Ethanol and Experimental Breast Cancer

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ALCOHOLISM: CI.MICAL
AND
EXPERIMENTAL RESEARCH
Vol.
21,
No.
2
April
1997
Ethanol and Experimental Breast Cancer:
A
Review
Keith Singletary
There is considerable evidence from epidemiological studies to sup-
port a positive association between alcohol intake and risk for breast
cancer. Yet, experimental evidence has provided less convincing
evidence to support this relationship, although much less attention
has been focused on elucidating the effect of ethanol on breast car-
cinogenesis in animal models. Although the number of reports are
limited, information on the effect of ethanol on mammary carcino-
genesis in spontaneous, chemically induced and metastatic models
has been published. In addition, a small number of reports provide
insights into an influence of ethanol on the physiological processes
associated with the initiation, promotion, and progression stages of
breast carcinogenesis in animals, as well as on the growth of human
breast cancer cells. This information from the literature is summa-
rized, and specific recommendations put forth
so
that greater
progress can be made in this controversial and complex research
area.
Key Words: Alcohol, Ethanol, Mammary Carcinogenesis.
UMEROUS EPIDEMIOLOGICAL studies support
N
the existence of a positive association between alco-
hol intake and risk for breast cancer.’,’ The building of a
consensus
on
this issue among the scientific community
could be strengthened if a plausible biological mechanism
for the breast cancer-enhancing effect of alcohol could be
demonstrated. Experimental studies have provided less
convincing evidence to support the positive ethanol-breast
cancer relationship, although much less attention has been
focused
on
elucidating the effect of ethanol
on
breast car-
cinogenesis in animal models. Nonetheless, some insights
into the biological action of ethanol relating to mammary
carcinogenesis can be gleaned from a review of the current
literature. Published reports evaluating the influence of
ethanol intake
on
several models of rodent mammary tu-
morigenesis and
on
physiological processes associated with
the initiation and promotion stages of mammary carcino-
genesis will be examined. The proliferative response of
several human breast cancer cell lines
to
ethanol will be
discussed. Lastly, suggestions for expanding research in this
area will be presented.
From the Department
of
Food Science and Human Nutrition and Division
Received
for
publication October
30,
1996; accepted November
20,
I996
This study was supported in part by the National Institute
on
Alcohol
Abuse and Alcoholism (Grant R29AA08584).
Reprint requests: Keith Singletaly, Ph. D., Department
of
Food Science and
Human Nutrition and Division
of
Num‘tional Sciences, University
of
Illinois,
905 South Goodwin Avenue, Urbana,
IL
61801.
of
Nutritional Sciences, University
of
Illinois, Urbana, Illinois.
Coprright
0
1997 by The Research Society
on
Alcoholism.
334
RODENT
MAMMARY
TUMOR
MODELS
A summary of published reports investigating the effect
of ethanol intake
on
mammary tumorigenesis in experi-
mental models is included in Table
1.
Spontaneous Mouse Mammaty Tumors
Mice of the
C3H
strain develop mammary tumors spon-
taneously, due to the transmission of an oncogenic RNA
virus transferred to offspring in the mother animal’s milk.
The influence of ethanol intake
on
spontaneous mammary
carcinogenesis in
C3H
mice has been reported in three
~tudies.~” One of the three reports indicated that animals
consuming ethanol at 12% (v/v) in drinking water exhibited
a significantly shorter latency period for mammary adeno-
carcinoma de~elopment.~ This effect was not observed in a
second study4 in which ethanol was given at
6%
v/v
in
drinking water. Hackney et al? observed that animals pro-
vided ethanol as 29% of calories in a defined diet, as 12%
(v/v) in drinking water, or by gavage at
4
@@day either
exhibited
no
change or a decrease in mammary tumor
development, compared with isocaloric controls. Some eth-
anol-fed animals in this latter report exhibited considerably
lower body weights than controls, which may have partly
contributed to the cancer-suppressing effect of ethanol
intake, compared with controls. The effects of lower in-
takes of ethanol
on
mammary tumor development have not
been examined in this model.
