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Nutrition and Cancer

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Colon Cancer Is Induced by a Single Low Dose of

Azoxymethane in Fasted-Refed Rats
Giovanna Caderni , Enrico Bollito , Robert M. Elashoff & Luciana Tessitore
Published online: 18 Nov 2009.

To cite this article: Giovanna Caderni , Enrico Bollito , Robert M. Elashoff & Luciana Tessitore (1999) Colon Cancer Is
Induced by a Single Low Dose of Azoxymethane in Fasted-Refed Rats, Nutrition and Cancer, 35:2, 137-142, DOI: 10.1207/
S15327914NC352_7

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 NUTRITION AND CANCER, 35(2), 137–142
Copyright © 1999, Lawrence Erlbaum Associates, Inc.
Colon Cancer Is Induced by a Single Low
Dose of Azoxymethane in Fasted-Refed Rats
Giovanna Caderni, Enrico Bollito, and Luciana Tessitore
Abstract: We reported previously that fasting-refeeding en-
hanced the growth of preneoplastic lesions in the colon of
rats induced by 20 mg/kg of azoxymethane (AOM). Here we
studied whether fasting-refeeding could also affect 1) the in-
duction of colon cancer by the same dose of AOM and 2) the
Downloaded by [University of Otago] at 19:49 28 December 2014
induction of foci by lower doses of AOM that do not induce
foci in fully fed rats. Fully fed and fasted-refed rats were
given AOM by single subcutaneous injection, and the devel-
opment of foci or tumors was evaluated three months or one
year later. The results of the long-term carcinogenesis ex-
periments showed that the total incidence of tumors was
increased in the fasted-refed rats. Moreover, although fully
fed rats developed foci only when injected with 7.5, 10, or 20
mg/kg of the carcinogen, a significant number of foci were
also induced by 5 mg/kg in fasted-refed rats. The crypt mul-
tiplicity of foci was also higher when rats were exposed to
fasting-refeeding, even when the number of foci was un-
changed. These data suggest that growth perturbations in-
duced by fasting-refeeding lead to the development of
preneoplastic lesions with doses of AOM too low to trigger
foci in fully fed rats and produce enhanced sensitivity to the
development of intestinal tumors.
Introduction
Dietary habits can influence the evolution of neoplasia in
animals and humans (1–3). Many epidemiological and ex-
perimental studies have provided evidence of the protective
role of chronic caloric restriction against many types of
spontaneous and chemically induced tumors. On the con-
trary, cycles of caloric restriction followed by ad libitum
feeding have been reported to enhance liver and mammary
carcinogenesis (4,5).
We reported previously that a period of complete food
withdrawal followed by one day of refeeding before initia-
tion is able to sustain the development of foci, nodules, and
hepatocarcinoma induced by a subnecrogenic (noninitiating)
dose of diethylnitrosamine (6,7). We also showed that cycles
G. Caderni is affiliated with the Dipartimento di Farmacologia, Università degli Studi di Firenze, Firenze, Italy. E. Bollito is affiliated with the Sezione di
Istologia ed Anatomia Patologica, Ospedale San Luigi Gonzaga, Orbassano, Torino, Italy. L. Tessitore is affiliated with the Dipartimento di Scienze Mediche,
Università del Piedmonte Orientale “Amedeo Avogadro,” Italy.
of fasting-refeeding during the promotion phase of
carcinogenesis enhanced the growth of liver nodules
selectable by the resistant hepatocyte model of Solt and
Farber in rats (8) and mammary cancer induced by
methylnitrosourea (9) and dimethylbenz[a]anthracene (10).
We recently demonstrated that rats starved for four days,
then refed (fasted-refed rats) and treated with 20 mg/kg body
wt of azoxymethane (AOM) developed larger preneoplastic
lesions [aberrant crypt foci (ACF)] than fully fed rats, partic-
ularly in the distal part of the colon and rectum (11); we ob-
served a depression of cell proliferation during fasting and
an overshoot in the subsequent refeeding, which led to a
high number of S phase cells the day of AOM administration
and thereafter. These results suggested that the apoptosis in-
duced by fasting might represent a cell proliferative stimulus
during the subsequent refeeding, leading to a higher number
of cells susceptible to DNA damage on the day of carcino-
gen treatment and higher probability of repairing the damage
thereafter.
The present study examines whether a four-day fast fol-
lowed by refeeding before initiation with 20 mg/kg body wt
of AOM enhances colorectal cancer induction in rats. We
were also interested in studying whether a low dose of
AOM, probably insufficient to induce ACF in fully fed rats,
induces ACF in fasted-refed rats.
Material and Methods
Animals
Ninety-four (6-wk-old) male Fischer 344 rats weighing
about 130 g (Charles River, Como, Italy) were housed in
suspended plastic cages with wire bottoms in an animal
holding room at controlled temperature (21 ± 1°C), humidity
(70–80%), and lighting (lights on from 0800 to 2000, lights
off from 2000 to 0800). They were given free access to water
for the entire experimental period and regular chow pellets
(standard AIN-76 diet, Piccioni, Brescia, Italy) until the be-
 ginning of the experiment. The experimental procedure was Table 1. Body Weight of Animalsa,b
approved by the Bioethics Committee of the University of
Fully Fed Fasted-Refed
Turin. A grating impeded the rats’ access to feces or bedding
during fasting. The cages were covered with filter tops for Days
five days beginning on the day of carcinogen treatment. -5 134 ± 4 131 ± 3
-1 145 ± 4 95 ± 2‡
0 149 ± 4 112 ± 3‡
Treatment
4 147 ± 2 121 ± 3‡
7 151 ± 4 123 ± 4†
The animals, from which 2 groups of 44 rats each were 20 190 ± 4 170 ± 3*
randomly selected, were given a single injection of 5, 7.5, Months
10, or 20 mg/kg body wt sc of AOM (Sigma Chemical, Mi- 4 336 ± 5 328 ± 6
lan, Italy) in 0.9% NaCl solution (Figure 1). 6 347 ± 4 351 ± 6
Rats in Group 1 were fed ad libitum, whereas those in 8 357 ± 7 355 ± 10
10 361 ± 9 375 ± 7
Group 2 were fasted for four days, then refed; the AOM was 12 388 ± 11 398 ± 9
administered on the day of refeeding (Figure 1). For the
long-term carcinogenesis experiment, rats were treated with a: Values are means ± SEM (n = 6) expressed in g.
20 mg/kg of AOM; there were 20 rats in each group b: Statistical significance is as follows: *, p < 0.01; †, p < 0.005; ‡, p £
0.0005 vs. fully fed rats at same time (Student's t-test).
(fasted-refed or fully fed rats); 6 rats received only saline
and were used as controls. For the ACF experiments, there
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were 12 rats per dose of AOM: 6 were fasted-refed, and 6 crypt multiplicity (mean number of ACs forming each focus)
were fully fed. Animals were killed by CO2 asphyxiation were evaluated in the entire colorectal mucosa.
three months or one year later for evaluation of the develop-
ment of ACF or tumors, respectively. Analysis of Tumors

