[Cancer Research 60, 6856-6858, December 15, 2000]
© 2000 American Association for Cancer Research
Polyethylene Glycol 8000 and Colon Carcinogenesis: Inhibition in the F344 Rat, Promotion in the Min Mouse1
Dinaz Naigamwalla,
Marie C. Chia,
Thien T. Tran,
Alan Medline,
Kazy Hay,
Steven Gallinger and
W. Robert Bruce2
Departments of Nutritional Sciences [D. N., T. T. T., W. R. B.], and Laboratory Medicine and Pathobiology [M. C. C., A. M.], University of Toronto, Toronto, Ontario, M5S 3E2 Canada, and Samuel Lunenfeld Research Institute, Department of Surgery, Mount Sinai Hospital, Toronto, M5G 1X5 Canada [K. H., S. G.]
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ABSTRACT
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It
has recently been reported that 5% polyethylene glycol 8000 (PEG 8000;
Mr 8000) in the diet markedly inhibits the
development of colonic tumors in carcinogen-treated rats. To assess the
possible use of this agent as a preventive or treatment agent for
patients with familial adenomatous polyposis, we determined the effect
of PEG 8000 on spontaneous carcinogenesis in the Min mouse. PEG at a
5% concentration in the diet of Min mice did not affect the number of
small intestinal or cecal tumors but did increase the number of colon
tumors and the number of animals with colonic tumors (2 of 18
versus 12 of 22 animals; P < 0.001). Although the chemopreventive effect of PEG 8000 in rats is
remarkable, we suggest a cautious approach in long-term testing of PEG
as a chemopreventive agent for subjects at risk for colonic neoplasia.
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Introduction
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Corpet and Parnaud recently reported
that 5% (w/w) PEG3
8000 (Mr
8000) in the diet markedly inhibits the number and size of ACF, tumor
incidence, and tumor multiplicity in F344 rats given the colon
carcinogen AOM (1, 2, 3)
. They suggested that because of its
low toxicity, PEG might be considered a chemopreventive agent for human
colon cancer. The clinical effectiveness of PEG 8000 in colon cancer
prevention could be readily assessed in patients with FAP. Before
initiating such studies, however, we chose to evaluate the effect of
PEG on colon carcinogenesis in the Min mouse, a strain bearing a
mutation in the Apc gene similar to that observed in
patients with FAP (4)
.
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Materials and Methods
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The 40 three-week-old Min mice used for this study were progeny
of crosses of male Apc(+/-)Msh2(+/-) and female
Apc(+/+)Msh2(+/-) mice maintained at the Research Annex at
the Samuel Lunenfeld Research Institute, Mount Sinai Hospital (Toronto,
Canada) for studies of the combined effect of the Apc gene
with a defect in DNA mismatch repair [Msh2(-/-)]. Those
with genotype Apc(+/-)Msh2(+/-) and
Apc(+/-)Msh2(+/+) were identified (4)
and
transferred to microisolator cages and, after removing some of the
males for further crosses, randomized by genotype and sex to a control
diet (AIN76A; Dyets, Bethlehem, PA) or to the same diet with 5% (w/w)
PEG 8000 (Sigma, St. Louis, MO). In our colony,
Apc(+/-)Msh2(+/-) and Apc(+/-)Msh2(+/+) mice
are similar with respect to small intestine and colon polyps and rarely
develop colonic ACF (4)
. These two genotypes are also very
similar phenotypically to standard Min mice (4)
. The mice
were examined daily for evidence of toxicity until they were 100 days
of age, at which time, they were sacrificed by cervical dislocation.
Their small intestines, cecums, and colons were removed, cut open
longitudinally, stretched flat, fixed in formalin, and coded. The small
intestines and cecums were stained with 0.2% methylene blue and
examined under a dissecting microscope at x40 magnification to
determine the number of adenomas. The colons were examined in the same
way, but because many of the apparent tumors were large lymphoid
aggregates, all colonic tumors larger than 0.5 mm in diameter were
excised and examined histologically to determine whether they were
adenomatous polyps. Because the data for tumor number (and for ACF)
were skewed and not normally distributed, they were analyzed after a
square-root transformation, and results were given as mean and 95% CI.
When differences were detected by ANOVA, Student’s t tests
were used and considered significant when two-sided P were
0.05.
