Both suboptimal and elevated vitamin intake increase intestinal neoplasia and alter crypt fission in the ApcMin/+ mouse
O. Bashir1,
A.J. FitzGerald2 and
R.A. Goodlad2,4
1 Gastroenterology and 2 Histopathology Departments, Imperial College, Hammersmith Hospital, London and 3 Histopathology Unit, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London, UK
4 To whom correspondence should be addressed Email r.goodlad{at}cancer.org.uk
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Abstract
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The effects of vitamin deficiency on intestinal cancer are unclear, and even less is known about the consequences of excessive intake. We therefore investigated the actions of altered vitamin content on intestinal polyp development, cell proliferation and crypt fission in a mouse model of neoplasia. Ninety multiple intestinal neoplasia (ApcMin/+) mice and 90 wild-type littermates, 4 weeks old, were divided into six groups and fed either a control semi-synthetic diet, or the semi-synthetic diet with the vitamin content lowered to a third of the RDA or the semi-synthetic diet with the vitamin content increased 5-fold (except for retinol and folate, which were doubled). The number and size of polyps in the small and large intestines were scored after 8 weeks on the diets, as was cell proliferation (native mitoses per crypt) and crypt fission. The small intestines of the low and high vitamin groups were heavier than the controls. There were significantly more polyps and the tumour burden was higher in both the low and the high vitamin groups (P < 0.02). Proliferation was slightly reduced by the vitamin alteration and crypt fission was significantly increased in the ApcMin/+ mice when compared with the wild-type (P < 0.001). Fission indices were decreased by vitamin alteration in the small intestine, and increased in the colon, but only in the ApcMin/+ mice. The effects of vitamin alteration on polyp number were most pronounced in the proximal intestine, which is also the site of maximum crypt fission. Both vitamin deficiency and over-supplementation can markedly enhance polyp number and tumour burden.
Abbreviations: Apc, adenomatous polyposis coli; FAP, familial adenomatous polyposis; Min, multiple intestinal neoplasia
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Introduction
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There are large differences in the risk of cancer in different parts of the world, which can be attributed to changes in the environment, especially in the diet (1). The high rates of cell renewal found in the gastrointestinal system, and its intimate exposure to foodstuffs and products of digestion render the colon particularly prone to neoplasia; it has been estimated that as much as 80% of colon cancer is diet related (2). Many of these differences were originally attributed to the benefits of dietary fibre, but several recent studies have cast serious doubt on the hypothesis of Burkitt (3) that the benefits of an unrefined (non-Western) diet are due to dietary fibre (4,5); however such (high fibre) diets are in addition usually rich in fruit and vegetables, which in turn are rich in many other micronutrients, including the various vitamins.
In addition to the classical deficiency diseases, sub-optimal or inadequate intake of several vitamins is linked to several chronic diseases; including coronary heart disease and cancer (6).
Dietary advice is still divided, as recently exemplified by one (USA) study recommending to medical practitioners that it appears prudent for all adults to take vitamin supplements (7), whilst the same week another (UK) article addressing a similar audience concluded that, with a few exceptions, regular consumption will probably do no good (8) and reiterating the traditional dietary advice that if the diet is adequate, there is no benefit of supplements.
The evolution of effective cellular strategies to detect and detoxify metabolites of molecular oxygen (reactive oxygen species) is a prerequisite of living in an oxygenated environment (9). Unfortunately, the nature of free radical transfer is such that antioxidants can also act as pro-oxidants, in addition, recent studies have shown that some antioxidants can moderate growth signalling pathways (9). Thus, the effects of excess vitamin intake, as may occur in people taking high-dose supplements (many of which contain considerably more than the daily allowance) has been considered to be a cause for concern. This has led to plans from the European Parliament to curtail the public right to take high dose supplements and the UK Food Standards Agency has recently published extensive guidelines on upper limits for vitamin and mineral intake (10).
Some of the earliest changes in colonic carcinogenesis are not those expected from environmental mutagens, suggesting that the effects of diet on the progression of cancer are promotional rather than mutagenic (11). Increased cell proliferation is a hallmark of carcinogenesis and is generally considered as one of the early events in colon cancer (12). Intestinal cell mass can also be increased by the relatively poorly understood process of crypt fission, in which bifurcations in the base of the crypts unzip to create new crypts (13) and this process is implicated in the clonal spread of mutated crypts in carcinogenesis (14) and sporadic human colorectal adenomas and hyperplastic polyps can grow by crypt fission (15,16). Thus, while cell proliferation and crypt fission are essential components of gut renewal and defence, excess proliferation has been associated with increased cancer risk (17) and increased proliferation and crypt fission are seen in crypts isolated from human adenomas and hyperplastic polyps (18).
