The Journal of Nutrition Vol. 128 No. 2 February 1998, pp. 175-179

Increases in the Concentrations of Available Iron in Response to Dietary Iron Supplementation Are Associated with Changes in Crypt Cell Proliferation in Rat Large Intestine^1,2,3

Elizabeth K. Lund⁴, S. Gabrielle Wharf, Susan J. Fairweather-Tait, and Ian T. Johnson

Department of Nutrition Diet and Health, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom

	ABSTRACT

Abstract Introduction Methods Results Discussion References

High concentrations of iron in the diet have been shown to increase chemically induced colorectal tumors in rats. It is therefore important to understand the influence of dietary iron on the concentration of unabsorbed iron in the large intestine and its distribution between soluble and insoluble pools in the luminal compartment. We sought to investigate this issue and to establish whether iron modifies mucosal cell proliferation, which is thought to influence initiation and progression through the adenoma carcinoma sequence. In the first experiment, four groups of seven rats were fed diets at two concentrations of iron, 29 and 102 mg/kg, with or without the addition of 2.5 g phytic acid/kg. The concentrations of iron in the contents of the large bowel extractable with water ("free iron") or a buffered EDTA solution ("exchangeable iron") were determined. The concentration of freely soluble iron increased ~100% with iron supplementation in both the cecum and the colon, and there was an approximately five- to sixfold increase in exchangeable iron at both sites (P < 0.05). In a second experiment with identical feeding conditions, there was a significantly greater number of cell divisions per crypt in the colon of the high iron group and a significantly greater number of cell divisions in the upper part of the crypt in the cecum. The concentrations of free and exchangeable iron observed in colonic contents in this study are consistent with those reported by others to increase free radical production in fecal material. Further studies are required to determine whether the small changes in crypt cytokinetics are a consequence of oxidative mucosal damage.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Processed foods are supplemented frequently with iron as a preventative measure against iron deficiency (Fairweather-Tait 1997). However, recent concern that excess iron could be implicated in the high incidence of colon cancer in developed countries has raised questions as to the desirability of fortification practices (Knekt et al. 1994, Nelson et al. 1994). It was suggested that iron may increase the risk of cancer by a prooxidant-dependent mechanism described by Babbs (1989), Blakeborough et al. (1989) and Graf and Eaton (1993). Recent epidemiological studies provide evidence that both dietary intake of iron and elevated serum iron are risk factors for colorectal cancer in the U.S. population (Wurzelmann et al. 1996).

The large fraction of dietary iron that remains unabsorbed in the small intestine may enter the colon and participate, in conjunction with the intraluminal bacteria, in Fenton-type reactions, which increase the production of hydrogen peroxide and hydroxyl radicals at the mucosal surface (Babbs 1989). Hydrogen peroxide or iron may enter the colonocytes and increase the risk of DNA damage in a manner similar to that described for immune cells (Buttke and Sandstrom 1994) and thus act to increase the risk of mutations occurring either as initiating events or later in the adenoma carcinoma sequence. Alternatively, iron may be involved in the conversion of pro-carcinogen to carcinogen within the lumen of the colon (Babbs 1989). In most previous studies iron has been regarded as a tumor promoter. It has been shown to increase crypt cell proliferation in rats treated with 1,2 dimethylhydrazine (Nelson et al. 1989) and in rats fed high fat diets (Thompson and Zhang 1991). In this study we investigated the effects of dietary iron supplementation, with and without phytate, on the distribution of iron between bound and freely exchangeable iron pools in the fecal water of rats. We used intact microdissected crypts to explore any effects of changes in intraluminal iron concentration on the frequency and distribution of crypt cell mitosis. The concentrations of iron and phytate in the feed were designed to simulate the maximum levels that might be found in the human diet.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animal and diets.  Male Wistar rats 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 randomization into control and experimental groups, all rats were fed a control powdered purified diet containing 28.9 mg of iron as FeSO₄·7H₂O/kg dry diet. Body weight and food intake were recorded during the study, and feces were collected throughout the experimental feeding periods. The routine animal care and experimental procedures used in this study were all conducted in accordance with the statutory regulations and ethical guidelines of the United Kingdom Home Office.

