Full text


Nicolas Olea,I


Pedraza,1 AnaM. Soto, andCarlosSonnenschein4

'Laboratoryof Medical Investigation, Departmentof Radiology,SchoolofMedicine,2Department

Dentistry, and3Departmentof Nutrition, Schoolof Pharmacy, Universityof Granada, 18071 Granada, 4Department

andCellular Biology, Tufts University,School of Medicine, Boston,MA02111 USA


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The impact ofcertain estrogenic xenobi- otics on the development, health, and reproductive systems of wildlife has been clearly documented (1). As data accumu-

late, environmental xenobiotics are also being implicated inhuman infertility, geni- tal tract malformations, and increased can-

cer rates in estrogen target tissues (2,3). In 1936, Dodds and Lawson reported the estrogenicity ofsome diphenyl compounds containing two hydroxyl groups inpara

positions (4). Reid and Wilson (5) subse- quently confirmedtheestrogenicityof 4,4'- dihyroxydiphenylmethane derivatives. One such derivative, bearing two methyl groups

and knownas bisphenol-A, isa majorcom-

ponent ofepoxy resins. Bisphenol-A was

found to leach from autoclavable polycar- bonate laboratory flasks (6). Recently, we

demonstrated that food packed in lacquer- coated cans was active in a bioassay for


bisphenol-A released from the epoxy resinliningofthecanswasidentified as the estrogeniccontaminant(7).

Bisphenol-A is acommon ingredient in restorativematerials used in dentistry. Since the 1960s, when bisphenol-A diglycidyl methacrylate (bis-GMA)-based restorative materials were first used in odontology, many studies have assessed the effects of resins on pulpal injury (8) and their cyto-

toxic properties (9-11). Little attentionwas

paid, however,to thesystemichealtheffects of these chemicals or their monomers (12,13). Some studies focused on the alky- latingproperties of the glycidaldehyde por-

tion of bisphenol-A diglycidylether (BADGE)(14). Forexample, the formation of glycidaldehyde adducts in adenine residues was demonstrated in mice after cutaneous treatment with BADGE (15).




:.... ...:

Other studiesexaminedthe



carcinogenic properties

of epoxy resi monomers,with






restoratih materials used in

dentistry ("composites'

consist oftwo major components: a


resin matrixandan






fillers a.

known as "sealants." Tooth-colore restorative materials are





purposes, to


missir tooth structuresandto


tooth cob and contour. The resin matrix is initial presentasafluidmonomerthatisconver

ed into a

rigid polymer by

a free radica initiated reaction ofaddition. The












ultraviolet or visible


in tI presenceofa


Becauseof the low





required by



concerns have been expresse about the


of chemicals froi


material, which is


released after


(16). In vitrostudii have shown that mostof the


ized monomers of bis-GMA had leache 24 hrafter


In addition,curedcon

posites placed

in the oral cavity ai



and chemicall, Enzymatic



methacrylate together

with mechanical forces, coi

tribute to the breakdown of




which are



persi tently degraded (11]).


significant portion

of theuncured


material that isswallowed co be absorbed


the intestine. Climie et

(19,20) studied the metabolic


of 14C-BADGE in mice after the or

administration of a single dose and found that 90% of the radioactivity waseliminat- ed in feces and urine during the first 3 days of the experiment. Interestingly, a small

k amount of BADGE (- 5%) underwent F oxidative dealkylation to yield glycidalde- hyde (which has alkylating properties) and V bisphenol-A, among other by-products (20). The systemic behavior of thedipheno- lic derivative is poorly known and needs further investigation.

The purpose of this research was to determine whether bis-GMA-based restora- tive resins and their components haveestro- Ld genic activity in an in culture assay. We


demonstrate here that the sealant and resin


components bisphenol-A and bisphenol-A ve dimethacrylate are estrogenic and may rep- 1)) resent an additional source ofxenoestrogen In exposure in humans.


re Methods



line and culture conditions. MCF7 Id human breast cancer cells originally estab- ig lished by Soule and colleagues (21) were at or passages 100-106 postcloning at the time ly of study. For routine maintenance, cells


were grown in Dulbecco's modification of


Eagle's medium (DME) supplemented with y- 5% fetal bovine serum (FBS; PAA Labor li- und Forschungs Ges, MBH, Linz, Austria) Lt- in an atmosphere of 5%C02/95% airunder


saturating humidity at


Plasma-derived human serum and r- removal of sex steroids. Plasma-derived

n- human serum was prepared from expired


plasma by adding calcium chlorideto a final m concentration of 30 mM to facilitate clot


formation. We removed sex steroids from es serum by charcoal-dextran stripping (22).

r- Briefly, a suspension of 5% charcoal (Norit

n- re y.




or an



Address correspondence to N. Olea.Departmentof Radiology, School of Medicine, Universidad de Granada, 18071 Granada, Spain.

