Carcinogenesis vol.17 no.2 pp.209-212, 1996
Inhibition of 7,12-dimethylbenz[a]anthracene-induced lung
tumorigenesis in A/J mice by food restriction is reversed by
adrenalectomy
Laura L.Pashko and Arthur CSchwartz1*2
Department of Microbiology and ' Fels Institute for Cancer Research and
Molecular Biology, Temple University School of Medicine, Philadelphia, PA
19140, USA
2
To whom correspondence should be addressed
Prior work has demonstrated that food restriction of
mice markedly suppresses 12-0-tetradecanoylphorbol-13acetate (TPA) promotion of skin papillomas and adrenalectomy prior to initiating food restriction completely reverses
the tumor inhibitory effect of underfeeding. In the present
experiment the effect of food restriction, with or without
prior adrenalectomy, on 7,12-dimethylbenz[a]anthracene
(DMBA)-induced lung tumor development in A/J mice was
explored. Food restriction (27%), beginning 3 weeks after
a single oral dose of 0.5 mg DMBA and continued for
the duration of the experiment (14 weeks), significantly
inhibited lung adenoma development, whereas adrenalectomy 2 weeks before initiating food restriction abolished the
tumor inhibitory effect of underfeeding and also enhanced
tumor development in the ad libitum fed mice. Plasma
corticosterone levels were significantly elevated in foodrestricted A/J mice, whereas plasma dehydroepiandrosterone (DHEA) levels showed no apparent change. These
studies suggest that adrenal gland secretory products may
play a general role in the tumor preventive effect of food
restriction in laboratory mice.
Introduction
Food restriction of laboratory mice and rats is probably the
most effective regimen known for the prevention of cancer
in these genera. Underfeeding retards the development of
spontaneous (1-3), chemically induced (1,4) and radiationinduced tumors (5,6). Although it has been known for over
50 years that food restriction inhibits tumorigenesis, the
mechanism of this effect is obscure (7). Recently we reported
that food restriction of CD-I mice inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA*) promotion of skin papillomas,
whereas adrenalectomy prior to initiating food restriction
completely reverses inhibition of tumor development (8). Since
both glucocorticoid steroids (9,10) and dehydroepiandrosterone
(DHEA) (11) block TPA promotion of skin tumors, we have
postulated that elevated levels of adrenocortical steroids in
response to reduced food intake mediate the tumor inhibitory
effect of food restriction (12).
We have used a carcinogen-induced lung adenoma model
in A/J mice to further explore the effect of adrenalectomy on
the tumor inhibitory effect of food restriction. The lungs of
strain A mice are extremely susceptible to induction of tumors
with carcinogenic agents (13) and both spontaneous lung
•Abbreviations: TPA, 12-O-tetradecanoylphorbol-13-acetate;
dehydroepiandrosterone; DMBA, 7,12-dimethylbenzfa]anthracene.
© Oxford University Press
DHEA,
tumorigenesis in this strain and lung tumorigenesis enhanced
by asbestos treatment are inhibited by caloric restriction (14).
The induced pulmonary tumors occur directly underneath the
pleura and are readily quantitated on the external surface of
the lung following fixation (13).
Using this lung tumor model we now report that food
restriction of A/J mice following a single administration
of 7,12-dimethylbenz[a]anthracene (DMBA) significantly
reduces the number of induced lung adenomas; adrenalectomy
prior to initiating food reduction completely abolishes the
tumor inhibitory effect of underfeeding.
Materials and methods
Induction of tumors and adrenalectomy
Male A/J-Jax mice were obtained from the Jackson Laboratory (Bar Harbor,
ME) at 7 weeks of age. Mice were initially housed in the Fels Animal Facility
at five animals per cage in 11.5X7X5 inch plastic shoebox cages on
hardwood bedding with ad libitum access to Purina 5015 chow. After 1 week
acclimatization to the facility mice were treated per os with 0.5 mg DMBA
(ChemSyn Laboratories, Lenexa, KS) in 0.2 ml sesame oil. All mice were
treated with DMBA between the hours of 1 and 3 p.m.
