a IX:AJINH

Medical Hypothesis: Xenoestrogens As PreventableCauses of Breast Cancer

WolffDevra Lee Davis, H. Leon Bradio Mary,Tracey Woodruff,' DavidG. Hoet,5and Hoda Anton-CUNW'Office of the Assistant Secretary for Health, Department of Health and Human Services,Washington, DC 20201 USA; 2Strang Cornell Cancer Research Laboratory, Cornell UniversityMedical Center, Ithaca, NY 10021 USA; 3Department of Environmental and CommunityMedicine, Mt. Sinai Medical Center, City University of New York, New York, NY 10029 USA;4Institute for Health Policy Research, University of California, San Francisco, CA 94109 USA;5Department of Biometrics and Epidemiology, Medical University of South Carolina, SC29425 USA; 6Epidemiology Program, Department of Medicine, University of California,Irvine, CA 92717 USA

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Experimental EvidenceA number of lines of evidence attest to theability of xenobiotic materials to affectestrogen production (9). Ovariectomy,which reduces endogenous hormones,inhibits the progression of chemicallyinduced mammary tumors, whereas rein-troduction of estrogen by implantationstimulates tumor development. Moreover,rat mammary cancers depend on both lateand early exposure to estrogen and pro-lactin (11).

Experimental studies indicate thatestradiol metabolism proceeds primarilyvia two mutually exclusive pathways, eachof which is affected by xenobiotic expo-sures: pathway I to 2-hydroxyestrone (2-OHEI), which has minimal estrogenicactivity and is nongenotoxic, or pathway IIto 16a-OHEI, a fully potent estrogenwhich is also genotoxic (12). Breast cancerrisk appears to be linked with these twopathways. Substances that elevate pathwayII or inhibit pathway I increase risk, where-as those that inhibit pathway II or elevatepathway I decrease risk. Thus, dimethylbenzanthracene (DMBA), benzo[a]pyrene(BaP), oncogenes, and tumor virus expo-sure inhibit pathway I and also inducemammary tumors (13). Dietary supple-ments that increase pathway I, such asindole-3-carbinol, also decrease mammarytumor incidence (14), but those thatinduce pathway II, such as alcohol, tend toincrease carcinogenic response (10,15). Inaddition to affecting pathway II, alcoholconsumption has been shown to induceP450IIE and decrease P4501A1, subfami-lies of enzymes key to the metabolism ofother potential carcinogens (15).

As to the role of dietary factors in estro-gen production, a number of studies indi-cate that both fat and fiber intake modu-lates estrogen metabolism. Linoleic andarachidonic acids increase pathway II (12).A high-fiber diet, along with greater physi-

Address correspondence to D.L. Davis, Office ofthe Assistant Secretary for Health, DHHS, 200Independence Avenue SW, Washington, DC20201 USA.Members of the Breast Cancer Prevention Col-laborative Work Group provided constructivecomments on the development of this hypothesisat meetings of the International Society forEnvironmental Epidemiology held at the NationalInstitute for Public Health in Cuernavaca, Mexico,August 1992, and at a workshop sponsored byNIEHS and Health and Welfare Canada, held atthe National Research Council, Washington, DC,December 1992. Support for this research was pro-vided to the authors by NIEHS, the NationalCancer Institute, and Health and Welfare Canada.Received 12 May 1993; accepted 28 June 1993.

Environmental Health Perspectives372

cal exercise, reduces enterohepatic resorp-tion of fecal estrogens (16). Severe caloricrestriction results in major increases inpathway I and reductions in pathway II(1X.A variety of in vitro and in vivo studies

have documented the estrogenicity ofsomelipophilic, bioconcentrated xenobiotics;that is, their ability to stimulate estrogenand function as xenoestrogens (Table 1).Estrogens promote breast cell proliferation(18) and hypertrophy of other secondarysex organs (19). Similar effects have beenobserved in fish, birds, and wildlife (9).Xenoestrogenic substances include severallipophilic, persistent compounds for whichhuman, food chain, and wildlife exposureshave been widespread (20). These chemi-cals include a number of chlorinatedorganics, such as o,p'-DDT, an isomer ofthe pesticide DDT, chlordecone, hep-tachlor, and other pesticides, polychlori-nated biphenyls (PCBs), triazine herbicide,and a number of polycyclic aromatichydrocarbons (PAH), compounds derivedfrom petroleum products, fossil fuels, andproducts of incomplete combustion. Inaddition, some pharmaceutical agents suchas diethylstilbestrol (DES) are direct estro-gens, and others, such as tamoxifen, mimicestrogen in premenopausal women butblock estrogen in postmenopausal women(21). Still other pharmaceuticals, such ascimetidine, inhibit pathway I and stimu-late breast cell proliferation (22). Animalsexposed to ambient levels of commonlyencountered compounds one at a time,such as Aroclor 1254, a PCB, showed noadverse effects. But animals dosed withmixtures of ambient levels of commonlyencountered groundwater contaminants,including PCBs and ubiquitous viruses,developed a variety of adverse effects ongrowth and reproduction (23).