This work was reported in part at the SETAC- Europe meeting held in Copenhagen, Denmark, 25-28 June 1995. We thank Karen Shashokfor improving the English of the manuscript.Thiswork was supported by grant 94/1551 from theFondode Investigaciones Sanitarias (FIS), the Spanish Ministry of Health and the Consejeriade Salud, JuntadeAndalucia(to N.O.), NationalInstitutesof Health grant CA13410 (to C.S.), CA55574, and NSF-DCB-9105594 (toA.M.S.).

Received 9 August 1995; accepted 21 November 1995.


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A, Sigma Chemical Co., St. Louis, Missouri) with 0.5% dextran T-70 (Pharmacia-LKB, Uppsala, Sweden) was prepared. Aliquots of the charcoal-dextran suspensionof avolumesimilar to the serum aliquottobe processedwere centrifuged at 1iOOg for 10 min. Supernatants were aspi- rated, andserum aliquots weremixed with the charcoal pellets. This charcoal-serum mixture was maintained in suspension by rolling at 6 cycles/min at 37°C for 1 hr.

The suspension was centrifugedat 10OOg for20 min, and thesupernatant wasthen filtered through a 0.20-pm filter (Gelman Sciences,Ann Arbor, Michigan). Charcoal dextran-treated human serum (CDHuS) wasstoredat-20°Cuntilneeded.

Cellproliferationexperiments. Weused MCF7 cells in the E-screen test ofestro- genicityaccordingto a technique slightly modified (23) from that originally described by Soto et al. (24). Briefly, cells were trypsinized and plated in 24-well plates (Limbro,McLean,Virginia) atinitial concentrations of10,000 cells perwell in 5% FBS in DME. Cells were allowed to attach for 24hr, then the seeding medium was replaced with 10% CDHuS-supple- mented phenol red-free DME. Different concentrations ofthe testcompound were added, and theassaywasstopped after 144 hrby removingmedium fromwells, fixing the cells, andstaining them with sulforho- damine-B (SRB). The staining technique was modified from that described by Skehanetal.(25). Briefly, cellsweretreated with cold10%trichloraceticacid andincu- batedat4°C for30min, washed fivetimes with tap water, and left to dry.

Trichloroacetic-fixed cellswere stained for 10 minwith0.4% (wt:vol) SRB dissolved in 1% acetic acid. Wellswere rinsed with 1% acetic acidand air dried. Bound dye wassolubilized with 10 mMTrisbase(pH 10.5) in a shaker for 20 min. Finally, aliquotswere transferred to a96-well plate and readin aTitertek Multiscan apparatus (Flow, Irvine, California) at492 nm. We verified the linearity of the SRB assayby cellnumber before cellgrowthexperiments.

Results are expressedas means ± SDs.

We normalized mean cell numbers from each experimenttothe steroid-free control culturestocorrectfordifferencesintheini- tial seeding density. Differences between the xenoestrogen and


groups were assessed by analysis ofvariance and the aposterioriScheffe's test. Ap-value <


Estrogenandprogesterone receptormea- surements. MCF7 cells were seeded in T- 25 flasks in 5% FBS-supplemented DME.

On the following day, the medium was

changed to 10% CDHuS-supplemented

phenolred-free DME medium, and estradi- ol-17l3 or the chemicals to be tested were added. One group ofcells received vehicle alone.After 72 hr, the culture medium was discarded and cells were frozen in liquid N2. To extract receptormolecules,weincu- bated cells at 40C for 30 minwith 1 ml extraction buffer (0.5 M KCl, 10 mM potassiumphosphate, 1.5 mM EDTA,and 1 mM monothioglycerol, pH 7.4), accord- ing to a technique previously described in detail(26). Aftercentrifugationtopelletthe celldebris,estrogenand progesterone recep- tors were measured in a


aliquot of the supernatant byenzyme immunoassay using theAbbott ER and PgR enzyme immunoassay monoclonal kits (Abbott Diagnostic, Wiesbaden, Germany) accord- ing tothemanufacturer'sinstructions.

Estrogen-induced cell type-specific pro- teins. We measuredcathepsin-D and pS2 intheculture medium ofMCF7cellswith the ELSA-CATH-D and ELSA-PS2 immunoradiometric assays (CIS Bio International, Gif-sur-Yvette, France). The culture medium wascentrifuged at 1,200g for 10 min to eliminate floating and detached cells. Sampleswerekept frozenat -800Cuntil the assayswereconducted.

