The Estrogenic Activity of Phthalate Esters In VitroCatherine A. Harris,1 Pirkko Henttu,2 Malcolm G. Parker,2 and John P. Sumpter1'Department of Biology and Biochemistry, Brunel University, Uxbridge, Middlesex, United Kingdom; 2Molecular EndocrinologyLaboratory, Imperial Cancer Research Fund, London, United Kingdom

A lrge r f eed for erogenicati w recombiant'yeast screen. Asii* of ee wa tested for mtogenc t on es -responsvehuman b. AL a - ofothe commercially-avaiablerphthalates tested

5 x io1iiem-Mr1~otiwlI h hhltsta were etoei nteyatsrewere also on the i c cer cells. Di(2-ethylht4l) phhalate (DEHP)`showed no ai ein i as. A number of metabolites were tested,induding :p ha monohhelh , mono--octylphtber~~~all were found totkislZiu-c. Oneof the phthalaret ditri-deyl hthalnaj(DTD o se w w anotheli

try e a reat uFbii in tiiy contie 5% the erdho-iso-mer iseno hitXIt ilkwly thth presetnoftte standardwa responsibe f wort on of andsnse carve-whc wasnoto hanatrtVe aspeta adntle s llev~intedwit .,'bisphenol A-4in .the yeast scren

hence TP4 ntwa4y h a iviisofsnpemno. fBi,DP

activitie oftenrue oeapoiaeyadtv.I umr,asalnme FPhalates are walesrgnci ih.No data has ye ee ulihdont whethedr:these are-also:esroeic in nsvo., this will require tests using different,classe -ofvreatsand differet routes-

of exposure. Key wornis contmntdsandrds-, estrogenicity, MCF-7, neaoiss hhaae,recombinant yev screen, ZR-75. HealthP 5:802811 (1997).

..: ~~~...!.....

In recent years there have been a plethora ofpublications discussing man-made estrogen-mimicking chemicals, the so-called xenoe-strogens. Reports of declining semen quali-ty (1) have been followed by hypothesesthat this phenomenon may be linked to anincrease in the exposure of humans toxenoestrogens, specifically in utero (2).Suspect chemicals originate from a varietyof backgrounds, many being anthropogenicin origin, such as pesticides, detergents, andplasticizers. One of the earliest endocrinedisruptors to be identified was the pesticideDDT, the effects of which are discussed byFry and Toone (3). Other man-madechemicals have since been recognized aspossessing estrogenic properties. For exam-ple, 4-nonylphenol is the degradation prod-uct of one group of nonionic surfactants,the nonylphenol polyethoxylates, and expo-sure to it has been demonstrated to induceestrogenic effects both in vitro (4-6) and invivo (7). However, naturally occurringxenoestrogens-including phytoestrogens,such as coumestrol and genistein, andmycoestrogens, such as zearalenone-alsoexist; these may be found in plant foodstuffs, to which humans have always beenexposed (8).

Phthalates are just one of the manyclasses of chemicals that have been implicat-ed as having estrogenic properties. Evidenceof the estrogenic behavior of certain phtha-lates in vitro has previously been reported(9-11). Furthermore, an in vivo study hasshown the adverse effects of butyl benzylphthalate (BBP) on rat testes size and spermproduction (12). A report concerning an invivo multigenerational study investigatingthe reproductive toxicity of dibutyl phtha-late (DBP) in Sprague-Dawley rats hasrecently been published. In this study, Wineet al. (13) found that a number of reproduc-tive parameters were adversely affected byexposure to DBP administered via feed andthat, critically, the second generationappeared more adversely affected than thefirst generation in that most of the Fl maleswere infertile. The mechanisms underpin-ning these adverse reproductive effects areunclear presently, but one possibility is thatsome phthalates are estrogenic in vivo andhence disrupt normal male development.

Phthalates are essentially used as plasti-cizers in the production of polymericmaterials such as polyvinyl chloride (PVC),imparting flexibility and workability, bothduring the manufacturing process and to

the end product. When used in this way,they are not chemically bound to the prod-uct (14) and may therefore leach into thesurrounding medium (15).

Phthalates are produced in extremelylarge volumes [the most widely used beingdi(2-ethylhexyl) phthalate (DEHP), at400-500 thousand tons per annum inEurope alone; see Table 1] and have beenidentified in all environmental compart-ments. For example, they have been reportedin water, sediment, air and biota sampledfrom the Gulf of Mexico (16), and riverwater and sewage effluent samples from theGreater Manchester area, United Kingdom(1/). Food samples contaminated withphthalates have also been reported (18-21).The lipophilic nature of these chemicals indi-cates that fatty foods such as cream, cheese,and butter are most likely to be subject tocontamination. Sharman et al. (21) discov-ered levels ofup to 1 14 mg/kg total phthalatein cheese samples; however, the majority ofsamples contained 0.6-3.0 mg/kg DEHPand 4-20 mg/kg total phthalate. The authorssuggested that these high levels might havearisen from environmental sources (for exam-ple, from the wrappers surrounding thecheese) rather than as a result of the dilutedpresence of the contaminant in the raw com-modity, followed by its distillation in thefatty phase (21). Although these chemicals areno longer used in most direct contact foodplastics and use in such materials has beenregulated for many years based on toxicologi-cal data available and the fat content of thefood concerned (22), it is possible that othersources of contamination during the manu-facturing process, and from certain printinginks and adhesives used in packaging, maycontribute to levels of phthalates found inmore recendy sampled foods (19).

