estriol prevention of mammary carcinoma induced by 7 ... - … · estriol to estrone plus...

(CANCER RESEARCH 35, 1341 1353, May 1975]

Estriol Prevention of Mammary Carcinoma Induced by7,12-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. Nebraska68105

SUMMARY

The concentration of estrogenic, androgenic, progesta-tional, and adrenocortical steroid hormones in body fluidsof mature intact Sprague-Dawley female rats was increasedby s.c. implantation of 5 to 7 mg NaCl pellets containing 1to 20% steroid 48 hr before administration p.o. of either7,12-dimethylbenz(a)anthracene or procarbazine. The incidence of rats developing one or more mammary carcinomasin each treated group was compared to that observed in simultaneously treated groups receiving only the carcinogen,steroid, or no treatment whatsoever, with weekly observation of all rats until palpably growing tumors were biopsiedand proven carcinomatous or until death occurred fromother causes determined by autopsy. A total of 105 untreated or steroid-implanted rats followed to death (234 to256 days median observation) developed no breast carcinomas. Rats fed either of the carcinogens developed initialevidence of breast carcinoma, after 136 to 156 days medianobservation, in 51 to 57% of 318 total treated rats. Non-breast carcinomas and sarcomas developed in 5 to 10% ofthe carcinogen-treated rats.

Estriol administered as 0.15 to 0.60 mg/pellet reim-planted every 2 months reduced breast carcinoma incidenceto as low as 5 to 7% of 106 rats given dimethylbenzan-thracene or procarbazine (p < 0.001) during a medianobservation period of 202 to 232 days. The incidence ofnonbreast neoplasms was not significantly altered. Breasttumor incidence noted following implantation of 0.50 to1.14 mg estrone and 17/3-estradiol in 80 dimethylbenzan-thracene-treated rats was reduced to 22 to 25%, compared to45 to 47% in carcinogen controls; more significant breastcancer reduction was observed by estrone treatment, insimilar doses, following procarbazine administration. 16-Epiestriol also reduced breast cancer incidence to one-halfthat observed in carcinogen-treated controls. Estrone 3-sul-fate, 0.88 mg/pellet, reduced breast tumor incidence afterprocarbazine administration, but it was ineffective in alower dose. D-Equilenin, 6-dehydroesterone, 16-keto-17/3-estradiol, 2-hydroxy-17/3-estradiol, 2-hydroxyestriol, es-tradiol-17Â«, 16,17-epiestriol, 17-epiestriol, testosterone,progesterone, corticosterone, dehydroepiandrosterone, and

1Supported in part by National Cancer Institute GrantCA 10626-01,02 from the United States Department of Health. Education,and Welfare: by the Department of Internal Medicine Memorial CancerResearch Funds of the University of Nebraska Medical Center in Omaha:and by research grants from Carnrick Laboratories, Cedar Knolls, N. J.

Received September 27, 1973: accepted February 3, 1975.

hexestrol, 0.60 mg/pellet, did not alter breast cancerincidence in 220 additional rats. Estrogen-treated ratsusually sustained a mean 0.6 to 8.9% reduction of bodygrowth for the first 6 to 8 months of observation; this didnot correlate with the breast carcinoma-suppressive activities of individual steroids.

Single implantation of 0.60 mg estriol 48 hr beforedimethylbenzanthracene p.o. or sustained implantationevery 2 months of 10% estriol pellets beginning 24 hr aftercarcinogen exposure failed significantly to alter mammarycarcinoma development after dimethylben/anthracene administration.

Inhibition of mammary carcinogenesis induced by thesetwo dissimilar carcinogens in intact mature rats usingsustained low dosages of estriol support previously published biochemical and epidemiological data suggesting aninverse relationship between the renal excretion ratio ofestriol to estrone plus 17/3-estradiol and risk of breast cancerin women. On a mg per kg per day basis, the calculateddaily absorption of estriol from the pellets by the rats isconsiderably less than that contributed by the humanfetoplacental unit to the mother during pregnancy (adeterrent of breast cancer risk) and is within a well-tolerated p.o. dosage range in the postmenopausal humanfemale in clinical trials.

INTRODUCTION

Multiple genetic and endocrine factors contribute toincreased risk of human breast carcinogenesis, such asfemale sex, Caucasian racial background, breast carcinomain premenopausal Ist-degree relatives, and duration ofovarian function; pregnancy, marked by a 1000-fold increase in estriol production, is notably protective (26, 34).Controversy exists as to the etiological significance ofreduced estriol excretion noted in 20% of nonpregnanthealthy Caucasians compared to 60% of patients withprecancerous and malignant breast disease that we havereported (27, 32). Premenopausal Asiatic women withone-sixth the breast carcinoma risk of Caucasians commonly excrete a higher proportion of estriol relative toestrone and estradiol in their urine (28, 34). Wide variationin estriol excretion by healthy Caucasian premenopausalwomen may reflect several phenotypic mutant variants forthe endogenous production of estriol (28), which appearsusceptible to genotypic quantitation by leukocyte incubation with tritiated precursors of estriol in vitro (29).

MAY 1975 1341

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H. M. Lemon

The present investigation was planned to explore thepossible carcinogenic or anticarcinogenic function not onlyof estriol but also of other metabolites of estradiol hydrox-ylated at C-2, C-6. C-16 and of nonestrogenic steroids.Some estrogen metabolites resemble estriol in alteringphysiological response to estradiol and are classified among"impeded" estrogens (16, 27, 51). Commencing in 1967, we

adapted the model system of rat mammary carcinogenesisdeveloped by Dao (5) and Muggins el al. (14, 15) to ourneeds, by superimposing upon the normal ovarian estradiolsecretory activity (44), required for DM BA2 induction ofmammary carcinogenesis, periodic s.c. implantation of0.06- to 1.3-mg doses of the test steroid (Table 1). Mammary carcinogenesis induced in the female Sprague-Dawleyrat by this agent has many similarities to human mammarycarcinogenesis, especially relating to the near identity ofestrogen receptor proteins and the response of these tumorsto various types of endocrine therapy (Table 2). Furthermore, the rat offered an acceptable model for determiningthe potential chronic toxicity and tolerance of various dosesof steroid metabolites, which might have significant inhibitory effect upon mammary carcinogenesis, and also besuitable for human clinical trials. PC was later substitutedfor DMBA as the carcinogenic agent to determine whetherthe inhibition of breast carcinogenesis by estriol was relatedto the chemical structure or possible endocrine activity ofeither carcinogen (13).