Competitive binding assays. Cytosol from immature, female rat uteri was pre- paredat aprotein concentrationofapprox- imately 2 mg/ml in phosphate buffer.

Aliquotsofthis 105,000g supernatant were thenincubatedwithvariousconcentrations ofbisphenol-A, bisphenol-A dimethacry- late, BADGE, bis-GMA, and 3 nM [3H]estradiol for 16hr at0-40C. The free and bound fractionswere separated with the charcoal-dextran technique. We calcu- lated the relative binding ability (RBA) of eachcompetitorastheratioof theconcen- tration of radioinert estradiol/competitor required to inhibit 50% of the specific [3H]estradiol binding, with the affinity of estradiolsetat100%.

Determination of componentsin resin- basedrestorative material We analyzed four commercial bis-GMA-based compos- ite resins using an HPLC technique.

Aliquots of50-100 mg ofeach sample weresuspended in 1 mlethanol and chro- matographed using aLichrocart MerckS5 ODS column (20 x 0.4 cm; Merck, Darmstadt, Germany) at aflow rate of1 ml/min in a Perkin-Elmer 250binary LC chromatograph with aPerkin-Elmer 235 diode arraydetector 235 equiped with a 20-pl loop injector (Waters 717 Plus Autosampler). The gradientwas: at time= 0 min, 100% phaseA(acetonitrile-water, 1:1) and0%phaseB (acetonitrile);attime

= 15 min, 100%phaseBand0%phaseA;

and attime = 17 min, 100%phaseAand

0%phaseB.Theelutionprofilewas moni- tored at 280 and254nm.

Determination ofbisphenol-A and relatedcompoundsinsaliva. Forthedini- calstudy, 18 healthy men and women (age range 18-25 years, mean age 20 years) were treated. The experiment was conducted in accordance with the Helsinki Declaration.

Asealant was applied to 12 molars accord- ing to a standard technique that included visible light asthe activator of the curing agents.Samples of whole saliva were collect- edby having subjects spit for 1 hr before and 1 hr after treatment into preweighed glass flasks containing 10 ml ethanol. The amount of commercial bis-GMA-based sealant usedwas approximately 50 mg per subject. After collection, saliva samples were centrifuged (1OOOg, 10 min) and passed through a 0.45-pm pore filter. Aliquots of 20 plwere chromatographed byHPLC as described above and by GC/MS. Saliva samples were also tested for estrogenicity in the MCF7 breast cancer assay.

Spectroscopy. Massspectraof extracted composite resins, filtered saliva samples, and technical grade bisphenol-A, dimethacrylate of bisphenol-A, and BADGE were obtained in a mass spec- trometry system (Fisson VG Platform-II, Manchester, UK) operating at an ion source temperature of 200°C in a Fisson GC 8000 SM Autospec chromatograph (Milan, Italy). A 15-mmethyl silicon col- umn(OV-P)wasusedat aflowrateof1.2 ml/min, with heliumasthecarriergas.The oven temperature was 80-320°C, with a graded increase of 10°C/min. Relative retention times of all thecomponents ana- lyzedwere 16.2 minforbisphenol-A; 20.5 minforBADGE,and21.2minforbisphe- nol-Adimethacrylate.

Steroidsandchemicalcompoundstested Estradiol-17g was obtained from Sigma.

Bisphenol-A, bisphenol-A dimeth-acrylate, BADGE, and bis-GMA were obtained from Aldrich (Aldrich-Chemie, Albuch, Germany). Chemicals were dissolved in ethanol to afinal concentration of1 mM andstoredat-20°C.

Theresinbased-materialswetestedwere Tetric(Ivoclar-Vivadent, Schaan,Liechten- stein), Charisma (Kulzer, Wehrheim, Germany), Pekalux (Bayer, Germany), identifiedascomposites 1,2and 3, respec- tively, and two different batches of the sealant Delton (Ash/Dentsply, Konstanz, Germany).Allweredilutedinphenol red- free DMEimmediately before use in the cultureassay. Thefinal ethanolconcentra- tioninthe culture mediumdidnotexceed 0.1%. We obtained [2,4,6,7-3H]estradiol (103 Bq/mmol) from Amersham (Buckinghamshire, England).



