 to 4 groups composed of 8 to 18 group of rats. rats each, 1 of which was fed carcinogen, with the others All histological sections of tumors were initially read given steroid pellets plus carcinogen. without knowledge of the treatment received by the rat. The development of 1 (or more) breast carcinoma(s) per They were again reviewed at the time of this report to rat sufficed for our objectives to determine cancer incidence confirm the pathological variants of breast carcinoma rats, so that these rates were sacrificed to save expense. The recognized according to the classification developed by results are reported as the number of rats in each group Murad and von Haam (43). Fibroadenomas and lactational developing 1 (or more) breast carcinoma(s) out of the total hyperplasia. which was usually grossly readily distinguisha number treated and alive 30 days after carcinogen adminis ble from carcinoma, were not included among the malig-

MAY 1975 1343

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1975 American Association for Cancer Research. H. M. Lemon nant neoplasms. Between 5 and 10% of mammary nodules Estrone 3-Sulfate. This conjugate is the principal estrogen less than 5 mm in diameter failed to grow during several recovered from human plasma. Tested in 10%pellet concen weeks of observation or regressed totally, a common finding tration (equivalent to 7.4% estrone), it was not effective in in these tumors (Table 2). On the other hand, once a tumor preventing breast neoplasms (Table 3). In 14.1% pellet had reached 5 to 10mm in diameter, we seldom encountered concentration (equivalent to 10.4% estrone), possible inhi regression except after castration, and it was at this size that bition of breast tumor development after PC feeding was most biopsies were made. Only pathologically verified noted in a single experiment. carcinomas were included in the statistics. No unusual All 3 of these estrogenic steroids similar to other incidence of tumor regression was noted in most of the estrogens reduced the rate of body growth by 3 to 6.6% steroid-treated groups, suggesting that the end results were compared to that noted in the rats receiving only carcino significantly affected by nonintrusion of spontaneously gens (Table 4). No significant correlation existed between regressed neoplasms. reduction in weight and inhibition of breast tumor forma Statistical analysis by x2 evaluation based upon a 4-fold tion by these and other estrogens. Neither of the 2 table of the number of rats with and without breast carci carcinogens used affected the rate of growth of the rats, in noma in the carcinogen versus steroid-plus-carcinogen- comparison with simultaneously observed untreated con treated group was used to determine significance of differ trols. No other systemic effects were noted from these small ences. Since cancer induction rates by these agents vary with estrogen doses, with the longevity of the steroid-implanted time and each batch of animals, only those groups simulta controls equivalent to that observed among untreated rats neously receiving the 2 treatment protocols were included (Table 3). for analysis of significance of differences in incidence rates. Estriol. The 2 principal pathways for estradici or estrone The Yale's correction for small numbers was used through metabolism involve hydroxylation at C-16 to produce estriol out in x2 analysis. or an epimer and at C-2 yielding 2-hydroxyestradiol, 2-hydroxyestrone, or 2-hydroxyestriol, which are methyl RESULTS ated in the kidney and liver prior to excretion (22, 27). Estrogenic Steriods Estriol in 10% pellet concentration reimplanted every 2 months proved to be the most inhibitory compound of any 17/3-Estradiol. This primary ovarian estrogen (27) was of the 17 tested in the entire series of experiments, reducing tested in 5 experiments in 1 to 10% pellet concentration for the development of palpable breast carcinoma to only 5 to its effect upon DMBA carcinogenesis (Table 3). No breast 7% of the treated rats (Table 5). No inhibition of tumor tumors appeared in untreated or steroid implanted controls formation followed 1% pellet implantation. Single implan observed for as long as 300 days median observation. The tation of 10% estriol pellets 2 days before DMBA adminis incidence of DMBA-induced mammary tumors was re tration in 2 experiments resulted in breast cancers in 20 out duced by nearly 50% in rats implanted with 10%but not 1% of 34 rats, within a median period of 83 to 111 days (range, pellets, and the median time of tumorigenesis was delayed 37 to 187 days), similar to the incidence of breast carcino by 34 days, compared to those receiving only the carcino mas observed in the DMBA-treated rats. Four multiple gen. This difference in incidence, while suggestive, did not primary breast carcinomas occurred. Serial implantation of achieve statistical significance. In 20% pellets, 17/3-estradiol 10% estriol pellets every 2 months commencing 24 hr after did not reduce significantly breast carcinoma incidence in a DMBA administration has resulted in 4 of 11 rats with single experiment, but it delayed onset of tumors by 147 breast carcinomas after 270+ days in 1 experiment, with days to a median of 221 days. another rat with 3 primary breast carcinomas. When 17/3-estradiol in 10% pellets was implanted only a Estriol was most inhibitory against PC-induced mam single time 2 days prior to DMBA administration, 7 of 10 mary carcinoma development, with highly significant reduc recipients developed mammary carcinomas in a median tion of breast tumors after implantation of 2.5% pellets period of 241 days (range of 65 to 297 days). yielding only a 16% incidence of rats with cancer. At this Estrone. Estrone equilibrates reversibly with 17/3- dosage no inhibition of DMBA-induced tumors occurred. estradiol in vivo, depending upon the oxidation-reduction The median survival of estriol-protected rats treated with potential and availability of pyridine nucleotide respiratory either carcinogen was in all cases somewhat shorter than cofactors, and might be expected to have a somewhat that noted in either untreated or steroid-treated controls. In similar degree of activity affecting carcinogenesis as es- all cases the survival of the protected rats considerably tradiol (27). In 6 experiments with 1 to 10% pellet concen exceeded that noted in the carcinogen-treated rats, which tration, estrone closely resembled 17/3-estradiol in its an- was shortened by the earlier biopsy and sacrifice of the ticarcinogenic effect upon DMBA induction of breast larger number of tumor-bearing animals. cancers, with a 50% reduction of their incidence compared Excluded from these incidence figures are 1 transient to DMBA controls (Table 3). Highly significant inhibition mammary tumor in the PC-treated rats and 8 transient of PC-induced mammary carcinogenesis was observed after tumors that totally regressed spontaneously within a few 10% estrone pellets with minimal prolongation of median weeks after discovery in the estriol implanted-plus-carcino- life-span of the implanted animals beyond that of untreated gen-treated (5 after DMBA and 3 after PC) rats. Five of controls. No spontaneous tumor regressions were observed these 8 transient regressing tumors were noted in rats either in rats receiving estrone or 17/3-estradiol in the implanted with the lowest estriol doses of 1.0 to 2.5% per DMBA- or PC-treated groups. pellet.