Experiment 1.  Twenty-eight male Wistar rats were fed control diet for 3 d and then randomly divided into four groups of seven. During a further 5-d feeding period, one group of rats continued to be fed the control diet⁵ while another group was fed the control diet with phytic acid added (2.5 g sodium phytate/kg diet). A further two groups received a high iron diet (102 mg of iron as FeSO₄·7H₂O/kg dry weight diet) with or without the addition of sodium phytate (2.5 g/kg diet). After 5 d, rats were killed by injection of pentobarbitone followed by cervical dislocation, the abdomen was opened, and the contents of the cecum and colon collected for the immediate analysis of free and available iron concentrations.

Experiment 2.  Twenty-eight male Wistar rats were fed control diet for 3 d, randomly divided into four groups of seven, and fed the control and experimental diets as previously described. Feces were collected throughout the experimental period. After 5 d, the rats were killed as before and the abdomen opened. The liver and entire large bowel were excised and ~5 mm of tissue was removed from the midpoint of the large intestine and from the tip of the cecum and collected into fixative (25:75 acetic acid:ethanol). The contents of the cecum and colon were collected, weighed, and frozen before analysis of total iron concentrations.

Analysis of total intraluminal and hepatic iron.  For the determination of total iron, samples of diets, cecal and large intestinal contents, feces, and liver were analyzed by atomic absorption spectrometry (AAS). Subsamples of the frozen material were freeze dried and then ashed in silica crucibles at 480°C for 48 h. The ash was dissolved in warm concentrated hydrochloric acid, and the solution diluted to an appropriate volume with distilled water.

Analysis of free and exchangeable intraluminal iron.  The quantity of free iron in the cecal and colonic contents of rats fed the two levels of iron was determined by mixing a preweighed sample with water, centrifuging for 30 min at room temperature (6000 g), and collecting the supernatant. This was repeated, and the two supernatants were combined to give a final volume of 10 mL·g sample¹. Readily exchangeable iron then was assessed on the same sample by washing twice more in a buffered EDTA solution (10 mmol/L tris HCl, 1 mmol/L EDTA pH 7.0; 5 mL buffer/g fecal sample). The supernatants were combined and the iron content of the resultant solutions were measured by flame atomic absorption spectrophotometry using a PU 9100X AAS (Philips, Eindhoven, The Netherlands). Values were calculated from a standard curve and analytical accuracy confirmed using NBS Standard Reference Material 8431-Mixed Diet (Office of Standard Reference Materials, Washington, D.C.). The water content of the fecal samples was calculated from a subsample by weighing before and after freeze drying.

Cell proliferation.  The effect of iron on large intestinal mucosal cell turnover 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. (1994). The tissue samples taken from the midsection of the colon and from the tip of the cecum were stored in 25:75 acetic acid:ethanol before staining with Feulgen's reagent. Single rows of crypts were dissected out using a binocular microscope and lightly flattened under a cover slip. The number and position of mitotic cells in each crypt were determined by reference to an eye piece graticule, and the data was recorded on a spread sheet. The average number of mitoses per crypt and the position of each mitotic figure relative to total crypt length were calculated for 10 crypts per animal at each site, and the average number of mitotic figures in each third of the crypt was found for each sample.

Statistics.  Mean values were compared using two-way analysis of variance, and significance (P  0.05) was assessed with the general linear model (GLM), using the Minitab statistics package (State College, PA). Values are means ± SEM.

	RESULTS

Abstract Introduction Methods Results Discussion References

Food intake, growth rates and the level of hepatic iron were assessed in the first experiment. The composition of the diets had no significant effect on the food intake or growth rate of the rats during the test period (Table 1), but the addition of iron to the diet caused a significant increase in hepatic iron stores. Although iron was added to the diet at only 80 mg elemental iron per kilogram, the concentration of total iron in the cecal contents, as measured in experiment 2, reached levels in the order of 5200 mg/kg dry weight (Table 2). This was presumably due to the concentrating effect caused by the relatively poor absorption of iron and the more or less complete absorption of other components of the diet.