Estrogenic Activity ofResin-based Composites

The addition of estradiol-17g toCDHuS- supplemented medium increased thenum-

bers of MCF7 cells in culture. Maximum proliferative effectwasobtained atestradi- ol-17f concentrationsof10pM andhigher (Fig. IA). Cell yieldswere sixfoldgreater

thanin control cultures after 6days (mean

±SD,6.67± 1.21; n= 15experiments). In the absence of estradiol-17l3, cells prolifer- atedminimally.

Four bis-GMA-based composite resins

were assayed before polymerization using the MCF7 proliferation test. Composites and sealantswere preparedin ethanol ata

concentration of 100 and 50 mg/ml, respectively, and assayedatarangeofdilu- tions (1/100 to 1/106). The cell yield obtained with 5 pg/ml sealant sample was

sixfoldgreaterthan in controlcultures. The magnitude of this proliferative effect was similartothat obtained withestradiol-1713.

Cell toxicity was observed at concentra-

tionsof50pg/ml and higher (Fig. 1B).

In contrast to the sealant sample, the three resin-based composites assayed did

not induce MCF7 cell proliferation at a

maximal concentration of1 mg/ml. These commercial formulations contain alarge proportion ofinorganic fillerparticles (50-85% by weight of the composite).

Stocksuspensions were prepared without discardingtheinorganic portion.




0 0


Identification ofEstrogenic Substances fromComposite Resins

Figure 2 shows the HPLC profile ofan unpolymerized sealant that was positive in the proliferation test. Relative retention timesof the components identified were: t= 2.35 min orbisphenol-A; t= 7.35 minfor BADGE; t= 8.55 min for bis-GMA; and t = 11.85 minfor dimethacrylate of bisphenol- A. These components were quantifiedby calibrationcurvesmadeafterelutingknown amounts (1


to 1 mM) ofthe puresub- stance [bisphenol-A, y= (2.92 x 1011)x+ (9.075 x 106), r= 0.998; bis-GMA,y= (1.04x 1011)x+ (0.670 x 106), r= 0.999;

BADGE, y= (1.12 x 1011)x+ (0.470 x 106), r = 0.999, and bisphenol-A dimethacrylate,y= (2.18x 10'1)x+ (8.782 x106), r=0.995].Mass spectrometric analy- sis confirmed thepresence ofbisphenol-A and dimethacrylate of bisphenol-A in all samples for which HPLCchromatograms showed thecorresponding peaks(Fig. 3).

To determine whether the monomer bisphenol-Acan be cleaved from compos- ites and from their main oligomers (dimethacrylate of bisphenol-A, bis-GMA, and BADGE), these were hydrolyzed in alkaline(pH= 13) and acidic media (pH= 1) after heating (100'C for 30 min), and then chromatographed as indicated. Table 1 summarizes the componentsidentifiedin the chromatograms. Extensive breakdown of the oligomers into smaller compounds suchasbisphenol-Awasobserved.







1013 1o-ll Concentration(M)

Estrogenic ActivityofSome Componentsof Resin-based RestorativeComposites

Bisphenol-A and its dimethacrylate were estrogenicwhen assayed in the breast can- cercell proliferation assay. The concentra- tionsrequired to produce maximumprolif- eration of MCF7 cells were 10,000-fold higher than those ofestradiol-17f; (Fig. 4).

MCF7 cells were grown in the presenceof bisphenol-Aorbisphenol-A dimethacrylate and the estrogen antagonisthydroxytamox- ifen; the proliferative effect of 0.1 pM bisphenol-A and bisphenol-A dimethacry- late disappearedwhen cells were grown in thepresenceofa 10-fold higherconcentra- tionof the antagonist(data not shown).

Bis-GMA was negative in the estro- genicity test. A proliferative effect of BADGE was observed at a concentration of 10 pM (Fig. 5). Oligomers from both compoundsbecame activeintheprolifera- tive test after hydrolysis (100°C for 30 min, in alkaline or acidic medium).

Bisphenol-Aand bisphenol-A dimethacry- late were identified in chromatograms when oligomers were found to be estro- genicafterhydrolysis.

ProgesteroneReceptorInduction MCF7 cells have receptors for estradiol- 17f (estrogen receptor; ER) and proges- terone (PgR). The basal concentration of


0.05 0.5 5 50 500


Figure1.Cellproliferationof MCF7 cells. Cellsgrowingin10%charcoal dextran-treated humanserum- supplemented mediumwereexposedfor144hrto (A)estradiol-17Band(B)bis-GMA-based sealant, sampleno. 1.Points representquadruplicate cultures;bracketsindicateSDs. Ordinates represent cell numbers(experimental/control);abscissas represent concentration of thetestcompound. Estradiol-171 waseffectiveat>1pM(0.272pg/ml).*Valuessignificantlydifferent fromcontrol,p<0.001.

