Carcinogen-DNA adducts in human breast epithelial cells

Article abstract of DOI:10.1002/em.10060

Diet and environmental exposures are often regarded as significant etiologic factors in human breast cancer. Chemicals that may be involved in these exposures include heterocyclic amines, aromatic amines, and polycyclic aromatic hydrocarbons, which also serve as strong mammary carcinogens in different animal models. In this study, we chose to quantify the major DNA adducts derived from one member of each of these classes of carcinogens, that is, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), 4-aminobiphenyl (ABP), and benzo[a]pyrene (B[a]P), respectively, in DNA isolated from exfoliated ductal epithelial cells in human breast milk. Milk was collected from healthy, nonsmoking mothers. The isolated DNA was digested to 3′ nucleotides and subjected to ³²P-postlabeling. Adduct enrichment was achieved using Oasis Sep-Paks and the analyses were conducted by HPLC using radiometric detection. Critical to the analyses were the syntheses of bis(phosphate) standards for the C8-dG adducts of PhIP and ABP, and the N²-dG adduct of B[a]P, which were added to each reaction as UV markers. Of the 64 samples analyzed, adducts were found in 31 samples. Thirty samples contained detectable levels of PhIP adducts, with a mean value of 4.7 adducts/10⁷ nucleotides; 18 were positive for ABP adducts with a mean value of 4.7 adducts/ 10⁷ nucleotides; and 13 were bund to contain B[a]P adducts with a mean level of 1.9 adducts/ 10⁷ nucleotides. These data indicate that women are exposed to several classes of dietary and environmental carcinogens and that these carcinogens react with DNA in breast ductal epithelial cells, the cells from which most breast cancers arise.

Full text of DOI:10.1002/em.10060

Environmental and Molecular Mutagenesis 39:184–192 (2002)
Carcinogen–DNA Adducts in Human Breast
Epithelial Cells
K. Gorlewska-Roberts,1 B. Green,1 M. Fares,1 C.B. Ambrosone,2
and F.F. Kadlubar1*
1National Center for Toxicological Research, Jefferson, Arkansas
2Mt. Sinai School of Medicine, New York, New York
Diet and environmental exposures are often re- cal to the analyses were the syntheses of bis(phos-
garded as significant etiologic factors in human phate) standards for the C8-dG adducts of PhIP
breast cancer. Chemicals that may be involved in and ABP, and the N2-dG adduct of B[a]P, which
these exposures include heterocyclic amines, aro- were added to each reaction as UV markers. Of
matic amines, and polycyclic aromatic hydrocar- the 64 samples analyzed, adducts were found in
bons, which also serve as strong mammary carcin- 31 samples. Thirty samples contained detectable
ogens in different animal models. In this study, we levels of PhIP adducts, with a mean value of 4.7
chose to quantify the major DNA adducts derived adducts/107 nucleotides; 18 were positive for
from one member of each of these classes of ABP adducts with a mean value of 4.7 adducts/
carcinogens, that is, 2-amino-1-methyl-6-phenylimi- 107 nucleotides; and 13 were found to contain
dazo[4,5-b]pyridine (PhIP), 4-aminobiphenyl (ABP), B[a]P adducts with a mean level of 1.9 adducts/
and benzo[a]pyrene (B[a]P), respectively, in DNA 107 nucleotides. These data indicate that women
isolated from exfoliated ductal epithelial cells in are exposed to several classes of dietary and en-
human breast milk. Milk was collected from vironmental carcinogens and that these carcino-
healthy, nonsmoking mothers. The isolated DNA gens react with DNA in breast ductal epithelial
was digested to 3Ј nucleotides and subjected to cells, the cells from which most breast cancers
32P-postlabeling. Adduct enrichment was achieved arise. Environ. Mol. Mutagen. 39:184–192,
using Oasis Sep-Paks and the analyses were con- 2002.