One week after DMBA treatment the mice were either adrenalectomized
or sham operated. The mice were anesthetized with Metofane and a midline
incision was made on the back of each mouse. The skin was loosened from
the abdominal wall, an incision was made in the abdominal wall on each side
and the adrenal glands were removed. The abdominal wall was sutured with
3.0 mm gut suture material and the outer skin was closed with 9 mm wound
clips. Sham-operated mice were treated identically except that the adrenal
glands were not removed. The mice were given 0.5 ml 0.9% NaCl i.p. to
minimize the effect of dehydration due to surgery. Mice were kept warm
(each cage was placed on a heating pad) following surgery to minimize
hypothermia. Each mouse was kept in a cage on a heating pad until it was
moving freely after surgery. Adrenalectomized mice were given ad libitum
access to 1% NaCl immediately after surgery and throughout the remainder
of the experiment. Following adrenalectomy or sham operation al] mice were
singly housed in 9.4X5.4X5.1 inch plastic shoebox cages for the duration of
the experiment.
Food restriction
Groups of mice were started on a food restriction regimen 2 weeks after
adrenalectomy or sham operation. Mice were divided into four groups: (i)
sham-operated, ad libitum fed, 30 mice; (ii) sham-operated, food-restricted,
33 mice; (iii) adrenalectomized, ad libitum fed, 32 mice; (iv) adrenalectomized,
food-restncted, 43 mice. Food consumption was determined weekly, with
correction made for food lost in the bedding, as described previously (15).
The mice were weighed approximately biweekly on a Sartorius balance (model
1409-M8-1) equipped with an animal weighing program. Food-restricted mice
received daily allotments at -11:30 a.m. of Purina chow that were 27% less
than the mean quantity of food consumed by the ad libitum fed groups.
Tumor counts
Fourteen weeks after DMBA treatment mice were killed with an overdose of
CO?. The lungs were excised, rinsed in 0.9% NaCl and fixed in Fekete's
fixative (a modification of Tellyesniczky's fluid containing 10 ml 21-40%
formaldehyde, 5 ml glacial acetic acid, 100 ml 70% alcohol), which brings
out clearly the smallest tumor nodules in the lung (16). Tumors were counted
with the aid of Bausch and Lomb 3.5 x and 7X hand magnifiers.
We performed a square root transformation on the number of tumors in
each group in order to more closely approximate the normality assumption
required by classical ANOVA. The four groups are the treatment combinations
of two factors each at two levels: sham-operated versus adrenalectomized and
ad libitum versus food-restricted. The data were then analyzed using the
program PROC GLM of SAS statistical software (17).
209
LX.Pasbko and A.CSchwartz
30 -|
Table I. Effect of food restriction and adrenalectomy on lung tumor
development
Sham-operated, ad libitum fed
Sham-operated, food-restricted
Adrenalectomized, ad libitum fed
Adrenalectomized, food-restricted
Number
of mice
Lung tumors
(mean ± SEM)
30
29
28
26
2.03±0.29*
0.52±0.10
4.46±0.83 b
4.65 ±0.97°
Each value represents the mean ± SEM of the total number of lung tumors
per mouse for each group. Virtually all of the tumors examined
histologically (96%) were type II alveolar adenomas.
•Significantly greater than sham-operated, food-restricted, P < 0.008.
''Significantly greater than sham-operated, ad libitum fed, P < 0.007.
c
Significantly greater than sham-operated, food-restricted, P < 0.001.
28
26 ~ 24 -
fS
22 20
\
18 -
10
Plasma corticosteronc and DHEA levels
In order to assess the effect of food restriction on plasma corticosterone and
DHEA levels, 6-week-old A/J-Jax mice were obtained from the Jackson
Laboratory. After 2 weeks of acclimatization to the Fels Animal Facility mice
were individually housed and either fed ad libitum or food restricted to -32%
less food than the ad libitum fed group.