Figure2.Chromatogramof sealantsampleno.1, whichwasestrogenicinthe breastcancerprolif- eration bioassayshown in Figure1. Peaksat retention times2.3, 7.3, 8.6,and 11.9minwere identified as 1) bisphenol-A,2) bis-GMA, 3) BADGE,and4) bisphenol-A dimethacrylate.



ER inthesecells was 183±29fmol/mg of extracted protein (Fig. 6A). Estradiol-171 decreased theconcentration ofERby50%

and increased that ofPgR The basal PgR value was 15.5 ± 4.3 fmol/mg protein, closetothe lower limit of detection of the monoclonal antibody assay (Fig. 6B).

Estradiol-17l (1 nM) significantly increasedPgRnearly 15-foldoverthecon- trol value.

TreatmentofMCF7cells with concen- trations of0.1 pM and higher ofbisphe- nol-Adimethacrylate resulted in asignifi- cant increasein PgRlevels. Bisphenol-A treatmentalso increased PgRlevels (Fig.

6B). However, no reduction ofER levels wasobserved when bisphenol-Aorbisphe- nol-Adimethacrylateinduced PgR Secretion



and Cathepsin-D

Cathepsin-Dand pS2 accumulation in the culture medium reflected increases in cell number during theexperiments.The secre- tionofpS2byMCF7 cellswassignificant- ly increased by >0.1 nMestradiol-17g (- 3.5-fold increase over controls). The basal concentration ofpS2 (55.4 ± 15.2 ngIl6 cells) increasedto 202.2± 19.1ng/10 cells after treatment with 10 pM bisphenol-A dimethacrylate; this was similar to the effect of estradiol-17L onpS2 secretion

(Fig. 7A).

The basalconcentrationofcathepsin-D secreted by MCF7 cells was 8.9 ± 2.9 pmol/106 cells. Estradiol-1781treatment (1 and 10 nM) resultedin amodest 1.7-fold increase in the accumulation ofcathepsin- D in the culture medium. Bisphenol-A dimethacrylate (1 and 10 pM) increased cathepsin-D secretion only 1.6-fold (Fig.

7B). Thesechangeswere notsignificant.

Binding Affinity

of Composite Resin


for the Estrogen Receptor

The binding affinity of bisphenol-A, bisphenol-A dimethacrylate, BADGE, and bis-GMA for the cytosol ERwas deter- mined by competitive binding analysis.

The receptor relative binding affinities (RBA) were 0.012 and 0.0033 forbisphe- nol-A and bisphenol-A dimethacrylate, respectively, versus 100 for estradiol-


Bis-GMA and BADGE did not compete with estradiol forbindingto theER, even at concentrations a million-fold higher than


(Fig. 8).

Analysis and Quantification of





Eighteen saliva samples taken before and after sealant treatment were chromato- graphed usingthe technique describedin Methods. Figure 9 shows the chromato-


100 r


0D U 70 W0 90 IlUU I IU 120 130 1IW IU 1W 17U 1WU 190 200 21U 22U 2d3 240 2W

Figure 3. Mass spectra of(A)puregrade bisphenol-A and(B)bisphenol-Afrom a sealant sample.

Table1.Identificationofthe components in thecomposites and sealants"

pg/100 mg commercialproduct

Product Bisphenol-A

andpH Bis-GMA BADGE dimethacrylate Bisphenol-A


7 45.8 75.0 29.2 7.4

13 32.8 117.0 4.6 84.4

1 49.4 164.8 6.4 18.2


7 105.0 19.6 ND 67.7

13 16.7 1.7 186.0 53.1

1 0.4 ND 1.8 49.9


7 18.1 10.0 ND 0.5

13 53.2 24.2 878.4 ND

1 46.8 140.0 ND ND


7 22.0 3.2 ND 4.2

13 1809.4 38.6 ND ND

1 1802.0 5.2 3.6 11.4

Abbreviations:bis-GMA, bisphenol-Adigicidylethermethacrylate;BADGE,bisphenol-Adiglcidylether;ND, notdetectable.

8Sampleswerehydrolyzedinalkaline(pH=13)and acidic media(pH=1)afterheating(100'Cfor 30min) andthenchromatographedasindicated in Methods. ThecontentsbeforehydrolysisareindicatedaspH=7.






0 ~~~~~~~~~~~~~~~~0

l0-10 lo's 108 100 100

Concentration(M) Concentration(M)

Figure4.Cell proliferationin MCF7 cells. Cells'growingin 10% charcoaldextran-treated human serum-

supplementedmediumwereexposedfor 144 hrto(A)bisphenol-Aand(B)bisphenol-A dimethacrylate.