ducted by HPLC using radiometric detection. Criti-
Published 2002 Wiley-Liss, Inc.†
Key words: DNA adducts; epithelial cells; breast milk; carcinogens
INTRODUCTION
al., 1998]. Heterocyclic amines are food contaminants
formed during the cooking of proteinaceous foods such as
meat and fish at high temperatures [Nagao et al., 1994;
Snyderwine, 1994]. Aromatic amines and polycyclic aro-
matic hydrocarbons are widely distributed in the environ-
ment and are present in fossil fuel products, tobacco smoke
[Pieraccini et al., 1992], air [Otson et al., 1987], and water
[Nukaya et al., 1997]. These carcinogens are known to
cause mammary tumors in animal models [Dunnick et al.,
1995].
Breast cancer is the major cause of cancer death among
women and has the highest cancer incidence and cancer
death rate worldwide [Higginson et al., 1992]. There are
demographic variations in the incidence of breast cancer
with Western countries having higher rates of the disease
than, for example, countries in the Far East [Parkin et al.,
1999]. Genetic predisposition accounts for only a small part
of cases [Henderson, 1993], although studies in migrant
populations show changing patterns of breast cancer inci-
dence, suggesting an important role of environmental
factors in the etiology of this disease. Hormones play an
important role as determinants of breast cancer risk [Feigel-
son and Henderson, 1996], but diet and environmental ex-
posures to carcinogenic compounds are regarded as signif-
icant etiologic factors of breast cancer [El-Bayoumy, 1992;
Morris and Seifter, 1992; Snyderwine, 1994; Josephy, 1996;
Wolff et al., 1996; Eldrige et al., 1997].
Carcinogens may directly react with cellular DNA, but
more often parent compounds are metabolized to more
reactive species. If there is excessive exposure, increased
Grant sponsor: Oak Ridge Institute for Science and Education.
*Correspondence to: Fred F. Kadlubar, National Center for Toxicological
Research, HFT 100, 3900 NCTR Road, Jefferson, AR 72079. E-mail:
fkadlubar@nctr.fda.gov
Genotoxic compounds implicated in human breast carci-
Received 21 November 2001; provisionally accepted 26 November 2001;
nogenesis include members of the most common classes of and in final form 29 November 2001
chemical carcinogens such as heterocyclic amines, aromatic
amines, and polycyclic aromatic hydrocarbons [Zheng et
Published 2002 Wiley-Liss, Inc. †This article is a US Government work and,
as such, is in the public domain in the United States of America.

Carcinogen–DNA Adducts in HBECs
185
activation, or decreased detoxification, some of the reactive breast milk and/or circulating blood [Martin et al., 1996]
metabolites may bind to DNA, forming DNA adducts, and and they are also capable of metabolic activation of these
lead to the initiation of the carcinogenic cascade [Kadlubar, compounds. It is known that breast carcinomas commonly
1987, 1994]. The major sites of DNA damage are C8 and N2 arise from mammary ductal epithelial cells.
atoms of guanine [Kadlubar et al., 1980; Turesky et al.,
It has been shown that human breast milk contains nu-
1992] and mutagenic consequences are mainly transver- merous cell types, including epithelial cells [Lawrence,
sions and frameshift mutations [Schut and Snyderwine, 1985]. The recovery of these cells from fresh milk provides
1999].
a noninvasive method for obtaining cellular DNA for study
Adduct levels are primarily a function of exposure and of genetic damage (i.e., DNA adduction). Adduct identifi-
polymorphism in genes involved in carcinogen metabolism cation leads to recognition of the agents responsible for the
as well as DNA repair. Bioactivation usually involves two initiation of breast cancer. Moreover, evaluation of adduct
or more enzymatic reactions, typically oxidation or hy- levels as a function of exposure and genetic polymorphisms
droxylation, followed by conjugation or hydrolysis. Certain could help in better understanding breast cancer etiology.