We thus investigated the effects of both low vitamin diets and high vitamin diets on polyp development, cell proliferation and crypt fission in a mouse model of intestinal cancer. We used the ApcMin/+ (multiple intestinal neoplasia) mice (19), which develop spontaneous polyps due to disruption of the Apc gene. Mutation of the Apc gene leads to the production of a truncated Apc protein and the movement of ß-catenin to the nucleus, which can then stimulate cell division and alter cell-to-cell adhesion (20). Inherited APC mutation leads to familial polyposis coli (FAP) in humans and its loss is a common and early change in sporadic human cancer.
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Materials and methods
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Study protocol
Three groups of wild-type mice and three groups of female ApcMin/+ (C57BL/6 J-ApcMin/+) mice, aged 4 weeks, were fed a control semi-synthetic diet, the semi-synthetic diet with its vitamin content lowered to a third of the mouse RDA (21) and the semi-synthetic supplemented to give five times the RDA of vitamins (except for retinol and folate, which were only doubled; see Table I). There were 30 mice/group and the diets were given for 8 weeks.
Mice
The ApcMin/+ heterozygote mice were originally obtained as a gift from Amy R.Moser (McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI). Male mice were back-crossed to female C57BL/6 J and the resultant embryos were transferred by aseptic hysterectomy to foster mothers in specific pathogen free isolators. All breeding was subsequently by brother-(C57BL/6 J-ApcMin/+) sister (C57BL/6 J) mating. Genotyping was carried out by a PCR-based method, using three primers including an internal control for normal mouse DNA. All procedures, including mutant and transgenic breeding and breeding of ApcMin/+ mice were approved by the Cancer Research UK and Imperial College School of Medicine Animal Ethics Committees and covered by the appropriate licences under the Home Office Animal Procedures Act, 1986.
Autopsy
At autopsy, the stomachs, small intestines, caecae and colons were rinsed and weighed, as were the pancreatica and spleens. The small bowel was divided into three equal sections; namely, proximal (SB1), middle (SB2) and distal (SB3), which were then dissected longitudinally and spread onto filter paper, as was the entire colon. The gut preparations were then fixed in Carnoy's fluid for 3 h and stored in 70% ethanol.
The intestines were later assessed, under 20x magnification, for polyp number and size (mean of two largest diameters measured with digital callipers). Number and diameter data were used to calculate the tumour burden as the summation of the volumes of the individual polyps. After evaluation the small bowel sections were rolled up into a Swiss-roll embedded in paraffin wax and sectioned at 4 µm for subsequent blinded evaluation by a qualified histopathologist.
Assessment of proliferation/fission
Assessment of proliferation and fission was performed using previously validated methods (22,23). Briefly, representative samples of tissue from the proximal, mid and distal small intestine and colon (taken from positions 10, 50 and 90% of the total length of the small bowel or colon) were hydrated, hydrolysed and stained with the Feulgen reaction. The mucosal crypts were gently teased apart under a dissection microscope and the numbers of mitoses per crypt (mean of 20 crypts) and crypt fission events per 200 crypts were then determined in this microdissected tissue. All samples were counted in a blinded fashion.
Statistics
Results are presented as the mean ± standard error of the mean. Data were tested by a two-way analysis of variance using a general linear model using Minitab Statistical Software (release 10.5 Xtra Minitab, Coventry, UK). Two factors, ApcMin/a and vitamin status classified the data. If the presence of one factor alters the effect of the other, this is indicated by a significant interaction effect. For the small bowel and the colon data the effect of site in the sector was included as a third variable.
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Results
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Wet weights of tissues
The different diets had no effect on body weight or body weight gain, but the ApcMin/+ mice were 13% lighter than the wild-type (P < 0.001). The ApcMin/+ mice had significantly (P < 0.001) heavier stomachs, small intestines caecae and colons (Figure 1 and Table II). Vitamin alteration had no effect on the weight of the stomach, caecum and colon, but significantly increased the weight of the small intestine by 9% and the pancreas by 12 and 17%, respectively, for the low and high vitamin diets (P < 0.002). The ApcMin/+ mice had much heavier spleens (attributable to intestinal blood loss) and this was greatly accentuated by vitamin alteration, as shown by a significant interaction effect (P < 0.002).