Analysis of Number and Multiplicity of ACF
The entire colon and rectum were removed, flushed with
cold saline solution (0.9% NaCl), slit open from cecum to
anus, and fixed in 19% buffered formalin, then stained with
methylene blue, as described by Bird (12). The colorectal mu-
cosa was assessed for aberrant crypts (AC) with a light micro-
scope. ACF were then classified as small (ACF of 1, 2, 3, or 4
crypts), medium (ACF of 5, 6, or 7 crypts), or large (ACF of
³8 crypts) to facilitate comparison of the results with the data
previously reported (11). The total number of ACF and the
All animals killed one year after AOM treatment were
autopsied, and the internal organs, external ear canal, sali-
vary glands, and mammary glands were carefully examined.
At autopsy, all tumor-bearing areas and areas suspected of
lesions were coded by location, then excised, fixed in 10%
formalin, and sectioned for histological examination. The
incidence and size of tumors were evaluated; the areas of tu-
mors were calculated by the product of the largest perpen-
dicular diameters per p/4; the organs were sectioned for
determination of any metastatic cancer growth; the livers
were also analyzed for g-glutamyltransferase.

Figure 1. Rats were given a single injection of 5, 7.5, 10, or 20 mg/kg sc azoxymethane (AOM) and killed 3 or 12 mo thereafter.