enzymes involved in the metabolism of carcinogens are
In this study we quantified, by means of 32P-postlabeling-
expressed in normal breast tissue [Lee et al., 1996; Williams HPLC, the major DNA adducts derived from each of three
and Philips, 2001]. Polycyclic aromatic hydrocarbons are classes of carcinogens, that is, 2-amino-1-methyl-6-pheny-
activated by cytochromes P4501A1 and P4501B1, and re- limidazo[4,5-b]pyridine (PhIP), 4-aminobiphenyl (ABP),
active intermediates are detoxified by glutathione-S-trans- and benzo[a]pyrene (B[a]P), in DNA isolated from exfoli-
ferases. Heterocyclic and aromatic amines are either directly ated epithelial cells from human breast milk (Fig. 1). Crit-
detoxified or activated to more potent carcinogens by N- ical to the analysis was the synthesis of bis(phosphate)
acetyltransferases (NAT1 and NAT2). In addition, lactoper- standards for the C8-dG adducts of PhIP and ABP, and
oxidase secreted by the mammary ductal epithelial cells and N2-dG adduct of B[a]P, which were used as UV markers.
myeloperoxidase found in neutrophils may play significant
roles in the bioactivation of carcinogens in breast tissue
[Josephy, 1996].
MATERIALS AND METHODS
Chemicals
The human breast contains a high percentage of adipose
tissue and it has been postulated that lipid-soluble carcino-
gens may accumulate in this tissue. However, this factor is
not sufficient, given that other lipophilic carcinogens are not
mammary specific. It has been postulated that tissue-spe-
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was pur-
chased from Toronto Research Chemicals (Toronto, Ontario, Canada),
4-aminobiphenyl was purchased from Aldrich (Milwaukee, WI), and (Ϯ)-
cific metabolic activation of carcinogens, along with the r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene
(BPDE) was purchased from ChemSyn Laboratories (Lenexa, KS). Aden-
ability of breast tissue to accumulate carcinogens, accounts
for this organ specificity [Josephy, 1996]. Epithelial cells
lining the mammary ducts, or suspended in adipose tissue,
osine 5Ј-[␥-32P]triphosphate with an original specific activity of about 7000
Ci/mmol was obtained from ICN Biomedicals (Irvine, CA), and T4
polynucleotide kinase (PNK) from USB (Cleveland, OH). Unless stated
are vulnerable to DNA damage because they are exposed to
compounds accumulated in adipose tissue and present in Louis, MO).
otherwise, all other materials were obtained from Sigma Chemicals (St.
Fig. 1. Chemical structures of the carcinogens discussed in the text.

186
Gorlewska et al.
Fig. 2. Schemes of synthesis of bis(phosphate) C8-dG adducts of PhIP (A) and ABP (B) and the N2-dG adduct
of BPDE (C).

Carcinogen–DNA Adducts in HBECs
187
Fig. 2. Continued
the bis(pyridinium) salt. Bis(pyridinium)cyanoethylphosphate was eluted
with water, lyophilized, and kept under vacuum in a dessicator.
Instrumentation
A Waters (Milford, MA) HPLC system was equipped with Model 6000E
pumps, a Model 717 autosampler, and a Model 996 photodiode array
detector. Radioactivity was measured on-line with a Radiomatics Model
A-500 FLO-ONE radioactivity detector using a 0.1-ml TRLSC cell and
Ultima-FLO-M scintillation fluid (Packard, Palo Alto, CA). The analytical
system had a precolumn New Guard™ holder with a 7-␮ RP-18 cartridge
(Perkin Elmer Cetus Instruments, Norwalk, CT) for diverting normal
nucleotides. As a main column, a Delta Pak 5-␮ C18-100A, 4.6 mm id ϫ
150 mm (Waters), was used. Adduct separation was performed by eluting
at 0.5 ml/min with a linear gradient of 1.2 M ammonium formate (pH 4.5)
and acetonitrile. The elution profile was as follows: 0–35% acetonitrile
(0–70 min), 35–90% acetonitrile (70–75 min), 90% acetonitrile (75–78
min), 90–50% acetonitrile (78–85 min), and 50–0% acetonitrile (86–87
min). The high concentration of ammonium formate was used to reduce the
32P background and to diminish the peak tailing.