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Fig. 1. The effects of the various treatments on tissue weighs. The tables show the results of analysis of variance.
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Fig. 3. The effects of the various treatments on cell proliferation in the proximal small intestine and colon. The tables show the results of analysis of variance.
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Fig. 4. The effects of the various treatments on crypt fission in the proximal small intestine and colon. The tables show the results of analysis of variance.
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Polyp formation
No polyps were found in the wild-type mice, thus only the results for the ApcMin/+ mice are shown in Figure 2. Both reduced and increased vitamin content resulted in a 70 or 80% increase in polyp number in the proximal third (SB1) of the small intestine (P < 0.001). This effect persisted throughout the small bowel, but was less pronounced in the mid small bowel (SB2) where the differences were 28 and 48% (P < 0.05 and P < 0.01 for the low and the high vitamin group, respectively). The differences in the SB3 were not significant. When the data from all the small intestine was considered there were 23% more small intestinal polyps in the low vitamin group (P = 0.014) and 32% more in the high vitamin group (P < 0.001). No significant differences in the large bowel polyp number were observed, but the number of polyps was relatively low in the colon. The mean diameter of polyps in the various sites did not differ between groups so that the differences in tumour burden were very similar to those of the polyp counts.

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Fig. 2. The effects of the various treatments on small bowel and colon polyp numbers. SB1 refers to the proximal third, SB2 the mid third and SB3 the distal third of the small bowel. The statistical probability of differences between the modified vitamin diets and the semi-synthetic control group are shown where * = P < 0.05 and *** = P < 0.001.
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Cell proliferation
Small intestine. Small decreases in proliferation were seen in the small intestine with vitamin alteration (Figure 3a), especially in the proximal small intestine, where mitoses decreased by 9% in the low vitamin group and 8% in the high (P = 0.058 and P < 0.001, respectively). Little change was seen in the mid small intestine. In the distal small intestine there was a significant interaction effect (P = 0.05) in the low vitamin groups, as the wild-type had decreased proliferation while that in the ApcMin/+ increased. A 17% decease in proliferation was associated with both groups on the high vitamin diet (P = 0.032). When the small intestine as a whole was considered there was a small decrease (
8%) in proliferation in both the low and high vitamin groups (P = 0.045) and there also appeared to be a ApcMin/+ effect (P = 0.053). In addition, there was a very statistically significant proximo-distal gradient in proliferation (P < 0.001).
Colon. Low vitamin content had no significant effect on proliferation in the colon (Figure 3b), but mitotic counts decreased significantly proximo-distally (P < 0.002) and the effect of ApcMin/+ status varied between sites as was shown by a significant interaction effect between ApcMin/+ status and site (P = 0.012). Thus, ApcMin/+ mice had increased mitotic counts when compared with the wild-types in the proximal and distal colon (P = 0.027 and 0.02) but slightly decreased counts in the mid colon.
High vitamin content slightly decreased proliferation in ApcMin/+ and in wild-type mice (P < 0.025). The site in the colon also influenced mitoses, with the distal sites having lower mitotic activities (P = 0.033)
Crypt fission
Small intestine. Crypt fission was more than doubled in the proximal and mid small intestine of the ApcMin/+ mice when compared with the wild-types; however, in the distal small intestine there was a decrease in fission in the ApcMin/+ mice, as indicated by a significant ApcMin/+ by site interaction (P < 0.001, Figure 4a). Multivariate analysis indicated that the low vitamin diet had no effect on fission, but there were highly significant effects of ApcMin/+ status and of site (P < 0.001).
The high vitamin diet caused significant decreases in fission in both the ApcMin/+ and the wild-type (P < 0.001) and there were significant effects of site and an ApcMin/+ by site interaction as the effects of ApcMin/+ status were not seen in the distal small intestine.
Colon. In the proximal colon (Figure 4b) the low and the high vitamin diets significantly increased fission indices 3-fold, but only in the ApcMin/+ mice, as was demonstrated by the interaction effects (P < 0.05). Indications of similar actions were seen in the other colon sites, but did not reach statistical significance. When all the colon data were considered for the control and low vitamin groups, the effect of ApcMin/+ status was significant at P < 0.001, diet was P = 0.056, site had no effect and the ApcMin/+ by diet interaction was significant at P < 0.002. Multivariate analysis of the control and high vitamin group showed no significant effect of ApcMin/+ status and site was significant at P = 0.047.