1344 CANCER RESEARCH VOL. 35

Table 3 Mammary carcinoma incidence after estrone and 17d-eslradiol intplantatinn

No. of Carcino- exper- Untreated pellet 10%pellet pellet 10%pellet controls170-EstradiolSteroid genie agent iments treated8/17(47)."136+ carcinogen +carcinogen8/32(25).170days.0.580.65+x207/13(54), carcinogen only

DMBA.23620 5 0/10." 1.6203 0/4,300 mgdays'DMBA. days5/11(45),74 days.days, 0.53mg0.063 mg15/22 mg'4/12(33),221

120 mgEstrone days9/20(45).159 days.Â«1.14 mg8/36(22),182days.0.500.76

DMBA.0/13,22820 7 2.2(68). 0/6.207 mgdaysPC. days10/15(66).1 days.85 mg0.065mg8.522/35(63)days. 0.63

mg1/14(7),147days.0.540.66

2mgEstrone 50 37days4/5,

mg8/12(67),142days.0.65

3-sulfate DMBA.220 80days4/7. mgPC.

mg2/8,

160 179days5/13(38),187 135days.0.64 mgPC. mgI/

150 12Â»(8).179days.0.88 mgTotal days45/88(51)20%

mg4/12(33)

incidence 0/23Carcinogen- 28/114(26)1% 0/10PNS'NSNS0.004NSNSNS " Incidence of tumors compared in carcinogen-treated to that in 10%steroid pellet-plus-carcinogen-treated using Yale's correction. *Number of rats with tumor/total number of rats treated. ' Median days of observation. " Numbers in parentheses, percentage of rats with tumor. ' mg estrogen per pellet. ' NS. no significant difference in incidence between carcinogen-treated and estrogen-plus-carcinogen-treated. Â«Onesurvivor free of tumor after 310 days. * 14.1% pellet concentration equivalent to 10.4%estrone.

Multiple breast carcinomas verified by biopsy were noted multiple primary breast carcinomas were observed in the 6 in 10 to 75% of DMBA-treated tumor-bearing animals, at developing breast cancers after estriol pellet implantation the time of initial biopsy and sacrifice of the tumor-bearing plus PC treatment; both of these were noted in rats receiving rats. Twelve of the 48 rats fed only DMBA had 2 to 4 2.5% estriol pellets. Therefore, adequate circulating concen multiple primary breast adenocarcinomas in the estriol trations of estriol sustained by 10to 20% pellet implantation experiments (Table 5). No multiple primary breast adeno every 2 months not only reduced the incidence of breast carcinomas were noted among rats protected by 10 to 20% cancer-bearing rats by 90% following either of these car estriol pellet implants prior to DMBA therapy, in the 4 cinogens compared to the carcinogen-treated controls but developing breast tumors. Six multiple primary neoplasms also abolished the appearance of multiple palpable primary with 2 to 6 breast tumors per rat were noted in the 26 carcinomas that grew progressively. tumor-bearing rats receiving 1.0 to 2.5% estriol pellets prior 16-Epiestriol. Following either DMBA or PC adminis to DMBA feeding. tration, only 18 to 19% of rats implanted with 0.6 mg After PC feeding, 7 out of 29 tumor-bearing rats 16-epiestriol developed breast cancers; however, the car developed 2 to 3 primary breast cancers, and 1 spontaneous cinogen-treated controls developed breast cancers in only 33 regression of a breast tumor was noted. Two rats with to 40%, requiring further tests to establish significance of