3؅,5؅-dG-bis(phosphate) (pdGp) Synthesis
N2-Isobutyryl dG was dissolved in a minimal volume of pyridine. Five
equivalents of bis(pyridinium)cyanoethylphosphate and 10 equivalents of
dicyclohexylcarbodiimide were added and stirred at room temperature
overnight. The reaction was quenched with water and filtered. The filtrate
was evaporated and 2 M ammonium hydroxide at 55°C was used to
deblock the N2 and PO4 groups. Ammonium hydroxide was evaporated
and the final product was purified on a 1-ml C18 precolumn.
Synthesis of the Standards
pdGp-PhIP
N-Hydroxy PhIP and the N-acetoxy derivative of N-hydroxy PhIP were
synthesized as described by Lin et al. [1992]. The adduct standard was
synthesized as follows (Fig. 2A). pdGp (25 mM) was dissolved in 0.1 mM
potassium citrate, pH 7, and ethanol. The mixture was cooled on ice and
flushed with argon for 2 min and 0.5 ml of 5 mM AcO–PhIP was added.
The reaction was flushed with argon and placed at 37°C for 4 hr. Ascorbic
acid (1 mg) was added and ethanol was removed by flushing with argon.
Water (0.5 ml) was added and the reaction mixture was extracted twice
with argon-saturated ether and once with argon-saturated ethyl acetate.
Ethyl acetate was removed by flushing with argon and the product was
purified by HPLC.
Chemical Syntheses
Bis(pyridinium)cyanoethylphosphate Synthesis
A Dowex 50 (Supelco, St. Louis, MO) pyridinium column was prepared
by washing with 1 N HCl, rinsing with water, and passing 1 M pyridine in
water through the column until the effluent ran clear. The column was then
washed with high-purity water until pyridine was no longer eluting. Barium
cyanoethylphosphate in water (one equivalent of barium cyanoethylphos-
phate per two equivalents of resin) was passed through the column forming

188
Gorlewska et al.
pdGp-ABP
Hydrolysis of DNA, Adduct Enrichment,
and 32P-Postlabeling
First, N-trifluoroacetyl-N-acetoxy-4-aminobiphenyl (TFAA) was pre-
pared as a reactant from 25 mg of N-hydroxy-4-aminobiphenyl by dissolv-
ing in argon-saturated anhydrous ethyl acetate and cooling on ice. Triflu-
oroacetic anhydride (27 ␮l) was added and left at room temperature for 45
min. The ethyl acetate was evaporated to dryness and the residue was
redissolved in 2.5 ml of argon-saturated anhydrous ethyl acetate and cooled
on ice. Anhydrous pyridine (50 ␮l) and acetic anhydride (25 ␮l) were
added and set at room temperature for 30 min. The TFAA solution was
quickly evaporated to dryness and redissolved in 1 ml of absolute ethanol.
The adduct standard was then prepared (Fig. 2B). pdGp (25 mM) was
dissolved in 4 mM potassium citrate, pH 7, cooled on ice, and flushed with
argon for 2 min. A 1-ml aliquot of the freshly prepared TFAA–ABP was
added and the reaction was flushed with argon and placed at 37°C with
continuous mixing for 10 min and then mixing every 10 min for up to 1 hr.
Ascorbic acid (20 mg) was added and ethanol was removed by flushing
with argon. The reaction was extracted twice with water-saturated ether
and once with water-saturated ethyl acetate. Ethyl acetate was removed by
flushing with argon and the final product was purified by semipreparative
HPLC and kept frozen.
DNA (15–30 ␮g) was hydrolyzed in 10 mM sodium succinate (pH 6)
buffer by adding a solution (1 ␮l/␮g DNA) of 0.2 U/␮l micrococcal
nuclease (MN) and 3.6 mU/␮l spleen phosphodiesterase (SPD), and was
incubated at 37°C overnight. The MN/SPD had been dialyzed about 10 hr
twice against water. The enrichment of the adducted nucleotides was
performed on 1 ml HLB Oasis Sep-Paks (Waters) in digestion buffer.
