effects of coumestrol on estrogen receptor function

Effects of Coumestrol on Estrogen ReceptorFunction and Uterine Growth in OvariectomizedRatsBarry M. Markaverich, Brett Webb, Charles L. Densmore, and Rebecca R.GregoryDepartment of Cell Biology and Center for Comparative Medicine, Baylor College ofMedicine, Houston, TX 77030 USA

.Isoflavonoids andrelated compo s.uha|s. co>umestrol he. asstcaily b.encitego-rized as phytoestgens because t.es envi-ronmenti .yderived sb sind to the.estrogen or (E.R) and increase uterinewet weight in immature rats.and mise.Assessme' t of di binding affinities of.isoflavoidfr R and subsequeitlcson uteriegro tisuges t compoundsare less acti s tha-nestrdo, l andtherefore myq redce the risk of deopingbreast or prostate cancer in humans by pre-venting estriol binding to ER With the.renewed' interest in the shre.t'isipsbetween' i estgercauseHeand. preven , we ass ;40essedthe efetof the .p estrogenn. co~ujm. e.strul. onuterotropic response in theimm nature,

ovartoinizdldrat Our studies.d.emostrat-ed that in thianimal model, cometrol isa atypica. .stro.n. at.d....o... n.s.lteuterine cellthprlsL doghd acute(sbctaneusi~neci) orchonc mutiple injectio ot. orlly via drinking wter)administration. of coumestrol significantlyincreased uterine wet and dry weighs, thephytoestrogen failed to increase uterine*DNA content T ck of teMstrg nic

t.his phyoairgn.to cas cytosolic ERdep.l..n..u.l ..E.... cu a o he*stimulation.:of.nuclear.typeI .s ih.... .. .... 'h

chaacerstcalyprecedeesrgictmuation of cellla DNA synthes and li-dion.In fact, s ubutaneous'or oral come-stro.,rea i;e case an atypca Idiceidctin of C solic ER out orre-*sn..~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. ...........lei..tsponding cyooi deltio a 'nidncar

accumulation of this receptor,4and-thisincreased thersensitivityof the uterus to sub-sequent stimulation by estradiol. Theseresultsin the immature, oaic1oie ratcontestt wh studies of inrat i treGas~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.. ...t... .t. .may..be .a...o ent in the.. ....t.rogeic:respne opl~ytesrgs suc as coume-..strol in intactamals. Consequenldy, thepotential. estrenicity of phytoeskrogensrequires. care. reassessment in intact -andovariectomized animals before theimpact oftheenvnwsfentl eivdsusane on

reprodui u onan. ... .........ce can beAd~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. .".^...b... .e.s..realizd, I~Vwei:coumesrletrogren

receptor, phytoestrogen, rat uterine growtype I [ H.es iol binding sites. EnvironHealhbP.eplI 103:574-581 (1995)

Bioflavonoids represent a class of naturallyoccurring plant pigments that humans con-sume daily in gram quantities (1,2). A highcorrelation exists between the intake ofbioflavonoid-rich diets and a lower inci-dence of stomach, colon, breast, andprostate cancer in man (3-7). For these rea-sons, some investigators have suggested thatconsumption of weakly estrogenicisoflavonoids such as equol, daidzein, andcoumestrol may prevent estrogen-depen-dent breast and prostate cancers by compet-ing with more active, endogenous estrogenssuch as estradiol for estrogen receptor (ER)in these target tissues (5-7). Conversely,studies by our laboratory and others haveshown that flavonoids such as luteolin andquercetin bind with high affinity (Kd 1-5nM) to nuclear type II [3H]estradiol bind-ing sites, but not ER, and this is correlatedwith the antagonism of estrogenic responsein the rat uterus, the inhibition of breast,ovarian, pancreatic, and colon cancer cellproliferation in vitro and estrogen-indepen-dent mammary tumor growth in mice(8-13). Therefore, it is not surprising that anaturally occurring flavonoid metabolite,methyl p-hydroxyphenyllactate (MeHPLA),has been identified as an endogenous cell-growth-regulating agent and the natural lig-and for the type II site (14). On the basis ofthese studies demonstrating mixedagonist/antagonist (estrogenic/antiestro-genic) activities of flavonoids andisoflavonoids in estrogen-responsive tissues,it is likely that dietarily derived flavonoidsand/or their metabolites which interacteither with ER or type II sites may pro-foundly affect reproductive function and theincidence of estrogen-dependent breast andprostate cancer (3-.

Although isoflavonoids such as coume-strol have demonstrated estrogenic activityin a variety of experimental systems, most ofthese studies involved feeding these com-pounds to intact animals for periods of timeranging from days to weeks and subsequent-ly determining uterine wet and/or dryweights (15-19). Consequently, theuterotropic response profiles to phytoestro-gens have not been extensively evaluated inovariectomized animals, in which the con-

tribution of ovarian steroids to the overallnet uterine hypertrophy, hyperplasia, andDNA synthesis has been eliminated. Recentstudies have shown that oral administrationof coumestrol to intact, immature rats failedto antagonize estrogenic stimulation ofuterine growth, and, in fact, coumestroltreatment increased uterine weight in thesestudies, suggesting that this isoflavonoidpossessed estrogenic activity (15-17).However, because these studies involved thetreatment of intact, immature female micewith coumestrol over a period of days, it ispossible that coumestrol modulatedgonadotropin secretion and/or ovariansteroidogenesis, which may have been par-tially responsible for the observed estrogenicresponse. A recent report by Yamazaki (20)demonstrating that the isoflavonoid ipri-flavone is estrogenic in intact, but notovariectomized, immature rats also supportsthis hypothesis (20).

Our studies described here weredesigned to assess the estrogenic activity ofparenterally and orally administeredcoumestrol in the immature, ovariec-tomized rat to rule out the effects of ovari-an estrogen on uterotropic response pat-terns. These data demonstrate that at thedose levels and treatment conditions used,coumestrol behaved as an atypical estrogen,failing to cause significant cytosolic-deple-tion and nuclear accumulation of ER oruterine hyperplasia and DNA synthesis inthe ovariectomized rat, even though uterinewet and dry weights were elevated abovecontrol levels. Therefore, the true estro-genic activity of coumestrol, and perhapsother well-known phytoestrogens, requirescareful evaluation in ovariectomized ani-mals, particularly in view of the fact thatemphasis is currently being directed towarddefining the relationships between phytoe-strogen exposure and neoplasia in estrogen-responsive tissues such as the mammarygland, uterine endometrium, and prostate.

Materials and MethodsChemicals. Coumestrol was purchasedfrom Eastman Kodak (Rochester, NewYork) and genestein and daidzein werepurchased from Indofine (Somerville, New

Address correspondence to B.M. Markaverich,Department of Cell Biology, Baylor College ofMedicine, One Baylor Plaza, Houston, TX 77030USA.This research was supported by grants from theNational Institute for Environmental HealthScience (ES-05477) and the National CancerInstitute (CA-35490 and CA-55590).Received 3 October 1994; accepted 27 February1995.