MATERIALS AND METHODS

RaCs and Their Maintenance. Throughout the study,intact female Sprague-Dawley rats were obtained from asingle source (Sasco, Inc., Omaha, Nebr.) at 50 to 55 daysof age. They were randomized upon receipt and housedindividually in suspended cages. They were fed Wayne LabBlox (South Omaha Terminal Co., Omaha, Nebr.) andmunicipal water, with temperature maintained within22-23Â° and 40 to 50% relative humidity. Prophylactic

hormone therapy was achieved by implanting s.c. in theposterior nuchal region 5- to 7-mg pellets once every 1 or 2months under light ether anesthesia. The 1st pellet implantation was 48 hr before administration by gavage of thecarcinogen, again under light ether anesthesia. The ratswere all examined for tumors and weighed every 1 to 2weeks. After 1 or more tumors were identified in themammary areas and their size(s) were measured, they werereexamined after 1 to 2 weeks to confirm the continuedgrowth of the tumor(s). Firm enlarging masses were biop-sied and the animals were sacrificed upon pathologicalverification of mammary carcinomas, as defined by Muradand von Haam (43). Animals with nonbreast tumors wereobserved until near death, when they were sacrificed andnecropsied. Non-tumor-bearing rats were reimplanted withsteroids according to protocol, until death, when they werenecropsied. At this time, gross examination was conductedof the dissected axillary and inguinal mammary pads, along

Table 1Compounds tested

Trivia] name Chemical name

17Â«-Estradiol17rf-Estradiol

EstroneEstriol16-Epiestriol17-Epiestriol16,17-Epiestriol16-Ketoestradiol2-Hvdroxyestradiol2-HydroxyestriolD-Equilenin6-Dehydroestrone

CorticosteroneDehydroepiandros-

teroneTestosteroneProgesteroneEstrone 3-sulfateHexestrol

Estra-l.3.5(10)-triene-3,17Â«-diolEstra-1.3.5(10)-triene-3,l7/3-diol3-Hydroxyestra-1,3,5( 10)-trien-17-oneEstra-1.3.5( 10)-triene-3.16Â«. 17/3-triolEstra-1,3,5( IO)-triene-3,16/3.170-lriolEstra-1,3,5( IO)-triene-3,16a, 17a-triolEstra-1,3,5( 10)-triene-3,16/3,17a-triol3.17j3-Dihydroxyestra-1.3.5(10)-trien-16-oneEstra-1,3,5( IO)-triene-2,3,17/3-triolEstra-1,3,5( 10)-2,3,16a. 17/i-tetrol3-Hydroxyestra-1,3.5( 10),6,8-pentaen-17-one3-Hydroxyestra-1.3.5( I0).6-tetraen-17-one1li3.21-Dihydroxypregn-4-ene-3.20-dione3d-H \droxyandrost-5-en-17-one

17-Hydroxyandrost-4-en-3-onePregn-4-ene-3.20-dione17-Oxoestra-l,3,5(IO)-trien-3-yl sulfate

p.p'-(1.2-Diethylethylene)diphenol

2The abbreviations used

thracene: PC, procarbazine.are: DMBA. 7.12-dimethvlbenz(a)an-

with gross examination of the heart, lungs, and abdomen. Insacrificed animals, the uterus was dissected free and itsweight was recovered as an index of estrogenic response.

Steroid Incorporation into Pellets. The pellets were constructed in a Forbes pellet press (9), from 1 to 20% mixturesof the authentic crystalline steroid with crystalline NaCl.They were prepared a few days before use, weighed, andkept in a dessicator until implantation. Their weight usuallyvaried between 5 and 7 mg and they could be identified aslong as 2 to 3 weeks after implantation, at necropsy. Thedynamics of steroid release from such pellets has beendescribed (10) and, in the case of 100% estrogen pellets,hormone release continues for at least 90 to 100 days in therat. Pellet reimplantation was carried out every 2 monthsfor the 2.5 to 20% pellets and monthly for the 1% pellets.The percentage composition of nonestrogenic steroids orsynthetic nonsteroidal estrogens in pellets was adjusted toprovide equimolar activity compared to estriol. Steroidswere obtained from commercial sources. Estrogens werescreened for identity and purity by thin-layer silica gelchromatography using 5% ethanol, 47.5% ethyl acetate,47.5% cyclohexane liquid phase, and UV spotting tomeasure RF values.

Carcinogens. DMBA was obtained from Eastman KodakCo., Rochester, N. Y., and PC was from the NationalCancer Institute, Bethesda, Md. Both were administered byflexible gavage tubes under light ether anesthesia. DMBAwas given in a 20-mg dose in 1.0 ml sesame oil for most ofthe experiments, producing a 10 to 40% mortality fromadrenocortical necrosis (17). Increasing the sesame oilvehicle to 2.0 ml without changing the DMBA doseabolished any mortality consequent upon carcinogen administration within the 1st 2 to 3 weeks of observation andthis volume was adopted. PC was kept in crystalline formuntil just before use at 4Â°,when it was made up to 50-70-mg/ml individual rat doses in distilled H2O, prepared in5.0-ml batches immediately before administration. Nomortality resulted from PC administration at any time, but

1342 CANCER RESEARCH VOL. 35



EstriÃ³!Prevention of Induced Rat Breast Carcinoma

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

Rat Human

Sex incidence

Effect of prior castration on car-cinogenesis

Estrogen replacement therapy incastrate

Pregnancy

Role of pituitary in carcinogene-sis

Evidence of viral cocarcinogene-sis

Tumor histogenesis

Pathology

Multiple primary tumors

MetastasisLocal invasionDistant

Spontaneous regression

Estrophile proteins

Tumor response to castration oradrenalectomy

Hypophysectomy

Prolactin

Rare in males (1.4. 5, 7. 8) 1:IOOM:F(34)