Pointsrepresentquadruplicatecultures;brackets indicateSDs. *Valuessignificantlydifferentfromcon-


3.5 3.5


pH pH

_1_ _ __

.52.5 5 .5


2.0 20

o 0_g __

LE 1.5 LE 1.5

1.0 1.0

0.5 0.5

0.0 0.0

10-9 10.7 105 109 10.7 10,5

Concentration(M) Concentration(M)

Figure5. Cell proliferation in MCF7 cells. Cellsgrowingin 10%charcoal dextran-treated human serum-

supplementedmediumwereexposedfor 144 hrto(A)bis-GMA and (B)BADGE andtoproductsobtained byhydrolytictreatmentsof thesecompoundsinalkaline(pH=13)and acidic medium(pH=1).Points rep- resentquadruplicatecultures; brackets indicate SDs. *Values significantlydifferent from control,p<


graphic profile ofa representative saliva sample from apatientselected at random after treatment with 50 mg of the bis- GMA-based sealant. Peaks in the chro- matogram corresponded to Bis-GMA, BADGE, bisphenol-A, and dimethacrylate ofbisphenol-A. Variations in the chro- matographic profiles were seen between sealant batches and between patients who received sealant from the same batch.

Monomers ofcompositecomponentswere

measuredbyHPLCinsaliva collected 1 hr after treatment (Table 2). Aftertreatment, all saliva samples contained variable amounts ofbisphenol-A ranging from 90 to 931 pg; this was confirmed by mass spectrometry.

Subjects were treated with a fixed amountofapproximately50 mgofsealant, and theproportionoffree, unpolymerized material collected during 1 hrafter treat- ment never exceeded 2% of the total

_ 200



,=ooE 15(




'I'S 201


*0.CD E 151


nM gM gM


1 10 1 10 1 10

nM gM gtM


Figure6.(A) Estrogen(ER) and(B)progesterone (PgR)receptors in MCF7 cells.CellsinT25 flasks were incubated in10% charcoal dextran-treated humanserumfor 72 hr with1and 10 nM ofestra-

diol-17B.Aparallelsetof flaskswasexposedto1 and 10 pM bisphenol-A or bisphenol-A dimethacrylate. Controls received the vehicle alone. At the end of theexperiment,the medium wasaspiratedandflaskswerekeptinliquid N2 until assayed. ER and PgR weremeasured in extracted cells withamonoclonalantibodytech- nique as indicated in Methods. Results are expressed asfemtomole permilligram ofextract- ed protein ±SD. *Values significantly different fromcontrol,p<0.001.

amount of sealant applied. Composite components were not observed in any of thesaliva samples collected before treat- ment. Asubject initially selected for treat- menthad been treated with tooth sealant2 years earlier; chromatograms demonstrated thepresence ofbisphenol-A (66.4


and bisphenol-A dimethacrylate (49.2






D co0

a. 100










1 10 i 10 1 10 1 10 1 10 1 10

nM PiM igM nM JiM JIM

Concentration Concentration

Figure 7. Accumulation of(A) pS2and(B) cathepsin-Dinculture medium. MCF7 cellswere grownin 10%

charcoal dextran-treated humanserum-supplemented medium andexposed for144hrto1 and 10nM estradiol-17B. Parallelcultureswereexposedto1and 10pM bisphenol-Aorbisphenol-A dimethacrylate.

Controlsreceivedthevehiclealone.*Valuessignificantly different from control,p <0.001.

hersaliva before thesecondtreatment.The results from this subject were excluded fromanalysis.

Samples containing the highest

amounts ofbisphenol-A and bisphenol-A dimethacrylate wereestrogenic inthe pro-

liferationtest. Figure 10showstheprolifer- ativepatternofasample containing231 jig bisphenol-A in 27 ml ofcollected saliva plus 10mlofethanol.Analiquot of100pi ofthissamplediluted in1mlof theculture mediumwas positive inthe estrogenicity assay.Theestimatedamountofbisphenol- A assayed was 0.62 pg/ml. Because the

presence ofendogenous estrogens could

not be ruled out, saliva samples taken before the sealant was appliedwere also assayed;noneshowedestrogenicity.