A number of xenoestrogenic com-pounds induce or promote breast cancerexperimentally (24). Primary carcinogene-sis may also occur with other xenobioticsbecause human breast epithelial and fibro-blastic cells metabolize them to carcino-genic electrophiles (25). Table 2 summa-rizes the research on mammary carcino-genicity of several important, widely usedtoxic substances. These indude o,p '-DDT,some isomers of PCB, benzene, and severalPAHs, including methylcholanthrene,dibenz[ah]anthracence, and DMBA (26-28).PAHs produce increased intracellular oxi-dation and chromosomal breaks or generearrangements (29).

Atrazine is of special interest, as it is atriazine herbicide that is one of the mostcommonly used pesticides in the UnitedStates today, and it is widely found ingroundwater (30). Atrazine is a stable com-pound that is retained in the abdominal fat

Table 1. Experimental evidence on the estrogenicity of some chlorinated organics

Chemicalo,p-DDT

o,p'-DDT

Methoxychlor

Chlordecone(kepone)

B-Hexachloro-cyclohexane

PCBs

Kepone(chlordecone),heptachlor,chlordane,Aroclor 1221

Atrazine

In vivo evidenceInitiated implantationand maintainedpregnancy in rats

Uterotropic (causesincreased uterineweight) in rats

Initated implantationand maintainedpregnancy in rats

Initiated implantationand maintainedpregnancy in rats

ReferenceJohnson et al.,1992 (67)

In vitro evidenceInhibits the bindingof [3HJestradiol torat uterinecytosolic estrogenreceptor

Welch et al., Inhibits the binding1969 (69) of 13HJestradiol to

rat uterine cytosolicestrogen receptor

Johnson et al.,1992 (67)

Johnson et al.,1992 (67)

Induction of cystosolicprogesterone receptor;redistribution ofestrogen receptors

Estrogen receptorbinding to probesEnhanced proliferativepotency in humanbreast estrogen-sensitive MCF7 cells

Hormone releaseincreased

ReferenceNelson,1974 (68)

Nelson,1974 (68)

Coosen andVan Velsen,1989(70)

Korach et al.,1988 (71)Soto et al.,1992 (19)

Ghinea et al.,1988 (72)

Table 2 Experimental evidence on mammary carcinogenesis of some chlorinated organics

Chemical Animal evidence ReferenceOrganochlorinesDDT Accelerator of mammary Scribner and

tumors in male mice treated Mottet, 1981 (73)with 2-acetamidophenanthrene

Triazines Increased incidence of mammary Pinter et al.,Atrazine tumors in male rats 1990 (32)

(750 ppm for 126 weeks)Benzene Breast cancer, oral and respiratory routes Maltoni et al.,

1989 (74)Polycyclic aromatic hydrocarbonsBenzola]pyrene Mammary tumors, gastrointestinal route Huggins and Yang,

1962 (75)Dibenz[ahlanthracene Mammary tumors, gastrointestinal route Snell and Steard,

1962 (76)

of chickens for a week following 1 week offeeding (31). It induces exceptionally rarebreast tumors in male animals and alsoproduces reproductive organ tumors (32)and endocrinological effects.

Epidemiologic EvidenceTwo recent epidemiologic studies havehighlighted the importance of pre-natalexposures to estrogen for breast cancer.One investigation found that women bornto mothers with pre-edampsia, and there-fore lower estrogen levels during pregnan-cy, had significandy reduced risks of devel-

oping breast cancer compared with con-trols. In this same analysis, women born tomothers with elevated estrogen duringpregnancy sustained an increased risk ofbreast cancer that was not statistically sig-nificant but may have biological impor-tance (33).A second study corroborates the role of

prenatal exposures to estrogen. Dizygotictwins, whose mothers generally have higherlevels of estrogens, have higher rates ofbreast cancer. Among all twins, the oddsratio associated with one of the twins beingmale (1.54) was higher than those associat-

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Table 3. Case-control studies on chlorinated organics and breast cancer