Reduced tumor incidence by 80 90% (5. 7. 8. 14, 19) Reduces cancer risk by 60 70<Ãd̄epending upon age(26. 34)

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

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

Required (5. 7. 40. 42. 45, 50) Required (26. 34)

Not proven Not proven

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

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

In 40 80%(4. 7,43)

Common (1.4. 5, 14. 15.43)Very rare (I. 4, 5. 14, 15,43)

In 10 20n;

CommonIn 50 70%

Rare but documentedUp to 20% tumors (1, 4. 5, 7. 8. 14, 40)

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)

Tumors with estrophile proteins regress (23. 40. 41) Some but not all tumors with estrophile proteinsregress (20. 21. 48)

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

Restores tumor growth after castration-induced Possibly sensitive to prolactinregression (39. 40. 44)

the interval required for mammary carcinogenesis wasconsiderably longer than that required for DMBA activity.

Experimental Design. Initial experimental protocols included groups of 4 to 12 rats assigned as untreated controls,steroid-implanted controls, carcinogen-treated rats, andsteroid-implanted rats followed by carcinogen feeding. Asexperience developed, and no breast carcinomas were notedin over 100 rats observed during their natural life-span in the1st 2 control groups, these were no longer included in theroutine design of experiments. Most of the later experimentsinvolved comparison of 2 to 4 groups composed of 8 to 18rats each, 1 of which was fed carcinogen, with the othersgiven steroid pellets plus carcinogen.

The development of 1 (or more) breast carcinoma(s) perrat sufficed for our objectives to determine cancer incidencerats, so that these rates were sacrificed to save expense. Theresults are reported as the number of rats in each groupdeveloping 1 (or more) breast carcinoma(s) out of the totalnumber treated and alive 30 days after carcinogen adminis

tration. The median duration in days of observation of allrats in each group was also determined: premature mortalityfrom infectious disease was the major factor reducing tumorincidence in carcinogen-treated groups, owing to the 120- to150-day median latent period of carcinogenesis. All breastcarcinomas were diagnosed between the 40th and 370th dayafter administration of either carcinogen. The mean ofmean weights of steroid in pellet implants every 1 to 2months has also been included in the tables when appropriate, to indicate the actual dosage administered to eachgroup of rats.

All histological sections of tumors were initially readwithout knowledge of the treatment received by the rat.They were again reviewed at the time of this report toconfirm the pathological variants of breast carcinomarecognized according to the classification developed byMurad and von Haam (43). Fibroadenomas and lactationalhyperplasia. which was usually grossly readily distinguishable from carcinoma, were not included among the malig-

MAY 1975 1343



H. M. Lemon

nant neoplasms. Between 5 and 10% of mammary nodulesless than 5 mm in diameter failed to grow during severalweeks of observation or regressed totally, a common findingin these tumors (Table 2). On the other hand, once a tumorhad reached 5 to 10 mm in diameter, we seldom encounteredregression except after castration, and it was at this size thatmost biopsies were made. Only pathologically verifiedcarcinomas were included in the statistics. No unusualincidence of tumor regression was noted in most of thesteroid-treated groups, suggesting that the end results weresignificantly affected by nonintrusion of spontaneouslyregressed neoplasms.

Statistical analysis by x2 evaluation based upon a 4-foldtable of the number of rats with and without breast carcinoma in the carcinogen versus steroid-plus-carcinogen-treated group was used to determine significance of differences. Since cancer induction rates by these agents vary withtime and each batch of animals, only those groups simultaneously receiving the 2 treatment protocols were includedfor analysis of significance of differences in incidence rates.The Yale's correction for small numbers was used throughout in x2 analysis.

RESULTSEstrogenic Steriods

17/3-Estradiol. This primary ovarian estrogen (27) wastested in 5 experiments in 1 to 10% pellet concentration forits effect upon DMBA carcinogenesis (Table 3). No breasttumors appeared in untreated or steroid implanted controlsobserved for as long as 300 days median observation. Theincidence of DMBA-induced mammary tumors was reduced by nearly 50% in rats implanted with 10% but not 1%pellets, and the median time of tumorigenesis was delayedby 34 days, compared to those receiving only the carcinogen. This difference in incidence, while suggestive, did notachieve statistical significance. In 20% pellets, 17/3-estradioldid not reduce significantly breast carcinoma incidence in asingle experiment, but it delayed onset of tumors by 147days to a median of 221 days.

When 17/3-estradiol in 10% pellets was implanted only asingle time 2 days prior to DMBA administration, 7 of 10recipients developed mammary carcinomas in a medianperiod of 241 days (range of 65 to 297 days).

Estrone. Estrone equilibrates reversibly with 17/3-estradiol in vivo, depending upon the oxidation-reductionpotential and availability of pyridine nucleotide respiratorycofactors, and might be expected to have a somewhatsimilar degree of activity affecting carcinogenesis as es-tradiol (27). In 6 experiments with 1 to 10% pellet concentration, estrone closely resembled 17/3-estradiol in its an-ticarcinogenic effect upon DMBA induction of breastcancers, with a 50% reduction of their incidence comparedto DMBA controls (Table 3). Highly significant inhibitionof PC-induced mammary carcinogenesis was observed after10% estrone pellets with minimal prolongation of medianlife-span of the implanted animals beyond that of untreatedcontrols. No spontaneous tumor regressions were observedeither in rats receiving estrone or 17/3-estradiol in theDMBA- or PC-treated groups.

Estrone 3-Sulfate. This conjugate is the principal estrogenrecovered from human plasma. Tested in 10%pellet concentration (equivalent to 7.4% estrone), it was not effective inpreventing breast neoplasms (Table 3). In 14.1% pelletconcentration (equivalent to 10.4% estrone), possible inhibition of breast tumor development after PC feeding wasnoted in a single experiment.