Chemically Induced Rat Mammary Tumors
The induction of mammary tumors in female rats by
either the indirect-acting polycyclic aromatic hydrocarbon
7,12-dimethylbenz(a)anthracene
(DMBA) or the direct-
acting N-methyl-N-nitrosourea (MNU) has provided one of
the most widely used models to study the process of mam-
mary cancer formation.6 Use of this model allows for the
examination of an effect of ethanol exposure
on
specific
stages
of
carcinogenesis. The initiation stage of the cancer
process in this model is characterized by a carcinogen-
induced, heritable, genetic change in target cell DNA. A
chemical carcinogen binds to specific DNA bases forming a
carcinogen-DNA adduct, that, if left unrepaired, can lead
to mutations or other genetic lesions.’ DMBA, in contrast
to MNU, requires metabolic activation to electrophilic di-
hydrodiol epoxide intermediates by specific cytochrome
P-450s
and epoxide hydrolase before covalent binding to
DNA. During the promotion stage, an initiated cell selec-
tively proliferates under the appropriate stimulus to form a
Alcohol
Clin
Erp
Res,
Vol21,
No
2, 1997:
pp
334-339

ETHANOL AND EXPERIMENTAL BREAST CANCER
335
Table
1.
Summary of Reports in Which the Influence of Ethanol on Experimental Mammary Tumorigenesis Was Evaluated
Animal (strain)' (davs)t modelt ETOH Dose6 controls levels Dietll examined# ethanol11 Ref.
Age Tumor Pair-fed Blood ethanol Stage Tumor response to
-
Mouse (C3H/St)
Mouse (C3H/St)
Mouse (C3H/Ou)
Mouse (C3H/Ou)
Mouse (C3H/Ou)
Rat (S-D)
Rat (S-D)
Rat (S-D)
Rat (S-D)
Rat (S-D)
Rat (S-D)
Rat (S-D)
Rat (S-D)
Rat (S-D)
Rat (S-D)
Rat (Fisher 344)
Rat (Fisher 344)
Weanl. Spon
Weanl. Spon.
42 Spon.
42 Spon.
42 Spon.
21 DMBA
21 DMBA
40 DMBA
35 DMBA
30 DMBA
25 DMBA
63 DMBA
37 MNU
28 MNU
58 MNU
70-84 MADE106
mammary
adenocarc.
70-84 MADBIO6
mammary
adenocarc.
12% v/v (Dr)
6% v/v (Dr)
12% w/v (Dr)
29% of cal. (Dt)
20% of cal. (Dt)
10-35% of cal. (Dt)
5%
v/v (Dr)
20% of cal. (Dt)
lo%,
20% of cal. (Dt)
15%.
30% of cal. (Dt)
4 g/kg
(G)
3.5 9-7.0 g/kg
(G)
3.5 9-7.0 g/kg
(G)
15%, 20%, 30% of cal. (Dt)
15%,
20%, 30% of cal.
(Dt)
35% of cal. (Dt)
1.5
g, 2.5 g, 3.5 g/kg
(IP)
No
No
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
23-86 mg/dl
163 mg/dl
136-341 mg/dl
-
C
C
S
S
S
S
I+P
S
I+P
C I+P
I
S
I
S
I
S
P
-
-
-
-
-
I
-
S
I
S
P
S
M
C
M
1
Latency
No effect
t
Latency
No effect
No effect
No effect
No effect
1
Tumor incidence
No.
of tumorslrat
Tumor incidence
7
Tumor incidence (20%
of cal. only)
t
Tumor incidence
(15%
of cal. only)
7
No. of turnors/rat (7.0/kg
T
No. of adenocarc./rat
(15%
of cal. only)
T
No. of adenocarchat
(15% and 20% of cal. only)
T
No. of lung metastases
only)
t
No. of lung metastases
(2.5 g, 3.5 g/kg only)
3
4
5
5
5
8
8
9
10
11
11
11
10
12
12
13
13
'
All rodents were female except for those in Ref. 13. S-D, Sprague-Dawley.
t
Age at which ethanol administration commenced. Weanl.. weanling.
+
Spon., spontaneous; adenocarc., adenocarcinoma.
J
Method of delivery in parentheses: Dr, drinking water; Dt, diet;
G,
gavage; IP, intraperitoneal injection; ETOH, ethanol; cal., calories.
1
C, commercial chow;
S,
semipurified or defined.
#
Stage of carcinogenesis when ethanol administered:
I+P,
initiation
+
promotion;
I,
initiation;
P,
promotion; M, metastasis or progression.