Matching; No. of cases/ Relative riskReference confounding controls Analytes (odds ratio)Albert et al. 1982 (77) None 8/7 DDT,a HCH,a dieldrin,(tumor vs. normal heptachlorepoxidemammary tissue)

Wassermann et al., None 9/5 DDT,a DDD,a PCB,-1976 (78) HCH, dieldrin,(tumor vs. normal heptachlorepoxidemammary tissue)

Unger et al., 1984 (48) No matching; age 14/21 DDE, PCB(biopsy)

Unger et al., 1984 (48) No matching; age 18/35 PCB(autopsy)

Falck et al., 1992 (49) No matching; age, 20/20 DDE,b DDT, PCB,bheight, weight, heptachlorepoxide,smoking oxychlordane,

trans-nonachlor,HCB, b-HCH

Mussalo-Rauhamaa No matching; age, 44/33 DDT, PCB, HCB,HCH,b 10.5 (>0.1 ppm)etal., 1990(50) parity, weight, heptachlorepoxide,

height, occupation, chlordane,smoking, fish meals trans-nonachlor, PAH

Wolff et al., 1993 (51) Age, menopausal 58/171 DDE,b PCB 4status, day of cycle, (1 Oth-90thenrollment date, body percentile)mass, age atmenarche, parity,family history, pastbenign disease,lactation, smoking,alcohol use, race

HCH, hexachlorocyclohexane; HCB, hexachlorobiphenyl.aAlbert et al. (77) and Wasserman et al. (78) found differences between malignant and normal breasttissue forthese residues.bAssociated with elevated risk.

ed with both twins being female (1.3) andwas highest in premenopausal women withtwin brothers (2.88) (34). This may be dueto the fact that developing, hormonallyaffected organs in sisters of twin brothersare exposed to relatively more circulatingprenatal estrogens than are twin sisters.Prenatal imprinting may prime estrogenreceptors for subsequent response or pro-vide irreversible programming of the distri-bution of cytochrome P450s and other ofthe terminal enzymes of the microsomaland mitochondrial mixed-function oxi-dase, electron transport chains.

Most of the known risk factors forbreast cancer, which at best account for30% of cases (2) are linked with total life-time exposure to reproductive hormones(35). Aside from genetics and radiation inpremenopausal breast cancer, these riskfactors include age, age at menarche,menopause, and first full-term pregnancy,total calorie intake, family history, height,radiation exposure, and alcohol intake(36). Artificial reduction of hormones byovariectomy before age 35 reduces humanbreast cancer risk (36).

Diet influences estrogen production invarious ways. A diet of proportionallyhigher total calories relative to body masscan also alter the metabolism of estrogen inthe gut and stimulate earlier onset ofmenses. However, obese, premenopausalwomen who are amenorrheic have reducedrisks of breast cancer, whereas obese post-menopausal women have higher risks, pre-sumably due to the ability of body fat toenhance production of estrogens aftermenopause when ovarian production ofestrogen diminishes rapidly (2). Severerestriction of caloric intake appears toreduce the risk of breast cancer (37), possi-bly by delaying the onset of menses or oth-erwise reducing total lifetime estrogenexposure.

The role of dietary fat for breast cancerremains the subject of a lively debate andinconsistent results. A meta-analysis of 12case-control studies reported that the riskof breast cancer increased with increasingdietary fat and included some studies withmore than a twofold difference (38).Others have found no such association(39).

Direct epidemiologic evidence linkingchlorinated organics to breast cancer rangesfrom suggestive geographic and cross-sec-tional mortality analyses to case-controlstudies. A recent analysis of chemical plantworkers found more than a twofold in-crease in breast cancer in female workersexposed to dioxin contamination (40).Elevated breast cancer rates were alsodetected in an analysis of women exposedto extensive environmental contaminationwith PCBs in Japan (41). In contrast,other studies have been negative (42,43).

Geographic and other large-scale analy-ses have suggested a link between breastcancer and PAH or chlorinated organiccompounds but have not involved anydirect measures of exposure (16). Signif-icantly elevated rates of breast cancer havebeen detected in one area of Minnesotawhere water was contaminated with PAHs(44,45). Women who work in the chemicalindustry (46) or who live near hazardouswaste sites are also reported to have higherrates of breast cancer (47). Another studyreported a temporal association between adecrease in population exposure to chlori-nated organics and a reduction in breastcancer mortality rates (16).

