Proc. Natl Acad. Sci. USAVol. 78, No. 10, pp. 6251-6255, October 1981Cell Biology

Metabolism of benzo[a]pyrene by human mammary epithelial cells:Toxicity and DNA adduct formation

(polycyclic aromatic hydrocarbons/carcinogenesis)

MARTHA R. STAMPFER*t, JAMES C. BARTHOLOMEWt, HELENE S. SMITH*t, AND JACK C. BARTLEY*§*Peralta Cancer Research Institute, Peralta Hospital, 3023 Summit Street, Oakland, California 94609; tDonner Laboratory and WMelvin Calvin Laboratory,Lawrence Berkeley Laboratory, University of Califomia, Berkeley, California 94720

Communicated by Melvin Calvin, June 8, 1981

ABSTRACT Pure cultures ofhuman breast epithelial cells andof fibroblastic cells in early passage provided the opportunity toask whether either cell type had the capability for metabolizingchemical carcinogens and, ifso, was the fate ofthe metabolic prod-ucts compatible with chemical carcinogens being a factor in theinitiation of breast cancer in women. For this purpose, cells wereexposed to benzo[a]pyrene (BaP), and (i) the influence on growthpotential and (ii) the extent, type, and persistence of adducts be-tween the metabolites of BaP and DNA were measured. Com-pared with fibroblasts, inhibition of growth by epithelial cells was50-100 times more sensitive to BaP. Because of this differentialsensitivity, epithelial cells were exposed to 0.4 FM BaP and fi-broblasts were exposed to 4.0 FM BaP in the studies of DNA ad-duct formation. Separation by high-pressure liquid chromatog-raphy of adducts between (±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP diol epoxide) and nucleosidesfrom purified DNA revealed that epithelial cells contained mod-ified DNA within 6 hr after adding BaP. Adducts between the 7Ranti stereoisomer of BaP diol epoxide and deoxyguanosine pre-dominated at all times. syn BaP diol epoxide adducts with deoxy-guanosine and what appeared to be BaP diol epoxide adducts withdeoxycytidine were consistently present but at much lower fre-quency. All three types of BaP diol epoxide-DNA adducts per-sisted in epithelial cells for 72 hr in BaP-free medium. No adductswere detected in fibroblastic cultures until 96 hr after first ex-posure to BaP. At this time, the type and extent of BaP diol epox-ide-DNA adduct formation was similar to that in epithelial cellsexposed to one-tenth the dose of BaP. The type, extent, rate offormation, and persistence of the adducts in human breast epi-thelial cells was similar to that in cells transformable by exposureto BaP, an indication that they may be targets for chemically in-duced carcinogenesis.

The ubiquitous presence of benzo[a]pyrene (BaP) as an envi-ronmental pollutant from the incomplete combustion of fossilfuels (1-3) and its potency as a mutagen (2) and carcinogen (3-9)after activation are well documented. The (±)-7,8-dihydroxy-9, 10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrenes (BaP-diol ep-oxides) are the set of metabolites most strongly linked to car-cinogenic transformation (3, 4). Of the four isomeric forms ofBaP diol epoxide, the 7/3,8a-dihydroxy-9a, lOa-epoxy-7,8,-9, 10-tetrahydrobenzo[a]pyrene (anti BaP diol epoxide), partic-ularly the (7R) or (+) enantiomer, is the most carcinogenic inrodent model systems (7, 8), and formation of adducts betweenthis metabolite and deoxyguanosine of DNA predominates incells susceptible to carcinogenic transformation (9, 10).

Most malignant tumors in humans are ofepithelial origin, butit has been difficult to study BaP metabolism and metabolitebinding to macromolecules in normal human epithelial cells

because of the inability to propagate this cell type in culture.Evidence of BaP utilization and macromolecular adduct for-mation in cultures of epithelial organs of human origin (e.g.,refs. 11-16) has been reported, but important questions remain:which cell type is responsible for formation of the putative ul-timate carcinogen; do other cell types present have a role inproducing the precursors of this carcinogen, and how is the cellat risk, the epithelial cell, affected by the metabolites of BaP?Studies ofBaP metabolism in epithelial cell lines of both humanand animal origin are useful in indicating possible metabolicfates ofBaP in various systems (e.g., refs. 10, 16-18). However,results from cell lines may not accurately reflect the metabolicproperties of normal cells. Furthermore, epithelial cells fromeach organ system and species may form a distinctly differentset of BaP metabolites with a different carcinogenic potential(18-20). Therefore, an accurate appraisal of the importance ofBaP and its metabolites in transformation of human cells mayrequire growth in culture ofpure populations of normal humanepithelial cells from the different organ systems.A system is now available for investigation ofBaP metabolism