Normal nucleotides were removed by washing with water and adducted
nucleotides were eluted in three column volumes of methanol. Samples
were evaporated to dryness, [5Ј-32P]-phosphorylated using polynucleotide
kinase (2 U) and [␥-32P]ATP (100 ␮Ci, ϳ2 ␮M) in 50 mM Tris–HCl
buffer (pH 7.6) containing 10 mM MgCl2 and 10 mM 2-mercaptoethanol
in a total volume of 20 ␮l, and were incubated at 37°C for 45 min. HPLC
analyses of the 32P-postlabeled DNA adducts were performed by injecting
the total 32P-postlabeled mixture containing three standards as UV markers
into the HPLC. Adducts were identified by UV–Vis and chromatographic
comparison with the synthesized standards. Recovery experiments using
PhIP–DNA, ABP–DNA, and BPDE–DNA standards from the IARC-
NCTR repository were conducted routinely and ranged from 70 to 90%;
therefore, we did not correct the values obtained because interindividual
variability was much greater.
pdGp-BPDE
The adduct standard was synthesized as follows (Fig. 2C): an 11 mM
solution of BPDE in tetrahydrofuran (300 ␮l) was added to an eightfold
molar excess of pdGp, dissolved in the same volume of 10 mM sodium
citrate (pH 6), and the mixture was stirred overnight at room temperature.
Following the evaporation of solvent, water was added to a final volume of
1 ml and the unbound BPDE derivatives were removed by repeated
extractions with diethyl ether. The pdGp-N2-BPDE was isolated by re-
versed-phase HPLC.
RESULTS
The representative standards for the three classes of car-
cinogens, with chemical structures shown in Figure 1, were
synthesized as described under Materials and Methods, that
is, bis(phosphates) for C8-dG adducts of PhIP and ABP and
the N2-dG adduct of B[a]P. The schemes for the synthesis of
these adducts are shown in Figure 2. These standards were
used as UV–Vis markers.
Mass spectra were obtained for each synthesized standard to confirm
purity and identity.
Isolation of Epithelial Cells From Human Breast Milk
DNA was isolated from epithelial cells as described un-
der Materials and Methods and the total amounts recovered
were in the range of 1–55 ␮g. DNA samples were then
examined for the presence of hydrophobic DNA adducts.
After digestion of DNA, adduct enrichment was achieved
using Oasis Sep-Paks and 32P-postlabeling was performed
according to the method developed by Gupta and cowork-
ers, as reviewed by Beach and Gupta [1992]. Previously
synthesized standards were added to each sample and the
analyses were conducted by means of HPLC, which has
advantages over TLC in time, efficiency, analytical capac-
ity, and adaptability. Also, interpretation of data from
HPLC is possible as soon as the separation is finished,
commonly 1–2 hr after the injection. Radioactivity and
UV–Vis chromatograms of one of the analyzed samples are
presented in Figure 3. Radioactivity profiles showed three
adducts (a, b, and c) that correspond to synthesized adduct
standards shown on the UV chromatogram (Fig. 3B),
After Human Subjects Approval had been obtained for this study, milk
samples were collected from healthy, nonsmoking mothers and a question-
naire was administered to gather information on demographic factors,
breast-feeding history and habits, and possible exposures to putative breast
carcinogens such as aromatic and heterocyclic amines and polycyclic
aromatic hydrocarbons. Milk was collected at 4–6 weeks postpartum, a
time at which an optimal yield of epithelial cells can be obtained [Thomp-
son et al., 1998]. Epithelial cells were isolated according to the procedure
described by Thompson et al. [1998]. In brief, human milk samples were
diluted 3:1 with Krebs–Henseleit buffer and 1% bovine serum albumin
(w/v) and centrifuged at 500g for 10 min. Fat and supernatant were
removed by aspiration. After washing, the cell pellets were incubated for 2
hr at 37°C. Epithelial cells were separated from the cell pellet through
adherence of monocytes to glass and stored at Ϫ80°C.