All 3 of these estrogenic steroids similar to otherestrogens reduced the rate of body growth by 3 to 6.6%compared to that noted in the rats receiving only carcinogens (Table 4). No significant correlation existed betweenreduction in weight and inhibition of breast tumor formation by these and other estrogens. Neither of the 2carcinogens used affected the rate of growth of the rats, incomparison with simultaneously observed untreated controls. No other systemic effects were noted from these smallestrogen doses, with the longevity of the steroid-implantedcontrols equivalent to that observed among untreated rats(Table 3).

Estriol. The 2 principal pathways for estradici or estronemetabolism involve hydroxylation at C-16 to produce estriolor an epimer and at C-2 yielding 2-hydroxyestradiol,2-hydroxyestrone, or 2-hydroxyestriol, which are methylated in the kidney and liver prior to excretion (22, 27).Estriol in 10% pellet concentration reimplanted every 2months proved to be the most inhibitory compound of anyof the 17 tested in the entire series of experiments, reducingthe development of palpable breast carcinoma to only 5 to7% of the treated rats (Table 5). No inhibition of tumorformation followed 1% pellet implantation. Single implantation of 10% estriol pellets 2 days before DMBA administration in 2 experiments resulted in breast cancers in 20 outof 34 rats, within a median period of 83 to 111 days (range,37 to 187 days), similar to the incidence of breast carcinomas observed in the DMBA-treated rats. Four multipleprimary breast carcinomas occurred. Serial implantation of10% estriol pellets every 2 months commencing 24 hr afterDMBA administration has resulted in 4 of 11 rats withbreast carcinomas after 270+ days in 1 experiment, withanother rat with 3 primary breast carcinomas.

Estriol was most inhibitory against PC-induced mammary carcinoma development, with highly significant reduction of breast tumors after implantation of 2.5% pelletsyielding only a 16% incidence of rats with cancer. At thisdosage no inhibition of DMBA-induced tumors occurred.

The median survival of estriol-protected rats treated witheither carcinogen was in all cases somewhat shorter thanthat noted in either untreated or steroid-treated controls. Inall cases the survival of the protected rats considerablyexceeded that noted in the carcinogen-treated rats, whichwas shortened by the earlier biopsy and sacrifice of thelarger number of tumor-bearing animals.

Excluded from these incidence figures are 1 transientmammary tumor in the PC-treated rats and 8 transienttumors that totally regressed spontaneously within a fewweeks after discovery in the estriol implanted-plus-carcino-gen-treated (5 after DMBA and 3 after PC) rats. Five ofthese 8 transient regressing tumors were noted in ratsimplanted with the lowest estriol doses of 1.0 to 2.5% perpellet.




Estriol Prevention of induced Rat Breast Carcinoma

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

No. ofCarcino- exper- Untreated

Steroid genie agent imentscontrols170-Estradiol

DMBA. 5 0/10."23620mgdays'DMBA.

120mgEstrone

DMBA. 70/13,22820mgdaysPC.

502mgEstrone

3-sulfate DMBA.220mgPC.

160mgPC.

150mgTotal

incidence 0/23Carcinogen-

treated8/17(47)."136

days5/11(45),74

days9/20(45).159

days10/15(66).1

37days4/5,

80days4/7.

179days5/13(38),187

days45/88(51)20%

pellet 10%pellet+ carcinogen +carcinogen8/32(25).170days.0.580.65

mg'4/12(33),221

days.Â«1.14mg8/36(22),182days.0.500.76

mg1/14(7),147days.0.540.66

mg8/12(67),142days.0.65

mg2/8,

135days.0.64

mgI/

12Â»(8).179days.0.88

mg4/12(33)

28/114(26)1%

pellet 10%pellet+ carcinogen onlyx207/13(54),

0/4,3001.6203days.days,0.53mg0.063

mg15/22

0/6.2072.2(68).days.85

days. 0.63mg0.065mg8.522/35(63)

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«One survivor free of tumor after 310 days.* 14.1% pellet concentration equivalent to 10.4%estrone.

Multiple breast carcinomas verified by biopsy were notedin 10 to 75% of DMBA-treated tumor-bearing animals, atthe time of initial biopsy and sacrifice of the tumor-bearingrats. Twelve of the 48 rats fed only DMBA had 2 to 4multiple primary breast adenocarcinomas in the estriolexperiments (Table 5). No multiple primary breast adenocarcinomas were noted among rats protected by 10 to 20%estriol pellet implants prior to DMBA therapy, in the 4developing breast tumors. Six multiple primary neoplasmswith 2 to 6 breast tumors per rat were noted in the 26tumor-bearing rats receiving 1.0 to 2.5% estriol pellets priorto DMBA feeding.

After PC feeding, 7 out of 29 tumor-bearing ratsdeveloped 2 to 3 primary breast cancers, and 1 spontaneousregression of a breast tumor was noted. Two rats with

multiple primary breast carcinomas were observed in the 6developing breast cancers after estriol pellet implantationplus PC treatment; both of these were noted in rats receiving2.5% estriol pellets. Therefore, adequate circulating concentrations of estriol sustained by 10to 20% pellet implantationevery 2 months not only reduced the incidence of breastcancer-bearing rats by 90% following either of these carcinogens compared to the carcinogen-treated controls butalso abolished the appearance of multiple palpable primarycarcinomas that grew progressively.

16-Epiestriol. Following either DMBA or PC administration, only 18 to 19% of rats implanted with 0.6 mg16-epiestriol developed breast cancers; however, the carcinogen-treated controls developed breast cancers in only 33to 40%, requiring further tests to establish significance of

MAY 1975 1345



H. M. Lemon

Table 4Body 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 ofbreast cancer induction = -0.151.