Although the total iron concentration in the cecum calculated from the wet:dry weight ratio of the contents reached 1.4 mg/g wet weight, equivalent to a concentration of 25 mmol/L, by far the major proportion of this iron was not freely eluted by water or EDTA and was therefore not in solution. The free and exchangeable fractions of iron in the cecum and colon, determined in experiment 1, are illustrated in Figure 1. A small but significant increase in the concentration of free iron was detected, both in the cecum and colon, by the additional iron in the diet. The level of exchangeable iron was increased approximately five- to sixfold in the cecum and colon by dietary supplementation (P < 0.001). There was no effect of phytate on free or exchangeable iron in the colon and a small but significant effect on free iron concentrations in the cecum.

View larger version (29K): [in this window] [in a new window]   Fig 1. The concentration of "available" iron; both "free iron," that which is readily soluble in water, and "exchangeable iron," that which can be extracted into an EDTA buffer, in the large intestine of rats fed control diet (29 mg iron/kg) or control plus phytic acid (2.5 g/kg diet) or high iron (102 mg Fe/kg diet) with and without phytic acid in experiment 1. Values are expressed as means ± SEM, n = 7. Two-way ANOVA indicated that iron and phytate affected free iron concentration (P < 0.01), whereas only iron affected exchangeable iron (P < 0.001).

The numbers of mitotic figures in colonic and cecal crypts are given in Figure 2, and the proportions of those mitoses occurring in the upper part of the crypt are shown in Figure 3. The analysis of variance revealed a significant effect of iron supplementation on numbers of mitoses per crypt in the colon only (P < 0.05). In the cecum, the percentage of mitotic figures occurring in the top third of the crypts was significantly higher in rats receiving additional dietary iron than in controls (Fig. 3; P < 0.01), and there was evidence of an interactive effect with phytate (Fig. 3; P < 0.05).

View larger version (23K): [in this window] [in a new window]   Fig 2. The number of mitotic cells/crypt in cecum and colon of rats fed control diet (29 mg/kg) or high iron diet (102 mg Fe/kg) with or without phytic acid (2.5 g/kg) in experiment 2. Values are expressed as means ± SEM, n = 6 or 7. Two-way ANOVA indicated that iron affected crypt cell proliferation only in the colon (P < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig 3. The spatial distribution of mitotic cells in cecal and colonic crypts of rats fed control diet (29 mg/kg) or high iron diet (102 mg Fe/kg) with or without phytic acid (2.5 g/kg) in experiment 2. Values are expressed as means ± SEM, n = 6 or 7 for the number of mitotic cells in the top third of the crypt, expressed as a percentage of the total number of mitotic cells in the whole crypt. Two-way ANOVA indicated that iron affected the distribution of mitoses in the cecum (P < 0.01), and there was evidence of an interaction between iron and phytate in the cecum and colon (P < 0.05).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The present study establishes that iron supplementation greatly increases the level of iron in the fecal stream in the rat. Under the conditions of the present study, the definition of "available" iron includes iron chelated with small organic molecules that render it soluble and therefore available for participation in reactions leading to free radical generation in the lumen (Babbs 1989). Although the level of iron in the colonic contents reached a theoretical concentration of ~25 mmol/L in the iron supplemented rats, only ~0.4 mmol were present as "available" iron. However, this value is of the same order of magnitude as that reported by previous workers for the contents of the distal small intestine of the rat (Simpson et al. 1992). The increases in free (20-90 µmol/kg wet weight) and exchangeable iron (20-200 µmol/kg wet weight) in the cecum in response to supplementation correspond to the range (0-100 µmol/L) over which Babbs (1989) has shown a sigmoidal relationship between free radical generation in vitro and the concentration of Fe-EDTA in the media. In the colon of control rats, the total concentration of available iron was close to the level required for maximal free radical production in vitro (Babbs 1989). However, the studies of Babbs (1989) were carried out using EDTA as the chelating agent. In the same report, it was shown that naturally occurring intraluminal chelators such as bilirubin are effective in vitro but require higher iron concentrations to support a similar level of free radical generation. Thus the changes in iron concentration in vivo induced by dietary manipulation appear to fall within the range where a significant effect on the level of intraluminal free radical production is feasible.