The F344 rats (26 male, 5-week-old rats obtained from Harlan
Sprague-Dawley, Inc., Indianapolis, IN) were housed individually in
wire-bottomed cages at the Division of Comparative Medicine, University
of Toronto (Toronto, Canada). After acclimatization on rodent chow
(Ralston Purina International, Strathroy, Canada) for 7 days, they were
initiated with a single dose of AOM (20 mg/kg body weight, i.p.;
Sigma). Seven days later, they were randomized by weight to the AIN-76A
diet or to the 5% PEG diet. They were sacrificed approximately 100
days after initiation, and their colons were removed, coded, and
assessed for ACF as described previously (5
, 6)
. The
colons were flushed clean of feces and other debris with Kreb’s
solution, cut longitudinally, fixed flat (mucosal side up) in 10%
formalin, and coded for blinding. After fixation, they were stained
lightly with 0.2% methylene blue and examined at x40 magnification.
ACF were distinguished on the basis of their enlarged crypts, increased
pericryptal space, and dark stain and scored for the number of
ACF/colon and ACF multiplicity (aberrant crypts/ACF).
The SWR mice (24 male, 6-week-old mice; Harlan Sprague-Dawley, Inc.)
were housed in microisolator cages at the Department of Comparative
Medicine, University of Toronto. After acclimatization, they were
initiated with three doses of AOM (10 mg/kg) given at weekly intervals.
One week after the last dose, they were randomized with regard to
diets; they were sacrificed at 100 days, and their colons were scored
for ACF. Some animals had macroscopic polyps larger than 0.5 mm in
diameter that were confirmed to be adenomas by histological
examination.
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Results and Discussion
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PEG 8000 produced no visible evidence of toxicity and did not
affect weight gain (data not shown). The average numbers of tumors in
the small intestine and cecum were not affected by dietary PEG or by
Msh2 genotype or sex (Table 1
,
columns 4 and 5). By contrast, an initial examination of the data for
colon tumors (Table 1
, column 7) showed that more animals receiving PEG
had adenomatous tumors than control animals (12 of 22 animals
versus 2 of 18 animals). Logistic regression using sex,
genotype, and diet as predictors supported this impression. It showed
that genotype was not significant (P = 0.27),
whereas diet (PEG) was very significant (P < 0.001), as was sex (P = 0.002). Because the
data for male animals were limited in number and significantly
different, a separate analysis for the females was relevant. Again,
logistic analysis indicated no interaction between diet and genotype
(P = 0.18), a significant effect of diet
(P < 0.001), and a nonsignificant effect of
genotype (P = 0.32). Thus, there was no
effect of PEG on the tumorigenesis process in the small bowel or cecum,
but there was an increase in the fraction of animals with colon tumors,
as well as in the total number of colon tumors/animal (Table 1
, column
6) in the PEG-treated female Min mice.
This unexpected result regarding colon tumors in Min mice led us to
reexamine the effect of PEG 8000 on AOM-induced ACF in the rat. The
results (Fig. 1
, top) confirm
the striking protective effect of dietary PEG 8000 on ACF observed by
Corpet and Parnaud (1
, 2)
. The average number of ACF per
colon was reduced from 62.0 to 14.8 (CIs = 56.3–68.1 to
10.8–19.4; P < 0.001) per colon. The number
of large ACF (greater than 4 aberrant crypts/ACF in size) was reduced
from 22.5 to 0.62 (20.8–24.2 to 0.11–1.55; P < 0.001). A decrease in the average size of ACF did not reach
statistical significance [3.11 (2.95–3.29) versus 2.31
(2.00–2.63); P = 0.055]. A replication of
this experiment with animals sacrificed at 40 days rather than 100 days
after initiation confirmed that PEG exerted its effect early in
carcinogenesis. The number of ACF/colon was reduced from 37.7 to 6.9
(24.6–53.6 to 3.9–10.7; P < 0.001).
The marked difference in response of carcinogen-treated rats and the
Min mice to PEG 8000 prompted us to evaluate the effect of PEG 8000 on
a mouse strain susceptible to AOM colon carcinogenesis
(7)
. The results (Fig. 1
, bottom) showed no
difference between the average number of ACF/colon in the animals
receiving PEG 8000 and those on the control diet [2.89 (1.74–4.32)
versus 2.46 (1.28–4.04); P = 0.54]. Adenomatous polyps were observed in animals receiving PEG 8000
but not in control diet-fed animals, although their average number per
colon did not reach statistical significance (0.42 ± 0.29 to 0; P = 0.21).
