Effects of Alcohol in Mouse Neoplasia
ALFRED
S. KETCHAM,
HILDA
WEXLER,
AND NATHAN
MANTEL
(Surgery Branch and the Biometry Branch, National Cancer Institute, National Institutes of Health,
Public Health [email protected]. Department of Health, FAucetion, and Welfare, Bethesda, Maryland)
SUMMARY
Mice survive indefinitely on a regimen of @O
per cent alcohol by volume in their
water as their only source of fluid intake. This induces marked but reversible changes
in liver fat appearance. Over a period of 15 months on the regimen no evidence of
tumorigenesis was found. The alcohol regimen did not alter the growth or stimulate
the metastatic spread of a transplanted tumor, nor did it interfere with the reduction
in metastatic spread due to surgical removal of the tumor. It is concluded that, if alco
hol plays a role in mouse neoplasia, it is one dependent on factors missing from the
current experiments,
Clinical experience has indicated that the in
gestion of alcohol may play a role in the etiology
of carcinoma of the oral cavity and pharynx.
Various investigators
(1, 10, 1@, 14) including
Wynder (15, 16, 18) have attempted to document
this clinical impression. Observing the high fre
quency of patients with oral carcinoma admitted
to the Surgery Branch of the National Cancer In
stitute who had a significant history of alcohol
consumption, we became interested in investigat
ing the effects of this drug and in elucidating, if
possible, its mechanism of action. The study we
conducted was directed toward detecting either
direct carcinogenic effects of alcohol or its stimu
lating effects on themetast.atic
processes of trans
planted tumors in mice. Laboratory
investiga
tions of the direct effects of alcohol upon the ani
mal host have been reported (@, 3, 8, 9, 11), but
we had found
none relating
to its effect
upon
the
growth and metastases of transplanted
tumors.
The results of this present study were primarily
negative. The purpose of this presentation is to de
tail the circumstances
sults.
and nature
of negative
re
MATERIALS
AND METHODS
In each of two shorter-term
experiments
(Ex
periments 1 and @)
and two long-term experiments
(Experiments
3 and 4), 6- to 8-week-old CDBA/
@F1
female mice were housed in colonies and main
tamed on either alcohol (20 per cent by volume)
or water as their only source of fluid intake. Food
Received for publication
September
27, 1962.
was offered ad libitum. Food intake and consump
tion of liquid were recorded for all groups. In the
first
short-term
experiment
animals
were
main
tamed on alcohol for 1 month and in the second
short-term experiment for 6 months. In the two
definitive experiments mice were kept on alcohol
for 1 year. At the end of the period on alcohol all
mice were given an inoculation in the right hind
leg of 0.05 cc. of Cloudman S-91 tumor inoculuin,
prepared by the cytosieve technic (13).
The 1-month and 6-month alcohol groups were
divided randomly into control groups and into
groups in which the primary tumors were removed
at 21 days after tumor inoculation. Primary tu
mor growth was measured periodically, and all
animals were sacrificed for evaluation of metas@
tases
at
42
days
after
tumor
inoculation.
Animals were individually housed. At the end
of the initial period (1 year) on alcohol, half of
the alcohol-treated
animals were left on alcohol,
and half were placed on tap water. Similarly, half
of the water-fed control animals were left on water
and half transferred to 20 per cent alcohol as their
only source of fluid intake. When the tumor
reached a size of 1.5—2cm. (28 days after tumor
inoculation), the groups were further subdivided,
with half of each group having the primary tumor
bearing leg amputated. The others had the tumor
bearing leg left intact (controls). Still 28 days
later, or 56 days following tumor implantation, at
a time when control tumor-bearing animals were
starting to die, all mice were sacrificed and au
topsied. Specific evaluation was made as to the
667
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668
Cancer Re8earch
number and size of pulmonary metastases. Spleen
and body weights were recorded. Histological ex
aminations were made of the liver, spleen, and
lungs. The technics of tumor inoculum preparation,
leg amputation,
and counting and sizing of pul
monary metastases have been previously described
(5—7).
There were groups receiving alcohol only prior
to tumor implant, only following tumor implant,
and both prior and subsequent to tumor implant.
This could help differentiate between the direct
effects of alcohol on the metastatic
process and
those mediated through its effect on the host.
RESULTS
It was shown by repeated trials that only at a
20 per cent level of concentration
of alcohol would
all the mice survive a 3..month period in which
they lost weight; eventually, however, they adjust
ed and were capable of being maintained indefi
nitely at this alcohol percentage.
Throughout
the entire
study
period
no animal
developed a spontaneous tumor or was found, at
autopsy, to have a tumor—induced or spontaneous
—other than that related to the tumor inocula
tion made into the leg or its resulting metastases.
The results of all experiments
were negative
and showed no tumor-stimulating
or tumorigenic
effects. Because of the possibility that the periods
of exposure to alcohol in earlier experiments were
too short (1 month and 6 months) for the effects
of alcohol to become evident, we did not interpret
the negative results as conclusive.
Table 1 shows the experimental results relating
to the effects of alcohol on the metastatic spread
of the Cloudman S-91 melanoma. In S per cent of
instances, perhaps owing to the advanced age of
mice at the time of transplant,
primary tumor
takes were unsuccessful.
It was noted that the usual effects of amputa
tion previously reported (7) showed up in all
groups and were also apparent in the shorter-term
experiments. Fewer mice with their primary tu
mors amputated
developed pulmonary
metas
tases; the metastases that did develop, though
fewer in number, were generally larger in size.