Environmental Health Perspectives

'.,, 7

574

i 1D ]lE 32 && U&iUimJersey). The purity of the flavonoids wasdetermined to be greater than 99% byHPLC analysis using a pBondapak CH8column (Waters/Millipore, Milford,Massachusetts) eluted with water:methanolby standard procedures in our laboratory(9). Estradiol and diethylstilbestrol wereobtained from Sigma (St. Louis, Missouri)and 2, 4, 6, 7-[3H]estradiol (112 Ci/mmole) was purchased from AmershamRadiochemicals (Boston, Massachusetts).

Animals and treatment. Immature (21-day-old) Sprague-Dawley female rats(Holtzman Laboratories, Madison,Wisconsin) were ovariectomized underMetofane anesthesia by standard proce-dures and allowed to recover 7-10 daysbefore treatment. Animals were housed instainless-steel cages under controlled con-ditions consisting of 12 hr of light daily(lights on at 0700 hr), and food and waterwere provided ad libitum. We separatedthe rats into treatment groups consisting offive or six animals per group and injectedthem subcutaneously or treated them oral-ly with the indicated dose levels of estradi-ol or coumestrol dissolved in saline-2.0%Tween 80 (injection) or tap water-2.0%Tween 80 vehicle (oral dosing studies)under the conditions described in the textand figure legends. This method of oraladministration is remarkably consistentthroughout the treatment period, and phy-toestrogens were delivered for weeks with-out significant effects on body weights orother signs of generalized, nonspecific sys-temic cytotoxicity. In these experiments,the 30- to 40-day-old rats typically con-sumed 25.8 ± 4.4 mL of vehicle or coume-strol solution per day. At a concentrationof 50 pg coumestrol/mL of tap water-Tween-80 vehicle, this represented a doseof 1.29 ± 0.22 mg of coumestrol per dayper animal and doses in excess of -13mg/rat/day (500 pg/mL drinking water;-286 mg/kg body weight) can be readilydelivered by this procedure. Animals weresacrificed by cervical dislocation and theuteri were removed, stripped of extraneoustissue, weighed, and stored in saline at 40Cfor biochemical analysis. To obtain dryweights, uteri from some of the animals(five to six per treatment group) were driedin an oven at 70°C for 16-24 hr until con-stant weights were obtained.

Tissue homogenization and fractiona-tion. For biochemical analyses, we homog-enized uteri from the control and treatedanimals in ice-cold TE buffer (10 mMTris, 1.5 mM EDTA, pH 7.4 at 22°C) inKontes ground-glass homogenizers in avolume equivalent to 100 mg fresh uterinewet weight equivalents per milliliter andcentrifuged the homogenate at 800g for 20min in a Beckman GH-3 rotor to obtainthe low-speed cytosol (supernatant) and

nuclear pellet fractions (21,22). Thecytosol was centrifuged at 40,000g for 30min in a Beckman JA 20 rotor to obtainthe high-speed cytosol fraction, and equiv-alent results are routinely obtained with200,000g cytosol preparations. We washedthe nuclear pellet fraction three times byresuspension and centrifugation (800g for7 min) in TE buffer before resuspension inthe same buffer and analysis for ER or typeII sites by [ 3H]estradiol exchange asdescribed below.

Measurement of cytosolic and nuclearER by [3H]estradiol exchange. We dilutedcytosol and nuclear fractions from controland treated animals to 20 mg fresh uterinewet weight equivalents/mL in TE bufferand brought them to 10 mM with dithio-threitol (21,22). The preparations wereincubated at 40C for 140 min in the pres-ence of the reducing agent to eliminateinterference from type II sites (21,22).Aliquots (250 pL) of the cytosol or nuclearsuspensions were incubated (cytosol, 300Cfor 30 min; nuclei, 300C for 30 min) in thepresence of a wide range of [3H]estradiolconcentrations (0.4 to 10 nM, total bind-ing) ± 300-fold excess diethylstilbestrol(0.12-3.0 pM, nonspecific binding). Afterthis incubation, we incubated cytosol frac-tions (40C for 15 min) with hydroxylapetite(HAP) and washed the HAP bound proteinby resuspension and centrifugation (800gfor 5 min) to separate bound and free[3H]estradiol. Nuclear suspensions were alsowashed by resuspension and centrifugationto remove free [3H]estradiol (21,22). Bound[3H]estradiol was extracted from the final,washed HAP or nuclear pellets with ethanoland specific [3H]estradiol binding evaluatedby Scatchard analysis (21,22). Results wereexpressed as ER sites per cell assuming thatmammalian tissues contain approximately 7pg of DNA per cell nucleus (23). UterineDNA content was estimated by the methodof Burton (24).

In studies where the binding affinity ofthe various phytoestrogens for ER wasassessed, uterine cytosol fractions fromovariectomized rats were prepared exactlyas described above and diluted to 20mg/mL in TE buffer containing 10 mMdithiothreitol. After preincubation (140min at 40C) in the presence of reducingagent to eliminate interference fromcytosol type II sites (21), aliquots of thecytosol were incubated (370C for 30 min)in triplicate in the presence of 10 nM[3H]estradiol ± the indicated concentra-tions (0.1 nM-10.0 pM) of daidzein,genistein, coumestrol, or diethylstilbestrol,and bound and free steroid were separatedby HAP adsorption as described above. Wedetermined [3H]estradiol binding to ER inthese studies in the absence (100% bound)or presence of the competitor as previously

described (8). In a typical experiment,100% bound represented approximately5000 cpm.

Assessment of isoflavonoid bindingaffinity for nuclear type II sites. Sincenuclear type II site stimulation appears tobe involved in target cell response to estro-genic hormones, and flavonoids such asluteolin, quercetin, and pelargonidinappear to inhibit cellular proliferationthrough type II site binding interactions(8), we determined the binding affinities ofisoflavonoids such as coumestrol, daidzein,and genistein for type II sites in rat uterinenuclei. For these studies, uterine nuclearfractions from estradiol-implanted rats (8)were prepared in TE buffer, diluted to afinal volume equivalent to 20 mg uterinewet weight equivalents/mL, and incubated(40C for 60 minutes) in the presence of 20nM [3H]estradiol ± the indicated concen-trations of competitor (1.0 nM-20.0 pM)as described in the figure legends. Afterincubation, the nuclear suspensions werewashed by resuspension in TE buffer andcentrifugRation to remove free steroid, andbound [ H]estradiol was determined byliquid scintillation counting exactly as pre-viously described (8). A value of 100%bound in the absence of competitor repre-sented approximately 25, 000 cpm.

Measurement of cytosolic and nucleartype II sites by [3H estradiol exchange. Inexperiments where it was necessary toquantitate treatment effects on the levels ofcytosolic and nuclear type II binding sites,these subcellular fractions were preparedexactly as described above for the type IIsite competition assays (21,22). We quan-tified cytosolic type II sites by saturationanalysis using the hydroxylapetite adsorp-tion-[3H]estradiol exchange assay(HAA-[3H]estradiol exchange) previouslydeveloped by our laboratory for measure-ment of type II sites without interferencefrom endogenous ligands such asMeHPLA (22,25). Briefly, cytosol prepara-tions from controls and coumestrol-treatedanimals were incubated with hydroxyl-apetite (HAP) at 40C for 15 min to allowthe type II site to bind to the HAP. Thepellet bound protein was washed threetimes by resuspension in TE buffer andcentrifugation (800g for 7 min), and thefinal washed pellet was resuspended in TEbuffer and aliquots incubated (220C for 16hr) in the presence of a wide range (0.4nM-40 nM) of [3H]estradiol concentra-tions in the absence (total binding) or pres-ence (nonspecific binding) of 300-foldexcess diethylstilbestrol (22,25). Similarly,the washed nuclear pellet fractions fromthese uteri were incubated (40C for 60min) with [3H]estradiol ± diethylstilbes-trol, and specific binding to type II sites incytosol and nuclear fractions was deter-

Volume 103, Number 6, June 1995 575

mined on the basis of uterine DNA con-tent (sites/cell) as previously described indetail (23). DNA was estimated by themethod of Burton (24).