As Table 3 reveals, most of the negativeanalytic studies have involved fewer than10 cases and have had only about a 30%chance of detecting a twofold difference inrisk (48). Several more recent studies haveprovided evidence that chlorinated organ-ics are linked to breast cancer. In onestudy, the breast fat of women with cancerat biopsy had about 40% more of somechlorinated pesticides such as metabolitesof DDT and elevated levels of PCB (49).About 50% more hexachlorocyclohexane(50) was detected in pooled blood frombreast cancer cases compared to a pooledreference group, controlling for age andparity.

Further resolution of the role of chlori-nated organics comes from a well-designedstudy that matched on several factors andadjusted for a number of confounders.This recent nested case-control study of 58prospectively gathered cases was drawnfrom a cohort of 14,000 women. Womenat the 90th percentile of DDE in serumhad a fourfold greater risk of breast cancercompared to those at the 10th percentile(51). PCB levels also differed, but this dif-ference was not statistically significant.Two other case-control studies are alsogermane involving ovarian cancer, which isalso hormonally mediated. A hospital-based case-control study in Italy detected asignificant link between herbicides andovarian cancer but could not pinpoint spe-cific exposures (52). A population-basedcase-control study which provides bettercharacterization of exposure and controls

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for relevant reproductive risk factors notesthat women exposed to chlorinated organictriazine herbicides through farmwork for10 or more years have a relative risk of 2.7for ovarian cancer (53).

HypothesisA variety of experimental and human evi-dence indicates that the greater the totallifetime exposure to bioavailable estrogen,the greater the risk for breast cancer. Inlight of the pivotal role of estrogen, wehypothesize that exposure to some xenoe-strogens elevates endogenous hormone lev-els, especially 16x-OHE1, which stimulatebreast cell proliferation and thereby induceor promote breast cancer. Xenobiotic com-pounds could alter bioactive estrogen orprogesterone in breast cancer cases or oth-erwise induce mammary carcinogenesisthrough the P450 enzymes. Those withgenetic susceptibility to breast cancermight be exquisitely sensitive to the prolif-erative effects of xenoestrogenic substancesin the environment.

Xenoestrogenic compounds may workthrough several estrogen-related mecha-nisms or through other mechanisms unre-lated to estrogen (Fig. 1). Environmentalchemicals may be directly estrogenic, as iso,p'-DDT. Or they may alter estradiolmetabolism, or otherwise enhance the pro-duction of 16a-OHE1 (pathway II) orreduce the production of 2-OHEI (path-way I), as occurs with some drugs such ascimetidine. In addition, potentiating fac-tors such as alcohol and fat may affectestrogen hydroxylation. Other breast car-cinogens appear to operate completely out-side of estrogen pathways as direct carcino-gens. For example, fluoroscopic radiationinduces mammary carcinoma experimen-tally and in humans, probably by directstimulation of proliferating ductal cells ordirect mutation of stem cells.

Whatever the pathway to breast cancer,genetic factors are ultimately involved inthe expression of this highly heterogenousdisease. Younger women are more affectedby inherited genetic factors, as hereditarybreast cancer accounts for 36% of breastcancer cases ages 20-29, but only 1% ofcases over age 80 (54). In some familieswith the rare, autosomal dominant Li-Fraumeni syndrome (55), a high breastcancer risk is due to the inheritance of aspecific cancer-predisposing mutation onchromosome 17q2l (56,57). Results of arecent population study suggest that morethan 6.5 women per 1000 with breast can-cer are carriers of another inherited breastcancer-predisposing mutation in a series ofcodons on chromosome 17 (54).

As the proportion of inherited breastcancer in the population is small (58),most breast cancers result from acquired

mutations, possibly stemming from xenoe-strogens or from environmental carcino-gens that affect pathways other than estro-gen. This hypothesis suggests that it isimportant to study genetic-environmentalinteractions to identify environmental fac-tors that influence gene expression in thegreat majority of acquired mutations.

Two lines of evidence are pinpointingthe location of cancer-predisposing geneson chromosome 17 in both inherited andsomatic mutations for breast cancer: genelinkage studies in families with multipleaffected members and allelic compositionstudies comparing tumors with normal tis-sues for loss of heterozygosity (Fig. 1).Two genes have recently been implicatedin the development of hereditary breastcancer, the tumor-suppressor gene p53(56-60), which maps to chromosome17pl3.1, and the breast-cancer-suppressorgene BRCA1 (61), which was originallyidentified by linkage to the anonymousDNA marker D17S74 at 17q21 (62) andappears to be involved in the largest pro-portion of inherited breast cancer. Fornoninherited breast cancer, the role ofxenoestrogenic or xenobiotic exposures foroncogene activation or gene suppressionneeds to be carefully evaluated.