138 Nutrition and Cancer 1999

 Intestinal tumors induced by AOM were classified histo-
logically into adenomas and carcinomas; adenocarcinomas
were clearly malignant tumors, showing invasion through
the muscularis mucosae.
Values are means ± SEM, and differences between groups
are analyzed by Student’s t-test and c2 test. A difference was
considered statistically significant when p £ 0.05.
Results
Table 1 shows that fasted rats quickly lost body weight,
then recovered during refeeding. There was no significant
difference in body weight between the two groups four
months after the dietary perturbation (Table 1).
Long-Term Carcinogenesis Experiment
Rats were killed for tumor determination one year after
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the two groups. The development of small bowel tumors, all
of which were adenocarcinomas, was similar in the two ex-
perimental groups for incidence, multiplicity (Table 2), and
area (Table 3).
No significant difference was seen in the incidence of
extraintestinal tumors between the two experimental groups
(Table 2). The extraintestinal tumors induced by AOM in the
fully fed rats were as follows: 3 primary g-glutamyltrans-
ferase-positive liver cancers, leading to a liver weight-to-
body weight ratio of 17.47 ± 4.9 vs. 3.34 ± 0.09, one
squamous cell carcinoma in the skin of the abdominal wall
and another at the base of the external ear canal, and one
mammary fibroadenoma. Fasted-refed rats developed five
squamous cell carcinomas at the base of the external ear ca-
nal, two originating from sebaceous and three from salivary
glands, and one liver adenoma.
No metastases were observed except for one peritoneal
metastasis from a colon adenocarcinoma in a fully fed ani-
mal. None of the six control rats, which were not given

treatment with the carcinogen. At death, tumors were found

in the colon, in the small bowel, and also in extraintestinal
organs (see below). The incidence of total intestinal cancers
was significantly higher in fasted-refed rats than in fully fed
rats (p £ 0.05, c2 test). In fasted-refed rats, most colorectal
tumors were malignant, and 9 of 11 were located in the distal
segments of the colon or in the rectum; in the fully fed rats,
the two colorectal cancers were in the ascending colon (Ta-
ble 2). No difference was observed in tumor multiplicity
(Table 2) or in size of colorectal tumors (Table 3) between
AOM, developed any kind of tumor during the experimental
observation time.
Experiments on the Induction of ACF
We were also interested in studying whether a low dose
of AOM, probably insufficient to induce ACF in fully fed
rats, might induce ACF in fasted-refed rats; this hypothesis
was confirmed (Table 4). Although at the lowest dose of

Table 2. Effect of Fasting-Refeeding on Tumor Incidence and Tumor Multiplicitya,b

Colon-Rectum
Total Small
Total Intestinal Total Colon Rectum Adenoma Adenocarcinoma Bowel Extraintestinal

Tumor incidence, %

Fully fed 40 25 15 15 5 5 10 10 30
(8/20) (5/20) (3/20) (3/20) (1/20) (1/20) (2/20) (2/20) (6/20)
Fasted-refed 75* 65† 50† 45* 5 10 40* 15 30
(15/20) (13/20) (10/20) (9/20) (1/20) (2/20) (8/20) (3/20) (6/20)

Tumor multiplicity, tumors/tumor-bearing rat

Fully fed 1.75 ± 0.25 1.67 ± 0.20 1.33 ± 0.21 1 1 2 1 1±0

Fasted-refed 1.75 ± 0.20 1.50 ± 0.16 1.4 ± 0.19 1 2 1.37 ± 0.28 1±0 1±0

a: Values are means ± SEM (n = 6). Azoxymethane (AOM, 20 mg/kg) was administered on Day 0 to fully fed and previously fasted and refed rats (see Figure
1); 78% and 0% of colon tumors were found in left side in fasted-refed and fully fed rats, respectively.
b: Statistical significance is as follows: *, p < 0.05; †, p < 0.025 vs. fully fed rats at the same time (c2 test).

Table 3. Effect of Fasting-Refeeding on Tumor Area

Colorectal Tumors
Small Bowel
Total Left Right Rectum Tumors

Fully fed 0.67 ± 0.28 0.49 ± .30 1.35 ± 0 6.21 ± 3.25

(5) (4) (1) (2)
Fasted-refed 0.70 ± 0.13 0.70 ± 0.16 0.73 ± 0.32 0.55 ± 0 2.78 ± 1.19
(15) (11) (3) (1) (3)

a: Values are means ± SEM in cm2; numbers in parentheses represent number of tumors.