MAY 1975 1345

Table 4 Body weight reduction of estrogen-plus-carcinogen-treated ram in relation to inhihilion of mammary carcinogenesis Spearman rank order correlation coefficient between mean body weight change and incidence of breast cancer induction = -0.151.

ofobservation(wtwtchange (%)from every2 carcino incidenceof ofexperiments5522122631121123Durationmos.)(mos.)g666668846g26463Meangen-treatedcontrols-4.2-6.3-8.9-3.0-6.2-4.4-0.6-5.2-6.1-5.2-1.2-8.4-6.6+breastcarcinoma571315171819222530404857666794 Treatment10%

PC10%estriolestriol + DMBA20% + DMBA10-estriol + PC10%14.1% estrone 3-sulfate + PC10%D-equilenin + PC10%16-epiestriol + DMBA10%16-epiestriol + DMBA10%estrone + DMBA6-dchvdroestrone170-estradiol + PC10% + PC10%I7a-estradiol t DMBA10%hexestrol + DMBA2-Hydroxyestriol16.17-epiestriol + DMBA10% + 2.8-6.6-7.4% BA10%estrone 3-sulfate + DM 17a-estradiol + DMBANo. ihe difference in incidence rates (Table 5). ments for optimum suppression of DMBA-induced mam This steroid caused the least reduction of body growth, mary carcinogenesis, with the 16Â«position providing maxi compared to that seen in recipients only of the carcinogen mal inhibition. used, of any of the estrogenic compounds (Table 4). Neither 2-Hydroxy-170-estradiol and 2-Hydroxyestriol. Oxida 16-epiestriol nor estriol altered the incidence of rats with tion of the 2nd carbon atom on ring A of the estrogen breast carcinomas, following monthly reimplantation of 1% nucleus modifies greatly the physiological activity of this pellets. If the stress of repeated anesthesia and pellet representative of the 2nd major pathway of estradiol implantation in any way were involved in the tumor-inhibi metabolism in man (27). Despite retention of some physio tory activities of these compounds, one would have expected logical activities classifying it among the "impeded" estro some reduction in tumor incidence in these groups. gens, 2-hydroxyestradiol was inactive in 10% pellet concen 17Â«-Estradiol. This unnatural stereoisomer of 17/3- tration in altering breast tumor incidence or in delaying estradiol may displace the latter, from intranuclear binding tumorigenesis (Table 6). 2-Hydroxyestriol in similar dosage of the estrogen receptor complex to uterine DNA (2). In the was also inactive. 10% pellet concentration tested, breast carcinomas devel o-Equilenin. This major equine urinary estrogen resem oped rapidly following DMBA administration in all recipi bles estrone except for the presence of 2 unsaturated C=C ents except 1 in the 3 experiments (Table 6). Multiple bonds in ring B of the steroid nucleus. Either alone in 10% primary breast carcinomas were noted in several animals. pellet concentration or in 21% concentration combined with 17Â«Epimers of Estriol. Neither of the unnatural stereoi- its L isomer, it has not significantly decreased breast somers of estriol with a 17Â«hydroxyl group inhibited the carcinoma incidence following DMBA administration incidence of mammary carcinoma induced by DMBA (Table 6); further experiments in PC-treated rats will be (Table 6). These findings, along with the negative results needed to assess its possible inhibition of breast neoplasia. observed with 17Â«-estradiol,suggest that the 17/3-hydroxyl However, 6-dehydroestrone with a single unsaturated C=C group that is required for uterine and mammary estrophile bond in ring B exhibited no antimammary carcinogenic protein binding is essential for mammary carcinogenesis activity in PC-treated rats. inhibition by estrogens. Five separate primary breast can Hexestrol. This nonsteroidal estrogen binds with high cers developed in 1 rat receiving 16,17-epiestriol and a affinity to rat uterine estrogen receptors and to some double primary in one of the carcinogen controls in this neoplastic human breast carcinomata in vivo (26, 27). In group of experiments. pellet concentrations equimolar to estriol, it has not signifi Ih-Ke(o-l7,5-fstradml. This oxidation product of es- cantly reduced DMBA-induced breast carcinoma incidence tradiol is one of the precursors of estriol and 16-epiestriol in (Table 6). Multiple breast cancers appeared in 3 hexestrol- human metabolism, but it was completely devoid of inhibi treated rats and in 1 DMBA-treated control; 1 spontaneous tory activity against DMBA mammary carcinogenesis breast tumor regression was noted in the latter group. (Table 6). These results suggest that only a C-16 hydroxyl Nonestrogenic Steroids. None of the nonestrogenic ste group will satisfy the steroid-protein structural require roids tested thus far in 10% pellet concentrations have