Address correspondence to C.A. Harris, Departmentof Biology and Biochemistry, Brunel University,Uxbridge, Middlesex, UB8 3PH, U.K.We are very grateful to the Natural EnvironmentResearch Council for funding this work (project no.R05761). We thank BP Chemical Ltd., EXXONChemical Ltd., and Monsanto Europe SA., who sup-plied us with commercial preparations of phthalateesters, Rob Bos and Monsanto Europe S.A. for theprovision of various metabolites, Dow Europe S.A.for the synthesis and donation of o,p'-bisphenol A,and the Ministry of Agriculture, Fisheries and Food(Fisheries Laboratory) for the analysis of chemicalstandards. We also thank David Cadogan of ECPIfor arranging for the chemical analysis ofDTDP.Received 18 February 1997; accepted 1 May 1997.

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Articles - Estrogenicity of phthalate esters in vitro

The possibility of such extensively usedchemicals as the phthalates having a detri-mental influence on reproductive systems, ofeither humans or wildlife, clearly causespublic concern, as is evident from the con-siderable media coverage of this issue.However, when phthalates are discussed,they are often mistakenly referred to as a sin-gle group of chemicals, with the assumptionthat they all have similar properties, forexample estrogenic activity. In this paper weinvestigate the ability of individual phthalateesters to produce an estrogenic response invitro and attempt to relate this factor to theiroccurrence as environmental contaminants,as a partial contribution to an assessment oftheir risk as endocrine disruptors.

Materials and MethodsChemicals tested. 170-estradiol was pur-chased from Sigma, Poole, United Kingdom.

Thirty-five phthalates, encompassing avariety of structural and behavioral differ-ences and including the major phthalates ofcommercial importance, were purchasedfrom Greyhound Chemservice, Merseyside,United Kindgom (Table 1). These were of97-99% purity.

For comparison, a number of commer-cial preparations were also obtained as giftsfrom companies as follows: dibutyl phtha-late (DBP, 99.7% pure), diisobutyl phtha-late (DIBP, 99.6% pure), diethyl phthalate(DEP, >99.7% pure), and dioctyl phthalate(DOP, 99.9% pure), from BP ChemicalLtd., Hull, United Kingdom; diisodecylphthalate (DIDP, 99.9%) and diisononylphthalate (DINP, 99.9%) from EXXONChemical Ltd., Fareham, United Kingdom;ditridecyl phthalate (DTDP) from EXXONChemical Ltd., Courbevoie, France; andbutyl benzyl phthalate (BBP, >98.5%) fromMonsanto Europe S.A., Louvain-la-Neuve,Belgium. Purity of these preparations isgiven as provided by the company.

Various phthalate metabolites weredonated by R. Bos of the Department ofToxicology, University of Nijmegen, TheNetherlands. These were mono-hexyl phtha-late (MHP), mono-ethylhexyl phthalate(MEHP), mono-pentyl phthalate (MPP),mono-n-octyl phthalate (MnOP) andmetabolites V, VI, and IX of DEHP (23).Also donated (by Monsanto Europe S.A.)were the primary metabolites of BBP, mono-butyl phthalate and mono-benzyl phthalate.

4-Nonylphenol, supplied by SchenectadyInternational Inc. (Schenectady, NY), bisphe-nol A (Aldrich, Poole, U.K.), o,p'-DDT(Greyhound Chemservice, Merseyside,U.K.), and genistein (Sigma, Poole, U.K.)were tested in the recombinant yeast screenonly, in order to demonstrate the activity andpotency of some known xenoestrogens.

Table 1. All the parent phthalate esters tested using the recombinant yeast screen

Phthalate name Abbreviation European consumption (tons/annum)Bis(2-ethylhexyl) phthalate DEHP 400,000-500,000Diisononyl phthalatea DINP 100,000-200,000Diisodecyl phthalate DIDP 100,000-200,000Butyl benzyl phthalatea BBP 20,000-50,000Dibutyl phthalatea DBP 20,000-40,000Diisobutyl phthalatea DIBP 20,000-40,000Ditridecyl phthalate8 DTDP 3,000-10,000Diethyl phthalatea DEP 10,000-20,000 (with DMP)Dimethyl phthalate DMP 10,000-20,000 (with DEP)Diisohexyl phthalate DIHP <2,000Diundecyl phthalate DUP <2,000Butyl decyl phthalate BDcP <1,000Butyl octyl phthalate BOP <1,000Dicyclohexyl phthalate DCHP <1,000Dihexyl phthalate DHP Not aloneDi-n-octyl phthalate DnOP Not aloneBis(2-n-butoxyethyl) phthalate DBoEP NegligibleBis[2-(2-ethoxyethoxy)ethyl] phthalate DEoEoEP NegligibleBis(ethoxyethyl) phthalate DEoEP NegligibleBis(2-ethylhexyl) hexahydrophthalate DEHhP NegligibleBis(2-ethylhexyl) isophthalate DEHIP NegligibleBis(methoxyethyl) phthalate DMoEP NegligibleButyl cyclohexyl phthalatea BCHP NegligibleButyl 2-ethylhexyl phthalate BEHP NegligibleButyl isodecyl phthalate BIDP NegligibleDiamyl phthalate DPeP NegligibleDimethyl isophthalate DMIP NegligibleDioctyl isophthalate DOIP NegligibleDiphenyl phthalatea DPhP Negligible2-Ethylhexyl isodecyl phthalate EHIDP NegligibleHexyl decyl phthalate HDP NegligibleHexyl 2-ethylhexyl phthalate HEHP NegligibleIsodecyl tridecyl phthalate IDTDP NegligibleOctyl isodecyl phthalate OIDP NegligibleIsohexylbenzyl phthalatea IHBP Negligible

A ballpark consumption figure for each phthalate is given; "not alone" indicates that these particularchemicals are not used individually but only in mixtures, in conjunction with other phthalates.aPhthalates found to possess estrogenic activity in the screen.