Statistical analyses. Where indicated,the experimental results are expressed asthe mean ± SEM. The data presented inthe various figures in this manuscript wereanalyzed statistically by the appropriateone-way or two-way analysis of variance(fixed treatment models) and Duncan'snew multiple range test on the treatmentmeans as described in detail (26).

ResultsTo correlate phytoestrogen binding interac-tions with biological response, we evaluatedthe binding affinities of these compoundswith ER and type II sites in rat uterinenuclear fractions. The data in Figure 1demonstrate that coumestrol (Kd -180nM), daidzein (Kd >1000 nM), and genis-tein (Kd -180 nM) bind to the ER (Fig.IA) with relatively low affinities asdescribed by numerous laboratories for var-ious tissues (16,26). This is consistent withthe fact that these compounds are weak orshort-acting estrogens with substantially lessbiological activity than long-acting estrogenssuch as estradiol (28). Coumestrol (Kd -10

180 EliM__11__.140

120

a100

60480

20

20

.01 .1 1 10 100 1,000 10.000 100,000Competitor nM)

140

-120

' 00

6080

*60

w.4020

0.1 1 10 100 1,000 10,000 100,000Competitor Concentration (nM)

U Daidzein aGenistein * Luteolin* Foemononetin A DES * Coumestrol

Figure 1. Bioflavonoid competition for (A) estro-gen receptor (ER) and (B) type 11 binding sites inrat uterine cytosol and nuclei. Uterine cytosol andnuclear fractions were incubated in the presenceof [3Hlestradiol (10 nM, ER; 20 nM, type 11) plus orminus the indicated concentrations of competitorunder conditions optimum for each of the respec-tive binding sites as described in methods. DES,diethylstilbestrol.

nM), daidzein (Kd -5 nM), and genistein(Kd -5 nM) displayed higher binding affini-ties for type II sites than for the ER (Fig. 1),and the apparent binding affinities of thesethree isoflavonoids for nuclear type II sitesare similar to those determined for luteolinand quercetin, which bind to nuclear type IIsites (but not ER) with very high affinity(Ad - 1-5 nM) and inhibit estrogen stimula-tion of uterine growth in the rat and mam-mary tumor growth in the mouse (8,9).

Because coumestrol displayed higheraffinity for ER than daidzein and hasrecently been described as an estrogen inthe intact, immature female rat, we focusedour efforts on the characterization of theestrogenic activity of coumestrol in theimmature, ovariectomized rat uterinemodel system. Dose-response studiesdemonstrated that a single injection ofcoumestrol in doses ranging from 50 to200 pg resulted in a significant increase inuterine wet weight relative to control (Fig.2), and the response obtained with100-200 pg of coumestrol was equivalentto that obtained after a single injection of 1pg estradiol-17f9. Therefore, it appearedthat in the immature, ovariectomized rat,coumestrol treatment increased uterine wetweight 24 hr after a single injection in amanner similar to that described for long-acting estrogens such as estradiol (28,29).

The data in Figure 3 represent the tem-poral effects of coumestrol on uterine wetweight and DNA content after the injec-tion of 100 pg of this phytoestrogen. Asexpected, based on previous studies sug-gesting that coumestrol has estrogenicactivity (15-1/), uterine wet weight wasincreased within 4 hr after coumestroltreatment, and this response was sustainedfor 24 hr, declining to control levels by 62hr after injection. This observation wasconsistent with that reported for activeestrogens such as estradiol, where sustain-

E801o a

-40 j

~j20

0 Control ig 50 gg 100 gg 200 igTreatment

Figure 2. Effects of coumestrol and estradiol onuterine growth. Immature, ovariectomized ratsreceived a single subcutaneous injection of vehi-cle (controls) or the indicated concentrations ofestradiol or coumestrol. Uterine wet weights weredeermi.ne ,4hr -after InMecto( No great

response was subsequently obtained with a singlesubcutaneous injection of 500 pg coumestrol. Dataare means ± SEM.

ing uterine wet weight beyond 24 hr afterinjection is typically associated with thestimulation of cellular DNA synthesis andtrue uterine growth (29). Much to our sur-prise, even though coumestrol treatmentincreased uterine wet and dry weights in amanner similar to that obtained with estra-diol, it failed to increase uterine DNA con-tent at 24 hr after injection. These findingssuggest that in the immature, ovariec-tomized rat, coumestrol behaves as anatypical estrogen, which stimulates cellularhypertrophy and perhaps protein synthesiswithout stimulating cellular hyperplasia,reflected by a doubling in DNA content(29). Therefore, coumestrol mimics estriol,estrone, and estradiol-17ct in this modelsystem by behaving as a short-acting estro-gen, capable of stimulating cellular hyper-trophy and not hyperplasia when adminis-tered as a single injection (28-31).

We also assessed the effects of multipleinjections of this phytoestrogen on uterinewet weight and DNA content 24 hr afterthe last injection (Fig. 4) because estrioland estradiol-17(x have been shown tostimulate uterine cellular hypertrophy andhyperplasia in the intact immature (29) oradult-ovariectomized (28,30,31) rats whenadministered by multiple injection or pel-let implant. Therefore, we expected thatsimilar results would be obtained aftermultiple injections of coumestrol. The datain Figure 4 clearly demonstrate that thiswas not the case. Although multiple injec-tions of coumestrol increased uterine wetweight relative to control, this responsewas not equivalent to that after following asingle injection of 1 pg estradiol (Fig. 4A).More importantly, estradiol treatmentnearly doubled uterine DNA content (Fig.4B), whereas neither single or multipleinjections of this phytoestrogen increaseduterine DNA content in this study. In fact,two injections of coumestrol may haveslightly reduced uterine DNA content rela-tive to control (Fig. 4B). These data fur-ther confirm that in the immature, ovariec-

- 15*a00CaOUer-v

12Hours

24 62

Figure 3. Temporal effects of coumestrol on uter-ine growth. Ovariectomized rats (four to six pergroup) were injected with vehicle (controls) orcoumestrol (100 pg), and uterine wet weights andDNA content were determined at the indicatedtimes following treatment. The data represent themeans for two separate experiments.


-..E-3cmlmmmz- M_

576

EA lI a - 9 D - .9 - 9* - * 9

tomized rat, coumestrol behaves as anatypical estrogen.