DNA Isolation
DNA was isolated from the frozen cell pellets using a QIAamp DNA
Mini Kit (Qiagen, Valencia, CA). Cell pellets were lysed using the cell
lysis solution included in the kit. After addition of the buffer and proteinase marked as A (ABP adduct), B (PhIP adduct), and C (BPDE
K, samples were pulse-vortexed and incubated for 10 min at 56°C. After
brief centrifugation, ethanol was added and samples were vortexed and
adduct).
Of the 71 samples analyzed, successful analysis was
performed for 64 samples, the results of which are presented
briefly centrifuged. Each sample was applied to the QIAamp spin column
and centrifuged, followed by washing with two buffers. DNA was eluted
in Table I. Of the 64 samples analyzed, 31 samples con-
tained radioactive peaks that clearly cochromatographed
from the column with the elution buffer and the DNA concentration was
determined spectrophotometrically using A260 (1.0 ϭ 50 ␮g/ml).

Carcinogen–DNA Adducts in HBECs
189
Fig. 3. HPLC/32P-radioactivity profile (A) and UV spectral detection (B) of DNA adducts from epithelial cells
from human breast milk coinjected with C8-dG-PhIP, C8-dG-ABP, and N2-dG-BPDE bis(phosphate) standards.
Three adducts detected in DNA (a, b, and c) correspond to adduct standards A (ABP adduct), B (PhIP adduct),
and C (BPDE adduct), respectively.
with one of the synthetic pdGp adduct standards (in 48% of carcinogenic processes but also reflect an exposure, biolog-
the samples from the studied group). DNA adduct levels,
determined by moles 32P-ATP incorporated/nucleotide,
ranged from about 1 to 100 adducts/108 nucleotides, with
two samples as high as 200 adducts/108 nucleotides and 500
adducts/108 nucleotides. In samples that were positive for
adducts using this method, adducts of ABP, PhIP, or B[a]P
were detected at varying levels. Thirty samples contained
detectable levels of PhIP adducts with a mean value of 4.7
adducts/107 nucleotides; 18 were positive for ABP adducts
with a mean value of 4.7 adducts/107 nucleotides; and 13
contained B[a]P adducts with mean level of 1.9 adducts/107
nucleotides.
ically effective dose, and local metabolic processes in-
volved in formation of reactive species capable of binding
to DNA and other macromolecules. The presence of car-
cinogen–DNA adducts in human breast tissue has already
been reported for smoking-related putative polycyclic aro-
matic hydrocarbons [Perera et al., 1995].
In this study we examined DNA isolated from mammary
epithelial cells from human breast milk for three hydropho-
bic DNA adducts. We showed the presence of DNA adducts
derived from members of three main groups of carcinogens:
heterocyclic amines, aromatic amines, and polycyclic aro-
matic hydrocarbons. We presented evidence for the detec-
tion of C8-dG adducts of PhIP and ABP, and the N2-dG
adduct of BPDE.
DISCUSSION
Breast milk, which provides a noninvasive source of
Carcinogen–DNA adducts are biological markers that
allow for identification of agents responsible for initiation of ductal epithelial cells for identification and quantification

190
Gorlewska et al.