It is known from tissue culture studies that low levels of pro-oxidants can increase cell proliferation (Dypbukt et al. 1994). Biologically available iron is essential for cell proliferation in culture (Furukawa 1992, Porreca et al. 1994), and it normally is required in the form of iron-transferrin, which is the major species present in plasma (Alcantara et al. 1991). Wurzelmann et al. (1996) recently have reported epidemiological data suggesting that humoral exposure to iron increases the risk of distal carcinoma in humans. The increase in hepatic iron stores occurring in response to the supplement in the present study raises the possibility that the colonic and cecal cells might be exposed to, and possibly stimulated by, increased concentrations of biologically available iron at the serosal side. It is not clear at present whether a diet high in iron can influence mucosal cell proliferation through such a mechanism. However, in humans, the levels of ferritin and transferrin in plasma would be expected to rise in parallel with hepatic iron stores (Hallberg 1982), and it is worth noting that abnormally high levels of plasma ferritin and transferrin have been observed in, for example, patients receiving prolonged parenteral nutrition (Ben-Hariz et al. 1993).

Intraluminal iron may stimulate an increase in cell proliferation directly, via participation in Fenton chemistry and hydrogen peroxide production (Dypbukt et al. 1994), through an increase in oxidative stress in the dividing cells as a result of hydrogen peroxide exposure or as a "repair response" to increased cell loss from the luminal surface. Finally, as proposed by Babbs (1989), generation of hydroxyl radicals via Fenton chemistry may increase the conversion of pro-carcinogens to carcinogens in the luminal contents. Increased crypt cell proliferation is considered to be an important risk factor for colon cancer, particularly when associated with a displacement of the proliferative zone toward the intestinal lumen (Lipkin 1988). We therefore looked for an association between intraluminal iron concentration and crypt cell proliferation in iron supplemented rats.

For both the cecum and the colon, the highest crypt mitotic rates occurred in the group given iron supplementation with no phytate, and the effect of iron supplementation was significant in the colon. In the cecum the increase in cell turnover in the iron supplemented groups was not statistically significant, but there was a significantly greater proportion of mitoses in the upper third of the crypt. A shift of the proliferation zone toward the luminal pole of the crypt is regarded as a risk factor for neoplasia, and it is interesting to note that Wurzelmann et al. (1996) recently have suggested that dietary iron may be a risk factor for carcinoma of the proximal colon in humans. Although the effects observed in the present study were small and seem unlikely to pose any increased risk of carcinogenesis in themselves, the response to longer term supplementation should be examined.

Previous studies have shown that levels of dietary iron in excess of 500 mg/kg can increase both the tumor yield induced by the chemical carcinogen dimethylhydrazine (DMH) in the rat colon (Nelson et al. 1989) and the mitotic and labelling indices in colonic crypts from mice (Thompson and Zhang 1991). However, in the latter study, fat was fed at 25% of total diet to increase the concentration of putative tumor promoters such as bile acids and fatty acids. The present evidence for a small increase in cell proliferation due to iron was obtained with relatively little fat (8%) in the diet and in the absence of a chemical carcinogen. Future studies to explore the effects of iron on intraluminal chemistry may need to be carried out over a higher range of lipid concentrations.

A number of studies have shown that phytate can reduce mucosal cell proliferation in the rat colon (Nielsen et al. 1987, Thompson and Zhang 1991). These earlier studies suggest that the protective effects of phytates can be explained at least partially by their iron binding capacity, although they also are known to bind bile acids. In the present study, there was some evidence of an increase in free iron in the cecum in response to phytate supplementation. It is not clear whether iron chelated by phytate is available for Fenton reactions, but there was no evidence of an effect of phytate on crypt cell proliferation.

We conclude that a fourfold increase in dietary iron causes significantly and disproportionately larger increases in both free and exchangeable iron in the large bowel of the rat. This pool of soluble and exchangeable iron may be available for reactions leading to adverse changes in free-radical mediated intraluminal chemistry. Future studies on the interrelationships among diet, fecal chemistry and mucosal cell physiology will need to take into account the level of dietary iron on its distribution between intraluminal pools.

	ACKNOWLEDGMENTS

The authors wish to thank Simon Deakin and Valerie Russell for their technical help.

	FOOTNOTES

Manuscript received 19 November 1996. Initial reviews completed 29 January 1997. Revision accepted 8 October 1997.

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