To explain the inability of coumestrolto stimulate uterine cellular hyperplasia(increased uterine DNA content) in theovariectomized rat, we assessed the effectsof this isoflavonoid on the intracellularcompartmentalization of ER in the uterusat various times after treatment. Althoughit is likely that ER is localized in the nucle-us and cytosolic ER is most likely an arti-fact generated during tissue homogeniza-tion (32-34), the assessment of ERdynamics (cytosolic ER depletion andnuclear ER accumulation) after estrogenadministration can be used to assess theestrogenicity of a variety of compounds(29,34). In the present studies, we injectedimmature, ovariectomized rats with a dose(100 pg) of coumestrol, which substantial-ly increased uterine wet weight withoutcausing cellular hyperplasia (Fig. 4) andthe levels of cytosolic and nuclear ER weremeasured as a function of time after treat-ment (Fig. 5). Again, coumestrol behavedas an atypical estrogen, failing to causemeasurable cytosolic ER depletion or sig-nificant nuclear ER accumulation. In fact,cytosolic ER levels were elevated above thetime zero control level within 4 hr aftercoumestrol treatment and reached a maxi-mum two- to threefold induction by 24 hr.

To ensure that the atypical effects ofcoumestrol on ER dynamics were not acharacteristic of the model system, wecompared the temporal effects of estradioland couumestrol on ER compartmentaliza-

75

Es50

s 25

tion in the rat uterus in immature, ovariec-tomized rats. The data in Figure 6 clearlydemonstrate that the cytosolic ER deple-tion and nuclear ER retention patternsafter estradiol injection were exactly asdescribed by our laboratory and others forthe rat uterine model system under a vari-ety of experimental conditions (29,35-41)and the level of cytosolic ER did not returnto control levels (0 hours) until 16-24 hrafter treatment. It is this sustained cytoso-lic ER depletion and nuclear ER occupan-cy that correlates with estrogenic stimula-tion of cellular hyperplasia and DNA syn-thesis (28,29). The failure of coumestrol tomimic these ER dynamics is likely respon-sible for the inability of this phytoestrogento stimulate DNA synthesis in these stud-ies. However, again, in this second experi-ment, coumestrol treatment did not causecytosolic depletion or nuclear ER accumu-lation/retention even though cytosolic ERlevels were elevated two- to threefold abovetime zero controls by 24 hr.

The aforementioned experiments sug-gested that coumestrol was an atypicalestrogen when administered subcutaneous-ly. Therefore, we evaluated the sustainedeffects of this compound on uterinegrowth in the rat after oral administrationin the drinking water. These studiesdemonstrated that 96 hr after treatment,orally administered coumestrol resulted ina dose-dependent increase in uterine wetand dry weight relative to controls at doselevels ranging from 5 to 100 pg/mL.Uterine wet and dry weights were essential-ly doubled by treatment with 100 pg ofcoumestrol/mL drinking water (-60 mg/kgbody weight/day; Fig. 7). Time studieswith the higher dose level of coumestrol(100 pg/mL drinking water) demonstratedthat the uterotropic response peakedbetween 72 and 96 hr after treatment, anduterine wet and dry weights were increased

20,000 LUMMuiuiOl

five- to sixfold relative to the time-zerovehicle controls (Fig. 8). More important,however, was the observation that uterineDNA content 72 hr after coumestrol treat-ment (242 pg/uterus) was nearly equiva-lent to the control value (217 pg/uterus),suggesting that coumestrol failed to stimu-late significant cellular hyperplasia andDNA synthesis even when administered ina sustained fashion under these experimen-tal conditions.

To further characterize the uterotropicresponse patterns to orally administeredcoumestrol, we also assessed the effects ofthis phytoestrogen on cytosolic and nuclearER and nuclear type II binding site levels72 hr after oral administration (Fig. 9).These data essentially confirmed the injec-tion studies (Figs. 5 and 6) in that oraladministration of coumestrol also failed tocause accumulation of nuclear ER ordeplete cytosolic ER. In fact, coumestroltreatment resulted in a three- to fourfoldinduction in the level of cytosolic ER, aswas the case for the coumestrol injectionstudies. That nuclear type II sites were notstimulated by coumestrol treatment (Fig. 9)is consistent with the observation that uter-ine hyperplasia and DNA synthesis was notstimulated by coumestrol under these con-ditions (see legend to Figure 8). Estrogenstimulation of nuclear type II sites in therat uterus is directly correlated with theinduction of uterine cellular DNA synthesisand true uterine growth under a wide vari-ety of experimental conditions (28-30,35).That cytosolic type II sites appeared to beslightly increased following coumestroltreatment (Fig. 8) is interesting; however,the relationship between this soluble[3H]estradiol binding site and uterotropic

Control 1x

Control 1x lx

Treatment

lx 2x

i. 15,000

a

5gL1,000

w

2x

Figure 4. Effects of multiple coumestrol injectionson uterine growth in the rat. Ovariectomized ratsreceived one daily injection of 10 pg estradiol orone or two daily injections of 100 pg coumestrolas indicated. Controls were injected with 2%Tween 80 in 0.9% saline vehicle. (A) Uterine wetweights and (B) DNA content were determined 24hr after the last injection. Data are means ± SEM.

0 20 40 60 80Hours after Injection

Figure 5. Effects of coumestrol on rat uterineestrogen receptor (ER) dynamics. Ovariectomizedrats were injected subcutaneously with 100 pgcoumestrol or vehicle (time 0 controls) and uterinecytosol and nuclear estrogen receptors wereassayed by [3H]estradiol exchange. Results areexpressed as sites per cell (23). Data are means +SEM.

10,000

5,000

Hours after Injection

Figure 6. Effects of estradiol and coumestrol onestrogen receptor (ER) dynamics in the rat uterus.Ovariectomized rats received a single injection ofestradiol (5 pg) or coumestrol (100 pg), and estro-gen receptors (ER) were measured in cytosol andnuclear fractions by [3H]estradiol exchange at theindicated times after injection. Results werebased on DNA content and were expressed assites/cell as described. In separate experimentsessentially identical ER responses were observedfollowing a 500 pg injection of coumestrol.

Volume 103, Number 6, June 1995

500

Ee 400

iCa..NC300

200

0o

577

E-C

, 2

AM

a

=:Is

50

Water Vehicle 5 ig 10 jg 50 jg 100 jg

Treatment

Figure 7. Dose response of rat uterus to coumestrol. Ovariectomized rats were given the indicated con-centrations of coumestrol dissolved in drinking water containing 2% Tween 80. Controls received wateror the 2% Tween-80 vehicle and uterine wet and dry weights were determined 96 hr after treatment.Data are means ± SEM.

response has not been evaluated.On the basis of the aforementioned

studies demonstrating that coumestroltreatment will stimulate cytosolic ER levelsin the rat uterus (Figs. 5, 6, and 9), we sus-

pected that subcutaneous or oral exposure

to this phytoestrogen may alter theuterotropic response to estrogenic steroids.To evaluate this possibility, immature,ovariectomized rats were treated orallywith vehicle (controls) or coumestrol (250pg/mL) for 5 days before receiving threedaily subcutaneous injections of variousdoses of estradiol (0.01-10 pg) and uterineweight was determined 24 hr after the lastinjection. Coumestrol pretreatmentincreased uterine sensitivity to estradiol as

the response of the coumestrol pretreateduterus to doses of estradiol greater than 0.1pg/day was significantly (p <0.05) greaterthan that observed in the vehicle pretreatedcontrols. Therefore, coumestrol inductionof ER during the 5-day pretreatment peri-od significantly enhanced uterine sensitivi-ty to estradiol.