PEG 8000 thus inhibits ACF growth in the initiated F344 rat, does not
inhibit ACF growth or carcinogenesis in the initiated SWR mouse, and
promotes colon carcinogenesis in the Min mouse.
The protective effect in the rat has been attributed to the
increased water content of the feces in animals consuming PEG (1
, 2)
. Under the conditions of our study, however, the moisture of
freshly collected feces was significantly increased by the consumption
of PEG 8000 in each of the models of colon carcinogenesis, ranging from
13.1 ± 2.4% to 21.6 ± 2.2% for the
Min mouse, from 25.3 ± 2.9% to 52.2 ± 2.0% for the rat, and from 13.5 ± 2.3% to
32.4 ± 2.3% for the SWR mouse, indicating that a
failure to increase fecal water was not responsible for the differences
in the effect of PEG. Fiber can reduce glycemic response, inhibit
insulin resistance, and affect carcinogenesis (8)
. We
found that dietary PEG, however, had no effect on glucose tolerance as
assessed by oral glucose tolerance and no effect on postprandial levels
of energy substrates (glucose, triglycerides, and free fatty acids) in
F344 rats (data not shown). The protective effect of PEG could be
associated with the development of ACF, which are not frequently
observed in the Min mouse. However, carcinogenesis in these mice is
inhibited by dietary changes that also affect ACF, by reductions in
dietary fat (9)
, by the addition of some fibers
(10)
, and by nonsteroidal anti-inflammatory drugs
(11)
, although the inhibition is not always observed with
changes in fat or fiber (12)
.
Finally, it is possible that the protective effect of PEG is a
consequence of its demulcent or soothing effect (13)
.
Compounds such as PEG are known to improve cell growth in tissue
culture (14)
and to facilitate membrane resealing after
injury (15
, 16) . High molecular weight PEGs are thought to
be confined to the lumen (17)
, where they might reduce
"spontaneous" injury to the surface epithelial barrier in the F344
rat that had received a single treatment with AOM. The colonic mucosal
barrier function in the Min mouse or repeatedly carcinogen-treated SWR
mouse may be compromised (18, 19, 20)
. In this situation, PEG
may not be confined to the surface but may interact with epithelial
cells deeper in the crypt or with cells in the lamina propria to
promote the carcinogenesis process.
FAP patients harbor small precursor lesions with Apc
mutations. It is our opinion that such individuals should not receive
PEG over long periods in clinical chemoprevention trials until the
effect of this remarkable agent is more completely understood.
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ACKNOWLEDGMENTS
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We thank Dr. D. Corpet (Sécurité des Aliments, INRA,
Ecole Nationale Vétérinaire de Toulouse, Toulouse,
France), Karen Parisien and Dr. Wendy Williams (Division of
Comparative Medicine, University of Toronto), and Colleen Ash (Samuel
Lunenfeld Research Institute, Mount Sinai Hospital) for genotyping the
Min mice; Drs. A. Giacca, D. J. A. Jenkins, and M. C.
Archer for discussion of the manuscript; and Dr. S. Minkin for
statistical advice.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by a Strategic Grant in Diet and
Cancer from the Cancer Research Society, Montreal, Canada. 
2 To whom requests for reprints should be
addressed, at Department of Nutritional Sciences, University of
Toronto, 150 College Street, Toronto, Ontario, M5S 3E2 Canada. Phone:
(416) 978-5425; Fax: (416) 978-5882; E-mail: wr.bruce{at}utoronto.ca
3 The abbreviations used are: PEG, polyethylene
glycol; ACF, aberrant crypt foci; AOM, azoxymethane; FAP, familial
adenomatous polyposis; CI, confidence interval.
Received 8/ 2/00.
Accepted 10/27/00.
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Top
ABSTRACT
Introduction
, data
for control, untreated animals; 
, data for animals given PEG 8000.
*, 0.01 < P < 0.05;
**, 0.001 < P < 0.01;
***, P < 0.001.