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Discussion
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The results of this study demonstrate that increased vitamin content [below those doses at which one would expect frank toxicity (24)] can increase polyp yield in an animal model and confirm that low vitamin intake can have adverse effects.
The actions of vitamins were first demonstrated by their several deficiency diseases, with one of the very first clinical trials being performed on the warship HMS Salisbury in 1747 by James Lind who discovered that the lack of something in citrus fruit caused scurvy. Since then our knowledge of vitamins in the prevention of deficiency disease has become well established, but there is now considerable evidence to suggest that suboptimal vitamin intake is involved in many other diseases.
There are several animal models for gastrointestinal cancer, including administration of pro-carcinogenic compounds such as dimethylhydrazine (DMH) (25) or using genetically modified animals that have increased spontaneous polyp development. We used multiple intestinal neoplasia (Min ApcMin/++) mice (26), which is generally agreed to be a useful model for the study of various factors on the early stages of intestinal cancer. ApcMin/+ mice are highly susceptible to spontaneous intestinal adenoma formation due to a mutation in the adenomatous polyposis coli (Apc) tumour suppresser gene (27), which is usually inactivated early in the carcinogenic process in man, both in sporadic colorectal cancer and in FAP (28). Although this is a useful model, it is important to remember the limitations of the use of such animals as polyp formation in APC deficient humans is mainly confined to the colon whereas the predominant site in the murine model is the small intestine. Nevertheless, the types of adenomas (intravillous adenoma) found in the small bowel are remarkably similar in mice and men (29).
The tissue weights showed a general increase in the ApcMin/+ mice in all sites of the gut, including the stomach. The pancreases were increased by both vitamin alterations, which could be a cause for concern. The effects on the weight of the spleen were very pronounced and reflect the blood loss and the tumour burden of the ApcMin/+ phenotype.
Spontaneous colon tumours have recently been reported in mice fed a western style diet, which was high in fat and low in calcium, vitamin D, folic acid, methionine, choline and vitamin B12 (30). This could perhaps be due to the low levels of (antioxidant) vitamins failing to neutralize reactive oxidizing species (ROS), which can damage DNA (31). ROS however play an important role in the immune system and can act as mediators of normal signalling processes, such as the nuclear factor of kappaB (NF-kappaB) pathway, and the signalling pathways that are involved in its activation are also important for cell proliferation, apoptosis and tumour development (32). An alternative explanation is that the diets were low in components that supply methyl-donor and cofactor groups, leading to reduction in S-adenosoyl-methionine and altered DNA methylation (5,30). Western-style diets have shown interactions with mouse genetics, as the combination of the diet with the absence of p21 led to a greater increase in polyps than either factor alone (33).
Deficiency or suboptimal levels of several vitamins can mimic radiation in damaging DNA by allowing single- and double-strand breaks, oxidative lesions, or both (34). Remedying such deficiencies could thus lead to a major improvement in health at a low cost (35). Unfortunately most nutritional supplements are usually taken by people who are health conscious and are already on an adequate diet. In the USA over 50% of the population are taking various forms of vitamin supplement (36). This may explain the lack of efficacy of human dietary vitamin intervention trials despite the promise shown in several experimental studies (37). The University of Oxford's clinical trial services unit has just spent £21 m on a 5-year study, which could not find any benefit for vitamin supplementation (38). The literature is as usual contradictory, as other studies have shown modest protective effects of vitamin use and interactions with other factors, for example; 15 years of a multivitamin containing folic acid, led to slightly decreased risk of colon cancer mortality but only in those consuming alcohol (39), but the same cohort study also showed a reduction in stomach cancer in participants with short duration of use (40).
ApcMin/+ mouse studies have shown whilst folate can suppress the development of ileal polyps when given early, at later time points, folate supplementation appears to have an opposite effect on ileal polyps (41). This would imply that folate might be able to block initiation phase of carcinogenesis while acting as a promoter for the later phases.
A recent study using the ApcMin/+ mouse found that, contrary to the author's expectations, retinoic acid stimulated both the formation and growth of polyps, perhaps due to direct transcriptional regulation (retinoic acid regulates at least 532 genes) (42). Polymorphism may alter the response to vitamins, for example a repeat polymorphism in the thymidylate synthase promoter, a key enzyme in folate metabolism, could explain why some people may have a 2-fold decreased risk of colon cancer, others may be at 1.5 times increased risk (43).