Vol. 35, No. 2 139

 Table 4. Effect of Fasting-Refeeding on the Number of ACF and Crypt Multiplicity of ACF Induced by AOMa,b
ACF/rat AC/ACF

AOM, mg/kg Fully fed Fasted-refed Fully fed Fasted-refed

5
Total 0.17 ± 0.15 3.50 ± 0.95§ 0.60 ± 0.46 3.17 ± 0.41§
Small 0.17 ± 0.15 3.25 ± 1.05‡ 0.60 ± 0.46 1.92 ± 0.43*
Medium 0 0.25 ± 0.09‡ 0 1.25 ± 0.82
7.5
Total 4.50 ± 0.79 12.0 ± 0.53|| 2.70 ± 0.02 3.19 ± 0.16‡
Small 4.50 ± 0.79 11.3 ± 0.18|| 2.70 ± 0.02 2.80 ± 0.14
Medium 0 0.70 ± 0.30† 3.10 ± 0.75 3.30 ± 0.89
10
Total 7.0 ± 0.72 11.0 ± 1.25‡ 2.53 ± 0.06 3.04 ± 0.13§
Small 7.0 ± 0.72 10.0 ± 0.80‡ 2.53 ± 0.06 2.83 ± 0.13*
Medium 0 1.0 ± 0.53* 0 2.10 ± 1.13*
20
Total 45.50 ± 1.25 37.50 ± 1.09|| 1.82 ± 0.47 4.78 ± 0.77‡
Small 39.80 ± 2.53 25.01 ± 1.90|| 0.57 ± 0.15 2.16 ± 0.19||
Medium 5.30 ± 0.60 8.0 ± 0.81† 2.57 ± 0.91 3.93 ± 0.90
Large 0.40 ± 0.28 4.50 ± 0.55|| 0.32 ± 0.36 8.26 ± 1.12||
Downloaded by [University of Otago] at 19:49 28 December 2014

a: Values are means ± SEM (n = 6). ACF, aberrant crypt foci; AC, aberrant crypts.
b: Statistical significance is as follows: *, p < 0.05; †, p < 0.025; ‡, p < 0.01; §, p £ 0.005; ||, p £ 0.0005 vs. fully fed rats at the same time (Student’s t-test).

AOM (5 mg/kg) only one of six fully fed rats developed
ACF, all the rats in the fasted-refed group did so (p £ 0.005,
c2 test). All the animals developed ACF when treated with
doses of AOM >5 mg/kg. Moreover, fasted-refed rats devel-
oped more ACF than fully fed rats after 7.5 or 10 mg/kg of
AOM (Table 4). The ACF found in the colorectal mucosa of
fasted-refed rats consisted of a higher number of ACs (crypt
multiplicity) (Table 4).
Discussion
The results of the present study indicate that 1)
fasted-refed rats treated with a single dose of 20 mg/kg body
wt of AOM are more susceptible to colon cancer than rats
that were fed ad libitum during carcinogenesis and 2) a low
dose of AOM insufficient to induce ACF in fully fed rats in-
duced ACF in all the fasted-refed rats. These results suggest
that the metabolic perturbation induced by fasting-refeeding
confers initiating properties to a noninitiating dose of AOM.
Carcinogenesis is a multistep process. It has been sug-
gested that colorectal cancer develops from ACF originating
from single ACs (13,14). As has been described by
McLellan and co-workers (17), the number of ACF consist-
ing of one or two ACs is transient, suggesting that they are
eliminated or undergo phenotypic reversion, as do preneo-
plastic lesions in the well-known liver model (15,16). Many
researchers (17–20) have also suggested that crypt multi-
plicity of ACF is a better predictor of colon cancer outcome
than the number of ACF. In line with these findings, in the
present study we observed that fasted-refed rats have a
higher tumor incidence in the distal colonic segments, where
a higher ACF multiplicity without changes in the number of
ACF had been observed previously (11). The fact that higher
crypt multiplicity in the fasted-refed rats corresponded to
higher tumor incidence but not to higher tumor multiplicity
is consistent with the remodeling of the preneoplastic lesions
(i.e., ACF), as described in detail elsewhere for hepatocarcino-
genesis (16).
On the basis of above data, we studied whether there was
a dose of AOM that does not induce preneoplastic lesions
but does have initiating capability in previously fasted-refed
rats.
Our results demonstrate that 5 mg/kg body wt of AOM is
not able to induce the development of ACF in ad libitum-fed
rats (ACF were only present in the colon-rectum of 1 of 6
rats), whereas fasting for four days followed by refeeding is
sufficient to confer initiating properties to this dose of AOM,
leading to the development of a remarkable number of ACF
per rat. Future experiments will verify whether the foci in-
duced by 5 mg/kg body wt of AOM in fasted-refed rats pro-
gress to carcinoma. The results obtained with a low dose of
AOM confirm those we obtained in a study of liver
carcinogenesis, when we showed that diethylnitrosamine at
20 mg/kg body wt induced the development of foci, nodules,
and hepatocarcinoma only in rats previously exposed to fast-
ing-refeeding (6,7).
Several possible mechanisms explain our data. Fasting-
refeeding determines alterations in cell growth, which may be
relevant to the carcinogenic process (11). Refeeding is the
mitogenic stimulus, induced by cell loss by apoptosis during
prolonged fasting (11). The consequent compensatory cell
proliferation favors the AOM initiation by making cells more
susceptible to carcinogen-induced DNA damage, fixing the
DNA lesions, enhancing the recombinatorial events, and cre-
ating hypomethylated sequences in the newly made DNA

