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Institute of Food Research, Norwich Research Park, Colney, Norwich, UK
2To whom correspondence should be addressed. E-mail: liz.lund{at}bbsrc.ac.uk
ABSTRACT
There is increasing evidence that excess dietary iron may be a risk factor for colorectal cancer. However, the majority of animal studies looking at possible mechanism have used unrealistically high concentrations of iron. The current study was designed to test whether chronic exposure to high levels of iron fortification affects the free radical generating capacity of the lumenal contents, mucosal lipid peroxidation and crypt cell proliferation. Rats were fed diets containing either 29 mg/kg or 102 mg/kg of elemental iron for 6 mo. The free radical generating capacity of lumenal contents was assessed using an in vitro assay. Crypt cell proliferation rate was measured in tissues taken from the cecum and colon, with the remaining tissue being used for the assessment of lipid peroxidation. Chronic feeding of iron did not increase crypt cell proliferation rate in either the colon or cecum, but it was associated with an increase in free radical generating capacity in the colon and increased lipid peroxidation, particularly in the cecum. These results may be relevant to epidemiological evidence showing that dietary iron is associated with the risk of proximal colon cancer in humans.
KEY WORDS: • iron • lipid peroxidation • colon • cecum • rats
The potential risks of an excess intake of iron, associated with an
increase in free radical generation were first proposed over 40 y
ago (1
) and, subsequently, a number of studies have shown
an increased risk of a variety of cancers associated with high levels
of transferrin saturation in serum (2
–6
). The cause of
the high serum iron may be dietary or due to poor control of iron
absorption or a combination. The hypothesis that high intakes of iron
may be related to an increased risk of colorectal cancer in humans has
been developed on the basis of epidemiological evidence showing an
association between high meat intake and cancer of the colon and rectum
(7
, 8
). Initial results from the National Health and
Nutrition Examination Study, a prospective study of over 10,000 people
in the United States, showed that men who had developed colon cancer in
the previous 10 y had significantly higher transferrin saturation
on recruitment compared with controls (9
), whereas
Freudenheim et al. (10
), using 277 patients with matched
controls, found that high iron intake was associated with a
significantly increased risk of rectal cancer. Most recently and
convincingly, data from 194 patients in the National Health and
Nutrition Examination Study trial not only demonstrated a link between
high body iron stores, but also showed a significant association
between high dietary iron and colon cancer in the proximal colon
(11
). Babbs (12
) proposed that the presence
of iron in the colon would act as a catalyst for the production of free
radicals by bacteria. We have shown that the feces of volunteers taking
an extra 18 mg of elemental iron/d as ferrous sulfate contained higher
concentrations of iron, in a form suitable to participate in free
radical generation, than in feces collected when on their normal diet.
This was associated with an increase in free radical generating
capacity of the fecal sample measured ex vivo (13
).
Previous animal studies concerned with colonic response to high dietary
iron intakes have shown that iron may act as a tumor promoter after
exposure to dimethylhydrazine or azoxymethane (14
),
whereas phytate has been shown to be protective, an effect presumed to
be due to its iron-binding capacity (15
). However,
phytate was also associated with an increase in apoptosis, providing an
alternative explanation for the protective effects of phytate.
Similarly, Thompson and Zhang (16
) found that mice fed
diets containing 535 mg/kg of elemental iron had a higher rate of cell
division than those fed an equivalent diet containing 35 mg/kg of
elemental iron. The addition of phytate (12 g/kg diet) reduced the rate
of cell division to control levels. These earlier studies used very
high levels of dietary iron fortification, which are unlikely to
reflect iron levels in the majority of the population. We have recently
shown that more moderate supplementation with iron, up to 100 mg of
Fe/kg of diet, caused a small but significant increase in epithelial
cell proliferation rate in the rat colon (17
) that was
associated with an increase in iron available for free radical
generation. The aim of this study was to investigate whether the
presence of higher concentrations of available iron in the colon was
associated with an increase in free radical generating capacity and
lipid peroxidation and whether the effect of moderate doses of iron on
crypt cell proliferation rate persisted when rats were fed a high iron
diet for >6 mo.