in normal human mammary epithelial cells; significantly, thiscell is involved in more than 99% of all human breast cancer(21). Mammary epithelial cells from human donors can now begrown in pure cultures for several passages (22). Pure culturesof mammary fibroblasts can also be obtained from the samespecimens, permitting a comparison of the fate of BaP in thesetwo mammary cell types. The present report focuses on theeffect of BaP on cell growth and the extent and type of DNAadducts formed in each type of breast cell.

MATERIALS AND METHODS

Chemicals. BaP, generally labeled with 3H (19-40 Ci/mmol; 1 Ci = 3.7 x 1010 becquerels), was purchased fromAmersham, and unlabeled BaP was from Aldrich. Both sub-strates were checked for purity by high-pressure liquid chro-matography (HPLC) prior to each use. Solvents for HPLC andfor isolation of DNA adducts were Omnisolve grade fromMatheson, Coleman and Bell. Standard adducts between BaPdiol epoxide and deoxycytidine, deoxyguanosine, and deoxy-adenosine were formed by reacting calf thymus DNA withenough racemic BaP diol epoxide to yield one adduct per 50base pairs (bp) (23). The DNA was digested, and nucleosideswith BaP diol epoxide adducts were purified by HPLC.

Abbreviations: BaP, benzo[a]pyrene; BaP diol epoxide, (+)-7,8-dihy-droxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; anti BaP diolepoxide, (+)-7,B,8a-dihydroxy-9a, lOa-epoxy-7,8,9, 10-tetrahydro-benzo[a]pyrene; syn BaP diol epoxide, (+)-7f3,8a-dihydroxy-9,8,10f-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; HPLC, high-pressure liq-uid chromatography; bp, base pair(s).§ To whom reprint requests should be addressed.

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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6252 Cell Biology: Stampfer et aL

Media and serum for cell culture were purchased fromGIBCO, and hormones and growth factors were from Sigma.

Cell Culture. The mammary epithelial cells and fibroblasticcells were isolated as described (22). Fibroblastic cells weregrown in a basal medium containing Ham's F-12 medium andDulbecco's modification of Eagle's medium, 1:1 (vol/vol), with5% (vol/vol) fetal calf serum and insulin (10 ,Lg/ml). The epi-thelial cells were grown in an enriched medium, designatedMM, containing 30% Ham's F-12 medium, 30% Dulbecco'smodified Eagle's medium, 1% fetal calf serum, 39% (vol/vol)conditioned medium from human epithelial cell lines, and sev-eral hormones and growth factors (22, 24). Primary cultures ofepithelial cells were initiated and subcultured as described (22).The mammary epithelial cells have been characterized by

their ultrastructural properties, dome formation, and the pres-ence of antigens specific for human mammary epithelial cells(22, 25). Examination of cells derived from one of the normalspecimens has shown a normal diploid chromosome numberand structure (S. Wolman, personal communication). On thebasis of the fibronectin pattern (24), the second- and third-pas-sage epithelial cell populations used in these experiments were98% pure; the remaining cells present had the morphologicalappearance ofmyoepithelial cells (22, 24). The fibroblastic cells,used at fourth or fifth passage, were free of epithelial cells be-cause epithelial cells are incapable of sustained growth in thebasal medium.

Inhibition of Cell Growth. The effect of BaP on cell growthwas determined by exposing duplicate T-25 flasks (Corning)containing sparse cultures of cells to various concentrations ofBaP dissolved in dimethyl sulfoxide (final concentration, 0.1).Control cultures received dimethyl sulfoxide alone. At the ini-tiation of each experiment, the number of viable cells in du-plicate flasks was determined by counting cells capable of ex-cluding trypan blue in a hemocytometer. After exposure to BaPfor 48 hr. the cells were refed with fresh medium without BaP.Cells were refed every 48 hr thereafter. The control cells ofbothtypes became confluent after 2-3 population doublings at 5-7days after initial exposure to BaP. At this time the number ofviable cells was determined on the basis of dye-excluding cells.