Epoxy resins of thebisphenol-A typehave

manyapplications such aslacquer coating infoodcans, indental, surgical, andpros-

theticdevices, andas additives inavariety of other plastic materials (27). Herewe demonstrate thatbisphenol-A and bisphe- nol-Adimethacrylate,componentsofcom-

mercialresin-basedcomposites and sealants used in dentistry, are estrogenic andmay represent additional sources of human


Commercial smooth-surface compos-

ites vary in monomer composition and thus have differentphysical properties and biological behaviors. We found that the

amount ofbisphenol-A and bisphenol-A dimethacrylatevaried between commercial

* Estradiol-173 A Bisphenol-A

* BADGE dimethacrylate

* Bisphsnol-A UBis-GMA






Figure9.Chromatogramofasaliva samplewith estrogenic activity. Peaks detected atretention times2.3, 7.4, 8.5,and11.9minwereidentifiedas1) bisphenol-A, 2) BADGE, 3) bis-GMA, and 4) bisphe- nol-Adimethacrylate, respectively.





Figure8.Competitive displacement of [3H1-estradi- ol from theestrogen receptorby estradiol-17 and bisphenol-A, bisphenol-A dimethacrylate, BADGE, andbis-GMA. The relative binding affinitiesof bisphenol-Aandbisphenol-A dimethacrylatewere

0.012 and0.0033,respectively.[3H]Estradiol con- centrationwas3nM,andthe totalbindingwas73 fmol/mgprotein; Kd=8.5x1011M.

composites and batches. Bisphenol-Awas identifiedinallthecompositesamples and batches analyzed, whereas methacrylate of bisphenol-A was identified only in the sealant. The oligomers BADGE and bis- GMAwere found in all three composites and in the sealant. Theproportionofthese compounds varied widelybetween com-

posites and batches. Vankerckhoven etal.

(28) studied 12 commercialrestorativebis-

Table2.Quantitative evaluation ofbisphenol-A identified in saliva obtained1hrafter the applica- tion of 50 mg of sealant

Sample. Bisphenol-Aa

no Sex Volume(ml) (pg) (pg/ml)

1 F 29 320.6 11.1

2 F 31 931.0 30.0

3 F 35 174.6 4.9

4 F 33 865.6 26.2

5 M 26 91.2 3.5

6 M 31 249.6 8.1

7 F 27 165.7 6.1

8 F 38 263.6 6.9

9 M 32 105.3 3.3

10 M 27 230.5 8.5

11 M 36 181.4 5.0

12 M 35 324.2 9.3

13 M 29 353.0 12.2

14 M 39 810.8 20.8

15 M 32 230.8 7.2

16 M 27 130.4 4.8

17 M 27 89.8 3.3

18 F 29 112.4 3.9

"Saliva sampleswere collected in glassflasks containing 10 mlethanol. Values of bisphenol-A concentrationrepresentthoseofundiluted saliva.

17 .~ ....11'Ssi.








1/10, 1/10, ~~~~1/10 Dilution factor

Figure10.Proliferative effect ofasaliva sample obtained 1 hraftertreatmentwith sealantno 1.

Pointsrepresentquadruplicate cultures;brackets indicate SDs. *Valuessignificantlydifferent from control,p<0.001.

GMA-based resinsandfound bis-GMAin all samples at concentrations ranging from 19 to 51% ofthe total composite weight.

Iso-bis-GMA was also present and repre- sented40% of the total percentage of bis- GMA. Bisphenol-A dimethacrylate was found in four commercial mixtures in amounts thatranged from 6to10% of the total composite weight. In addition to thesemonomers, othercompounds suchas light activators, phthalates, and benzoates, as well as a large amount ofinorganic fillers, are commom ingredients of compos- ites(28).

Onceappliedtotoothcavities, compos- ites andsealants are polymerized in situ.

The degree ofconversionof oligomersinto polymersvariesdependingonthe composi- tionoftheresin and its distance from the tooth surface. Conversion of 60-75% is expected with most common composites (16), although levels of curing as lowas 30% are found in the bottom offillings.

Lower levels ofconversion arethought to be associated with greater elution of free components fromthe composites (16). A stronginverse relationship has been found between theleaching of resin from bis- GMA-based composites and monomer conversion. Monomerleaching reached a plateauatconversion levelsof 60%(29).In nine commercial resins, the amount of residual bis-GMA measured at the end of the setting time was in the range of 0.4-1.2% of the original weight aftercur- ing.Amountsof the residualmonomers of up to one-tenth the initial releasewere elutedinto water over aperiod of14 days

(29). Highervalues were described by Ferracane and Condon (16), who found differencesinpercentage elutioninrelation to curing and double illumination. Our dataconfirm thesefigures: residual bisphe- nol-Ainsalivaafter curing ranged from 0.1 to2%of the 50 mginitially appliedtothe tooth surfaceinsamples taken during 1 hr after treatment.