MATERIALS AND METHODS
Male Wistar rats (95–125 g; A. Tuck & Son,
Battlesbridge, UK) were housed singly in wire-bottomed,
polypropylene cages, and kept in ventilated rooms at 21°C with a
12 h light: dark cycle. Tap water was freely available. Before
random assignment to control and experimental groups, all rats were fed
a control powdered purified diet, as described previously
(17
), containing 28.9 mg of iron as FeSO4
7H2O/kg of dry diet. The iron content was
determined by atomic absorption spectroscopy after preparation of
diets. Body weight and food intake were recorded during the study. The
routine animal care and experimental procedures were all conducted in
accordance with the statutory regulations and ethical guidelines of the
United Kingdom Home Office. Forty rats were fed a semisynthetic control
diet for 3 d before being randomly allocated to four groups of 10
rats. One group consumed the control diet for 26 wk, while another
group was fed the control diet with phytic acid added (sodium phytate,
2.5 g/kg of diet). An additional two groups received an
iron-supplemented diet (102 mg of iron as
FeSO4 7H2O/kg of dry diet)
with or without the addition of sodium phytate (2.5 g/kg of diet). Food
intake and weight gain were monitored at monthly intervals. At the end
of 6 mo, rats were killed, the cecum and colon removed and the contents
collected and used immediately to assess the free radical generating
capacity in the lumen using the in vitro incubation technique described
below. Lengths of tissue (
10 mm) from the proximal and distal colon
and an equivalent amount of tissue from the cecal tip were collected
into fixative (25:75 acetic acid:ethanol). The cecum and colon were
then everted and washed thoroughly in phosphate-buffered saline (pH
7.4, 4°C), placed on a sheet of plate glass resting on ice and the
mucosal layer was removed by scraping with a glass slide held at
45°C. Mucosal scrapes were placed in microfuge tubes
(Sarstedt, Nümbrecht, Germany), flushed with
N2 gas and stored at -20°C for subsequent
analysis of lipid peroxidation.
Samples were thawed and then homogenized in 1.5 mL of Tris-buffered
saline (pH 7.4, 4°C) by repeatedly passing through a 26-gauge needle.
A subsample was taken for protein analysis using an adaptation of the
method of Lowry (18
). Samples of the homogenate were
centrifuged at 14,000 x g for 5 min and 200 µL of
the supernatant analyzed for the presence of lipid peroxidation
products, malondialdehyde
(MDA)3
plus 4-hydroxy-2(E)-nonenal (4-HNE) using the Bioxytech LPO-586 method
(OXIS International S.A., Cedex, France). The results were standardized
relative to the protein content of the original homogenate. The effect
of dietary iron on free radical capacity of the lumenal contents of the
colon and cecum was explored using an assay, based on the reaction:
DMSO + hydroxyl radical 
methanesulfinic acid (MSA) + methyl radical
(19
, 20
). The total content of the cecum (0.5–1.7 g of wet
weight) or colon (1.1–2.5 g of wet weight) was incubated overnight in
9 mL of degassed Tris-buffered saline (pH 7.0) containing: DMSO
(0.7 mol/L), glucose (5.6 mmol/L) and Na2EDTA (50
mmol/L) at 37°C in a tube with a loosely fitted lid to allow escape
of gas while keeping oxygen tension low. The sample was then
centrifuged at 900 x g for 10 min, the supernatant
removed and the pH lowered to 1.0 for 10 min, using 12 mol/L of HCl.
The pH was then returned to 7.4, the sample centrifuged and the
supernatant stored at -20°C before batch analysis of the MSA content
(13
). The amount of MSA produced was standardized to the
calculated dry weight content of the sample.