1346 CANCER RESEARCH VOL. 35

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1975 American Association for Cancer Research. Estriol Prevention of Induced Rat Breast Carcinoma shown any promise of altering experimentally induced castration to pellet implantation was probably too brief breast cancer incidence in our rats (Table 7). None of these entirely to remove ovarian influence upon breast car- significantly altered body weight from that observed in cinogenesis. Further exploration of ratio of the steroids used carcinogen-treated controls. In 20% concentration, testos is needed to ascertain the dosage required to reproduce an terone pellets reduced tumor incidence in a single experi ovarian type of hormonal sensitivity of the breast to this ment and increased mean body weight by 8% at 2 months. carcinogen and to establish whether estriol is equally Further experiments in progress with 20% pellets of testos inhibitory under these circumstances. terone and progesterone have not reduced breast cancer Incidence of Nonbreast Carcinomas and Sarcomas. Both incidence. DMBA and PC-induced 5 to 10%incidence of sarcomas and Effect of Castration upon Breast Carcinogenesis Modified nonbreast carcinomas, especially epidermoid carcinomas of by Estrogen Pellet Implantation. Castration prior to DMBA the ear duct ( 1, 4, 5, 13) (Table 9). No significant alteration administration reduced breast tumor incidence markedly in in the incidence of these tumors occurred following estro- Sprague-Dawley females (Tables 2 and 8). The reduced genie steroid implantation prior to DMBA treatment, incidence of breast carcinomas was not significantly altered regardless of the inhibitory activity that any of these by 10 to 20% estriol pellet implantation prior to DMBA steroids possessed for mammary tumorigenesis. Nonestro- administration in castrate females. Simultaneous implanta genic steroids tested thus far also did not significantly affect tion of 10%estrone or 170-estradiol pellets, along with 20% nonbreast tumor incidence. estriol pellets in castrate females, did not significantly Following the use of breast tumor-inhibitory doses of change the low incidence of tumors observed in these estriol in PC-treated females, nonbreast neoplasms oc experiments (Table 8). The interval of 1 to 3 days from curred more frequently (16.7%). These were similar in type

Table 5 Mammarv carcinoma incidence after estriol and 16-epieslriol

No. of Carcino- ex- pellet 10% pellet pellet 20% genic peri- Untreated ogen- + carcin- pellet i + car + car pellet controlsEstriolSteroid agent ments treated29/48 ogencarcinogen2/16(13), cinogen6/7, cinogen20/36(56),114onlyx2"0/25,

DMBA.25420 13 0/23." (60),"155 2/27(7).237 75days.0.16mg5/31(16),224days.0.16mg1%2499.2'days, mgdays'PC, days18/29(62).156 202days, days.0.07 17.9Â«0.601.30mg24.5Â«11.5"0/4.0.001< days.1.30 mg6/7,68days.0.06mg10 0.62mg"" mg2/41(5).232days.0.63mg4/21

50570 0.001< mg16-Epiestriol days4/10(40).125 0.001NSNS

DMBA,320 (19).101days.0.60 2050.6Â«days.0.64mg0.6Â«P0.002< mgPC. days7/21(33),2

mg4/22(18).263days.0.590.63mg2.5%

602mgCarcin 15days20%

Total breast 0/23 58/108(54) 2/16(13) 12/111(11)11/38(29)26/43(61) 0/29 tumor inci dence " Number of rats with tumors/total number treated. " Yale's correction used for all X2 determinations for all tables. ' Median days of observation. '' Numbers in parentheses, percentage of rats with tumors. ' mg estrogen per pellet. ' X2 comparing incidence of tumors in carcinogen-treated to that in 20% steroid pellet-plus-carcinogen-treated. Â«X2comparing incidence of tumors in carcinogen-treated to that in 10% steroid pellet-plus-carcinogen-treated. " X2 comparing incidence of tumors in carcinogen-treated to that in 2.5% steroid pellet-plus-carcinogen-treated.

MAY 1975 1347

Table 6 Mammary carcinoma incidence after implantation of 9 other estrogens

of ex- Un- peri- treated pellet + 1%pellet 10% pellet SteroidI7a-Estradiol16,17-Epiestriol17-Epies-agentDMBA,20 mentscontrols3 treated6/7, x'18/19(94),'carcinogen + carcinogen only

0/1Â°1113 99 116 0/5,200 mgPC, days"2/2, days, 0.59 days, mg4/10,mg" 0.67

60mgDMBA.20 243 149 days5/10,82 mg8/14(57),days, 0.62

107 mgDMBA.20 days8/12(67), mg6/1days, 0.59

110 2' (50), 193 trioll6-Keto-17/3- mgDMBA.20 days7/14(50), mg12/19(63),days, 0.52

0/231 126 0/2,135 estradiol2-Hydroxy-l70-estradiol[)-Equilenin6-Dehydroestrone2-HydroxyestriolHexestrolTotalmgDMBA,20 130days4/7. days. 0.59 mg days. mg13/18 0.56

161 (72). 99 mgDMBA. days6/7.67 mg7/10(70).days, 0.60

0/111120/4Carcinogen- 86 20mgPC, days3/6, mg4/18(22),days, 0.58

60mgPC, 192 175 days3/6, mg3/10(30),days, 0.61

60mgDMBA.20 192 208 days5/11(45).74 mg8/12(67).days. 0.60

74 mgDMBA.20 days10/22(45).' mg12/25days, 0.62

(48),Â«100 mgNo. 107days56/98(57)10% mg95/167(57)days, 0.61

incidenceCarcinogenic- 0/7PNS'NSNSNSNSNSNSNSNSNS " Number of rats with tumors/total number tested. '' Median days of observation. ' Numbers in parentheses, percentage of rats treated. '' mg estrogen per pellet (mean). ' NS. no significant difference between incidence in carcinogen-treated versus estrogen-plus-carcinogen-treated by X â€¢¿ ' One survivor free of tumor after 360 days of observation. " Five survivors free of tumor after 210 days of observation.