Treatment10%

estriol +PC10%estriol+DMBA20%

estriol +DMBA10-14.1% estrone 3-sulfate +PC10%D-equilenin +PC10%16-epiestriol +PC10%16-epiestriol +DMBA10%estrone +DMBA10%170-estradiol +DMBA6-dchvdroestrone

+PC10%I7a-estradiol tPC10%

hexestrol +DMBA10%16.17-epiestriol +DMBA2-Hydroxyestriol

+DMBA10%estrone 3-sulfate + DMBA10%17a-estradiol + DMBANo.

ofexperiments5522122631121123Duration

ofobservation(wt

every2mos.)(mos.)g666668846g26463Mean

wtchange(%)from

carcinogen-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+

2.8-6.6-7.4%

incidenceofbreastcarcinoma571315171819222530404857666794

ihe difference in incidence rates (Table 5).This steroid caused the least reduction of body growth,

compared to that seen in recipients only of the carcinogenused, of any of the estrogenic compounds (Table 4). Neither16-epiestriol nor estriol altered the incidence of rats withbreast carcinomas, following monthly reimplantation of 1%pellets. If the stress of repeated anesthesia and pelletimplantation in any way were involved in the tumor-inhibitory activities of these compounds, one would have expectedsome reduction in tumor incidence in these groups.

17Â«-Estradiol. This unnatural stereoisomer of 17/3-estradiol may displace the latter, from intranuclear bindingof the estrogen receptor complex to uterine DNA (2). In the10% pellet concentration tested, breast carcinomas developed rapidly following DMBA administration in all recipients except 1 in the 3 experiments (Table 6). Multipleprimary breast carcinomas were noted in several animals.

17Â«Epimers of Estriol. Neither of the unnatural stereoi-somers of estriol with a 17Â«hydroxyl group inhibited theincidence of mammary carcinoma induced by DMBA(Table 6). These findings, along with the negative resultsobserved with 17Â«-estradiol,suggest that the 17/3-hydroxylgroup that is required for uterine and mammary estrophileprotein binding is essential for mammary carcinogenesisinhibition by estrogens. Five separate primary breast cancers developed in 1 rat receiving 16,17-epiestriol and adouble primary in one of the carcinogen controls in thisgroup of experiments.

Ih-Ke(o-l7,5-fstradml. This oxidation product of es-tradiol is one of the precursors of estriol and 16-epiestriol inhuman metabolism, but it was completely devoid of inhibitory activity against DMBA mammary carcinogenesis(Table 6). These results suggest that only a C-16 hydroxylgroup will satisfy the steroid-protein structural require

ments for optimum suppression of DMBA-induced mammary carcinogenesis, with the 16Â«position providing maximal inhibition.

2-Hydroxy-170-estradiol and 2-Hydroxyestriol. Oxidation of the 2nd carbon atom on ring A of the estrogennucleus modifies greatly the physiological activity of thisrepresentative of the 2nd major pathway of estradiolmetabolism in man (27). Despite retention of some physiological activities classifying it among the "impeded" estrogens, 2-hydroxyestradiol was inactive in 10% pellet concentration in altering breast tumor incidence or in delayingtumorigenesis (Table 6). 2-Hydroxyestriol in similar dosagewas also inactive.

o-Equilenin. This major equine urinary estrogen resembles estrone except for the presence of 2 unsaturated C=Cbonds in ring B of the steroid nucleus. Either alone in 10%pellet concentration or in 21% concentration combined withits L isomer, it has not significantly decreased breastcarcinoma incidence following DMBA administration(Table 6); further experiments in PC-treated rats will beneeded to assess its possible inhibition of breast neoplasia.However, 6-dehydroestrone with a single unsaturated C=Cbond in ring B exhibited no antimammary carcinogenicactivity in PC-treated rats.

Hexestrol. This nonsteroidal estrogen binds with highaffinity to rat uterine estrogen receptors and to someneoplastic human breast carcinomata in vivo (26, 27). Inpellet concentrations equimolar to estriol, it has not significantly reduced DMBA-induced breast carcinoma incidence(Table 6). Multiple breast cancers appeared in 3 hexestrol-treated rats and in 1 DMBA-treated control; 1 spontaneousbreast tumor regression was noted in the latter group.

Nonestrogenic Steroids. None of the nonestrogenic steroids tested thus far in 10% pellet concentrations have




shown any promise of altering experimentally inducedbreast cancer incidence in our rats (Table 7). None of thesesignificantly altered body weight from that observed incarcinogen-treated controls. In 20% concentration, testosterone pellets reduced tumor incidence in a single experiment and increased mean body weight by 8% at 2 months.Further experiments in progress with 20% pellets of testosterone and progesterone have not reduced breast cancerincidence.

Effect of Castration upon Breast Carcinogenesis Modifiedby Estrogen Pellet Implantation. Castration prior to DMBAadministration reduced breast tumor incidence markedly inSprague-Dawley females (Tables 2 and 8). The reducedincidence of breast carcinomas was not significantly alteredby 10 to 20% estriol pellet implantation prior to DMBAadministration in castrate females. Simultaneous implantation of 10%estrone or 170-estradiol pellets, along with 20%estriol pellets in castrate females, did not significantlychange the low incidence of tumors observed in theseexperiments (Table 8). The interval of 1 to 3 days from

Estriol Prevention of Induced Rat Breast Carcinoma

castration to pellet implantation was probably too briefentirely to remove ovarian influence upon breast car-cinogenesis. Further exploration of ratio of the steroids usedis needed to ascertain the dosage required to reproduce anovarian type of hormonal sensitivity of the breast to thiscarcinogen and to establish whether estriol is equallyinhibitory under these circumstances.

Incidence of Nonbreast Carcinomas and Sarcomas. BothDMBA and PC-induced 5 to 10% incidence of sarcomas andnonbreast carcinomas, especially epidermoid carcinomas ofthe ear duct ( 1, 4, 5, 13) (Table 9). No significant alterationin the incidence of these tumors occurred following estro-genie steroid implantation prior to DMBA treatment,regardless of the inhibitory activity that any of thesesteroids possessed for mammary tumorigenesis. Nonestro-genic steroids tested thus far also did not significantly affectnonbreast tumor incidence.

Following the use of breast tumor-inhibitory doses ofestriol in PC-treated females, nonbreast neoplasms occurred more frequently (16.7%). These were similar in type

Table 5Mammarv carcinoma incidence after estriol and 16-epieslriol

No.of

Carcino- ex-genic peri- Untreated

Steroid agent mentscontrolsEstriol

DMBA. 13 0/23."25420mgdays'PC,

50570mg16-Epiestriol

DMBA,320mgPC.