No substantial or consistent effect of alcohol on
the size or incidence of pulmonary
metastases
was found, in contrast to the somewhat consist
ent effect of amputation.
Data on average spleen
weights are informative,
since enlarged spleens
have been observed to be associated with dissemi
nated neoplasia (4). For the four control groups
terminally on water (but initially on water or al
cohol), average spleen weights range from 0.78 to
0.80 gm. The two control groups maintained con
tinuously on alcohol have average spleen weights
Vol. 23, June
1963
of 0.43 and 0.45 gm., while the two control groups
only terminally on alcohol have averages of 0.22
and 0.21 gm. The eight amputated
groups, irre
spective of alcohol consumption,
have average
spleen weights ranging only from 0.11 to 0.21 gm.
Spleen weights are clearly greater for nonampu
tated groups. The intermediate spleen weights for
nonamputated
controls continuously or terminally
on alcohol were associated with lesser body weight
gains,
or even
body
weight
losses.
Control
mice
continuously
on alcohol showed modest weight
gains and higher intermediate spleen weights. Con
trol mice only terminally on alcohol showed con
siderable weight loss and had comparatively
low
spleen weights.
Body weight changes of mice initially on al
cohol lagged behind mice on water. On their initial
exposure to water at the end of 1 year such mice
drank excessive amounts of water. In the ensuing
period the weight gains were greater than those
for mice continuously on alcohol. This contrasts
with the mice transferred from water to alcohol
which showed severe weight loss and were, by far,
the poorest appearing of the experimental groups.
The body weight changes occurred in parallel
fashion for bothcontroland
amputated groups. The
lesser average mouse weights for amputated groups
chiefly reflect the physical absence of the ampu
tated tumor-bearing
leg.
In time (2—4months) animals adjusted to the
alcohol; however, their daily intake of food and
liquid never equaled that of the water-fed controls.
Older mice, perhaps owing to the added insult of
tumor inoculation, tolerated a transfer to alcohol
very poorly. Ten per cent died prior to time of
amputation
or sacrifice and are not included in
Table 1.
Gross pathological changes other than spleno
megaly in nonamputated
groups were not ob
served. Microscopically,
tumor cells were not
found in organs other than the lungs. The liver sec
tions failed to reveal any evidence of cirrhosis.
The alcoholic mice, both control and amputated,
did exhibit changes in hepatic cell metamorphosis
as compared with the water-drinking
groups. In
both the water-to-alcohol
and alcohol-to-water
groups, there was minimal to moderate fatty in
filtration of the parenchymal
cells of the liver.
Animals continuously
on water had normal-ap
pearing liver cells. The fatty infiltration of the
liver cells was most extreme for the animals con
tinually on alcohol.'
1 We
wish
Anatomy
to
thank
Dr.
Branch, National
Peter
D.
Olch
Cancer Institute,
of
the
Pathologic
for making his
tological examinations and for noting the fatty changes in the
liver cells.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1963 American Association for Cancer Research.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1963 American Association for Cancer Research.
670
Cancer Research
Despite
DISCUSSION
the total observation
period of up to
Vol. 23, June
transferred off alcohol after a year, it would seem
that such damage may be reversible.
15 months in Experiments
3 and 4 and up to 3
months and 8 months in the two shorter-term
ex
periments, all results were negative concerning the
carcinogenic effects of alcohol. There was no mdi
cation that alcohol promotes or stimulates cancer
spread. Where the primary tumor had been surgi
cally removed, alcohol did not interfere with the
reduced frequency of metastases.
The correlation observed by the clinician be
tween alcohol intake and particularly
head and
neck cancer cannot be easily avoided. This animal
study
failed to show a relationship,
and this could
be for a variety of reasons. First, we have no clini
cal or laboratory proof that there is such a phe
nomenon. We do know, however, that both dm1cally and in the laboratory
different tumors do
not necessarily act similarly in response to modify
ing agents. It is possible that there are factors
present in the environment
of man, but absent
from that of the laboratory mouse, to account for
the difference. This is not unlikely if alcohol affects
only one of a chain of sequential changes necessary
to elicit neoplasia. Heavy alcohol consumption is
usually associated with altered and inadequate
eating habits—the laboratory mouse on alcohol,
though consuming less solid food, nevertheless re
ceived the same Purina chow as its water control.
Another factor in the environment
of man is to
bacco. The larger proportion of alcoholic patients
are also heavy smokers; a carcinogenic effect for
alcohol in conjunction with tobacco is thus a possi
bility. To this point it is of interest that tobacco
chewing and snuff-dipping have been suggested
to have a role in oral cancer (17).
Another possibility could lie in the difference
between the laboratory
alcohol used in these
studies and the whiskey or other alcoholic bever
ages consumed by humans. Whiskey contains, in
addition to alcohol, a wide variety of agents in
cluding aromatics, alkaloids, and even polycyclic
hydrocarbons.
The severe fatty changes observed in liver cells
of animals kept on alcohol are of interest. When
mice had been on alcohol for only a limited period
(8-12 weeks), these changes were only moderate.
Since
less severe
changes
were found
in mice
1963
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Effects of Alcohol in Mouse Neoplasia
Alfred S. Ketcham, Hilda Wexler and Nathan Mantel
Cancer Res 1963;23:667-670.
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