3.0

2.5

-2Cto

2.0

1.5

1.0

0.5

10-12 10-11 10-10 10-9 10-8 10-7 iO-6

Molar concentration

Figure 1. Estrogenic activity of some known environmental estrogens in the recombinant yeast screen.17,B-estradiol serially diluted from 10-8 M and ethanol were used as positive and negative controls,respectively. 4-Nonylphenol, o'p'-DDT, bisphenol A, and genistein are shown as standard curves seriallydiluted from 10-5 M.

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803

Articles - Harris et al.

3.0 1 2 1 11 S | |liigl|11Egt_go,p'-Bisphenol A was supplied by Dow- I1*I Il7p-estradioI -Europe S.A., Horgen, Switzerland, and was

l 11 1 _* Ethanol Etested in the recombinant yeast screen toI 31 !11II _1111_A DBP assess the possible significance of its presence

2. -il I DI 1NI _ |g !1I11 lBP _ DEHP as a contaminant in one of the phthalateA DINPi h samples and its effect on the apparent estro-A DINPi _genicity of that sample.

2.0 The recombinant yeast screen. Alln') chemicals were assessed for estrogenic

activity using a recombinant yeast screen.This is a cost-effective, sensitive, and spe-

(A cific process for detecting estrogenic activi-oc ty. The screen has been described and

extensively validated elsewhere [see__0 Routledge and Sumpter (24) for full

details]. Essentially, a gene for the humanestrogen receptor has been integrated intothe main yeast genome and is expressed in

0.5 a form capable of binding to estrogen10-12 10.11 10-10 io09 I o-B10-B7i~ 10-B i5 104 10f 102 response elements and controlling the

Molar concentration expression of the reporter gene Lac-Z. Thus,3.0 1 1 _v L .- .- on activation of the receptor, the lac-Z

B - * ...7e.tradio. ..gene is expressed, producing the enzyme* Ethanol galactosidase, which is secreted into the* DTDP medium where it causes a color change of

2.5 thBCHP the chromogenic substance chlorophenolA IHBP red-p-D-galactopyranoside (CPRG) from

yellow to red. The intensity of the red color2.0 __-- can be measured by absorbance.

1, -The screen is highly specific for estro-_

.. gens; androgens, progesterones and corti-.0 costeroids are either completely inactive in.0°i5_ the screen or very weakly active at very high

o: _-_ concentrations (24).Details of the preparation of medium

1.0 components and yeast stocks have been_ _ ~~~~~~~~~~~~~~~~~~~~publishedpreviously (24t).

Growth medium was prepared by adding5 ml 20% w/v glucose solution, 1.25 ml 4

0.5 mg/m1 L-aspartic acid solution, 0.5 ml vita-10.12 10-11 10.10 io-9 10- 10-7 104 10-5 1o-4 10-3 10-2 min solution, 0.4 ml 24 mg/ml L-threonine

Molar concentration solution, and 0.125 ml 20 niM copper (II)sulfate solution to 45 ml single strength min-

3.0 e691 imal medium. The yeast culture was then

* 17,-estradiol prepared by seeding 50 ml growth medium* Ethanol* DHPA DIHP - Figure 2. The estrogenic activity in the yeast

2.5... DMP screen of phthalate esters at concentrations rang-2.5 DUP ing from 10-3 M to 5 x 10-7 M, compared to 17p-estradiol (serially diluted from 10-8 M). A) Illustratesthe estrogenic activity of phthalates consumed in

20 major volumes in Europe. B) Illustrates the estro-genic activity of DEP and DTDP, which are usedcommercially in Europe, and DPhP, BCHP, and

Ca IHBP, which are of negligible commercial usage..0 . .1.5 C Portrays the lack of estrogenic activity observed

in the yeast screen when the cells were incubatedwith certain phthalates. Abbreviations: BBP, butylbenzyl phthalate; DBP, dibutyl phthalate; DIBP,diisobutyl phthalate; DEHP, bis(2-ethylhexyl) phtha-

1.0 late; DIOP, diisodecyl phthalate; DINP, diisononylphthalate; DEP, diethyl phthalate; DTDP, ditridecylphthalate; DPhP, diphenyl phthalate; BCHP, butylcyclohexyl phthalate; IHBP, isohexylbenzyl phtha-

0.5 late; DHP, dihexyl phthalate; DIHP, diisohexyl10-12 10-11 10-1O io-9 10-8 10-7 10- i10-5 10-4 103 10-2 phthalate; DMP, dimethyl phthalate; DUP, diunde-

Molar concentration cyl phthalate.

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Articles - Estrogenicity of phthalate esters in vitro

with 125 pl yeast stock and incubating thisovernight at 28°C on an orbital shaker. Assaymedium contained 0.5 ml 10 mg/mlchlorophenol red-f-D-galactopyranosideadded to 50 ml growth medium seeded with1 ml of the above yeast culture.

All glassware was thoroughly washed withsolvent. Test chemicals were made up inethanol to 2 x 10-2 M (phthalates), 2 x 104M (4-nonylphenol, bisphenol A, genistein,o,p'-DDT), or 2 x 10-7 M (17p3-estradiol)stock solutions and stored at 40C.