to those observed after DMBA administration. The increase free rats in this study (5, 7, 11, 14, 15, 19, 36, 45, 47, 49, 50). in incidence was not significant by x2 and may be attributed Huggins et al. (18) noted that 10 to 20 ^g estriol injected partly to the longer observation period of the protected rats. daily stimulated growth of a transplantable mammary fibroadenoma, while 100 jig/day inhibited its growth. Terenius (47) observed that 100 ^g estriol injected every 12 DISCUSSION hr for 12 days reduced the volume of DMBA-induced mammary tumors from 2670 cu mm in sham-injected Previous investigations of steroid action upon chemically carcinogen-fed rats to 110 cu mm after 120 days. Progester induced rat mammary carcinoma incidence utilized larger one-treated rats averaged 1460 cu mm tumor volume and intermittent doses over a shorter period of time than given testosterone-treated rats averaged 4370 cu mm. The small in this study. Few have demonstrated as marked inhibition est tumor volume of 31 cu mm occurred after treatment of mammary carcinogenesis as noted herein following with an estrogen antagonist. Mer 25, which like estriol adequate estriol therapy, despite shorter postcarcinogen decreased somatic growth rate. Progesterone pretreatment observation than the natural life-span follow-up of cancer- of rats during 25 days prior to carcinogen administration

1348 CANCER RESEARCH VOL. 35

Table 7 Mammary carcinoma incidence after implantation of nonestrogenic steroid'; The carcinogenic agent was DMBA. 20 nig.

of experi- Untreated Carcinogen- pellet pellet 1% pellet pellet + carcinogen only\'Â¿0/7, SteroidDehydroepian- mentstreated2 controls +carcinogen12/15(80).' +carcinogen4/5,

0/1Â° 4/6.112 172 drosteroneTestosteroneProgesteroneCorticosterone(12.7%')Totaldays"2 I06davs, days, 1.14mg"1/9, 1.23 mg4/6,67

3/5, 266 184 66 0/2.230 days2 days, 1.15 days, 0.63 days, days, 1.15 mg10% mg9/17(53). 0.060 mg mg0/9PNS"NSNSNS

3/5, 145 days1 146 days. 0.64mg4/7.Â«

6/7,67 92 days0/1 days, 0.77 mg20%

tumor inci 16/23(70)20% denceNo.

" Number of rats with tumor/total number of rats. '' Median days of observation. 1 Numbers in parentheses, percentage of rats with tumors. " mg steroid per pellet (mean). '' NS. no significant difference in incidence between carcinogen-treated and steroid-plus-carcinogen-treated. ' Corrected to equimolar concentration at 10% pellet concentration for estriol. * One survivor free of tumor after 300+ days.

Table 8 Mammary carcinomas induced in females 24 to 72 hr after castration The carcinogenic agent was DMBA, 20 mg.

of 20%steroid experi pellets + 20% steroid X20/16.pellets only SteroidEstriol10% ments833Untreatedcontrols0/10," treated3/21(14),' carcinogen3/12(25),

333 180 203 301 days. days"0/10Carcinogen-days3/21(14)10 days,0.61l.27mg"6/20' 0.62 1.19 mg0/16PNS'NSNS

estrone + 20% (30). 204 estriol10% days2/22Â« 17/3-estradiol + 20%estriolTotal (9), 292 days10

tumor incidenceNo.

" Number of rats with tumor/total number of rats treated. " Median days of observation. ' Numbers in parentheses, percentage of rats with tumor. '' mg estrogen per pellet (mean). ' NS, no significant difference in incidence rates between carcinogen-treated and steroid-plus-carcinogen-treated. ' Also epidermoid carcinoma of the ear. * Also epidermoid carcinoma of the ear and '.'parotid neoplasm. reduced DMBA-induced tumor growth (50), but others both before and after DMBA completely inhibited breast noted stimulatory activity upon mouse and rat mammary carcinogenesis for as long as 150 days of observation, with carcinogenesis (19, 42). Estradici benzoate, 20 /ug/day for marked impairment of weight gain (24). Breast cancer onset 20 days, completely suppressed further growth of DMBA- has been delayed as long as 241 days (median) (range, 65 to induced mammary tumors, reversible by daily injections of 297 days) by a single implant of 17/3-estradiol 48 hr prior to 28 IU ovine prolactin (39). Similar estradiol doses given DMBA treatment in this investigation. Since breast carci-

MAY 1975 1349

Table 9 Incidence of nonbreast neoplasms induced by carcinogens in relation to steroid therapy

Steroid inhibition of Nonbreast neoplasms mammary Carcinogen Steroid implanted 48 hr carcinogenesis administered before carcinogen Incidence Pathological diagnosis