DiscussionA major focus of our laboratory over thepast decade has been estrogen regulation ofnormal and abnormal cell growth and pro-

liferation. Our efforts have led to the iden-tification of a bioflavonoid metabolite(MeHPLA) as an important cell growthregulating agent (9,14). MeHPLA is an

endogenous ligand for nuclear type II sites(14), and occupancy of this site byMeHPLA and bioflavonoids such as lute-olin and quercetin appears to inhibit estro-

gen stimulation of uterine growth in therat and mammary tumor growth in mice(8,9,14). Subsequent studies demonstrate a

direct correlation exists between the occu-

pancy of type II sites by flavonoids such as

luteolin, quercetin, and dihydroxybenzyli-dine acetophenone and the inhibition ofbreast (8-10), colorectal (11), pancreatic(12, and ovarian cancer (13) as well as theinhibition of leukemia (42) and lym-phoblastoid cell proliferation (43). Thesebioflavonoids do not bind to the ER (8),suggesting that the antiestrogenic and/orinhibitory effects of these compounds on

cellular proliferation are mediated throughtype II sites. Therefore, it is likely thatdietarily derived bioflavonoids inhibit nor-

mal and abnormal cell growth through thismechanism as well.

Conversely, isoflavonoids such as

coumestrol, genistein, and daidzein havebeen described as estrogens, antiestrogens,and anticarcinogens because of their abili-ties to bind to ER in estrogen target cells(44-48), even though these compoundsinhibit the proliferation of ER negativebreast cancer cells (49). This formerassumption has led to the generally accept-ed hypothesis that consumption ofisoflavonoids may prevent breast or

prostate cancer because these phytoestro-gens compete with ovarian estradiol forER, thus reducing exposure to the more

active endogenous estrogenic hormones(7,46,48). This line of reasoning was

offered as an explanation as to whyJapanese women, who have higher circulat-ing levels of less potent, short-acting estro-

gens such as estriol and estrone, have a

lower incidence of breast cancer than theirAmerican counterparts (50). Althoughthese hypotheses regarding the protectiveeffects of the so-called weak or impededestrogens are logical, they are not necessari-

15E

._

10i

e5 M

0 20 40 60 80 100

Hours after Injection

Figure 8. Effects of orally administered coume-strol on uterine growth in the rat. Ovariectomizedrats were treated with 100 pg/mL coumestrol dis-solved in drinking water containing 2% Tween 80(vehicle). Controls received water-Tween 80vehicle and uterine wet and dry weights weredetermined at the indicated times (0-96 hr) aftertreatment. The uterine DNA content (not shownin graph) after 72 hr of coumestrol treatment (242pg/uterus) was not different from that measuredin uteri from controls (217 pg/uterus). Data aremeans ± SEM.

ly supported by animal studies. It is welldocumented that estriol and estradiol-17ciare very weak estrogens incapable of stimu-lating cellular hyperplasia, when exposure

is acute (single injection). However, chron-ic exposure to estriol or estradiol-17a viamultiple injection or subcutaneous implantcauses uterine cellular hypertrophy, hyper-plasia, and mammary cancer in rodents ina manner analogous to that achieved withestradiol (28-31,51). This is a likely para-

digm for coumestrol, daidzein, and genis-tein if their pharmacology and mechanismof action are similar to those of other shortacting estrogens (28-30,51).

Although some investigators have sug-

gested that coumestrol, daidzein, and otherrelated phytoestrogens may prevent cancer

because these compounds inhibit malig-nant cell proliferation in vitro (52,53) anddimethylbenz[a]anthracene (DMBA)induction of mammary tumors in the rat

(54), these phytoestrogens do not inhibitthe growth of established DMBA-inducedmammary tumors (55), and their effects onthe growth of other types of tumors in ani-mals remains to be established. In fact,there is a paucity of experimental datadirectly demonstrating that phytoestrogensinhibit tumor growth in vivo. On the otherhand, it is well documented that sustainedexposure to phytoestrogens during theneonatal period is associated with persis-tent vaginal cornification, cervico-vaginalpegs and downgrowths, and uterine squa-

mous metaplasia, which mimics thatobserved after exposure to diethylstibestrol(56,57). These results demonstrate thatphytoestrogens may hyperestrogenize tar-

get tissues under certain experimental con-

ditions in vivo. Therefore, although it is


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Figure 9. Effe~Cotsofl cue roI}sladministra-

tion on cytosolic and nuclear estrogen receptor(ER) and type 11 sites in the rat uterus. Uteri fromovariectomnized rats treated with vehicle (con-trols) or coumnestrol for 72 hr as described inFigure 8 were assayed for cytosolic and nuclearER and type 11 sites by 13Hlestradiol exchange.Results are expressed as binding sites/cell.

certainly possible that acute exposure tophytoestrogens may block carcinogenicinsult to prevent cancer (46-48,54), thesecompounds may also hyperestrogenize ER-containing target tissues after chronicexposure, and none of the parameters (doseand duration of exposure) involved inthese response profiles have been adequate-ly defined.

The present studies were performed tomore completely define the interactions ofisoflavonoids with ER and type II sites inthe ovariectomnized rat uterus and evaluatesubsequent effects on uterine growth andDNA content after acute or sustainedexposure to these phytoestrogens. Thefindings confirmed our hypothesis thatisoflavonoids bind to the ER with lowaffinity and therefore should demonstratevery little estrogenic activity when admin-istered acutely. In fact, the apparent disso-ciation constants of coumnestrol anddaidzein for rat uterine nuclear ER wereapproximately 180 nM and 1000 nM,respectively, whereas little competition wasobtained with genistein (Fig. 1). Based onthese binding affinities for ER, one has towonder whether endogenous levels ofeither coumestrol or genistein will reachconcentrations capable of occupying ERand eliciting estrogenic response undernormal physiological conditions. Althoughwe previously noted that someisoflavonoids and lignans failed to competesignificantly for nuclear type II sites beforebecoming insoluble in the binding assays(8), we used dimethylsulfoxide to solubilizethese compounds in the present studies toenhance their solubility in the aqueousbinding assay buffer. Under these condi-tions counestrol and daidzein competedfor[3Hnestradiol binding to nuclear type IIsites and displayed much higher affinitiesfor this protein (Fig. 1). That coumestroland daidzein displayed much higher affini-

ties for nuclear type II sites (Kd 5-10 nM)than for the ER suggests that type II sitesmight be occupied in vivo at concentra-tions where these compounds will not bindto ER. This is currently being evaluated.