The effect of iron on large intestinal mucosal cell proliferation was
assessed by measuring the mean number of mitotic figures per crypt in
10 isolated intact crypts, using a modification of the method described
by Matthew et al. (21
). The tissue samples taken from the
colon and from the tip of the cecum were stored in 25:75 acetic
acid:ethanol (-4°C), before staining chromatin with Feulgen’s
reagent. Single rows of crypts were microdissected and lightly
flattened beneath a cover slip. The number and position of mitotic
cells within each crypt were determined by reference to an eyepiece
graticule, and the average number of mitoses per crypt was calculated
for 10 crypts per rat at each site.
Data were analyzed by three-way ANOVA, using the general linear model with phytate, iron and intestinal site as factors at two levels (Minitab, State College, PA), followed by two-way ANOVA to determine the effects of iron and phytate within site. The significance of the F statistic for each factor and interactions between factors was determined from the ANOVA table, using P < 0.05 as evidence of a significant difference.
RESULTS
After 3 mo there was no effect of additional iron or the presence
of phytate on weight gain but by 6 mo, those rats fed 102 mg of iron/kg
in the diet compared with 29 mg/kg, and also containing phytate, had
gained significantly less weight (358 ± 8 g) than those on a
low iron diet with no phytate (440 ± 23 g). Those rats fed
29 mg of iron/kg with phytate and 102 mg of iron/kg with no phytate had
intermediate weight gains after 6 mo of 398 ± 10 g and 412
± 14 g, respectively. The amounts of the two lipid peroxides
(MDA + 4-HNE) expressed relative to the protein contents of the mucosal
scrapes in the four test groups used are shown in Figure 1
. Rats exposed to the high concentrations of iron had significantly more
lipid peroxides in the mucosa of both the cecum and colon. This effect
was independent of the phytate content of the diet. The free radical
generating capacity of cecal and colonic content was compared by
standardizing to the calculated dry weight of sample added to the
incubation medium (Fig. 2
). A high intake of dietary iron was significantly associated with an
increase in the free radical generating capacity of the colonic content
but there was no significant effect of phytate. In contrast, the free
radical generating capacity in the cecal contents was similar in all
groups; hence, there was a site-specific effect of iron. Although
phytate intake was associated with a small increase in MSA production
in the cecal contents, this was not significant (P = 0.06). The level of free radical generation was higher in the cecal
contents than in the colonic contents. Three-way ANOVA revealed
that neither the addition of iron nor phytate had an effect on crypt
cell proliferation rates but two-way analysis revealed that phytate
caused a small but insignificant increase in the numbers of mitotic
figures per crypt in both the proximal (P = 0.07) and
distal (P = 0.06) colon in rats fed control diets
(Table 1).
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DISCUSSION
Two recent studies suggest that dietary iron intake is
particularly associated with an increased risk of cancer in the
proximal large bowel, whereas high transferrin saturation is associated
with increased risk in the distal regions of the colon and in the
rectum (11
, 22
). The effects of high dietary iron intake
were, in these two recent studies, particularly marked in women, who
are a more likely target group for iron supplementation. We have
previously shown that iron fortification increased the amount of free
iron that would be available either for uptake into the cells lining
the large intestine or for free radical generation at the mucosal
surface (17
).
The present study shows that the presence of iron in the lumenal
contents of the colon is associated with an increased free radical
generating capacity of the chyme, as assessed using an in vitro assay.
Although the free radical generating capacity of the cecum was found to
be higher than in the colon, there was no effect of iron fortification,
suggesting that iron was not the rate-limiting factor for free
radical generation in the cecal contents. Indeed, our previous studies
showed that the concentration of available iron was lower in the cecum
than in the colon and less affected by iron fortification
(17
). It is interesting to note that the effect of iron on
free radical generating capacity of the rat colon contents parallels
our previous results (13
), using fecal samples from human
volunteers taking iron supplements (19 mg of iron/d).