None None 0/51 0

None DMBA None (Tables 3, 5 7) 10/196 5.1 hpidermoid carcinoma of ear 5 Leukemia 1 Sarcoma of uterus I Other 3 None DMBA 1.2 0.60 mg estrone, estrone 3-sulfate. 7/92 7.6 I7tf-estradiol (Table 3) 0.15 0.060 mg estrone. I7tf-estradiol. 3/76 4.0 Epidermoid carcinoma of ear II estriol, 16-epiestriol (Tables 3, 5) Fibrosarcoma of ear Sarcoma of lymphoma 0.60 mg 16-epiestriol (Table 5) 0/21 0 Epidermoid carcinoma of ear Lymphosarcoma 0.60 mg other estrogen metabolites 4/129 3.1 (Table 6) 1.20 0.60 mg nonestrogenic steroids 2/59 3.4 (Table 7) Subtotal 16/377 4.2 (mean) Significant DMBA 1.20 0.60 mg estriol (Table 5) 2/43 4.7 (p < 0.001)

None PC None (Tables 3. 5 6) 5/48 10.4 Endometrial carcinoma Epidermoid carcinoma of ear Sarcoma None PC 0.60 mg 17a-estradiol. estrone 3-sul- 6/52 11.5 Epidermoid carcinoma of ear fate (Table 3, 6) Sarcoma 0.60 mg 16-epiestriol (Table 5) Significant PC 0.15 0.60 mg estriol (Table 5) 12/72 16.7 Epidermoid carcinoma of ear 5 (p < O.(X)I) Misc. sarcomas 2 Adenocarcinoma? gut 1 Sarcoma of uterus, ovary 3 Adenocarcinoma of lung I nomas may develop up to 370 days after carcinogen, the out by histolÃ³gica! examination. Therefore, the final ob value of shorter observation periods in evaluating breast served incidence of mammary carcinoma in various rat cancer prophylactic agents is dubious. groups was conservative and was minimally influenced by With the technique used in this investigation, which lactational changes that develop in older virgin females. The permitted continuous absorption of varying doses of free incidence of mammary carcinoma in our carcinogen control steroids into body fluids, a 75 to 90% reduction in incidence groups fed a single 20-mg dose of DMBA was similar to of palpable or visible mammary carcinomas was achieved that observed by others who histologically confirmed all by 10 to 20% estriol pellets reimplanted every 2 months, malignant tumors (1, 4, 5, 7, 13, 36, 39, 47, 50). No differ commencing prior to feeding of either DMBA or PC. ence was observed in the histological types of mammary Tumor-free rats were observed until death from natural carcinomas between estriol-treated and non-estriol-treated causes, usually bronchopneumonia, which occurred usually rats, nor was there any significant incidence of spontaneous long after the age peak when most breast carcinomas remission of unbiopsied tumors to contribute materially to become manifest. A threshold of cancer-inhibitory activity the differences noted in tumor incidence. was noted when estriol concentration in pellets was reduced The 5 to 10% incidence of rats with sarcomas and to 1.0 to 2.5% with pellet reimplantation every 1 to 2 nonbreast carcinomas (chiefly squamous neoplasms arising months, respectively (Table 5). PC-induced mammary from the ear ducts) induced by both carcinogens was not carcinoma was more sensitive to estriol inhibition than that significantly altered by implantation of maximal doses of induced by DMBA, since significant inhibition of breast estriol or any other of the 17 tested steroids (Table 1). This tumorigenesis occurred after implantation of 2.5% pellets of suggests that the anticarcinogenic activity of estriol was estriol in PC-treated rats. With decreasing estriol dosage, specific for mammary neoplasms only and that estriol did the incidence of rats developing single as well as multiple not alter the endogenous metabolism, if any, of the carcino primary breast tumors increased to equal the incidence gens, thereby reducing their carcinogenic activity. Contin observed in rats fed carcinogens only. Every enlarging ued implantation of estriol pellets every 2 months was breast tumor was biopsied, and simple hyperplasia with or required for inhibition of carcinogenesis, since in 2 experi without lactation as a cause of breast enlargement was ruled ments no reduction of breast cancer incidence was demon-