602mgCarcin

ogen-treated29/48

(60),"155days18/29(62).156

days4/10(40).125

days7/21(33),2

15days20%

pellet 10%+ carcin- pellet i

ogencarcinogen2/16(13),

2/27(7).237202days,days.1.300.62mg""

mg2/41(5).232days.0.63mg4/21

(19).101days.0.60

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

pellet+ car

cinogen6/7,

75days.0.16mg5/31(16),224days.0.16mg1%

pellet+ car

cinogen20/36(56),114

days.0.07mg6/7,68days.0.06mg10

20%pelletonlyx2"0/25,

2499.2'days,17.9Â«0.601.30mg24.5Â«11.5"0/4.

2050.6Â«days.0.64mg0.6Â«P0.002<

0.001<

0.001<0.001NSNS

Total breasttumor incidence

0/23 58/108(54) 2/16(13) 12/111(11)11/38(29)26/43(61) 0/29

" 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«X2 comparing 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



H. M. Lemon

Table 6Mammary carcinoma incidence after implantation of 9 other estrogens

SteroidI7a-Estradiol16,17-Epiestriol17-Epies-

trioll6-Keto-17/3-

estradiol2-Hydroxy-l70-estradiol[)-Equilenin6-Dehydroestrone2-HydroxyestriolHexestrolTotal

incidenceCarcinogenic-

agentDMBA,20

mgPC,

60mgDMBA.20

mgDMBA.20

mgDMBA.20

mgDMBA,20

mgDMBA.

20mgPC,

60mgPC,

60mgDMBA.20

mgDMBA.20

mgNo.

ofex- Un-

peri- treatedmentscontrols3

0/1Â°1113

0/231

0/111120/4Carcinogen-

treated6/7,

99days"2/2,

243days5/10,82

days8/12(67),

110days7/14(50),

130days4/7.

161days6/7.67

days3/6,

192days3/6,

192days5/11(45).74

days10/22(45).'

107days56/98(57)10%

pellet + 1%pellet 10%pelletcarcinogen + carcinogen onlyx'18/19(94),'

116 0/5,200

days, 0.59 days,mg" 0.67mg4/10,

149days, 0.62mg8/14(57),

107days, 0.59mg6/1

2' (50), 193

days, 0.52mg12/19(63),

126 0/2,135days. 0.59 mg days.

0.56mg13/18

(72). 99days, 0.60mg7/10(70).

86days, 0.58mg4/18(22),

175days, 0.61mg3/10(30),

208days. 0.60mg8/12(67).

74days, 0.62mg12/25

(48),Â«100

days, 0.61mg95/167(57)

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 increasein incidence was not significant by x2 and may be attributedpartly to the longer observation period of the protected rats.

DISCUSSION

Previous investigations of steroid action upon chemicallyinduced rat mammary carcinoma incidence utilized largerintermittent doses over a shorter period of time than givenin this study. Few have demonstrated as marked inhibitionof mammary carcinogenesis as noted herein followingadequate estriol therapy, despite shorter postcarcinogenobservation than the natural life-span follow-up of cancer-

free rats in this study (5, 7, 11, 14, 15, 19, 36, 45, 47, 49, 50).Huggins et al. (18) noted that 10 to 20 ^g estriol injecteddaily stimulated growth of a transplantable mammaryfibroadenoma, while 100 jig/day inhibited its growth.Terenius (47) observed that 100 ^g estriol injected every 12hr for 12 days reduced the volume of DMBA-inducedmammary tumors from 2670 cu mm in sham-injectedcarcinogen-fed rats to 110 cu mm after 120 days. Progesterone-treated rats averaged 1460 cu mm tumor volume andtestosterone-treated rats averaged 4370 cu mm. The smallest tumor volume of 31 cu mm occurred after treatmentwith an estrogen antagonist. Mer 25, which like estrioldecreased somatic growth rate. Progesterone pretreatmentof rats during 25 days prior to carcinogen administration





Table 7Mammary carcinoma incidence after implantation of nonestrogenic steroid';

The carcinogenic agent was DMBA. 20 nig.

SteroidDehydroepian-

drosteroneTestosteroneProgesteroneCorticosterone(12.7%')Total

tumor incidenceNo.

ofexperi- Untreated Carcinogen-ments controlstreated2

0/1Â° 4/6.112days"2

3/5, 266days2

3/5, 145days1

6/7,67days0/1

16/23(70)20%

pellet+carcinogen12/15(80).'

I06davs,1.14mg"1/9,

184days, 1.15mg10%

pellet+carcinogen4/5,

66days, 0.63

mg9/17(53).

146 days.0.64mg4/7.Â«

92

days,0.77 mg20%

1% pellet pellet+ carcinogen only\'Â¿0/7,

172days,1.23

mg4/6,67

0/2.230days, days, 1.150.060 mg mg0/9PNS"NSNSNS

" 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 castrationThe carcinogenic agent was DMBA, 20 mg.

SteroidEstriol10%

estrone + 20%estriol10%

17/3-estradiol +20%estriolTotal

tumor incidenceNo.

ofexperiments833Untreatedcontrols0/10,"

333days"0/10Carcinogen-

treated3/21(14),'

180days3/21(14)10

20%steroidpellets +

carcinogen3/12(25),

203days,0.61l.27mg"6/20'

(30). 204days2/22Â«

(9), 292 days10

20% steroidpellets onlyX20/16.