Stock solutions were serially diluted inethanol, and 10 pl of each dilution was trans-ferred to a 96-well microtiter plate(Linbro/Titertek, ICN FLOW, Bucks,U.K). This gave a final concentration of 10-3M to 4.8 x 10-7 M for the phthalates andtheir metabolites, 10-5M to 4.8 x 10-9M forother xenoestrogens, or 10-8M to 4.8 x 10-12M for 170-estradiol. Solvent controls wereset up on each plate using 10-pl aliquots ofethanol. The ethanol was allowed to evapo-rate and 200-pl aliquots of assay medium(containing the yeast) was then added to eachwell. The plates were then sealed with auto-clave tape, shaken for 2 min on a titer-plateshaker, and incubated at 320C for 4-6 daysin a naturally ventilated oven (WTIB binder,BD-series; Jencons Scentific Ltd.,Bedfordshire, U.K.). Plates were shaken onday 1 of incubation and again approximately1 hr before taking absorbance readings (540nm for color and 620 nm for turbidity),using a Titertek Multiskan MCC/340 platereader (Life Sciences Int., Basingstoke, U.K).

Mammalian cells. For comparison, theproliferative effects of all commercially avail-able phthalates showing estrogenic activity inthe recombinant yeast screen, as well as thosethat were negative but of major volume use,were tested using two estrogen-responsivehuman breast cancer cell lines, MCF-7 andZR-75. As these cell lines are of human ori-gin, they may be of particular relevance whenconsidering the wide exposure of humans tothe phthalates, which are ubiquitous in theenvironment (25) and can be found in suchdomestic products as vinyl flooring, chil-dren's toys, printing inks, and cosmetics (26).

The phthalate samples used in theseassays were the analytical standards as sup-plied by Greyhound Chemservice. Cellswere cultured in phenol red-free mediumcontaining 5% v/v charcoal dextran strippedserum (DCC). They were then plated in 6-well microtiter plates (Falcon, BectonDickinson, Lincoln Park, NJ) into the afore-mentioned medium 3-4 days prior to com-mencing the experiment. For the MCF-7cells, medium was replaced with treatedmedium containing either 0.1% vehide sol-vent (ethanol) as a negative control, 10-8 M17,-estradiol as a positive control, or 10-5

M of each respective phthalate. Cells weretrypsinized and counted using a CoulterCounter (Coulter Electronics, Harpenden,Herts, U.K.) on days 0, 2, 5, 8, and 12.Treatments were duplicated and the experi-ment was repeated twice. For the ZR75cells, the treatments (control, 10-8 M, 10-10M, and 10-12 M 17P-estradiol and 10-5 M,10-6 M, and 10-7 M of individual phtha-lates) were done in triplicate. Cells werecounted at a single endpoint on day 11.

ResultsTable 1 lists the phthalate esters tested,together with their consumption figures in

Europe, to give an idea of their impor-tance relative to one another as industrialchemicals. Some phthalates generated adose-dependent increase in 0-galactosidaseproduction in the yeast screen, indicatingweak estrogenic activity.

In order to relate the significance of theactivity of the estrogenic phthalates to thatof other environmental estrogens, weassessed the response of the yeast screen toa range of environmental estrogens. Thechemicals tested were bisphenol A (anantioxidant), genistein (a phytoestrogen),4-nonylphenol (the degradation product ofa surfactant), and o,p'-DDT (a pesticide);

Table 2. Phthalates found to give an estrogenic response in the yeast screen

Chemical

173-Estradiol

Diethyl phthalate

Dibutyl phthalate

Diisobutyl phthalate

Butyl cyclohexyl phthalate

Butyl benzyl phthalate

Diphenyl phthalate

Isohexylbenzyl phthalate

Diisononyl phthalate

Ditridecyl phthalate

StructureOH

o

Ho0

110

,c,

0

0

oco0

110

c

011oco

6°0

0

0

lo . *o0

co

Approximatepotencya,b

0.0000005

0.0000001

0.0000001

0.000001

0.0000001

Maximumresponse (%)b

100

30

35

30

20

50

10

20

15

95

Potency and response relative to 17,-estradiol were calculated from data obtained on day 6 of the assay.Longer incubation times can increase the relative maximum response; thus, the values shown here applyonly to a specific set of conditions."lndicates the value was calculated at 25% of the maximum response.bAll data shown here were obtained using analytical standards.*Alkyl chains are composed of various branched isomers.

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*

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the results are shown in Figure 1. Thesechemicals were tested over a concentrationrange of 10-5 M to 5 x 10-9 M, and werefound to have potencies varying fromapproximately 104-105 times less than thatof the main natural estrogen, 17,-estradiol.

The estrogenic activities of the majorvolume usage phthalates (those exceeding20,000 ton/annum in Europe) in the yeastscreen are shown in Figure 2A. Of these sixmajor volume use phthalates, three pos-sessed estrogenic activity (BBP, DBP, and

DIBP), two didand one (DINP)the screen. The fthe most activelatter three are tindustry. Two (

produced for DIproducible behaiyeast screen. DImean responsewhich a detectaldase production

Figure 3. The activity of bisphenol A (rows A and B), o,p'-bisphenol A (ro%and H) in the recombinant yeast screen. Bisphenol A and the ortho-pariserially diluted (left to right) from 10-5 M. DTDP was serially diluted from 1C(10 pi ethanol added to each of these wells).