11
cal.. calories: adenocarc., adenocarcinoma.
preneoplastic lesion and ultimately a tumor. The progres-
sion stage is characterized by metastasis of the primary
tumor to distant tissues. The effect of ethanol on each of
these stages of experimental mammary carcinogenesis has
been examined to some degree.
The effect of ethanol provided during both the initiation
and promotion stages of chemically induced rat mammary
tumorigenesis was examined in
two
laboratorie~.~~~ No sig-
nificant enhancing effect was observed when ethanol was
provided at 20 to 35% of calories' or in the drinking water
(5%
v/v)~ both before and after DMBA dosing. In one of
the studies, it is unclear whether care was taken to prevent
an acute effect of ethanol on carcinogen metabolism at the
time of DMBA dosing,' which could result in impaired
DMBA activation and decreased tumor initiation. In the
other study: the amount of ethanol in the blood to which
the mammary gland was exposed may have been very mod-
est (mean
=
33 mudl).
There is evidence that ethanol intake was associated with
enhanced initiation of mammary carcinogenesis in three
Ethanol was provided before carcinogen dos-
ing, which would coincide with an effect on the initiation
stage of carcinogenesis. However, the mammary tumor
response to the quantity of ethanol consumed was not
consistent. Ethanol administered by gavage for
3
weeks at
3.5 and
7.0
g/kg body weight/day'' and in a liquid diet for
3
weeks as 20% of calories
(-8
to
9
g/kg/day)" before dosing
with DMBA was associated with an increase in mammary
tumor development, compared with isocaloric controls. Di-
etary ethanol intake at
10%
of calories had no effect on the
initiation stage of DMBA-induced mammary carcinogene-
sis." With MNU as the initiating agent, animals adminis-
tered ethanol by gavage at
7.0
g/kg, but not 3.5 g/kg'' and
ethanol at
15%
of calories
(-4
to
5
adday) but not at
20% or 30% of calories (12 to 13 g/kg)" exhibited an
increase in the formation of mammary tumors, compared
with isocaloric controls. The magnitude of the increases in
either mammary tumor incidence or multiplicity in these
studies ranged from 35 to
74%.
The reason for this incon-
sistent dose response to ethanol is not known. It is likely
that the lowest ethanol dose examined in one of these
experiments,
10%
of calories," was ineffective due to low
levels of ethanol circulating in the blood.
When ethanol is provided after administration of carcin-
ogen (i.e., during the promotion stage), there is evidence
that mammary carcinogenesis can be stimulated. However,
an inconsistent dose-response relationship also was ob-
served. Intake of dietary ethanol at 15% but not at 30% of
calories increased mammary tumor incidence
in
DMBA-
treated rats," compared with animals fed isocaloric control
diets. Similarly, ethanol consumption at 15% and 20%, but
not at 30% of calories,I2 increased the number of mam-
mary adenocarcinomas per MNU-treated rat, compared
with isocaloric controls. The magnitude of the increases in

336
SINGLETARY
either mammary tumor incidence or multiplicity in these
studies ranged from 32 to 91%.
Metastatic Mammaly Cancer
Tumor progression or metastatic spread
of
rat mammary
adenocarcinomas to the lung of rats was reported to be
increased 1.5-fold by chronic consumption of ethanol at
35%
of calories
(9
to 12 g/kg/day) for
5
weeks.I3 In this
study, acute administration (intraperitoneal) of ethanol at
2.5 and
3.5
glkgtday increased lung metastasis 2.5- and
3.4-fold, respectively, compared with controls. It would be
important to confirm this association, because it has been
reported recently that alcohol consumption by women aged
under
45
was directly related to increased risk for regional
and distant tumors, but not of earlier stage breast tumors.14
In general, there have been fewer animal studies examining
the role of diet
on
metastasis, compared with the initiation
and promotion stages
of
carcinogenesis. Evaluations of
dietary amino acid composition and lipid content on the
metastatic process have been re~orted.’~-’~
In summary, there is not evidence of enhanced formation
of spontaneous mammary tumors nor of DMBA-induced
mammary tumors when ethanol is consumed continuously
during both stages of tumor development. There is some
evidence that ethanol may be a weak cocarcinogen and a
weak promoter in the MNU- and DMBA-induced mam-
mary tumor models. However, the lack of consistent dose-
response relationships between ethanol intake and carcino-
genesis in these latter studies makes it difficult to draw firm
conclusions. Ethanol does seem to augment mammary tu-
mor progression in one published study. Additional confir-
mation of this enhancement of metastasis is needed.