DiscussionHow can we explain the fact that the gen-eration of women who had their childrenearlier in life and had more of them nowhave higher rates of breast cancer? Ob-viously, known risk factors do not com-pletely account for breast cancer patternsin industrialized countries (4). The greatpreponderance of these factors, such asearly age of menses, late age of menopause,reproductive history, and caloric intake rel-ative to body weight, can be linked to total

lifetime exposure to bioavailable estrogen.We need to identify xenobiotic factors thatdirectly or indirectly increase estrogenexposure. A number of studies are suggest-ed by this theory. Investigations of popula-tions with differing rates of disease andxenoestrogen exposures will permit us totest this hypothesis, as will analyses ofarchived materials from varied populations.A major series of case-control studies incountries with differing breast cancer ratesand chemical exposures should furtherassess the role of xenoestrogenic materialsby assaying markers of exposure to suspectxenoestrogens and early markers of biolog-ic effect. Special attention needs to be paidto the mechanisms by which environmen-tal exposures, including xenoestrogens,might activate oncogenes or suppress othergenes, teasing out the interaction betweengenetic susceptibility and environmentalfactors. In addition, laboratory studies inhuman cell culture and in vivo systems ofsuspect xenoestrogenic materials should beexpanded to screen chemicals for estro-genicity and to identify mechanisms bywhich these substances could affect estro-gen metabolism or otherwise promote thedevelopment of breast cancer. Preliminaryin vitro work has found that several pesti-cides enhance the production of the geno-toxic pathway II, 16a-OHEI, and reducethe yield of the nongenotoxic pathway I, 2-OHEI.

The xenoestrogenic hypothesis offers apossible resolution to several anomalies inbreast cancer research, including someinconsistent results about dietary fat andbreast cancer. Fat per se may be less impor-tant than xenoestrogenic contaminants infat at different stages in women's lives. Asto the postmenopausal period, one recentstudy found that 4 years after treatment,

Figure 1. Hypothesized pathways to breast cancer. OHE1, hydroxyestrone; LOH, loss of heterozygosity.

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breast cancer recurred in postmenopausalwomen with positive estrogen-responsivereceptors who ate diets high in fat, total fatintake, and saturated and polyunsaturatedfats, as assessed at time of diagnosis (63).We need to determine whether bioaccu-mulated lipophilic, xenoestrogenic sub-stances, such as PAHs and chlorinatedorganics, are elevated in these women, aswell as whether saturated animal fats bio-concentrate higher levels of such com-pounds than do polyunsaturated vegetablefats.

This hypothesis also may account forone of the discrepancies in cancer that wasfirst noted by the distinguished Danishresearcher Clemmensen. Commonly re-ferred to as Clemmensen's hook, the dis-crepancy occurs in the relationship be-tween age and breast cancer incidence forseveral countries. Incidence of breast can-cer rises monotonically with age up toabout age 45, after which the rate ofincrease forms a hook and levels off ordeclines for about 10 years and thenresumes an increasing, but more modestslope (64). The ages of this plateau corre-spond to the period of perimenopause,when the ovaries begin to produce lessestrogen and progestin. It is tempting tospeculate that the renewed surge in breastcancer after menopause, especially in obesewomen, might be linked with xenoestro-gens and with the production of endoge-nous estrogens, which would be greatest inthose with proportionally more body fat.

A recent series of studies on the possi-ble link of environmental hormones withmale reproductive disorders bears mention(65). Reported disorders of the male repro-ductive tract, including reduced spermcounts and testicular cancer, have increasedover the past several decades (66). Couldprenatal exposures to xenoestrogens beinvolved? Evidence from a number offields points to the profound importanceof endogenous and xenobiotic materials forthe production of estrogen and other hor-mones. Future research will need to spandisciplines to discern the extent to whichhuman activities affect hormones through-out our lives.

With respect to breast cancer, most ofthe confirmed risk factors, which relate toreproductive behavior and dietary factors,are not easily changed by social policy.Many of the proposed interventions toreduce breast cancer involve the lifelonguse of pharmaceutical agents or the advo-cacy of radical changes in diet, lifestyle, oreven reproductive behavior. As to the latterpoint, a generation of women that hasstruggled long for reproductive freedom isunlikely to embrace suggestions that con-strain reproductive choices. Research topinpoint preventable causes of breast can-

cer is critically needed. If xenoestrogens doplay a role in breast cancer, reductions inexposure will provide an opportunity forprimary prevention of this growing disease.

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