3.0

2.5

E

a

=ce

0.0

2.0

1.5

1.0

0.5

10-12 10>l o10-1 lo-,9 o la con-e r io-6

Molar concentration

Figure 4. Development of the butyl benzyl phthalate (BBP) standard curvcurve can be seen to be developing to an almost maximal response in this

I not (DEHP and DIDP), was reproduced in three separate assays, butbehaved unreproducibly in differed in a further three in which DINPformer three phthalates were appeared completely inactive (DINP i).of all those tested, and the The phthalates of relatively low or neg-he most extensively used in ligible use in Europe (29 different ones)dose-response curves were were assessed for estrogenic activity usingNP due to the slighdly unre- the yeast screen only. Relatively few ofvior of this chemical in the these (five in total) possessed any estrogenic[NP ii (Fig. 2A) shows the activity; all others were inactive, even at theof two standard curves in highest concentration tested (10-3 M) (Fig.ble increase in ,-galactosi- 2B, 2C). The results obtained from the fivewas observed. This pattern phthalates that showed estrogenic activity

are illustrated in Figure 2B. Of these, only* n 4 ea-i.. ....-i..two(DEP and DTDP) are used commer-

cially in Europe.All of the phthalates that showed activity

were very weak estrogens. The most potent,BBP, was approximately 1 million-fold lesspotent than estradiol (Table 2), making it

_-w N considerably less potent than other environ-- S 0 mental estrogens such as bisphenol A,

(8g_f_ @ i ; nonylphenol, and o,p'-DDT. When chemi-cals are so weakly estrogenic, it is entirely

- |i;i ti |XWrfeasiblethat it is not the chemical (in thiscase the phthalate) itself which is intrinsical-ly estrogenic, but rather that an impurity inthe chemical is estrogenic. Thus, beforelabeling a chemical as a weak estrogen, it isnecessary to exdude the possibility that thechemical is contaminated with an estrogenicimpurity. One way to address this issue is totest a number of samples, of different origin,of each phthalate possessing estrogenic activ-ity. If all samples of a phthalate possess thesame degree of estrogenic activity, it is likely

ws D and E), and DTDP (rows G that that particular phthalate is intrinsically-aisomer of this chemical were active, whereas if the different samples of al- M. Rows C and F are controls phthalate possess considerably different

potencies, it is then likely that the phthalateitself is not estrogenic, but that some sam-ples contain varying proportions of one ormore contaminants that are estrogenic.

3 __10To assess this possibility-that estrogeniccontaminants might be present in somephthalates-commercial preparations of allthe major volume usage phthalates, includingDTDP and DEP, were assessed for estrogenicactivity and their potencies compared to thatof their respective analytical standards (datanot shown). With one exception, no differ-ences were observed; the estrogenic activities

_==------ of the commercial preparations were equiva-lent to those of their respective analyticalstandards. However, contrary to the analyti-cal standard, the commercial preparation ofDTDP failed to produce a response, evenwhen present at 10-3 M. Both samples ofDTDP were subsequently analyzed by gelchromatography-mass spectrometry (GC-MS). The analytical standard (the active sam-ple) was found to contain 0.5% of the ortho-isomer of bisphenol A. The inactive prepara-

re over time. The BBP standard tion ofDTDP did not contain this chemical.yeast assay. A sample of o,p'-bisphenol A was then

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Articles - Estrogenicity of phthalate esters in vitro

obtained and its response in the yeast screenwas compared with that of the active DTDPsample. Figure 3 shows that o,p'-bisphenol Awas about 100 times more potent thanDTDP. Therefore, the presence of this chem-ical at just 0.5% in the DTDP sample wouldproduce a response equivalent to that seen.Thus, it is likely that this chemical (o,p'-bisphenol A) was responsible for the weakactivity observed in this phthalate sample (seeFig. 1); hence DTDP is not estrogenic.

The results shown in Figure 2A and 2Bshow that most of the active phthalates wereunable to produce a maximal response in theyeast assay; only DTDP did so. For example,the response to BBP (the most estrogenicphthalate) reached a plateau at approximately50% of the maximum response achieved with17,B-estradiol. To determine whether thismeans that most of the phthalates are onlypartial estrogen agonists or whether otherexplanations account for the submaximalresponses observed, a yeast screen containingBBP was incubated for longer than usual andthe response was monitored daily. The results(Fig. 4) show that on day 4 (the usual incuba-tion time for our yeast assays) BBP produceda shallow dose-response curve. However, byday 6, the response was greater. By day 13 thehighest concentration ofBBP had produced amaximal response. Note also that thedose-response curve to 1713-estradiol movedapproximately fourfold to the left betweendays 4 and 13 (i.e., the yeast screen becamemore sensitive), but the dose-response curvefor BBP moved considerably further. Thus,the potency of BBP increased somewhat withtime. For this reason, all the other phthalatedata shown in this paper was obtained fromyeast assays incubated for 6 days.

To assess whether the estrogenicresponses observed in the yeast assay werereproducible in other estrogen assays, activephthalates (plus the major volume usephthalates, DEHP and DIDP) were alsotested for their ability to stimulate prolifera-tion of MCF-7 and ZR-75 cells. The resultsfrom these assays (Fig. 5 and Fig. 6), whichare based on human breast cancer cell lines,are mostly comparable to those obtainedfrom the yeast screen. However, DEP andDTDP failed to induce proliferation of ZR-75 cells at 10-5, 10-6, or 10-7 M (Fig. SB)although they had been active in the yeastscreen, albeit only at higher concentrations.Using the ZR-75 cells, DINP at 10-5,10-6,and 10-7 M induced proliferation to a sig-nificantly greater extent than the control,which is in contrast to our findings for thischemical using the yeast screen. Growthcurves for all estrogenic phthalates (i.e.,those active in the yeast assay) and forDEHP and DIDP were obtained usingMCF-7 cells. The results (Fig. 6) showed

that BBP was considerably more mitogenicthan any of the other phthalates. DTDP,DIBP, and DBP were approximately equiv-alent in activity, and all the other phthalatestested showed relatively little activity. Allthese results are consistent with thoseobtained using the yeast assay.

Possible additive or synergistic effectsbetween the most potent phthalates wereinvestigated by incubating known concentra-tions of BBP, DBP, and 17[B-estradiol eitherindividually or as simple mixtures in the yeastscreen. The concentration of 170-estradiolused produced only a small response abovebackground (Fig. 7), so that if additivity orsynergism occurred, they could be observed

within the range of the assay. Two concentra-tions of each of the most active phthalates(BBP and DBP) were tested alone and incombination with 17p-estradiol. In all cases,the response obtained was very close to thatexpected if additivity had occurred (Fig. 7); inno case was the response significantly greaterthan predicted if additivity had occurred, thatis, no evidence ofsynergism was observed.