CARCINOGENESIS-ASSOCIATED PROCESSES
Few published reports have evaluated in experimental
models the biological mechanisms whereby ethanol could
modify the susceptibility of the mammary gland to carcino-
genesis or stimulate the growth of mammary cancer cells.
The formation and repair of target tissue carcinogen-
DNA adducts is considered to be an important process
associated with the initiation stage of carcin~genesis.~ It has
been reported that the
in
vivo formation of rat mammary
DMBA-DNA adducts determined by the 32P-postlabeling
assay was not increased by chronic ethanol intake at
20%
of
calories before DMBA administration.”
An
effect of
higher ethanol intakes remains to be determined. There is
some evidence that repair
of
DMBA-DNA adducts in the
rat mammary gland might be impaired in rats consuming
ethanol and treated with the acetaldehyde dehydrogenase
inhibitor disulfiram.’’ The influence of long-term ethanol
treatment on the biotransformation of the polycyclic aro-
matic hydrocarbon benzo(a)pyrene in female rat liver, but
not in the mammary gland, has been reported.20321
The developmental stage and rate of cell division of the
maturing rat mammary gland are believed to be important
determinants of susceptibility of the mammary gland
to
carcinogen-induced DNA damage.” Ethanol intake at
20
to 30% of calories by female rats has been reported to be
associated with a 19 to
45%
increase in the density of
mammary gland terminal end buds (TEB), the target struc-
tures from which mammary adenocarcinomas develop.’3.’4
It was noted in these reports that TEB cell proliferation
also increased by 21 to
48%
in response to ethanol feeding.
An increased quantity of TEB and a higher rate of TEB cell
division at the time of carcinogen exposure could lead to
enhanced tumor initiation. Likewise, greater quantities of
TEB structures and higher TEB DNA-labeling indices
as-
sociated with ethanol intake could contribute to an en-
hancement of the process of mammary tumor promo-
In the study by Yirmaya et al.,I3 increased mammary
tumor metastasis in animals administered ethanol was as-
sociated with an impairment of natural killer cell activity.
This suggests that compromised immune function may be
contributing to increased tumor progression in response to
the quantities of ethanol examined. The interaction be-
tween alcohol and immunity has been extensively reviewed
elsewhere in relation to other animal cancer models.27
The ovarian steroid hormones estrogen and progester-
one and pituitary prolactin can support the development
of
rodent mammary tumors.6 However, the effects
of
ethanol
intake
on
blood estrogen and progesterone levels in ro-
dents are difficult to reconcile with the enhancement of
tumor promotion. Chronic ethanol intake at
15
to 30% of
calories has been reported to have no effect
on
serum
estradiol
(E2)
level^,^^*^^
and at
36%
of calories is associ-
ated with a small but statistically significant decrease in
serum
E,.28
On the other hand, substantial decreases in
serum progesterone
(49
to
57%)
have been reported for
female rats consuming ethanol at >20% of
calorie^.^'*^^*^^
These lower blood concentrations
of
progesterone would
not be expected to support mammary carcinogenesis, be-
cause it has been observed that transformation of mam-
mary epithelial cells by MNU requires prolactin and pro-
gesterone,” and that active immunity to progesterone
inhibits MNU-induced mammary tumor de~elopment.~’
Of
interest, however, is the observation that female, oopho-
rectomized rats provided ethanol in drinking water
(5.5%
v/v) for
10
weeks exhibited significantly higher serum
E,
levels, compared with controls.31
Also,
bourbon congeners
have been observed to elicit an estrogenic response in
ovariectomized rats as determined by changes in uterine
mass and plasma luteinizing hormone levels.32 Whether
these estrogenic responses would be sufficient to stimulate
mammary tumor promotion is not known. In contrast to
estrogen and progesterone, published reports have more
consistently noted a positive association
of
ethanol intake
with circulating prolactin levels in female rodents.33-3s This
enhancement of circulating prolactin levels in response to
ethanol consumption may partly contribute to a mammary
tumor-promoting effect
of
ethanol.