Inaddition to bisphenol-A, present in 100%of the saliva samples, its methacry- late derivative was present in3 out of 18 samples. Both compounds wereidentified by chromatography and estrogenic activity asmeasuredinthe MCF7 breastcancercell proliferation test, the E-screen assay.

The oligomer bis-GMA was not estro- genicintheE-screenbioassay. Hydrolyzed BADGEand bis-GMAinalkaline or acidic media were estrogenic. Chromatographic analyses of hydrolyzed samples detected the presence of bisphenol-A and bisphenol-A dimethacrylate after treatment. The meta- bolic routes of systemic degradation of somecompositeresinshavebeen studiedin mice (15,19,20). Forexample, BADGE is metabolized by oxidative dealkylation to give aphenol diol derivative. Much atten- tionhas been giventothe smallerfragment of the cleavage process because of the mutagenic properties ofthe glycidylalde- hyde portion. However, the estrogenic activity ofthese derivatives has notbeen evaluated; their chemical similarityto some bisphenol-A derivatives with this property suggests the need for closer examination.

Moreover, little information is available about the potential estrogenicity of by- products ofthe environmental breakdown ofbiphenolic compounds. Lobosetal.(30) isolatedanovel bacteriumfrom thesludge ofa wastewatertreatmentplant ofaplastics manufacturing facility; theorganism was able todegrade bisphenol-Ato4-hydroxy- benzoic acid and4-hydroxyacetophenone (both monophenolic by-products) and to 2,2-bis(4-hydroxyphenyl)-1-propanol, and finallyto 2,3-bis(4-hydroxyphenyl)-1,2- propanediol. So far, the estrogenicity of thesebisphenol-A derivatives has not been explored, although the similarity of the lat- ter compoundto some stilbenes suggests thatitmaydisplay considerable activity.

Bisphenol-Ais toxictofishandinverte- bratesatconcentrationsof1.1-10 mg/l (>

4 pmol/l) (31). Inamousemodel, bisphe- nol-A induced photoallergiccontact der- matitis (32). Ifadministered during organogenesis, bisphenol-A is fetotoxic whenadministeredtopregnant mothers.A study of the effects ofbisphenol-Aon reproduction andfertility showedasignifi- cant reduction in seminal vesicleweight and sperm motility in treated male mice

(33). These effects were present in parents and in a second generation when offspring were maintained at the same exposure level as their parents (4.37-17.5 mg/kg body weight). In addition, bisphenol-A treat- ment resulted in a significant reduction in the number of litters per pair and in live pups perlitter.

Composites werediscovered tobe toxic in humans because lesions developed in unlined cavities (34,35). The concentra- tions to which cells and tissues are exposed in humans are unknown (11), but it was estimated thatseven components ofcom- posites, including bisphenol-A, were toxic in the 10-100


concentration range in an inculture assay (11). The sealant we tested was toxic to MCF7 breast cancer cells atconcentrations > 50 jig/ml ( -200 pmol/l of bisphenol-A). Lower concentra- tions were not toxic butwere fully estro- genicinthebioassay (Fig. 1B).

Human exposure to bisphenol-A and dimethacrylate of bisphenol-A has increased during the last 30 years because ofthe extensive use ofbisphenol-A-based resins. The use of sealants as an effective therapy and preventivetreatmentfor dental pits and fissures inadults and children has beenendorsed by European andU.S.agen- cies


Inviewof the documented exposure to bis-GMA-based composites and sealants used indental treatments for adults andchildren, theuseof thesexenoe- strogens shouldbereevaluated.


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Princeton, NJ:Princeton Scientific Publishing, 1992.

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3. Wolff MP, Toniolo P,LeeE,RiveraM,Dubin N. Blood levels oforganochlorine residues and risk of breast cancer.J Natl Cancer Inst 85:648-652 (1993).

4. Dodds EC, Lawson W. Syntheticestrogenic agentswithout thephenanthrene nucleus.


5. Reid EE,WilsonE.The relation of estrogenic activitytostructure in some4,4'-dihydroxyde- phenylmethanes. JAmChemSoc66:967-969 (1944).

6. Krishnan AV, Starhis P, PermuthSF, Tokes L, Feldman D.Bisphenol-A: anestrogenic sub- stance isreleased frompolycarbonate flasks during autoclaving. Endocrinology


7. BrotonsJA, Olea-Serrano MF, Villalobos M, Pedraza V, OleaN.Xenoestrogens released fromlacquer coating infood cans. Environ HealthPerspect103:608-612 (1995).

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