The ability of isoflavonoids to interactwith both the ER and nuclear type II sitessuggest that these compounds may displaymixed agonist/antagonist activities. Atlower concentrations (<100 nM) coume-strol may occupy nuclear type II sites (Kd10 nM) and inhibit cell proliferation, as wehave shown for luteolin and quercetin (8),whereas at higher concentrations (>100nM) coumestrol may occupy ER (Rd - 180nM), resulting in the stimulation of cellu-lar proliferation. This concept is consistentwith our observations that the binding ofthe ER complex in the nucleus results inthe estrogen-induced dissociation ofMeHPLA from nuclear type II sites, andsimilar events may occur after the bindingof isoflavonoid-ER complexes in the nucle-us as well (14,58). However, it is impor-tant to consider that although supraphysio-logical concentrations (pM) of coumestrolstimulate ER-dependent reporter genetranscription in MCF7 breast cancer cellsor HeLa cervical cancer cells in vitro(59,60), whether endogenous levels ofcoumestrol reach concentrations requiredfor ER (100-1000 nM) binding in vivounder physiological conditions remains tobe resolved.

Although recent studies demonstratethat oral administration of coumestrolincreased uterine wet and dry weight,nuclear ER levels, and uterine progesteronereceptor content in intact, immature rats,uterine DNA content was not determinedin these studies (15-17), and whethercoumestrol stimulated cellular hyperplasiaremains to be resolved. Therefore, eventhough coumestrol appeared to behave as acomplete estrogen in these experiments,the animals were dosed with the phytoe-strogen over a number of days, and it ispossible that ovarian-derived estrogen con-tributed to the observed stimulation ofuterine growth and progesterone receptorcontent as these animals reached puberty.The present studies using the immature,ovariectomized rat support this contention.Although coumestrol administration bysingle or multiple injection (Figs. 2-4) ororally in the drinking water (Figs. 7 and 8)increased uterine wet and dry weights rela-tive to control, even sustained exposure tohigh doses of this phytoestrogen failed toincrease uterine DNA content, suggestingthat uterine hyperplasia was not observed.This is a significant finding demonstratingthat increases in uterine wet and dryweight are not always indicative of uterinehyperplasia as reflected by a doubling inDNA content (28,29). It is more likely

that the coumestrol-induced increase inuterine wet and dry weight in our studiesin ovariectomized animals reflectedincreases in water and protein content.Studies by Yamazaki demonstrating thatthe isoflavonoid ipriflavone is estrogenic inintact, but not ovariectomized animals(20) support our findings with coumestroland confirm the hypothesis that ovarianestrogens contribute to the net estrogenicresponse of the uterus to isoflavonoids.

Further evidence that coumestrol maybe an atypical estrogen is provided by stud-ies where the effects of this phytoestrogenon ER function were assessed. In two sepa-rate experiments designed to evaluate thetemporal effects of coumestrol on ERdynamics and compartmentalization in theovariectomized rat uterus, injection of 100pg (or 500 pg; not shown) of coumestrolfailed to deplete cytosolic ER or causenuclear ER accumulation (Figs. 5 and 6)even though increases in uterine wet anddry weight were observed. These results arein sharp contrast to the data in Figure 6where injection of immature, ovariec-tomized rats with estradiol resulted in theclassical cytosolic ER depletion and nuclearER accumulation and retention patternswhich precede estradiol stimulation ofuterine growth in immature (28,29) oradult, ovariectomized rats (29-31,35).Since sustained nuclear occupancy by theER-estrogen complex is generally thoughtto be required for cellular hyperplasia andDNA synthesis (28-31), it is not surpris-ing that coumestrol failed to stimulateuterine cellular hyperplasia (DNA content)under these experimental conditions inovariectomized rats.

Even more surprising was the observa-tion that subcutaneous injection (Figs. 5and 6) or oral administration of coume-strol (Fig. 9) increased cytosolic ER levelstwo- to threefold without causing signifi-cant cytosolic depletion and nuclear accu-mulation of ER or induction of nucleartype II sites, which are characteristicresponses to estrogenic hormone adminis-tration (22,23,28). These latter twonuclear events are typically correlated withestrogenic stimulation of cellular DNAsynthesis and proliferation (28-30,35).These findings also imply that coumestrolmay be an atypical estrogen which doesnot modulate ER or type II site function ina manner analogous to that of estradiol(Figs. 6 and 9), estriol, or estradiol-17a(28-31) in the ovariectomized rat uterus.Consequently, although recent studies sug-gest nuclear ER are elevated in coumestrol-treated, intact, immature rats (15-17), thisER could have been occupied by ovarianestrogen and not necessarily coumestrol.

Although it is certainly possible thatmuch higher doses of coumestrol would


have significantly altered ER dynamics tostimulate true uterine growth in the pre-sent studies, injection of 500 pg of coume-strol under the conditions described inFigures 5 and 6 also failed to cause cytoso-lic ER depletion and/or nuclear ER accu-mulation and DNA synthesis (not shown).Nevertheless, 500 pg of coumestrol stimu-lated cytosolic ER in a manner similar tothat shown obtained with 100 pg of thisphytoestrogen (Fig 5). Therefore, increas-ing the dose level of coumestrol fivefold(-10 mg/kg body weight) failed to alter theresponse profiles at these short times (1-3days) after injection. Whether this increasein cytosolic ER concentration after coume-strol treatment reflects phytoestrogen-induced ER activation, ER phosphoryla-tion, and/or the stimulation of ER genetranscription remains to be resolved(61,62).

Regardless of the mechanism by whichcoumestrol increases ER concentration inthe uterus, it appears that this isoflavonoidmay enhance the sensitivity of this targettissue to estradiol. The data in Figure 10demonstrate that coumestrol pretreatmentsignificantly shifted the dose-responsecurve for estradiol, and it is likely thatcoumestrol induction of cytosolic ER asshown in Figures 5 and 6 was responsiblefor this increased sensitivity of the uterusto estradiol. Therefore, the mechanisms bywhich phytoestrogens such as coumestrol,daidzein, and genistein modulate estro-genic response and uterine growth may bemuch more complex than generallythought and may involve binding to ER,increasing the ER binding capacity ofestrogen target tissues such as the uterus

700 - _

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o 300

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Figure 10. Coumestrol effects on estradiol stimula-tion of uterine growth in the rat. Adult, ovariec-tomized rats were treated orally with vehicle (2%Tween 80) or coumnestrol (250 pg/mL) in the drink-ing water for 5 days before receiving three dailysubcutaneous injections of the indicated doses(0.1-10 pg) of estradiol. Animals were sacrificed24 hr after the last injection and uterine wetweights were determined. Results are expressedas the mearns ±. SEM and the resonseo to 1, 5 and

vehicle ~ ~ Esraicontrol

by causing ER activation or phosphoryla-tion (60,61) or by enhancing ovarianrelease of estrogen. If this is the case, onemight anticipate that the observed estro-genicity or antiestrogenicity of dietarilyderived phytoestrogens such as coumestrolmay be different in premenopausal andpostmenopausal women. This being thecase, it is difficult to speculate as towhether phytoestrogens such as coume-strol will prevent and/or protect againstcancer by competing with ovarian estro-gens for ER as suggested (5,6) or whethercontinuous consumption of antiestrogenicflavonoids such as luteolin and quercetinwhich inhibit the growth of a broad spec-trum of rodent (8,9) and human cancersin vitro and in vivo (10-13) will reducecancer incidence in humans by antago-nism at the level of the type II site. Studiesdesigned to accurately define the estro-genicity and antiestrogenicity of dietarilyderived isoflavonoids and flavonoids andpotential interactions with one anotherand endogenous estrogens and androgensin these experimental systems will berequired to adequately address these issues.