tion.75.26

ETHANOL AND EXPERIMENTAL BREAST CANCER
337
It is known that the susceptibility of mammary cells and
mammary cancer cells to estrogens and other hormones is
partly determined by hormone receptors in these cells.36
The effect of ethanol on estrogen and progesterone recep-
tors in normal and neoplastic mammary cells has not been
examined, although there
is
evidence that alcohol enhances
hepatocarcinogenesis induced by estrogen through modify-
ing estrogen and progesterone receptor
kinetic^.'^"'
Besides examining the effect of ethanol on pituitary and
ovarian hormones that could modulate breast cancer, ad-
ditional attention should be given to the influence of eth-
anol intake on adrenocortical steroids, particularly cortico-
sterone. Acute and chronic intakes of ethanol in rats have
been associated with increased serum corticosterone con-
centration~.~~ This is relevant, because there is compelling
recent data linking the suppression of chemically induced
lung and skin tumorigenesis in mice by food restriction to
elevated plasma corticosterone concentrations, a phenom-
enon that can be reversed by adrenale~tomy.~"'~' This has
not yet been demonstrated in a mammary tumor model.
Nonetheless, serum corticosterone levels in response to
graded intakes of ethanol by female rats and the resultant
impact on chemically induced mammary carcinogenesis
needs to be examined. It is possible that ethanol may have
the potential to either stimulate or inhibit chemically in-
duced mammary carcinogenesis, depending on the specific
endocrine response(s) stimulated and the ethanol dose at
which such responses occur.
HUMAN
BREAST
CANCER CELLS
Recently, the influence of ethanol concentration on the
growth
of
human breast cancer cells in vitro has provided
an additional system in which to examine the alcohol-
breast cancer relationship. Both estrogen receptor-positive
(ER+) and estrogen receptor-negative (ER-) cell lines
are widely used to study the process
of
human breast
carcin~genesis.~~ The ER+ human breast adenocarcinoma
cell line MCF-7 is the most widely used of human breast
cancer cells to study breast carcinogenesis. It has been
observed that ethanol added to cell cultures at concentra-
tions between
10
to
100
mM can selectively stimulate cell
proliferation
of
ER+ but not of ER- human breast cancer
cells.43 Compared with controls, cell proliferation in
MCF-7 (ER+) and ZR75.1 ER+ cell lines was stimulated
-2-fold in response to ethanol at a concentration of
100
mM. This increase in ER+ cell numbers at an ethanol
concentration of
100
mM was associated with an -2-fold
increase in the formation of intracellular CAMP. There was
no increase in cell number nor in intracellular CAMP con-
tent in the ER- cell lines BT-20 and MB-MDA-231 in
response to ethanol exposure.
No
change in the total con-
tent
of
ER in the
two
ER+ cell lines was observed after
addition of ethanol. These observations are noteworthy,
because there is recent evidence linking estrogen receptor
activity to cell signaling pathways in human breast cancer
cells.4J." Also, ethanol intake has been associated with
increased prevalence of ER+ breast tumors in one reportj6
and, in another study,j7 was observed to interact with
ERPR tumor subtypes and family history of breast cancer
in influencing breast cancer risk. Therefore, further confir-
mation
of
this effect
of
ethanol on human breast tumor
hormone receptor status should be a priority.
RESEARCH RECOMMENDATIONS
Currently, there is not sufficient experimental evidence
to establish ethanol unequivocally as a cocarcinogen or as a
promoter
of
experimental mammary carcinogenesis, nor
to
propose a clear biological mechanism for an effect of eth-
anol on mammary carcinogenesis. Nonetheless, it is possi-
ble for substantial progress to be made
if
several research
issues are addressed. First, the reason for the variable
mammary tumor responses to ethanol intake needs to be
understood. The diversity of experimental protocols used in
mammary tumor models could contribute to the variability
in responses. For instance, the amount
of
carcinogen ad-
ministered to animals has ranged from intermediate to very
high quantities. Care should be exercised in selecting the
carcinogen dose, because
it
has been reported, for example,
that DMBA at a sufficiently high dose can damage the
adrenals and reproductive tissues of rodents.48 The effect
of ethanol intake on chemically induced mammary tumor-
igenesis may depend on the carcinogen and dose used. In
this regard,
it
may be worthwhile to examine an effect
of
ethanol on the development of mammary tumors induced
by other newly identified mammary carcinogens, such as
the heterocyclic amine
2-amino-1-methyl-6-phenylimi-
dazo[4,5-b]pyridine
or
other polycyclic aromatic hydrocar-
bon~.~~~" The capacity of ethanol to modify the metabolism
and disposition of various mammary carcinogens that are
activated by diverse cytochrome P-450 isozymes has not
been thoroughly investigated.