The phthalate metabolites testedincluded 1) derivatives of the most abun-dant phthalate (DEHP), namely MEHPand metabolites V, VI, and IX (23); 2)MBzP and MBuP, which are primarymetabolites of the most estrogenic phtha-late (BBP); and 3) MHP, MnOP, and

800,000

0.0E

-

C.

600,000

400,000

200,000

0

800,000

.0E==0i

t = 0 Control 10-8 1-10 lo-2 10-5 10-10-610-7 10-5 10- 10- 10-6 10-7

Treatmnent

600,000

400,000

200,000

0

500,000

400,000

b.

E

C

t=0 Control 10- lo-1 lo-10 10-5 10-6 107

Treatment

10.5 10-6 10-7 10-5 10- 10'-7

300,000

200,000

100,000

t=0 Control 108- 10-10 101-2 1o-, 10- 10-l

Treataent

Figure 5. The proliferation of ZR-75 cells incubated with various phthalates and controls including time = 0 (t= 0), ethanol, and 173-estradiol (data obtained from three separate assays). A) Cells incubated with butylbenzyl phthalate (BBP), diisobutyl phthalate (DIBP), and dibutyl phthalate (DBP). B) Cells incubated withdiisononyl phthalate; (DINP), diethyl phthalate (DEP), and ditridecyl phthalate (DTDP). C) Cells incubatedwith bis(2-ethylhexyl) phthalate (DEHP), diisodecyl phthalate (DIDP), and dihexyl phthalate (DHP). A simpleANOVA was performed on the data, followed by the Bonferroni/Dunn test for multiple comparisons. Cellnumbers significantly greater than the control are denoted by *p<0.05; **p<0.01; #p<J.0OJ.

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800,000

600,0W0

400,000

200,000

0

0 5

lime

Figure 6. This figure depicts the proliferation of MCF-Iethanol, or iO-5 M of bis(2-ethylhexyl) phthalate (DEHP),(DIBP), diisononyl phthalate (DINP), butyl benzyl phthallate (DEP), or ditridecyl phthalate (DTDP) over a perioda

1.00

E

a0

.0

*0

0

0.75

0.50

0.25

0.00

Control A B C D E

Figure 7. The activity observed in the yeast screen whetons of butyl benzyl phthalate (BBP), dibutyl phthalate (Dmixtures. Abbreviations: A, 10-11 M 17,-estradiol; B, 10-41Actual absorbance represents the corrected absorbyeast minus that of the control). Theoretical absorbanividual treatments added together (the absorbance thain an additive manner).

MPeP. All were serially diluted from 10-3M to 4.8 x 10-7 M, and none showed anysigns of estrogenic activity in the yeastscreen (data not shown).

Table 2 summarizes the relative poten-cy and the magnitude of the responses(compared to 17p-estradiol) of all phtha- '-

lates that were active in the yeast screen,together with their structures.

g _ iof which is for DEHP, at up to 500,000tons/annum in Western Europe. Theworldwide production of another class ofchemicals, the alkylphenol polyethoxylates,was 360,000 tons/annum in the late 1980s(27), which puts into perspective the largescale use of phthalate esters as industrialchemicals, as well as their potential envi-ronmental importance.

In terms of their estrogenic behavior, itseems that those phthalates requiring furtherscrutiny indude 1) the shorter chain phtha-

_-_ 1 l lates, namely BBP, DBP, and DIBP, whichare used by industry in smaller quantities(Table 1), but are more estrogenically active;and 2) the longer chain phthalate DINP,which although extremely weakly estrogenicin vitro, is used in large quantities (up to

i(days) 200,000 tons/annum in Europe). The estro-genic behavior of the phthalates in these

7 cells incubated with 10-8 M 17(3-estradiol, 0.1% assays compares favorably to that previously1, diisodecyl phthalate (DIDP), diisobutyl phthalate reported (9-11), where the potency of BBPlate (BBP), dibutyl phthalate (DBP), diethyl phtha- (approximately 1 millionfold less potent thanif 12 days. estradiol in the yeast screen) was similar to

that reported by Soto et al. (10,11) in the E-SCREEN assay (3 millionfold less potentthan estradiol) and the relative strengths of

BDBP W. the phthalates reported to be estrogenic by' #_.Actual absorbance _ Jobling et al. (9) correspond to that observed-nTheoretical absorbance ~in the yeast screen (BBP>DBP). It must also

be noted that, generally speaking, the activi-ties of the phthalates in the recombinantyeast screen were reproduced in the mam-malian assays, thus implying that these arereal estrogenic effects, and not artifactual.There were occasional discrepancies betweenassays: DTDP and DEP were not found tobe mitogenic in the ZR-75 cell line, but theyhad shown slight mitogenic activity in theMCF-7 assay and a positive response in therecombinant yeast screen. The yeast cells aremore robust than mammalian cells and socould be exposed to higher concentrations ofphthalates with no adverse effects, hence, theobservation of activity at the higher c-oncen-

A+1B A+C A+D A+E B+E C+E trations applied in the yeast screen. The rea-Treatment sons for discrepancy between the two mam-

malian assays are undear, but may be a resultn yeast cells were incubated with single concentra- Of the enhanced proliferation of the MCF-7)BP), and 17p-estradiol, either individually or in simple cell line*in the presence of growth factorsM BBP; C, 10-5M BBP; D, 104 M DBP; E, i0-5 M DBP. ( theidesenceof growth asance figure (the absorbance read for the treated (the identity of which iS not known), asce is the corrected absorbance of the relevant indi- compared to the ZR-75 cell line, which isit would be expected if the two chemicals behaved more estrogen specific.