1350 CANCER RESEARCH VOL.35

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1975 American Association for Cancer Research. Estriol Prevention of Induced Rat Breast Carcinoma strated following only a single implantation 48 hr before Induced Rat Mammary Carcinomas, Autologous Muscle carcinogen administration: pellet absorption was estimated and Uteri, in preparation]. Dehydroepiandrosterone has no to occur over a 90-day period (10). Estriol, therefore, binding activity and was inactive in 20% pellet concentration probably does not block initiation of mammary car- in altering mammary carcinogenesis (Table 7). Although cinogenesis by an agent such as DMBA which acts within 1 estriol probably binds to mammary receptor protein in vivo, to 3 days upon the breast to induce carcinogenesis (6). suppression of DMBA-induced mammary carcinogenesis at Estriol probably interferes with the 2nd or promotional the site of receptor-complex binding to DNA appears stage of mammary carcinogenesis, in which continued unlikely. Polycyclic hydrocarbon carcinogens interfere with estradici stimulation of mammary duct and myoepithelial estrogenic cellular responses (7), but there has been no close cell proliferation (3, 35) is the major cocarcinogenic factor. association noted between these compounds and estrogens Illustrating this was the similar degree of reduction of for nuclear protein binding (12). mammary carcinogenesis achieved by preliminary castra High chronic doses of estrone and 17/8-estradiol alone are tion as achieved by estriol treatment (Table 8). In castrate carcinogenic in rodents (33,46). Estriol lacks any mammary females, substitution therapy with estrone or 17/3-estradiol carcinogenic activity resulting from maximally sustained was ineffective in restoring the expected incidence of duct epithelial proliferation (3) in castrate or intact rats or DMBA-induced breast tumors, as long as estriol was mice, but renal tumors have resulted (32, 46). In castrate coadministered, in contrast to the results of substitution C3Hf mice free of mammary tumor virus, 10 to 200 Â¿Â¿g therapy with estradiol or diethylstilbesterol alone (5, 7, 14, estriol applied cutaneously weekly for 60 weeks failed to 19, 33). elicit breast carcinomas, while 10 jig estrone weekly for a In intact female rats, neither of the 2 major estriol similar period of time resulted in 25% incidence of mice precursors, estrone and 17/3-estradiol, inhibited car with breast cancers, comparable to the incidence rate in cinogenesis in the breast as effectively as estriol. 17Â«- older breeding females of the same inbred strain (46). Estradiol was completely ineffective against carcinogenesis Weekly administration of 5 pg estrone along with 5 Â¿ig in spite of its binding to uterine estrogen receptor proteins estriol resulted in a single breast carcinoma (2.9% inci (27). Estrone and 17/3-estradiol (0.50 to 0.76 mg) reduced dence); when a mixture of 1.4 ^g 17/3-estradiol, 4.3 ^g of breast tumor incidence to 22 to 25% in DMBA-treated rats, estrone, and 4.3 //g estriol was similarly applied, the inci but 20% estradiol (1.14 mg) was even less inhibitory in 1 dence of breast carcinomas increased to that noted in breed ing females in Pullinger's studies, suggesting the critical experiment. Estrone decreased breast cancer incidence more significantly after PC treatment (Table 3). Rat liver, role of 17/3-estradiol in rodent mammary carcinogenesis. intestine, and kidney incubated in vitro with tritiated Estradiol maximally increases mitotic activity with in 170-estradiol along with the oxidative cofactors, NADPH creasing dosage in the castrate mouse uterus, while and nicotinamide, yield estriol as a major metabolite (31), estriol exhibits a flat dose-response curve, beyond which so that part of the anticarcinogenic activity of estradiol or mitotic activity does not increase (35). No studies are re estrone may be attributed to metabolism to estriol following ported using similar techniques for the rodent breast. implantation. Other estrone and 170-estradiol metabolites The effects of estriol implantation upon prolactin or such as 16-ketoestradiol or 2-hydroxyestradiol possessed no gonadotropin activity are unknown as yet; frequently ob anticarcinogenic activity (Table 6), and 16-epiestriol had served lactation in castrate and estrone-implanted rats was lesser anticarcinogenic activity (Table 5). Since these and uniformly minimal at necropsy in our estriol-treated groups. other inactive metabolites of estrone and estradiol implants The paucity of grossly detectable mammary tissue in were probably formed in vivo in addition to estriol, the estriol-implanted rats was noteworthy. lesser anticarcinogenic activity of estrone and estradiol in DMBA-induced mammary carcinomas are highly prolac the dosage tested may reflect the net response of tissues to a tin dependent, and in the work of Meites (39) and Welsch et variety of metabolites, many of which were inactive. Estriol al. (50) it has been suggested that estradiol blocks the metabolism to 2-hydroxyestriol occurs in vitro by mouse mammary end-organ response to prolactin, which should be liver (22), which also is ineffective as an antimammary elevated by high doses of estradiol. In women, lactationul carcinogen. The protective activity of estriol implantation activity begins after blood estrogen concentration decreases therefore results from circulation of the native steroid along toward normal following delivery. Prolactin as a cofactor in with inactive metabolites until renal and biliary excretion. human mammary carcinogenesis is currently being investi If estriol indeed accounted for most of the anticarcino gated. genic activity observed in this study, the mechanism might The reduction of somatic growth observed herein follow involve either local mammary effects or effects upon the ing estrogen implantation, which did not correlate with pituitary or other endocrine organs or both. Sustained high breast cancer inhibition, has been noted previously in rats estriol concentrations in extracellular fluid would probably and mice receiving estrogen therapy, including estriol (33, displace much of the ovarian-secreted estradiol bound to 46). Appetite and/or somatotropin secretion may be sup mammary estrophile receptor proteins and thereby alter the pressed to account for reduction of body growth by nature of the nuclear estrogen-receptor complex bound to estrogens. DNA (51). Estriol retains 70 to 100% of the binding activity In spite of numerous similarities between human mam of equimolar concentrations of 17/3-estradiol in vitro for rat mary carcinoma and chemically induced rat mammary mammary estrophile proteins [H. M. Lemon. Estriol and carcinoma (Table 2), the suppressive effects of estriol on Estradiol-17/8 Binding to 7,12-Dimethylbenz(a)anthracene mammary carcinogenesis may not be similar in the 2