REFERENCES

1. Kuhnau J. The flavonoids. A class of semi-essen-tial food components: their role in human nutri-tion. Wld Rev Nutr Diet 24:117-119 (1976).

2. Harborne JB, Mabry TJ, Mabry H. Theflavonoids. New York:Academic Press, 1975.

3. Haenszel W, Locke FB, Segi MA. Case-controlstudy of large bowel cancer in Japan. J NatlCancer Inst 64:17-22 (1980).

4. Graham W, Dayal H, Swanson, M. Diet in theepidemiology of cancer of the colon and rec-tum. J Natl Cancer Inst 51:709-714 (1978).

5. Adlercreutz H, Mousavi Y, Hockerstedt K.Diet and breast cancer. Acta Oncol31:175-181 (1992).

6. Sharma OP, Adlercreutz H, Strandberg JD,Zirkin BR, Coffey DS, Ewing LL. Soy ofdietary source plays a preventive role against thepathogenesis of prostatitis in rats. J SteroidBiochem Molec Biol 43:557-564 (1992).

7. Adlercreutz H, Markkanen H, Watanabe S.Plasma concentrations of phyto-oestrogens inJapanese men. Lancet 342:1209-1210 (1993).

8. Markaverich BM, Roberts RR, Alejandro MA,Johnson GA, Middleditch BS, Clark JH.Bioflavonoid interactions with rat uterine typeII binding sites and cell growth inhibition. JSteroid Biochem 30:71-78 (1988).

9. Markaverich BM, Gregory RR, Alejandro MA,Kittrell FS, Medina D, Clark JH, Varma M,Varma RS. Methyl p-hydroxyphenyllactate andnuclear type II binding sites in malignant cells:metabolic fate and mammary tumor growth.Cancer Res 50:1470-1478 (1990).

10. Scambia G, Ranelletti FO, Benedetti Panici P,Piantelli M, Bonanno G, DeVincenzo R,Ferrandina G, Pierelli L, Capelli A, Mancuso S.Quercetin inhibits the growth of multidrug-resistant estrogen-receptor negative MCF-7human breast cancer cell line expressing rype IIestrogen-binding sites. Cancer ChemotherPharmacol 28:255-258 (1991).

11. Piantelli M, Ricci R, Larocca LM, Capelli A,

Rizzo S, Scambia G, Ranelletti FO. Type IIestrogen binding sites in human colorectal car-cinoma. J Clin Pathol 43:1004-1006 (1990).

12. Carbone A, Ranelletti FO, Rinelli A, VecchioFM, Lauriola L, Piantelli M, Capelli A. Type IIestrogen receptor in the papillary cystic tumorof the pancreas. Am J Cancer Res 92:572-576(1989).

13. Scambia G, Ranelletti FO, Benedetti Panici P.Piantelli M, Bonanno G, DeVincenzo R,Ferrandina G, Rumi C, Larocca LM, ManuscoSX. Inhibitory effects of quercetin on OVCA433 cells and the presence of type II oestrogenbinding sites in primary ovarian tumors andcultured cells. Br J Cancer 62:942-946 (1989).

14. Markaverich BM, Gregory RR, Alejandro MA,Clark JH, Johnson GA, Middleditch BS.Methyl p-hydroxyphenyllactate: an inhibitor ofcell proliferation and an endogenous ligand fornuclear type II binding sites. J Biol Chem263:7203-7210 (1988).

15. Whitten PL, Naftolin F. Effects of phytoestro-gen diet on estrogen-dependent reproductiveprocesses in immature female rats. Steroids57:56-61 (1992).

16. Whiten PL, Russel E, Naftolin F. Effects of anormal, human-concentration, phytoestrogendiet on rat uterine growth. Steroids 57:98-106(1992).

17. Whitten PL, Russell E, Naftolin F. Influence ofphytoestrogen diets on estradiol action in theuterus. Steroids 59:443-449 (1994).

18. Kaziaro R, Dennedy JP, Cole ER, Sothwel-Keely PT. The oestrogenicity of equol in sheep.J Endocrinol 103:395-399 (1984).

19. Shutt DA. The effects of plant oestrogens onanimal reproduction. Endeavor 35:110-113(1976).

20. Yamazaki I. Effect of Ipriflavone on theresponse of the uterus and thyroid to estrogen.Lancet 342:1209-1210 (1993).

21. Markaverich BM, Upchurch S, Williams M,Clark JH. Heterogeneity of nuclear estrogen-binding sites in the rat uterus: a simple methodfor the quantitation of type I and type II sitesby [3H]estradiol exchange. Endocrinology109:62-69 (1981).

22. Markaverich BM, Adams NR, Roberts RR,Alejandro MA, Clark JH. Cytosol type II sitesin the rat uterus: interaction with an endoge-nous ligand. J Steroid Biochem 28:599-608(1987).

23. Markaverich BM, Roberts RR, Alejandro MA,Clark JH. The effect of low dose continuousexposure to estradiol on the estrogen receptor(type I) and nuclear type II sites. Endocrinology114:814-820 (1984).

24. Burton K. A study on the conditions and mech-anism of the diphenylamine reaction for colori-metric estimation of deoxyribonucleic acid.Biochem J 62:315-323 (1956).

25. Markaverich BM, Roberts RR, Alejandro MA,Clark JH. An endogenous inhibitor of[3H]estradiol binding in normal and malignanttissues. Cancer Res 44:1515-1519 (1984).

26. Steele RGD, Torrie JH. Principles and proce-dures of statistics. New York:McGraw Hill,1960.

27. Tang BY, Adams NR. Effect of equol onoestrogen receptors and on synthesis of DNAand protein in the immature rat uterus. JEndocrinol 85:291-297 (1980).

28. Clark JH, Paszko Z, Peck EJ Jr. Nuclear bind-ing and retention of the receptor estrogen com-plex: relation to the agonisric and antagonisticproperties of estriol. Endocrinology 100:91-96(1977).

580 Environmental Health Perspectives

I - * . ee -ee - g 9EMrCI

29. Clark JH, Peck EJ Jr. Female sex steroids,receptors and function. New York:Springer-Verlag, 1979.

30. Markaverich BM, Clark, JH. Two binding sitesfor estradiol in rat uterine nuclei: relationship touterotropic response. Endocrinology105:1458-1462 (1979).

31. Clark JH, Williams M, Upchurch S, ErikssonH, Helton E, Markaverich BM. Effects of estra-diol-17a on nuclear occupancy of the estrogenreceptor, stimulation of nuclear type II sites anduterine growth. J Steroid Biochem 16:323-328(1982).

32. King WJ, Greene GL. Monoclonal antibodieslocalize oestrogen receptor in the nuclei of tar-get cells. Nature 307:745-746 (1984).

33. Jordan VC, Tat AC, Lyman SD, Gosded B,Wolf MF, Bain RR, Welshons WB. Rat uterinegrowth and induction of progesterone receptorwithout estrogen receptor translocation.Endocrinology 116:1845-1857 (1985).