All active chemicals, however potent, aresaid to be active because they cause a

Discussion ~ response above the baseline. However, forIn this paper, we investigate the possible all active phthalates, only a partial doseestrogenic behavior of a large number of response was observed after the usual incu-phthalate esters in vitro. As far as we are bation time. For example, for DINP, theaware, this is the first paper to address indi- most used of all the active phthalates, thevidual estrogenic potencies for such a compre- maximum response was just 15% of thehensive spectrum of this dass ofchemicals. maximum response obtained with 17,B-

The phthalates studied are used by estradiol. A possible explanation for resultsindustry in variable amounts, the greatest such as these, which suggest partial agonistic

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behavior of the phthalates, is that thesechemicals were not fully solubilized in thewater-based medium employed in theseassays. This is a situation frequently encoun-tered when applying highly organic com-pounds to in vitro assays and is entirely feasi-ble since, generally speaking, the solubilityvalues for phthalates are lower than the con-centrations used in these trials. Thus, it isplausible that some of the phthalates testedare actually more potent than they appear tobe. However, it must be noted that thechemical treatments were added to themedium of the mammalian cell assays inethanol, thus leading to greater homogeneitythroughout, and still only a partial responsewas observed. Conversely, contamination ofa chemical with an estrogenic compoundcan imply a weak estrogenicity of the sub-stance in question when it is, in fact, thecontaminant that is generating the observedresponse and the chemical under investiga-tion is not estrogenically active. This phe-nomenon was detected in the case ofDTDP, where the weakly estrogenic prepa-ration was found to be contaminated withthe ortho-isomer of bisphenol A. Hence,caution must be applied when labeling achemical a weak estrogen, particularly if thechemical is not pure (which is usually thecase, especially with industrial chemicals).

It has been reported that there is a rela-tionship between the structure of a chemi-cal and its estrogenic behavior (28). Of thetotal number of phthalates tested in ourstudy, five possessed a secondary ring struc-ture (BBP, BCHP, DPhP, IHBP, DCHP);of these, the first four were all weakly estro-genic, albeit with varying potencies.However, by no means was this the key toestrogenicity. Of those considered to beestrogenically active, there were several thatpossessed alkyl side-chains, and of these, agreater maximum response was obtainedwith DBP, DIBP, and DEP than by thosewith a secondary ring structure. It appearedthat the majority of the active phthalateswere among the lower molecular weightspecies, but again there were inconsisten-cies with this observation, with many ofthe lighter phthalates being inactive in therecombinant yeast screen. It is thereforedifficult to deduce, from their two-dimen-sional structures alone, which phthalateesters will elicit estrogenic responses.

If a chemical exhibits only weak estro-genic activity in vitro, it does not necessarilyfollow that the effect of the same chemicalwill be insignificant when applied to awhole organism. Unfortunately, results ofthe nature obtained here cannot be directlyextrapolated to an in vivo situation. It is notknown at present whether any phthalatesare estrogenic in vivo, and it will be neces-

sary to test these chemicals in vivo via dif-ferent routes of exposure before reachingconclusions. Although in vitro assays give usan idea of the potential strength of a chemi-cal as a xenoestrogen, they cannot simulatechanges to the chemical within an organismand differences in the systems of individualorganisms, which may influence the poten-cy and/or bioavailability of the chemical.Metabolic processes will vary greatly,depending on the route of uptake and onthe characteristics of both the chemical andthe organism concerned.

Another difficulty in estimating theenvironmental hazard posed by phthalateesters is the lack of data documenting theexposure of humans or wildlife to thesechemicals. The fact that phthalates are usedin a wide variety of extensively used goodsis indisputable. It is also known that theycan exude from these products. For exam-ple, DBP has been found to leach fromdentures (29), as has DINP from milk tub-ing (30). Furtmann (31) has suggested thatthe main source of phthalates are the con-sumer products themselves and that there issome justification in the inference that, fol-lowing dumping or incineration of theseproducts, there are considerable phthalateemissions into the environment. The esti-mated total loss of phthalate esters inWestern Europe has been put at 7,740tons/annum, or approximately 1% of totalconsumption (32). However, the use ofsuch data in the analysis of environmentalhazard assessment for individual chemicalsis problematic because the data is general-ized and estimates refer to total phthalates.

By far the most frequently reportedphthalate, and that found at highest concen-trations in the environment, is DEHP. Thisis to be expected, considering its high usageand greater likelihood of persistence relativeto the shorter chain phthalates. For this rea-son, one would also expect DIDP andDINP to be apparent in environmentalsamples, but reports concerning thesephthalates are sparse. Other phthalates thathave been regularly documented in food(19,20), air (33,34), sediments (31,35), andriver water (36,37) include the lower molec-ular weight phthalates such as DMP, DEP,DBP, and BBP. These are less stable as plas-ticizers and are therefore liable to migratefrom a polymer matrix, particularly whenthis material is subjected to elevated temper-ature or surrounded by a lipophilic medium.For this reason, despite lower consumptionof these phthalates compared to the highermolecular weight species, it is perhaps notsurprising for them to be commonly detect-ed, albeit at very low concentrations, inenvironmental samples. The solubility andenvironmental persistence of individual