MAY 1975 1351

species. However, the dose of estriol required for human Dimethylbenz(a (anthracene and Estradici 17/3 in the Calf Uterus clinical trials of breast cancer prophylaxis may be estimated Cytosol. Steroids, 20: 499 514, 1972. from the concentrations used in this investigation. If linear 13. Heuson. J. C, and Heimann. R. Ibenzmethycin (Natulan). a Highly Effective Mammary Carcinogen in the Huggins System. European J. absorption of estriol is assumed from the 10%pellets which Cancer, 2: 385-386, 1966. ranged between 500 to 700 pg initial estriol content during their estimated 90- to 100-day life, a minimum absorption 14. Huggins. C.. Briziarelli. G., and Sutton. H. Rapid Induction of Mammary Cancer in the Rat and the Influence of Hormones on the rate of 5 to 7 ^g/day was released into extracellular fluid, or Tumors. J. Exptl. Med.. 109: 25 41, 1959. about 20 to 30 j/g/kg/day for a 250-g rat. One-quarter of 15. Huggins. C., Grand. L. C., and Brillantes. F. P. Mammary Cancer this dose was adequate for 75% inhibition of PC-induced Induced by a Single Feeding of Polvnuclear Hydrocarbons and Its breast carcinomas (Table 5). If allowance is made for Suppression. Nature, 189: 204-207, 1961. differences between parenteral and enteric absorption, es 16. Huggins, C., and Jensen, E. V. The Depression of Estrone Induced triol, 2.5 to 5.0 mg/day p.o., might be a suitable minimum Uterine Growth by Phenolic Estrogens with Oxygenated Functions at dose for prophylactic therapy in premenopausal women. Position 6 or 16:the Impeded Estrogens. J. Exptl. Med., 102: 335 346. Estriol, 2.5 to 5.0 mg/day p.o., has been well tolerated in 1955. 17. Huggins. C.. and Morii. S. Selective Adrenal Necrosis and Apoplexy menopausal and postmenopausal breast cancer patients, for Induced by 7,l2-Dimethylbenz(a)anthracene. J. Exptl. Med.. 114: as long as 11 months. Such a dosage is far less then the daily 741-761, 1961. estriol production of pregnant women, who excrete up to 30 18. Huggins. C., Torralba, Y., and Mainzer. K. Hormonal Influences on to 50 mg estriol per day. Mammary Tumors of the Rat. I. Acceleration of Growth of Trans These observations afford a basis for clinical trials of planted Fibroadenoma in Ovariectomized and Hypophysectomized estriol therapy for prevention of breast cancer in Caucasian Rats. J. Exptl. Med.. 104: 525-538, 1956. women, especially when nulliparity, familial history, pre- 19. Huggins. C., and Yang, N. C. Induction and Extinction of Mammary cancerous breast disease, or reduced endogenous estriol Cancer. Science. 137: 257 262. 1962. 20. James. F.. Braunsberg, H.. Irvine. W. T., and James, V. H. T. production suggest excessive risk. Radioactivity in Human Breast Tumors after Infusion of 3H-Oestradi- ol. Effect of Androgen Treatment. Steroids. 15: 669 678, 1970. 21. Jensen, E. V., Block, G. E.. Smith. S., and DeSombre. E. R. REFERENCES Hormonal Dependency of Breast Cancer. Recent Results Cancer Res. 42: 55-62, 1973. 1. Archer, F. L., and Orlando. R. A. Morphology, Natural History and 22. King, R. J. B. Oestriol Metabolism by Rat and Rabbit Liver Slices Enzyme Patterns in Mammary Tumors of the Rat Induced by Isolation of 2 Methoxy-oestriol and 2 Hydroxy-oestriol. Biochem. J., 7,12-Dimethylbcnz(a)anthracene. Cancer Res., 28: 217 224, 1968. 79: 355 361. 1961. 2. Barker. K. L.. and Anderson, J. M. Displacement of Estradici \lÃŸ 23. King, R. J. B.. Gordon, J.. Cowan, D. M., and Inman, D. R. The from the Uterus by Estradici 17Â«;Effectupon Template Capacity of Intranuclear Localization of (6 73H) Oestradiol \lÃŸinDimethylbenz- Uterine Chromatin. Endocrinology 83: 585 593, 1968. anthracene Induced Rat Mammary Adenocarcinoma and Other 3. Bresciani. F. Ovarian Steroid Control of Cell Proliferation in the Tissues. J. Endocrino!.. 36: 139 150, 1966. Mammary Gland and Cancer. In: P. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1975 American Association for Cancer Research. Estriol Prevention of Mammary Carcinoma Induced by 7,12-Dimethylbenzanthracene and Procarbazine

Henry M. Lemon

Cancer Res 1975;35:1341-1353.

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