The investigation of the effects of altered vitamin status in a more colon-specific mouse model, such as the DMH (44) model may thus be warranted. Information is needed on the actions of crypt fission at different stages of tumour formation and progression. Moreover, the actions of some agents on fission may also vary, as our studies on the effects of epidermal growth factor (EGF) (45) have shown that EGF initially elevates fission and then causes a long-term inhibition. The effects of time and the time course should be investigated.
Evidence that excess vitamins can be harmful is accumulating, as exemplified by the unexpected adverse effects of vitamin supplementation seen in the ß-carotene intervention studies in which an at-risk group (smokers) had more cancer that the controls (46,47). Potentially harmful effects of supplementation have been suggested by the results of other intervention studies (48).
Adverse effects (at sub toxic doses) are not totally unexpected, as while dietary antioxidants can significantly decrease the effects of reactive species, such as reactive oxygen and nitrogen species, the nature of the reversible oxidation-reduction (electron transfer) process is such that most antioxidants can also act as pro-oxidants (9). In addition, it is possible that some supplements interfere with the body's own production of antioxidants, for example vitamin C supplementation can lower total reductant capacity and the antioxidants produced by the body may be more effective in protecting against free-radical damage than vitamin C (49). The shape of the doseresponse curve for vitamin intake remains to be determined, but it could be shaped like the letter U or perhaps J, with adverse effects at both low and high intakes. Considering the range of intakes possible with dietary variations and especially with supplementation, there does not appear to be a large margin of error for several vitamins (and minerals).
The results of this study show that there was a modest but significant increase in cell proliferation in the ApcMin/+ mice, which is at odds with our previous report, but the large number of mice used in the present study would have given considerably more statistical power. More dramatic effects were seen on crypt fission in the ApcMin/+, although the response varied between sites. No significant change in polyp size was seen so that the burden data mirrored the polyp counts. More crypt fission was observed in the colon of the ApcMin/+ than in the wild-type mice and suggests that fission may indeed be involved in the process of polyp formation or size increase.
The vitamin alterations were associated with slightly reduced proliferation whilst these groups had more polyps indicating that reduced proliferation is not always protective as implied in some studies. Thus, the finding that folate supplementation decreases colonic mucosal cell proliferation in humans at high risk for colon cancer (50) may not be showing a benefit, especially as folate deficiency has been reported to reduce the development of colorectal cancer in rats (51).
Much more crypt fission was seen in the ApcMin/+ mice and they responded differently to the wild-type to altered vitamin content, thus in the small intestine high vitamins caused a decrease in proliferation, whilst in the colon fission was elevated by both changes, but only in the ApcMin/+ mice. A reduction in (small bowel) crypt fission has been reported in riboflavin deficient young rats, and this impaired the normal increase in villus number seen at weaning, leading to an adaptive increase in the depth of crypts and length of villi (52,53). In the present study low vitamin status only affected the colon.
There are likely to be several other factors involved but high rates of crypt fission could lead to tumour promotion once the 2nd hit had occurred. Crypt fission is an uncommon event in normal human colonic mucosa but common in crypts isolated from adenomas and hyperplastic polyps, leading to the suggestion that sporadic human colorectal adenomas and hyperplastic polyps clonally expand (grow) by crypt fission (18).
Both vitamin deficiency and excess can enhance intestinal polyp number suggesting a pivotal role for these factors in tumour initiation. The effects of vitamin alteration on polyp number were most pronounced in the proximal intestine and this study confirmed that the proximal regions of the small intestine and of the colon are the sites of maximum crypt fission (54). In human FAP invasive upper gastrointestinal adenocarcinoma patients the main site of cancer occurrence after colectomy is the duodenum then the pancreatic ampulla and stomach (55,56).
Thus, altered crypt fission as observed in the ApcMin/+ mice could possibly constitute a risk factor.
It is still generally agreed that a balanced diet with the recommended intake of fruit and vegetables should provide all the vitamins and minerals required. Such a diet will also contain many other potentially beneficial agents (phytoprotectants) associated with the prison walls of the plant material and should help to exclude some of the excesses of the modern high fat, high calorie Western diet (4). If such a diet cannot be taken there may be a case for rectifying sub-optimal vitamin intake by supplementation, but high doses should be avoided.
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Acknowledgments
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O.B. was funded by the Government of Libya and A.J.F. by the Biology and Biotechnology Science Research Council (BBSRC).
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Received November 10, 2003;
revised February 18, 2004;
accepted February 27, 2004.