phthalates is somewhat dependent upon thechain length of the phthalate concerned.[For a more detailed discussion of thebehavior of phthalates in the aquatic envi-ronment, see Furtmann (31)]. It must alsobe considered that these chemicals are notpresent in isolation in environmental sys-tems. In any one system, various mixtures oftoxic organic chemicals can be found. Forexample, a cocktail of trace organics wasdocumented in alligator eggs in LakeApopka, Florida (38). Phthalates themselveshave been found in environmental samplesalongside polychlorinated biphenyls, p,p'-DDT, and p,p'-DDE (34). Certain of thePCB congeners, for example 3,4,3',4'-tetra-chlorobiphenyl, have been identified asestrogen mimics (39), whereas p,p'-DDTand p,p'-DDE have both been reported topossess antiandrogenic properties (40). Inaddition, various combinations of phthalateshave been found to be present in environ-mental samples (37,41). With the possibilitythat any contaminated environmental sam-ple will contain more than one endocrinedisrupting chemical, it seems necessary toinvestigate whether the effect of a combina-tion of these chemicals will be additive,more than additive, or antagonistic. Thisissue was addressed by incubating simplecombinations of 17p-estradiol and twoestrogenic phthalates (BBP and DBP) in therecombinant yeast screen. Jobling et al. (9)found DBP and BBP, in the presence of170-estradiol, to have an agonistic, asopposed to antagonistic, effect on the stimu-lation of transcriptional activity in transfect-ed MCF-7 cells. We demonstrate in thispaper that the activity of combinations oftwo phthalates, DBP and BBP, at the con-centrations shown (Fig. 7) are, in fact,slightly less than additive. When thesechemicals were incubated in the presence of17p-estradiol (with BBP at a concentrationthat would induce a less than maximalresponse), the behavior of the combinationwas again additive rather than synergistic.

Another factor influencing the occur-rence of phthalates in the environment istheir potential for persisting and accumulat-ing in organic matrices. This would beexpected to be high because phthalates arehydrophobic chemicals; thus, it might bepossible to predict their environmental fatepattern based on that of other man-madeorganic chemicals. For example, the poly-chlorinated biphenyls (42) and 4-nonylphe-nol (43) bioaccumulate in organisms that areexposed to these chemicals over a period oftime, and they also biomagnify through thefood chain. However, phthalates appear tobe more readily metabolized than these per-sistent chemicals, particularly by enzymes inthe gut (44) and in sewage treatment works,

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although their rate of degradation doesappear to be influenced by the length of theirside chains (45,46). It is not known whetherthe yeast strain employed in the assays shownin this paper is capable of metabolizing com-plex organic chemicals, although methoxy-chlor has shown a positive response in therecombinant yeast screen (47); and it hasbeen reported that this chemical must bemetabolized before it becomes estrogenicallyactive (48), thus suggesting that the yeaststrain is capable of degrading certain organicchemicals. A small number of phthalatemetabolites were tested in the recombinantyeast screen, including monobutyl phthalate(the primary metabolite of DBP and DIBP)and monobenzyl phthalate (which, withmonobutyl phthalate, are the primarymetabolites of BBP). All metabolites testedwere inactive in this assay, suggesting that itis the parent compounds which are estro-genic. This is significant in that, as previous-ly discussed, the phthalates appear to bemetabolized following oral exposure, andhence the monoesters are more likely to bethe bioavailable form of phthalates.

It is conceivable that the route of expo-sure of an organism to phthalates is animportant parameter when consideringmetabolism of these chemicals in vivo. Itseems probable that phthalates are readilymetabolized in the gut, such that oral expo-sure would not lead to accumulation ofhigh concentrations of these chemicals.However, there is little data available on themetabolism of this group of chemicals fol-lowing inhalation or dermal exposure. It isperhaps necessary to investigate the fate ofphthalates within an organism followingadministration via these routes, judging bythe presence of these chemicals in a widearray of contact media. In addition, uptakevia the gills, hence directly into the bloodsystem, as occurs in fish, may elicit respons-es that other routes of exposure would not.

In summary, a small number of com-mercially available phthalate esters (BBP,DIBP, DBP, DEP, DINP) are capable ofacting as extremely weak estrogens in vitro.How this is relevant to the environmentcannot yet be directly estimated, partlybecause comprehensive data concerning theenvironmental fate and behavior of theseindividual phthalates is not available andpartly due to the impracticalities involvedwith extrapolating in vitro data to a wholeanimal situation. The phthalate most wide-ly used by the plastic industry, and thatreported on with greatest frequency, isDEHP. This phthalate did not show estro-genic activity in the assays employed in thispaper. Laboratory biodegradation studies,particularly of the shorter chain phthalates(that is, those which are the more potent

xenoestrogens), might imply that concen-trations in the environment as a whole, andwithin an organism, would not reach valueshigh enough to be of significant danger.Although the potential exists for the above-mentioned chemicals to generate adverseeffects when present within the system of anorganism, the concentrations and the con-ditions of exposure required to do so areunknown. Also note that this paper hasinvestigated one mechanism of action only,that is, the ability of phthalates to act asestrogen agonists. This may be just one ofmany pathways that might lead to adversereproductive effects in animals exposed tothese chemicals. The results of in vivoexperiments, such as those reported bySharpe et al. (12) and Wine et al. (13), maynot be due solely to the weak estrogenicactivities of the particular phthalates admin-istered, but may involve other, and possiblymore important, mechanisms of action. Forexample, DEHP has been recognized formany years to be a reproductive toxicant(49-52), yet this particular phthalatedemonstrated no estrogenic behavior in theassays employed in this study. It may alsotranspire that it is not simply a matter ofthe response of a parent organism to thechemical concerned, whether exposure isacute or chronic, but that any effect maynot be detected until subsequent genera-tions. This possibility has been very clearlydemonstrated by Wine et al. (13), whofound that adverse reproductive effectsinduced by DBP in Sprague-Dawley ratswere most pronounced in the second gener-ation although the mechanisms generatingthese responses are unknown.

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A a

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