301 days.0.62 1.19

mg0/16PNS'NSNS

" 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 othersnoted stimulatory activity upon mouse and rat mammarycarcinogenesis (19, 42). Estradici benzoate, 20 /ug/day for20 days, completely suppressed further growth of DMBA-induced mammary tumors, reversible by daily injections of28 IU ovine prolactin (39). Similar estradiol doses given

both before and after DMBA completely inhibited breastcarcinogenesis for as long as 150 days of observation, withmarked impairment of weight gain (24). Breast cancer onsethas been delayed as long as 241 days (median) (range, 65 to297 days) by a single implant of 17/3-estradiol 48 hr prior toDMBA treatment in this investigation. Since breast carci-

MAY 1975 1349



H. M. Lemon

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

Steroidinhibition ofmammary

carcinogenesisCarcinogen

administeredSteroid implanted 48 hr

before carcinogen

Nonbreast neoplasms

Incidence Pathological diagnosis

None

None

Significant(p < 0.001)

None

None

Significant(p < O.(X)I)

None None 0/51 0

DMBA None (Tables 3, 5 7) 10/196 5.1

DMBA 1.2 0.60 mg estrone, estrone 3-sulfate. 7/92 7.6I7tf-estradiol (Table 3)

0.15 0.060 mg estrone. I7tf-estradiol. 3/76 4.0estriol, 16-epiestriol (Tables 3, 5)

0.60 mg 16-epiestriol (Table 5) 0/21 0

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)DMBA 1.20 0.60 mg estriol (Table 5) 2/43 4.7

PC None (Tables 3. 5 6) 5/48 10.4

PC 0.60 mg 17a-estradiol. estrone 3-sul- 6/52 11.5fate (Table 3, 6)

0.60 mg 16-epiestriol (Table 5)PC 0.15 0.60 mg estriol (Table 5) 12/72 16.7

hpidermoid carcinoma of ear 5Leukemia 1Sarcoma of uterus IOther 3

Epidermoid carcinoma of ear IIFibrosarcoma of earSarcoma of lymphomaEpidermoid carcinoma of earLymphosarcoma

Endometrial carcinomaEpidermoid carcinoma of earSarcomaEpidermoid carcinoma of earSarcoma

Epidermoid carcinoma of ear 5Misc. sarcomas 2Adenocarcinoma? gut 1Sarcoma of uterus, ovary 3Adenocarcinoma of lung I

nomas may develop up to 370 days after carcinogen, thevalue of shorter observation periods in evaluating breastcancer prophylactic agents is dubious.

With the technique used in this investigation, whichpermitted continuous absorption of varying doses of freesteroids into body fluids, a 75 to 90% reduction in incidenceof palpable or visible mammary carcinomas was achievedby 10 to 20% estriol pellets reimplanted every 2 months,commencing prior to feeding of either DMBA or PC.Tumor-free rats were observed until death from naturalcauses, usually bronchopneumonia, which occurred usuallylong after the age peak when most breast carcinomasbecome manifest. A threshold of cancer-inhibitory activitywas noted when estriol concentration in pellets was reducedto 1.0 to 2.5% with pellet reimplantation every 1 to 2months, respectively (Table 5). PC-induced mammarycarcinoma was more sensitive to estriol inhibition than thatinduced by DMBA, since significant inhibition of breasttumorigenesis occurred after implantation of 2.5% pellets ofestriol in PC-treated rats. With decreasing estriol dosage,the incidence of rats developing single as well as multipleprimary breast tumors increased to equal the incidenceobserved in rats fed carcinogens only. Every enlargingbreast tumor was biopsied, and simple hyperplasia with orwithout lactation as a cause of breast enlargement was ruled

out by histolÃ³gica! examination. Therefore, the final observed incidence of mammary carcinoma in various ratgroups was conservative and was minimally influenced bylactational changes that develop in older virgin females. Theincidence of mammary carcinoma in our carcinogen controlgroups fed a single 20-mg dose of DMBA was similar tothat observed by others who histologically confirmed allmalignant tumors (1, 4, 5, 7, 13, 36, 39, 47, 50). No difference was observed in the histological types of mammarycarcinomas between estriol-treated and non-estriol-treatedrats, nor was there any significant incidence of spontaneousremission of unbiopsied tumors to contribute materially tothe differences noted in tumor incidence.

The 5 to 10% incidence of rats with sarcomas andnonbreast carcinomas (chiefly squamous neoplasms arisingfrom the ear ducts) induced by both carcinogens was notsignificantly altered by implantation of maximal doses ofestriol or any other of the 17 tested steroids (Table 1). Thissuggests that the anticarcinogenic activity of estriol wasspecific for mammary neoplasms only and that estriol didnot alter the endogenous metabolism, if any, of the carcinogens, thereby reducing their carcinogenic activity. Continued implantation of estriol pellets every 2 months wasrequired for inhibition of carcinogenesis, since in 2 experiments no reduction of breast cancer incidence was demon-

1350 CANCER RESEARCH VOL.35




strated following only a single implantation 48 hr beforecarcinogen administration: pellet absorption was estimatedto occur over a 90-day period (10). Estriol, therefore,probably does not block initiation of mammary car-cinogenesis by an agent such as DMBA which acts within 1to 3 days upon the breast to induce carcinogenesis (6).Estriol probably interferes with the 2nd or promotionalstage of mammary carcinogenesis, in which continuedestradici stimulation of mammary duct and myoepithelialcell proliferation (3, 35) is the major cocarcinogenic factor.Illustrating this was the similar degree of reduction ofmammary carcinogenesis achieved by preliminary castration as achieved by estriol treatment (Table 8). In castratefemales, substitution therapy with estrone or 17/3-estradiolwas ineffective in restoring the expected incidence ofDMBA-induced breast tumors, as long as estriol wascoadministered, in contrast to the results of substitutiontherapy with estradiol or diethylstilbesterol alone (5, 7, 14,19, 33).