Isolation of BaP Adducts of DNA. Subconfluent cultures inT-75 flasks (Corning) were exposed to [3H]BaP at concentra-tions of 0.4 ,uM (specific activity, 19 Ci/mmol) for epithelialcells and 4.0 ,uM (specific activity, 8 Ci/mmol) for fibroblasts.At the times indicated, medium was collected, and the cellswere washed twice with BaP-free medium and harvested bytrypsinization. To evaluate the persistence ofDNA adducts, cellcultures exposed to [3H]BaP for 24 hr were washed twice withBaP-free medium and then grown for an additional 72 hr in theabsence ofBaP with a medium change 24 hr before harvesting.The DNA from the harvested cells was isolated, purified, and

digested to constituent nucleosides as described (23). Nucleo-sides covalently bound to BaP metabolites were isolated fromfree nucleosides by elution of a Sephadex LH 20 column withwater and then with methanol. The material eluting with meth-anol was separated into individual derivatized nucleosides byHPLC (23). The extent ofadduct formation is expressed as pmolof BaP converted to material associated with specific bases.DNA concentration was determined by absorption at 260 nm.

Assay ofBaP in Media. The amount ofBaP remaining in themedia was determined by extracting a portion of each mediumthree times with ethyl acetate/acetone, 2:1 (vol/vol), containinga-tocopherol or hydroquinone as antioxidant and assaying theradioactivity ofthe BaP fraction isolated by HPLC. The originalmedia also were analyzed to ensure that the added radioactivityeluted in the BaP fraction from HPLC.HPLC. A Varian chromatograph, model number 5000, fitted

with an Altex ultrasphere column (250 x 4.6 mm) of octade-

cyltrimethoxysilane was used. For separation of adducts, thecolumn was eluted isocratically with 52% (vol/vol) methanol atroom temperature for 80 min and then linearly, increasing themethanol gradient to 100% methanol over the next 20 min. Theflow rate was 0.7 mlmin, and fractions were collected every60 sec throughout the run for determination ofradioactivity. Forisolation and assay of [3H]BaP in the medium, the column waseluted with a linearly increasing gradient of methanol (60-100%) for an hour with the same flow rate, detection system,and fraction collection protocol as with the adduct elutions.

RESULTSInhibition of Cell Growth. The epithelial cells derived from

four reduction mammoplasty specimens experienced growthinhibition after a 48-hr exposure to 0.04 ,ug of BaP per ml,whereas the growth of fibroblasts from the same donors showedinitial sensitivity to BaP at concentrations 20-50 times higher(Fig. 1). At concentrations above 1.0 pug of BaP per ml, the ep-ithelial cells could be observed to undergo one round of rep-lication after addition of BaP, followed by a complete cessationof mitotic activity. These cells appeared elongated and granular(Fig. 2b) compared to the untreated control cells (Fig. 2a). Atlower concentrations, the epithelial cells continued to dividebut at a reduced rate compared to controls. In contrast, the fi-broblastic cells (Fig. 2d) showed no obvious morphologicalchange even after exposure to 10 1kg of BaP per ml (Fig. 2 cand d), although growth was measurably inhibited at this con-centration (Fig. 1). All the treated fibroblastic cultures contin-ued to divide and reach confluence.DNA Adduct Formation. Separation on HPLC of the BaP

diol epoxide adducts of DNA nucleosides isolated from bothepithelial and fibroblastic cells revealed that the major nucleo-side involved in adduct formation eluted with standards derivedfrom deoxyguanosine and anti BaP diol epoxide (Fig. 3). A sub-sequent smaller peak coeluted with the deoxyguanosine adductof syn BaP diol epoxide. BaP diol epoxide adducts with deoxy-cytidine elute just prior to its adducts with deoxyguanosine(refs. 9 and 23; Fig. 3); therefore, this peak has tentatively beengiven that designation. No evidence of adducts between BaP

100

' 80

~o 600

, 400

Car,or

20

0.1 1.0 10

log BaP concentration, pg/mlFIG. 1. Effect of BaP on growth of human mammary cells. Flasks

(T-25) were seeded with 2-3 x 10i cells and incubated for 2-3 days.Duplicate cultures were trypsinized, and viable cells were counted.Fresh medium with BaP was added to the remaining flasks for an ad-ditional 48 hr. Cultures were refed with fresh medium, and cells wereallowed to grow until control cultures (dimethyl sulfoxide alone) be-came confluent (5-7 days). At this time, duplicate cultures at eachdrug concentration were counted in a hemocytometer for trypan blue-excluding cells. -, Epithelial cells; ----, fibroblasts. A, Donor H65;o, donor H60; e, donor H48; c, donor H51.