34. Welshons WV, Lieberman ME, Gorski J.Nuclear localization of unoccupied oestrogenreceptors. Nature 307:747-749 (1984).

35. Markaverich BM, Upchurch S, Clark JH.Progesterone and dexamethasone antagonism ofuterine growth: role for a second nuclear estro-gen binding site for estradiol in estrogen action.J Steroid Biochem 14:125-132 (1981).

36. Ruh TS, Baudendistal LJ. Different nuclearbinding sites for antiestrogen and estrogenreceptor complexes. Endocrinology100:420-426 (1977).

37. Ruh TS, Baudendistal LJ. Antiestrogen modu-lation of the salt-resistant nuclear estrogenreceptor. Endocrinology 102:1838-1846(1978).

38. Katzenellenbogen BS, Ferguson ER.Antiestrogen action in the uterus: biologicalineffectiveness of nuclear bound estradiol afterantiestrogen. Endocrinology 97:1-12 (1975).

39. Koseki Y, Zava D, Chamness GC, McGuireWL. Estrogen receptor translocation andreplenishment by the antiestrogen tamoxifen.Endocrinology 101:1104-1110 (1977).

40. Ferguson ER, Katzenellenbogen BS. A compar-ative study of antiestrogen action: temporal pat-terns of antagonism of estrogen stimulated uter-ine growth and effects on estrogen receptor lev-els. Endocrinology 100:1242-1251 (1977).

41. Lan NC, Katzenellenbogen BS. Temporal rela-tionships between hormone receptor bindingand biological responses in the uterus: studies

with short- and long-acting derivatives of estri-ol. Endocrinology 98:220-227 (1976).

42. Teofili L, Pierelli L, lovino MS, Leone G,Scambia G, DeVincenzo R, Beneditti-PanciniP, Menichella G, Macri E, Piantelli FO,Larocca LM. The combination of quercetin andcytosin arabinoside synergistically inhibitsleukemic cell growth. Leukemia Res16:497-503 (1992).

43. Scambia G, Ranelletti FO, Beneditti-Pancini P,Piantelli M, Rumi C, Battaglia F, Larocca LM,Capelli A, Manusco S. Type II estrogen bindingsites in a lymphoblastoid cell line and growthinhibitory effect of estrogen, antiestrogen andbioflavonoids. Int J Cancer 46:1112-1116(1990).

44. Murphy PA. Phytoestrogen content ofprocessed soybean products. Food Technol36:62-64 (1982).

45. Xu X, Wang H-J, Murphy PA, Cook L,Hendrich S. Daidzein is a more bioavailablesoymilk isoflavone than is genistein in adultwomen. J Nutr 124:825-832 (1994).

46. Setchell KDR, Adlercreutz H. Mammalian lig-nans and phytoestrogens. Recent studies ontheir formation, metabolism, and biological rolein health and disease. In: Role of the gut florain toxicity and cancer (Rowland IR, ed).London:Academic Press, 1988;315-345.

47. Fisher S, Gameron GS, Baldwin JK, JashewayDW, Patrick KE. Reactive oxygen in the tumorpromotion stage of skin carcinogenesis. Lipids23:592-597 (1988).

48. Adlercreutz H, Mousavi Y, Hockerstedt K. Dietand breast cancer. Acta Oncol 31:175-181(1992).

49. Peterson G, Barnes SX. Genistein inhibition ofthe growth of human breast cancer cells: inde-pendence from estrogen receptors and themulti-drug resistance gene. Biochem BiophysRes Commun 179:661-667 (1991).

50. Siiteri PK, Nisker JA, Hammond GL.Hormonal basis of risk factors for breast andendometrial cancer. In: Hormones and cancer(lacobeli S, ed). New York:Raven Press,1980;499-505.

51. Noble RL, Hochachka BC, King D.Spontaneous and estrogen-produced tumors inNb rats and their behavior after transplantation.Cancer Res 35:766-780 (1975).

52. Monti E, Sinha BK. Antiproliferative effect ofgenistein and adriamycin against estrogen-dependent and -independent human breast car-

cinoma cell lines. Anticancer Res 14:1221-1226 (1994).

53. Pagliacci MC, Spinozzi F, Migliorati G, FumiG, Smacchia M, Grignani F, Riccardi C,Nicoletti I. Genistein inhibits tumour cellgrowth in vitro but enhances mitochondrialreduction of tetrazolium salts: a further pitfallin the use of the MTT assay for evaluating cellgrowth and survival. Eur J Cancer29A:1573-1577 (1993).

54. Lamartininiere CA, Moore J, Holland M,Barnes S. Neonatal genistein chemopreventsmammary cancer. Proc Soc Exp Biol Med208:120-123 (1993).

55. Verdeal K, Brown RR, Richardson T, RyanDS. Affinity phytoestrogens for estradiol bind-ing proteins and effect of coumestrol on growthof 7,12-dimethylbenz[a]anthracene-induced ratmammary tumors. J Natl Cancer Inst64:285-290 (1980).

56. Burroughs CD, Mills KT, Bern HA.Reproductive tract abnormalities in female miceexposed neonatally to various doses of coume-strol. J Toxicol Environ Health 30:105-122(1990).

57. Burroughs CD, Bern HA, Stokstad EL.Prolonged vaginal cornification and otherchanges in mice treated neonatally with coume-strol, a plant estrogen. J Toxicol EnvironHealth 15:51-61 (1985).

58. Markaverich BM, Roberts RR, Finney RW,Clark JH. Preliminary characterization of anendogenous inhibitor of [3H]estradiol bindingin rat uterine nuclei. J Biol Chem258:11663-11671 (1983).

59. Miksicek RJ. Commonly occuring plantflavonoids have estrogenic activity. MolPharmacol 44:37-43 (1993).

60. Makela S, Davis VL, Tally WC, Korkman J,Salo L, Vihko R, Santti R, Korach KS. Dietaryestrogens act through estrogen receptor-mediat-ed processes and show no antiestrogenicity incultured breast cancer cells. Environ HealthPerspect 102:572-578 (1994).

61. Orti E, Bodwell J, Monk A. Phosphorylation ofsteroid hormone receptors. Endocr Rev13:105-128 (1993).

62. Migliaccio A, Pagano M, De Goed CJC, DiDomenico M, Castoria G. Phosphorylation andestradiol binding of estrogen receptor in hor-mone-dependent and hormone-independentGR mouse mammary tumors. Int J Cancer51:733-739 (1992).

"Mechanisms and Prevention of Environmentally Caused Cancers", a symposium presentedby The Lovelace Institutes, will be held October 21-25, 1995, in Santa Fe, New Mexico. The purpose ofthis symposium is to promote collaboration between scientists interested in the basic mechanisms ofenvironmentally-caused cancer and investigators focusing on preventing cancer development withchemo-intervention strategies. Dr. Bruce Ames (University of California) will be the keynote speaker.Other speakers include Dr. Eric Stanbridge (UC Irvine), Dr. Stephen Friend (Harvard), and Dr. GaryStoner (Ohio State University).

For further information, please contact:Alice M. Hannon, The Lovelace Institutes

2425 Ridgecrest Drive S.E.Albuquerque, NM 87108-5127


effects of coumestrol on estrogen receptor function

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