After 36 days of ad libitum feeding or food restriction mice were bled
between the hours of 3 and 5 p.m., a time when plasma corticosterone levels
are significantly elevated from the diurnal nadir (18). To minimize stress to
the mice on the day of plasma collection no personnel entered the animal
room prior to bleeding. Mice were removed from their cages, lightly
anesthetized with Metofane and bled from the orbital sinus using heparinized
Natelson blood collecting tubes. All mice were bled within 2 min of removal
from the cage. Blood (-250 jil from each mouse) was pooled from four mice
for each sample. Four samples were analyzed in each group (total of 16 mice).
Blood was kept on ice until the bleeding was finished. The samples were then
centrifuged at 3000 g for 15 min. The plasma was transferred to 1 ml plastic
tubes, frozen in liquid nitrogen and stored at -80°C.
Frozen samples were sent to Endocrine Sciences (Tarzana, CA) for
determination of corticosterone levels by radioimmunoassay and to Dr Gary
Gordon (Johns Hopkins University Medical School) for determination of
DHEA and DHEA sulfate levels by radioimmunoassay (19).
Histological examination of tumors
For histological examination of tumors the lungs were trimmed, embedded
in paraffin, sectioned to a thickness of 5 |im and stained with hematoxyhn
and eosin. Individual tumors were characterized as alveolar or bronchiolar
adenomas by morphological criteria (20). Tumor examination was performed
by Dr Thomas Van Winkle (University of Pennsylvania School of Veterinary
Medicine). A total of 28 lungs were examined (eight adrenalectomized, ad
libitum fed; seven adrenalectomized, food-restricted; seven sham-operated,
food-restricted; six sham-operated, ad libitum fed).
Results
As shown in Table I, food restriction of sham-operated animals
significantly inhibited lung tumor development (P < 0.008).
Adrenalectomy of mice 1 week after DMBA treatment and 2
weeks prior to initiating food restriction completely eliminated
suppression of lung tumor development produced by food
restriction. Adrenalectomy also enhanced the number of lung
tumors observed in the ad libitum fed animals (P < 0.007).
Droms et al. (21) also found that adrenalectomy of ad libitum
fed A/J mice significantly enhanced the number of lung
adenomas produced by a single injection of urethane.
Histological analysis of tumors revealed that virtually all
the tumors were type II alveolar adenomas. Of a total of 49
tumors examined 47 were alveolar adenomas and two were
bronchiolar adenomas (one in the adrenalectomized, foodrestricted group and one in the sham-operated, ad libitum
fed group).
At the termination of the experiment, 14 weeks after DMBA
treatment, the numbers of mice alive in each group were: (i)
sham-operated, ad libitum fed, 100% (30/30); (ii) sham210
16
12
18
Aft
(wctfcs)
Fig. 1. Weights of mice in each treatment group. Food restriction was
initiated 3 weeks following DMBA treatment and 2 weeks after
adrenalectomy or sham operation. Each point is the mean weight ± SD in
grams. All animals in each experimental group were weighed at each time
point Taking into consideration the loss of mice with time in three out of
four of the experimental groups the minimum number of mice weighed for
each point in each group were: sham-operated, ad libitum fed, 30; shamoperated, food-restricted, 29; adrenalectomized, ad libitum fed, 28;
adrenalectomized, food-restricted, 26. The average food consumption (mean
± SD g/mouse/day) for each group was: sham-operated, ad libitum fed,
3.45 ± 0.17 (n = 6); sham-operated, food-restricted, 2.52 ± 0.14 (n = 7);
adrenalectomized, ad libitum fed, 3.48 ± 0.11 (n = 7); adrenalectomized,
food-restricted, 2.54 ± 0.10 (n = 7).
Table II. Effect of food restriction on plasma corticosterone and DHEA
levels
Steroid concentration (ng/ml)
Ad libitum fed
Food-restricted
Corticosterone
DHEA
67±6
183±34*
1.06±0.14
O.82±O.I3
Eight-week-old male A/J mice were singly housed and fed ad libitum or
given daily allotments of 32% less food. After 36 days mice were bled
between 3 and 5 p.m. and plasma corticosterone and DHEA levels were
measured as described in Materials and methods. Blood (-250 |il from each
mouse) was pooled from four mice for each sample. Each value is the mean
± SD for four samples (total of 16 mice). The average food consumption
(mean ± SD, n = 4) was 3.57 ± 0.04 g/mouse/day for the ad libitum fed
and 2.44 ±0.17 g/mouse/day for the food-restricted group.