Proc. Nad Acad. Sci. USA 78 (1981)

Proc. Natd Acad. Sci. USA 78 (1981) 6253

A.:_s~~~~~Vr ._ 4

/..!*. ! i :~~~~

g S +- ~ ~~a.. ~ ~ / ,/ !l * t < a

At!*j+ @ :

FIG. 2. Appearance of human breast epithelial and fibroblastic cells exposed to BaP. Photomicrographs of epithelial cells at confluence (a),epithelial cells exposed to BaP (10 pg/ml) (b), fibroblastic cells at confluence (c), and fibroblastic cells exposed to BaP (10 mg/mb) (d).

diol epoxide and deoxyadenosine was found (retention time> 56 min). The earlier minor peaks in these chromatograms mayrepresent BaP tetrols intercalated into the DNA or oligonu-cleotides from incomplete digestion ofDNA (10).When the area within the BaP diol epoxide-nucleoside peaks

was integrated, 73-82% of the DNA adduct material from ep-ithelial cells (Table 1) was recovered in peaks coeluting with theanti and syn stereoisomers ofBaP diol epoxide covalently boundto deoxyguanosine and with the putative deoxycytidine adduct(Table 1). No radioactive peaks were detected by HPLC ofmaterial from fibroblastic cultures after 24 hr in the presenceof a 10-fold higher concentration of BaP (Fig. 3, Table 1). Sev-enty-two hours after removal of medium containing BaP, over60% of the total DNA adduct material from fibroblasts was re-coverable in peaks identifiable as adducts between BaP diolepoxide and deoxyguanosine or deoxycytidine (Table 1).

In the case of the epithelial cells, increasingly more materialwas found to be coeluting with the anti form ofBaP diol epoxidethan was recovered with the syn isomer (Fig. 3, Table 1); from16 hr onward, about 90% of the adducts with deoxyguanosinewere with the anti isomer (Table 1). After an additional 72 hr

in the absence of added BaP, the decrease in covalent bindingof both isomers to the DNA of epithelial cells was similar, in-dicating that the higher content ofanti BaP diol epoxide adductswas due to preferential formation, not persistence.

As with the effect of BaP on growth, the total quantity ofadducts on the bases of DNA after exposure to BaP was quitedifferent in the two cell types (Table 1). Binding of BaP me-tabolites to DNA (one per 107 base pairs) from epithelial cellscould be detected at doses as low as 0.04 ,uM BaP (0.01 Ag ofBaP per ml) for 24 hr (data not shown). At 0.4 LM BaP (0.1 tg/ml), the identifiable BaP diol epoxide-DNA adducts in epithe-lial cells amounted to a BaP metabolite for every 6-12 x 10'bp ofDNA after 6 hr (Table 1). After a 24-hr exposure, the fre-quency had increased to a mean of one adduct per 106 bp ofDNA, but by this time virtually all ofthe BaP had been utilizedby epithelial cells (Table 2).

Only the fibroblasts incubated a total of 96 hr after first ex-posure to BaP contained DNA with identifiable quantities ofBaP diol epoxide-DNA adducts (Fig. 3, Table 1). The relativepercentages and adduct per bp values ofBaP diol epoxide withdeoxycytidine and ofthe anti and syn isomers ofBaP diol epox-

Cell Biology: Stampfer et aL

6254 Cell Biology: Stampfer et al

2000

1500

1000

500

.