140 Nutrition and Cancer 1999

 (15). Consistently, refeeding after food restriction (21) or
complete fasting (22) depressed apoptosis and enhanced cell
proliferation in the rat liver foci. Fasting-refeeding could also
affect carcinogen metabolism. Hong and colleagues (23)
demonstrated that a two-day fast increases the activity of
cytochrome P450ac isolated in hepatic microsomes almost five-
fold. Because AOM metabolism occurs in hepatic micro-
somes (24), fasting-refeeding could increase the DNA
damage induced by the carcinogen by enhancing its metabo-
lism. There might also be a weakening of the defense mecha-
nisms. In this connection, it has been reported that the levels
of hepatic glutathione decreased to 70% after three days of
fasting (25). In our experimental conditions, a drop in the
glutathione level during fasting might determine the forma-
tion of more toxic molecules during AOM administration
and, consequently, increase the number of cells that are initi-
ated by whatever dose of AOM. An increase in lipid
peroxidation might occur during fasting and refeeding (26).
The radical species originating during lipid peroxidation
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might induce DNA damage and, by a process of summing
with the damage provoked by the AOM treatment, might lead
to a higher number of initiated cells.
Different types of dietary factors can play an important
role in the modulation of colorectal carcinogenesis. On the
basis of the epidemiological and clinical aspects of our re-
sults, future experiments will study the effect of a period of
fasting followed by refeeding with various diets to evaluate
any difference in the growth of colorectal tissue and its sus-
ceptibility to carcinogenesis. We will also attempt to clarify
the effect of fasting-refeeding on AOM metabolism, cellular
defense and error repair mechanisms, and lipid peroxidation.
Western countries are characterized by unbalanced di-
etary habits, such as periods of complete fasting followed by
high ad libitum refeeding, which, according to our results,
might make people more prone to the development of
colorectal cancer. We are daily in contact with various sub-
stances that can be carcinogenic and determine the develop-
ment of initiated cells in our tissues and organs. Unbalanced
dietary habits, such as days of fasting alternated with periods
of high food consumption, may confer carcinogenic proper-
ties to various substances/conditions with which we are
daily in contact (27,28). This dietary perturbation might also
stimulate any existing initiated cell toward the development
of preneoplastic lesions.
Acknowledgments and Notes
This work was supported by grants from Ministero dell’Università
della Ricerca Scientifica e Tecnologica (40% and 60% funds, Rome). Ad-
dress correspondence to Luciana Tessitore, Dipartimento di Scienze
Cliniche e Biologiche, Università di Torino, Ospedale San Luigi
Gonzaga, Regione Gonzole 10-10043 Orbassano, Torino, Italy.
Submitted 21 April 1999; accepted in final form 15 July 1999.
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