In intact female rats, neither of the 2 major estriolprecursors, estrone and 17/3-estradiol, inhibited carcinogenesis in the breast as effectively as estriol. 17Â«-Estradiol was completely ineffective against carcinogenesisin spite of its binding to uterine estrogen receptor proteins(27). Estrone and 17/3-estradiol (0.50 to 0.76 mg) reducedbreast tumor incidence to 22 to 25% in DMBA-treated rats,but 20% estradiol (1.14 mg) was even less inhibitory in 1experiment. Estrone decreased breast cancer incidence moresignificantly after PC treatment (Table 3). Rat liver,intestine, and kidney incubated in vitro with tritiated170-estradiol along with the oxidative cofactors, NADPHand nicotinamide, yield estriol as a major metabolite (31),so that part of the anticarcinogenic activity of estradiol orestrone may be attributed to metabolism to estriol followingimplantation. Other estrone and 170-estradiol metabolitessuch as 16-ketoestradiol or 2-hydroxyestradiol possessed noanticarcinogenic activity (Table 6), and 16-epiestriol hadlesser anticarcinogenic activity (Table 5). Since these andother inactive metabolites of estrone and estradiol implantswere probably formed in vivo in addition to estriol, thelesser anticarcinogenic activity of estrone and estradiol inthe dosage tested may reflect the net response of tissues to avariety of metabolites, many of which were inactive. Estriolmetabolism to 2-hydroxyestriol occurs in vitro by mouseliver (22), which also is ineffective as an antimammarycarcinogen. The protective activity of estriol implantationtherefore results from circulation of the native steroid alongwith inactive metabolites until renal and biliary excretion.

If estriol indeed accounted for most of the anticarcinogenic activity observed in this study, the mechanism mightinvolve either local mammary effects or effects upon thepituitary or other endocrine organs or both. Sustained highestriol concentrations in extracellular fluid would probablydisplace much of the ovarian-secreted estradiol bound tomammary estrophile receptor proteins and thereby alter thenature of the nuclear estrogen-receptor complex bound toDNA (51). Estriol retains 70 to 100% of the binding activityof equimolar concentrations of 17/3-estradiol in vitro for ratmammary estrophile proteins [H. M. Lemon. Estriol andEstradiol-17/8 Binding to 7,12-Dimethylbenz(a)anthracene

Induced Rat Mammary Carcinomas, Autologous Muscleand Uteri, in preparation]. Dehydroepiandrosterone has nobinding activity and was inactive in 20% pellet concentrationin altering mammary carcinogenesis (Table 7). Althoughestriol probably binds to mammary receptor protein in vivo,suppression of DMBA-induced mammary carcinogenesis atthe site of receptor-complex binding to DNA appearsunlikely. Polycyclic hydrocarbon carcinogens interfere withestrogenic cellular responses (7), but there has been no closeassociation noted between these compounds and estrogensfor nuclear protein binding (12).

High chronic doses of estrone and 17/8-estradiol alone arecarcinogenic in rodents (33,46). Estriol lacks any mammarycarcinogenic activity resulting from maximally sustainedduct epithelial proliferation (3) in castrate or intact rats ormice, but renal tumors have resulted (32, 46). In castrateC3Hf mice free of mammary tumor virus, 10 to 200 Â¿Â¿gestriol applied cutaneously weekly for 60 weeks failed toelicit breast carcinomas, while 10 jig estrone weekly for asimilar period of time resulted in 25% incidence of micewith breast cancers, comparable to the incidence rate inolder breeding females of the same inbred strain (46).Weekly administration of 5 pg estrone along with 5 Â¿igestriol resulted in a single breast carcinoma (2.9% incidence); when a mixture of 1.4 ^g 17/3-estradiol, 4.3 ^g ofestrone, and 4.3 //g estriol was similarly applied, the incidence of breast carcinomas increased to that noted in breeding females in Pullinger's studies, suggesting the critical

role of 17/3-estradiol in rodent mammary carcinogenesis.Estradiol maximally increases mitotic activity with increasing dosage in the castrate mouse uterus, whileestriol exhibits a flat dose-response curve, beyond whichmitotic activity does not increase (35). No studies are reported using similar techniques for the rodent breast.

The effects of estriol implantation upon prolactin orgonadotropin activity are unknown as yet; frequently observed lactation in castrate and estrone-implanted rats wasuniformly minimal at necropsy in our estriol-treated groups.The paucity of grossly detectable mammary tissue inestriol-implanted rats was noteworthy.

DMBA-induced mammary carcinomas are highly prolactin dependent, and in the work of Meites (39) and Welsch etal. (50) it has been suggested that estradiol blocks themammary end-organ response to prolactin, which should beelevated by high doses of estradiol. In women, lactationulactivity begins after blood estrogen concentration decreasestoward normal following delivery. Prolactin as a cofactor inhuman mammary carcinogenesis is currently being investigated.

The reduction of somatic growth observed herein following estrogen implantation, which did not correlate withbreast cancer inhibition, has been noted previously in ratsand mice receiving estrogen therapy, including estriol (33,46). Appetite and/or somatotropin secretion may be suppressed to account for reduction of body growth byestrogens.

In spite of numerous similarities between human mammary carcinoma and chemically induced rat mammarycarcinoma (Table 2), the suppressive effects of estriol onmammary carcinogenesis may not be similar in the 2

MAY 1975 1351



H. M. Lemon

species. However, the dose of estriol required for humanclinical trials of breast cancer prophylaxis may be estimatedfrom the concentrations used in this investigation. If linearabsorption of estriol is assumed from the 10% pellets whichranged between 500 to 700 pg initial estriol content duringtheir estimated 90- to 100-day life, a minimum absorptionrate of 5 to 7 ^g/day was released into extracellular fluid, orabout 20 to 30 j/g/kg/day for a 250-g rat. One-quarter ofthis dose was adequate for 75% inhibition of PC-inducedbreast carcinomas (Table 5). If allowance is made fordifferences between parenteral and enteric absorption, estriol, 2.5 to 5.0 mg/day p.o., might be a suitable minimumdose for prophylactic therapy in premenopausal women.Estriol, 2.5 to 5.0 mg/day p.o., has been well tolerated inmenopausal and postmenopausal breast cancer patients, foras long as 11 months. Such a dosage is far less then the dailyestriol production of pregnant women, who excrete up to 30to 50 mg estriol per day.

These observations afford a basis for clinical trials ofestriol therapy for prevention of breast cancer in Caucasianwomen, especially when nulliparity, familial history, pre-cancerous breast disease, or reduced endogenous estriolproduction suggest excessive risk.

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1975;35:1341-1353. Cancer Res Henry M. Lemon 7,12-Dimethylbenzanthracene and ProcarbazineEstriol Prevention of Mammary Carcinoma Induced by

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