& 1500

e 1000

5000

._.4

O00

1500

1000

500

06 20 40 60 80 0 20 40 60 80 100Retention time, min

FIG. 3. HPLC profiles of DNA adducts with metabolites of BaPformed in cultures of breast epithelial and fibroblastic cells incubatedwith ['H]BaP. The octadecyltrimethoxysilane columns were eluted is-ocratically at 52% methanol in water at a flow rate of 0.7 ml/min for80 min. Fractions were collected every minute for radioactive assay.(A-D) Profiles forDNA adducts from epithelial cells exposed to 0.4MMBaP for 6 hr (A), 16 hr (B), 24 hr (C), and 24 hr plus an additional 72hr in the absence of BaP (D). (E andF) Profiles for fibroblaste exposedto 4.0 MBaP for24 hr (E) plusan additional 72 hr in BaP-free medium(F). Based on retention times for known BaP diol epoxide-nucleosideadducts (dashed line in D) (23), adducts between deoxyguanosine andthe7R form of anti BaP diol epoxidewere recovered in fractions elutingat 30-40 min, those between deoxyguanosine and syn BaP diol epoxideat 41-45 min, and adducts tentatively identified between deoxycyti-dine and BaP diol epoxide at 27-30 min. Deoxyadenosine adducts hadretention times in excess of 56 min.

ide with deoxyguanosine were similar to the values observedin the DNA ofepithelial cells after a 16-hr exposure to one-tenththe concentration of BaP (Table 1).

Persistence ofDNA Adducts. In epithelial cells, the quantityof adducts between BaP metabolites and the bases ofDNA de-creased slightly during culture for an additional 72 hr in BaP-free medium (Table 1). Because <1 pmol of BaP per ml re-

mained at the end of the incubation (Table 2), substrate was notavailable for continued adduct formation. Hence, the persist-ence ofthe adducts indicates that few ofthe modified bases wereremoved from DNA of the mammary epithelial cells.

In contrast, persistence ofDNA adducts could not be testedin breast fibroblasts because covalent binding of BaP metabo-lites to fibroblastic DNA was not detectable during the 24-hrexposure to BaP-containing medium. Adducts with DNA were

found 72 hr later on fibroblasts in BaP-free medium (Table 1).The probable explanation for this phenomenon lies in the factthat after 24 hr of exposure to BaP, the fibroblastic cells stillcontained BaP intracellularly, even though the cultures were

washed repeatedly with BaP-free medium. This intracellularBaP would continue to be metabolized and, thereby, continue

Table 1. BaP diol epoxide-nucleoside adduct formation andpersistence in human mammary epithelial and fibroblasticcells exposed to BaP in culture

BaP diol Formation and persistence, adductDonor Cell epoxide per 1 x 106 bpno. type* adduct 6 hr 16 hr 24 hr +72 hrt

H48 EC dC 0.01 ND 0.05 0.03dG-anti 0.06 ND 0.71 0.57dG-syn 0.03 ND 0.08 0.06

H51 EC dC 0.01 ND 0.03 0.06dG-anti 0.10 ND 0.50 0.40dG-syn 0.05 ND 0.06 0.07

H97 EC dC 0.02 0.14 0.29 0.19dG-anti 0.04 0.50 1.19 0.75dG-syn 0.02 0.06 0.13 0.09

H51 FC dG-total <0.01 <0.01 <0.01 0.77H48 FC dC <0.01 <0.01 <0.01 0.19

dG-anti <0.01 <0.01 <0.01 0.45dG-8yn <0.01 <0.01 <0.01 0.08

The epithelial cells were exposed to 0.4 p.M BaP (0.1 pg/ml) for thetimes shown. The fibroblastic cells were treated with 10 times this con-centration. Adduct per million bp was calculated from the materialisolated on HPLC and the quantity of purified DNA analyzed.*EC, epithelial cell cultures exposed to 0.4 IAM BaP; FC, fibroblastsexposed to 4.0 p.M BaP; ND, not determined.

t Twenty-four hours in medium containing BaP followed by 72 hr inBaP-free medium.

to provide substrate for the formation of adducts with DNA.

DISCUSSIONThe development of techniques for isolating breast epithelialcells and growing them in culture for several passages has al-lowed us to demonstrate (i) that human mammary epithelialcells have the ability to metabolize BaP; (ii) that cell growth isinhibited by the metabolites; (iii) that the pattern of metabolicproducts results in the cellular lesion linked to carcinogenictransformation [i.e., formation of adducts between BaP diolepoxide and the bases of DNA (7-10)]; and (iv) that the DNAadducts, once formed, persist for several days.