•Significantly greater than ad libitum fed, P < 0.01, Student's r-tcsL
operated, food-restricted, 88% (29/33); (iii) adrenalectomized,
ad libitum fed, 88% (28/32); (iv) adrenalectomized, foodrestricted, 60% (26743). The increased mortality of the
adrenalectomized, food-restricted A/J mice is similar to that
experienced by CD-I mice (8) and reflects the greater fragility
of these animals to the stress of food deprivation.
Although the ad libitum fed, sham-operated animals consumed the same amount of food as the ad libitum fed,
adrenalectomized mice, the adrenalectomized mice gained less
weight. In contrast, the adrenalectomized, food-restricted mice
gained more weight than their non-adrenalectomized, foodrestricted counterparts (Figure 1). The reason for these alterations in the efficiency of food utilization in the various
treatment groups is not clear.
As shown in Table II, plasma corticosterone levels were
DMBA-lnduced tumorigenesis, food restriction and adrenalectomy
significantly elevated in A/J mice food restricted for 36 days;
DHEA plasma levels, in contrast, showed no apparent change.
Discussion
These experiments demonstrate that adrenalectomy prior to
initiating food restriction reverses the tumor inhibitory effect
of underfeeding in a mouse lung tumorigenesis model; together
with a similar finding in a mouse skin tumor model, these
results suggest that secretory products of the adrenal gland
may play a major role in mediating the tumor inhibitory effect
of food restriction.
Plasma corticosterone levels (blood drawn between 3 and
5 p.m.) were 2.7-fold higher in the A/J food-restricted mice
compared with ad libitum fed animals. We also observed a
1.7-fold elevation in plasma corticosterone levels (taken
between 9 and 11 a.m.) in CD-I mice which received 27%
less food than ad libitum fed controls for 11 weeks (8), as
well as a 3.3-fold elevation in plasma corticosterone levels
(taken between 9 and 11 a.m.) in CD-I mice receiving 43%
less food than ad libitum fed controls for 8 weeks (unpublished
observation). Klebanov et al. (18) found that 40% food
restriction of BALB/c mice for 4-6 weeks produced a 3-fold
rise in the diurnal elevation of plasma corticosterone levels,
as well as an attenuated inflammatory response in hindfoot
pads injected with carrageenan.
A/J mice that were food restricted and adrenalectomized
experienced a greater mortality throughout the lung tumorigenesis experiment than mice which were food restricted and
sham operated. We observed a similar effect of adrenalectomy
on survivorship of food-restricted CD-I mice (8). A critical role
of glucocorticoid hormones is to maintain glucose homeostasis
during periods of food deprivation (22) and this may account
for the increased fragility of food-restricted mice lacking
adrenal glands.
In previous experiments we found that the hyperplastic
response of mouse epidermis to topical treatment with the
tumor promoter TPA is abolished by 1 week of food restriction
prior to TPA treatment (8,23). This anti-hyperplastic effect of
food restriction is very likely critical to the anti-tumor promoting action of underfeeding in the mouse skin, since it is likely
that the hyperproliferative and tumor promoting actions of
TPA are causally linked (24).
We have quantitated the degree of epidermal hyperplasia
produced by TPA treatment by either measuring the specific
incorporation of [3H]thymidine into epidermal DNA 20 h after
TPA application or by determining the epidermal DNA content
of a 2X2 cm2 section of mouse skin 48 h after TPA treatment.