TABLE I. Carcinogen–DNA Adduct Levels Found in Analyzed Samplesa
Sample
ABP
PhIP
B[a]P
Sample
ABP
PhIP
B[a]P
2
329
330
332
333
334
335
336
337
339
340
341
342
343
344
345
346
347
348
349
350
351
352
354
355
356
357
359
360
362
363
364
366
369
370
373
384
No adducts
No adducts
Insufficient sample
No adducts
No adducts
10
No adducts
60
10
No adducts
No adducts
No adducts
No adducts
No adducts
No adducts
No adducts
No adducts
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
30
30
200
70
90
No adducts
Insufficient sample
1
30
10
30
2
8
No adducts
10
No adducts
10
Insufficient sample
10
50
20
7
5
9
Insufficient sample
5
300
No adducts
No adducts
7
10
9
2
4
1
4
3
1
20
3
1
20
7
20
Insufficient sample
Insufficient sample
No adducts
No adducts
No adducts
No adducts
40
300
No adducts
7
Insufficient sample
No adducts
No adducts
No adducts
10
No adducts
No adducts
90
70
500
20
60
20
80
60
10
2
2
80
30
No adducts
No adducts
No adducts
No adducts
1
1
5
2
No adducts
aDNA adduct levels/108 nucleotides.
of chemical exposures, was used in this study. It was DNA–carcinogen adducts was earlier demonstrated in hu-
previously found [Thompson et al., 2002] that human man breast tissue [Perera et al., 1995; Li et al., 1996] and in
breast milk exhibits mutagenic activity. The same study cultured human epithelial cells [Swaminathan et al., 1994].
showed that most of the mutagenicity is in the basic Our study showed not only that most of the breast milk
fraction of the milk, which implies that basic compounds samples that were positive for DNA adducts contained PhIP
such as aromatic and heterocyclic amines, and polycyclic adducts, but also that the levels of PhIP adducts were higher
aromatic hydrocarbons contribute to the observed mutage- than the levels of ABP and BPDE adducts. These findings
nicity. Other studies showed that these compounds are poten- are in accordance with recent studies on the detection of
tially mutagenic and carcinogenic to mammary epithelial cells PhIP in breast milk from healthy women [DeBruin et al.,
[El-Bayoumy, 1992; Ambrosone and Shields, 1999; Snyder- 2001]. Heterocyclic amines are dietary carcinogens that
wine, 1999; De Bruin et al., 2001]. Lipophilic compounds like could be metabolized by hepatic as well as mammary en-
aromatic and heterocyclic amines and polycyclic aromatic zymes. Studies by Toniolo et al. [1994] and Destefani et al.
hydrocarbons can bioaccumulate in fatty tissues such as adi- [2001] concluded that breast cancer risk increased signifi-
pose tissue of breast and can be further metabolized to DNA- cantly with consumption of meat. It was also determined
reactive derivatives.
that consumption of well-done meats increased breast can-
The study by Thompson et al. [2002] showed that epi- cer risk in a dose-dependent manner [Zheng et al., 1998].
thelial cells from human breast milk that had mutagenic This suggests that diet plays a significant role in breast
activity contained bulky DNA adducts. The presence of cancer formation, especially because the uptake of carcin-

Carcinogen–DNA Adducts in HBECs
191
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ogens from the diet is in most cases higher than that from
other sources (e.g., cigarette smoke).
The presence of ABP and BPDE adducts implies that
other environmental exposures, in addition to diet, are sig-
nificant etiologic factors in human breast cancer. However,
from the apparent frequency of the PhIP–DNA adduct ob-
served, among the different classes of carcinogens, the
heterocyclic amines may play the most important role in
etiology of breast cancer.
Metabolism of heterocyclic amines, aromatic amines, and
polycyclic aromatic hydrocarbons varies among individuals
and depends, in part, on genetic polymorphisms involved in
Phase I and/or Phase II metabolism. These include N-acetyl-
transferases (NAT1, NAT2), cytochrome P450 (CYP1A1,
CYP1A2), glutathione-S-transferases (GSTA1, GSTM1),
and phenol sulfotransferases. The association between met-
abolic genotype and other host factors and adduct formation
should be further investigated.
ACKNOWLEDGMENTS
The authors thank all of the mothers who donated their
time and breast milk for this study. This research was
supported, in part, by an appointment of K.G. to the
Postgraduate Research Participation Program at the Na-
tional Center for Toxicological Research administered by
the Oak Ridge Institute for Science and Education
through an interagency agreement between the U.S. De-
partment of Energy and the U.S. Food and Drug Admin-
istration.
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