Comparisons between these results and those from severalsources indicate that human mammary epithelial cells are pos-sible targets for chemically induced carcinogenesis. The extentof adduct formation in mammary epithelial cell DNA (one per106 bp) (9, 10, 26) and the types of DNA adducts present, in-cluding in some cases those with deoxycytidine (9-11, 16, 17,27, 28), are those found in cells in which transformation can beinduced by exposure to BaP in culture (5, 29-32). From thestandpoint of carcinogenic potential, the most important fate ofBaP appears to be adduct formation between deoxyguanosineand anti BaP diol epoxide, especially the 7R enantiomer (7-10).

Table 2. Concentration of BaP in culture media from humanmammary epithelial and fibroblastic cells exposed to ['H]BaP

Donor Cell BaP in culture media, nMno. type Initial 6 hr 16 hr 24 hr +72 hr*

H48 EC 400 148 ND 6 NDH51 EC 400 126 ND 11 NDH97 EC 400 163 63 10 0.8H48 FC 4000 2369 1809 1513 194

Cells were exposed to [(H]BaP for the times shown. EC, epithelialcell cultures exposed to 0.4 IAM BaP; FC, fibroblasts exposed to 4.0 IAMBaP; ND, not determined.* Twenty-four hours in medium containing BaP followed by 72 hr inBaP-free medium.

Proc. Nad Acad. Sci. USA 78 (1981)

Proc. Nati Acad. Sci. USA 78 (1981) 6255

This adduct was the most prevalent in DNA of human maimmaryepithelial cells treated with BaP.

Comparison with human fibroblasts from the same breastspecimens provides evidence that human mammary epithelialcells have a high capacity for this adduct formation: (i) adductsare formed at much lower concentrations of BaP,. (ii) the fre-quency of adduct formation per bp of DNA is equivalent to orhigher in. epithelial cells at lAoth the original concentration ofBaP, and (iii) the adducts are formed much faster by epithelialcells, within 6 hr after addition of BaP as compared with morethan 24 hr with fibroblastic cells.The persistence of modified DNA has also been related to

transformation (33-35). Even though 40% of BaP diol epoxideadducts with deoxyguanosine formed in DNA from 10T 1/2 cellsexposed to BaP can be removed in 68 hr (9), transformation bythe metabolic products ofpolycyclic aromatic hydrocarbons canbe demonstrated in these cells (5, 32). Once formed in humanmammary epithelial cells, the adducts persisted for over 96 hr(24 hr in the presence of BaP plus 72 hr in BaP-free medium),a property shared with cells transformable by polycyclic aro-matic hydrocarbons.

Recently, binding between metabolites of BaP and nucleo-sides ofDNA was reported in two human epithelial cell systems,freshly isolated cell aggregates ofmammary epithelial cells (36),and cell strains of keratinocytes (37). The adducts were not pos-itively identified, but both groups (36, 37) suggest that the elu-tion from LH20 columns was compatible with BaP diol epoxideadducts, consistent with the findings reported here.

Based on the rate, the extent, and the types ofadducts formedand their persistence, we conclude that ifbreast epithelial cellscome in contact with BaP under natural conditions, the meta-bolic events leading up to formation ofBaP diol epoxide adductsof DNA can take place entirely within that cell, and the extentof the reactions involved is comparable to that of cells suscep-tible to carcinogenic transformation. Therefore, chemical car-cinogens, particularly BaP, should not be minimized as possiblefactors in the initiation ofbreast cancer. This conclusion is sup-ported by studies of the induction of mammary adenocarcino-mas in rodents by chemical carcinogens, such as dimethylben-zanthracene, methylcholanthrene (38), and more recently, BaP(39). The induction and promotion of these chemically inducedadenocarcinomas are enhanced by factors such as high-fat diets,particularly those rich in polyunsaturated fatty acids (40, 41),and specific hormonal imbalances (42). The development ofhuman breast cancer likely involves an interaction betweenchemical carcinogens and these other factors. The availabilityof a system for cultivating human mammary epithelial cells pro-vides the opportunity to test this possibility.

We gratefully acknowledge Annie Pang for excellent technical assis-tance, Dr. Dezider Grunberger for helpful discussions, and Dr. AdelineHackett for her support and encouragement. This research was sup-ported by Grant CA-24844 from the National Cancer Institute, GrantCD-61B from the American Cancer Society, and Contracts 4425/003298 and W-7405-ENG-48 from the U.S. Department of Energy.

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Cell Biology: Stampfer et aL