Using both of these measurements of epidermal hyperplasia 1
week of food restriction prior to a single topical TPA application
completely abolished the observed hyperplasia. Adrenalectomy
3-7 days prior to initiating food restriction completely reversed
both the anti-hyperplastic effect and anti-tumor promoting
effect of food restriction (8). Since glucocorticoid steroids
(9,10) and DHEA (11) both block the hyperplastic and tumor
promoting effects of TPA, we have hypothesized that overproduction of these adrenocortical steroids in response to food
restriction may mediate the tumor suppressing effect of underfeeding (12). Both DHEA and glucocorticoids inhibit oxygen
free radical formation (12). Recent evidence suggests that
oxygen free radicals may be widely used messengers which
activate signal transduction pathways stimulating cellular proliferation (25,26). Overproduction of oxygen free radicals may
also damage critical macromolecules, such as DNA, lipids and
proteins, which could also enhance tumorigenesis (27,28).
Although the adrenalectomy experiments suggest an important role for the adrenal gland in mediating the tumor inhibitory
effect of food restriction, more specific evidence is required
to indicate a direct involvement of adrenocortical steroids. We
found previously that administration of the glucocorticoid
receptor antagonist mifepristone (RU-486) to CD-I mice during
an 8 day period of food restriction significantly reversed the
anti-hyperplastic effect of food restriction in TPA-treated
mouse epidermis (29). This effect of RU-486 suggests a direct
role for elevated corticosterone levels in mediating, at least in
part, the anti-hyperplastic effect of food restriction in TPAtreated mouse skin.
Plasma levels of DHEA and DHEA sulfate are much higher
in the human and various primate species than in several
rodent and domestic species examined (30). The level of
DHEA detected by us in A/J mouse plasma is ~20% of that
found in young men and women (31). DHEA sulfate levels in
mouse plasma were below the sensitivity of our assay and
< 1/300 of the level found in young humans (31). In contrast
to the consistently higher corticosterone plasma levels found
by us and others in food-restricted mice, we found no change
in plasma DHEA levels in food-restricted A/J mice. Thus
either DHEA plays no direct role in mediating the tumor
preventive and other age retarding effects of food restriction
in the mouse or a congener of DHEA, with the biological
properties of the native steroid, exists in the mouse and does
not significantly react with the antibody for DHEA in the
radioimmunoassay.
Since DHEA produces striking biological effects in laboratory rodents, many of which mimic the effects of food
restriction (32), including inhibition of DMBA-induced lung
tumor development in A/J mice (33), the latter possibility
would seem likely. However, our experiments on DHEA
plasma levels provide no direct support for the hypothesis that
elevated DHEA levels in food-restricted mice contribute to the
tumor inhibitory and age retarding effects of food restriction.
The results of these experiments in both skin and lung
tumorigenesis models strongly suggest that secretory products
of the adrenal gland play a major role in mediating the tumor
inhibitory effect of food restriction in mice. The observation
that administration of the glucocorticoid antagonist RU-486 to
CD-I mice significantly reverses the anti-hyperplastic effect
of food restriction in TPA-treated epidermis suggests that one
of the adrenal gland secretory products is corticosterone
(29). The adrenocortical glucocorticoids possess marked antiinflammatory action (34) and the inflammatory process, with
its attendant infiltration of affected tissue by neutrophils and
macrophages, may be an important source of oxygen free
radicals. Chronic inflammation has been linked to the development of several cancers in humans, including cancer of the
bladder (35), stomach (36), colon (37) and pancreas (38).
There is increasing evidence that the regular use of aspirin
may significantly reduce the risk of colorectal cancer (39).
Further research is needed to determine if elevated corticosterone levels unequivocally contribute to the tumor
suppressing effect of food restriction in mice and what role
other adrenal secretory products, such as DHEA, may have in
this phenomenon.
Acknowledgements
We thank Dr B.Holland and Ms Jiang Yu (Temple University) for assistance
with statistical analysis of the data and Mr M.Paden for excellent technical
211
L.L.Pashko and A.CSchwartz
assistance. This work was supported by NIH grants CA 60471 and CA 12227.
All animaJ care in these experiments was in accordance with the guidelines
of the Institutional Animal Use and Care Committee of Temple University.
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Received on August 16, 1995; revised on October 20, 1995; accepted on
October 30, 1995