In contrast to the effects of iron on free radical generation in vitro,
the effect of iron on lipid peroxidation was more marked in the cecum
than in the colon. This may be because of the more acidic environment
of the cecum, which would support greater uptake of iron into the
epithelial cells lining the colonic tract. The pH dependency of iron
transport into Caco 2 cells has been reported recently
(23
) and we have previously shown that uptake of iron into
rat colon cells shows similar kinetics to that seen in the small
intestine (unpublished results). This would suggest that the presence
of iron within the cell poses more risk than any effect on free radical
generation in the external milieu. Evidence to support this hypothesis
is provided by tissue culture studies showing that the effects of
exposure of cells to hydrogen peroxide are ameliorated by the presence
of iron in a nonabsorbable form but exacerbated when iron is presented
in an absorbable form (24
). Although body iron stores were
not measured in this study, we found higher concentration of liver iron
in our earlier short-term study (17
), which suggests
that there would have been higher circulating concentrations of iron in
this study. However, iron uptake into intestinal cells from the
basolateral side is tightly regulated throughout the gut and unlikely
to result in increased lipid peroxidation within the mucosal epithelial
cells. Alternatively, the smaller increase in lipid peroxidation in the
colon may be due to intrinsic anti-oxidant properties of the cell
membranes associated with the presence of free monounsaturated fatty
acids (25
). Although lipid peroxidation does not
necessarily mean that there is an increased risk of DNA oxidative
damage, it suggests that cells are under a greater level of stress
(26
). Although Soyars and Fischer (27
), using
more moderate dietary iron concentrations (450 mg/kg) than those used
previously, showed no effect of iron on cell proliferation or the
development of aberrant crypt foci in rats that had received
azoxymethane, Davis and Feng (28
) showed an increase in
aberrant crypt foci (ACF) with iron fortification at levels as
low as 140 mg/kg. Soyars and Fischer (27
) were also unable
to show any increase in lipid peroxidation in the whole colon, even
after 10 wk of dietary manipulation, in rats exposed to the higher
concentrations of iron. The lack of consistency with the present report
may reflect the difference in sampling site, the use of a different
strain of rat or the period of fortification used.
Phytate had no significant effect on free radical generation in samples
from either site, although it is a highly effective chelating agent in
the prevention of Fenton-driven free radical production. The rather
surprising lack of effect of phytate suggests that it is either rapidly
metabolized by the cecal bacteria or absorbed before or soon after
reaching the large intestine. However, the addition of phytate to the
diet increased crypt cell proliferation in the colon, particularly in
rats fed the control iron diet. This is in contrast to the reduction in
labeling indices in mice fed phytate, at higher concentrations than in
the present study, after 3 d of consuming test diets
(16
). Although exposure to low levels of hydrogen
peroxide, as a model for oxidative stress, have been associated with
increases in cell division (29
), the effect of phytate
does not seem to be related to free radical generation in this study.
An alternative hypothesis, suggested by Shamsuddin (30
),
is that phytate is absorbed and its metabolites (inositol trisphosphate
and inositol tetraphosphate (IP4) act as
intracellular signaling molecules to increase free intracellular
calcium and cell division. The results of the current study provide
evidence that excess dietary iron, taken over an extended period, may
pose a significantly greater level of oxidative stress to the mucosal
cells of the colon and particularly to those lining the cecum,
equivalent to the proximal colon in human subjects. This is the site
suggested by epidemiological studies to be most vulnerable to excess
iron intake.
ACKNOWLEDGMENTS
We thank Simon Deakin and Valerie Russell for their technical help.
FOOTNOTES
1 This work was funded by The Ministry of
Agriculture, Fisheries and Food, England and Wales. 
3 Abbreviations used: 4-HNE,
4-hydroxy-2(E)-nonenal; MDA, malondialdehyde; MSA, methanesulfinic
acid.
Manuscript received 4 May 2001. Revision accepted 7 August 2001.
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