Another potential source
of
variability is the method
chosen to provide ethanol and the quantity given to ani-
mals. In published reports examining the relationship of
ethanol intake to mammary carcinogenesis in rodents, eth-
anol has been administered by gavage, in the drinking
water, and as part
of
a defined liquid diet. In addition, the
amount of ethanol provided to animals has generally been
at one dose and at a level of 20% of calories and higher.
The rate of ethanol absorption, level and duration of eth-
anol in the blood, and subsequent effects on body metab-
olism could vary considerably depending on the amount of
ethanol consumed and the methods used to provide it.
Future experiments should examine more than one dose
of
ethanol, including low to high levels. It would be valuable to
examine ethanol intakes at doses
<30
to
36%
of calories,
because the substantial reduction in food and caloric intake
routinely observed for rodents consuming high quantities
of ethanol, as well as for their pair-fed controls, may con-
found an effect of ethanol on mammary tumorigenesis. It

33x
SINGLETARY
has been observed that rats fed ethanol at <20% of calories
consume quantities of diet nearly identical to ad libitum-
fed controls.'' This is an important issue, because there is
abundant evidence that dietary restriction can effectively
inhibit mammary tumorigenesis in
Also,
it
would be preferable that ethanol be delivered along with
defined, semipurified diets
so
that the content of macronu-
trients, micronutrients, and total calories can remain con-
sistent between control and ethanol-treated groups. Using
defined diets would have the additional benefit of eliminat-
ing the phytochemicals that are present in cereal-based
natural product diets. These phytochemicals are capable of
modifying and thus causing variability in tumor responses
independent of ethanol. Lastly, it would aid interlaboratory
comparisons for investigators to report blood ethanol con-
centrations accompanying ethanol feeding protocols. One
laboratory, for example, reported that provision of ethanol
in liquid diets at concentrations from
15
to 30% of calories
resulted in proportionate increases in blood ethanol from
23 to
86
rngld1.l'
Mammary gland and mammary tumor growth are re-
sponsive not only to numerous hormones, but also to di-
verse growth The relationship between ethanol
dose and the actions of relevant mammary-associated
growth factors, such as epidermal growth factor and insu-
lin-like growth factor, needs to be examined in more detail,
because there is evidence that ethanol is capable of influ-
encing growth factor regulation and actions in other tis-
sue~.~~-~~
Other mammary cancer models may need to be used to
resolve this complex research issue. For example, several
xenograft models59 could be used to determine in vivo
whether and how ethanol can modulate human breast can-
cer growth. Both the promotion and progression stages of
human breast cancer cell growth in response to ethanol can
be analyzed in these immunodeficient mice.60 Although a
more detailed understanding
of
the action of ethanol on
the individual biochemical and molecular steps associated
with breast cancer is a priority at this time, the future use of
transgenic mammary tumor models could provide valuable
mechanistic insights.61 Various strains are available that
differ in their expression of growth factors and their recep-
tors, cell-cycle genes, oncogenes, and tumor-suppressor
genes.
Compared with the progress made in the past two de-
cades in determining the relationship between alcohol and
human breast cancer risk, much less information is avail-
able from experimental models of breast cancer. The de-
velopment of a biologically plausible mechanism for an
enhancing effect of ethanol on breast cancer in animal
models would assist in the interpretation of the epidemio-
logical data and improve the consensus of opinion on this
controversial topic. Progress toward a clear, biological un-
derstanding can be made as improvements in experimental
protocols are implemented, as investigators use a combina-
tion of well-characterized in vivo and in vitro model systems
currently available, and as dose-response relationships be-
tween ethanol intake and multiple metabolic and neuroen-
docrine factors modulating mammary gland development
and mammary carcinogenesis are elucidated.
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