Fabrice H F Pierre

Sylviane Taché

Claude R Petit

Roelof Van Der Meer

Denis E Corpet

Meat and cancer: haemoglobin and haemin in a low-calcium diet promote colorectal carcinogenesis at the

Keywords: Colorectal carcinogenesis, Meat, Haem, Aberrant crypt foci, Rat ACF, Aberrant crypt foci, HO-1, Haem-hoxygenase-1, LOO . , Alkylperoxyl radicals, MACF, Major ACF, MDA, Malondialdehyde, NOC, N-nitroso compounds, TBARs, Thiobarbituric acid reactive substances

Introduction

Colorectal cancer is the second most common cause of cancer death in affluent countries. Dietary modifications might reduce the incidence by more than 70%, but the necessary changes are not precisely known [START_REF] Cummings | Diet and the prevention of cancer[END_REF]. People in western countries are advised to reduce their intake of red meat [START_REF]). Food, nutrition and the prevention of cancer: a global perspective[END_REF], although epidemiological results are not entirely consistent. The recent dose-response meta-analysis of epidemiological studies by Norat et al. suggests that red meat and processed meat intakes are associated with increased risks of colorectal cancer.

The mean relative risk associated with the consumption of 120 g/day of red meat is 1.24 (95% confidence interval, 1.08-1.41). The risk fraction attributable to current levels of red meat intake is about 10-25% in region where red meat intake is high. If average red meat intake is reduced to 70 g/week in these regions, colorectal cancer risk would hypothetically decrease by 7-24% [START_REF] Norat | Meat consumption and colorectal cancer risk: doseresponse meta-analysis of epidemiological studies[END_REF]. In contrast, white meat (e.g., chicken) and fish intakes are not associated with risk.

Surprisingly, several studies show that rats or mice fed a beef-based diet have fewer tumours than control animals fed a casein or a soy-protein based diet. The evidence from animal studies is far from conclusive [START_REF] Parnaud | Colorectal cancer: controversial role of meat consumption[END_REF]. Studies testing more specifically the effect of meat components (fat, protein and iron), on colorectal carcinogenesis yielded conflicting results. Many studies tested the effect of saturated fat on the incidence of colon cancer: whereas several studies show that beef tallow promotes cancer in F344 rats [START_REF] Mori | Beef tallow, but not perilla or corn oil, promotion of rat prostate and intestinal carcinogenesis by 3,2'-dimethyl-4-aminobiphenyl[END_REF][START_REF] Reddy | Effect of a diet with high levels of protein and fat on colon carcinogenesis in F344 rats treated with 1,2-dimethylhydrazine[END_REF], no promotion was observed in SD rats [START_REF] Nauss | Effect of alterations in the quality and quantity of dietary fat on 1,2-dimethylhydrazine-induced colon tumorigenesis in rats[END_REF][START_REF] Zhao | Quantitative review of studies of dietary fat and rat colon carcinoma[END_REF]. Furthermore, the hypothesis that saturated fat enhances faecal bile acid excretion, a speculated promoting stimulus, is not supported by experimental studies in rats and in volunteers [START_REF] Gallaher | Beef tallow, but not corn bran or soybean polysaccharide, reduces large intestinal and fecal bile acid concentrations in rats[END_REF][START_REF] Suzuki | Effect of low-fat, high-fat, and fiber-supplemented high-fat diets on colon cancer risk factors in feces of healthy subjects[END_REF].

Similarly, no clear picture emerges from the studies on protein-rich [START_REF] Mcintosh | Dairy proteins protect against dimethylhydrazine-induced intestinal cancers in rats[END_REF] and high-iron diets. For instance, a diet fortified with iron may, or may not, enhance colon carcinogenesis [START_REF] Soyars | Iron supplementation does not affect cell proliferation or aberrant crypt foci development in the colon of sprague-dawley rats[END_REF][START_REF] Davis | Dietary copper, manganese and iron affect the formation of aberrant crypts in colon of rats administered 3,2'-dimethyl-4-aminobiphenyl[END_REF]. In addition, meat can deliver specific carcinogens to the colon: N-nitroso compounds (NOC) and heterocyclic amines. Red meat consumption enhances the excretion of NOC in the faeces of volunteers [START_REF] Bingham | Does increased endogenous formation of N-nitroso compounds in the human colon explain the association between red meat and colon cancer?[END_REF][START_REF] Hughes | Dose-dependent effect of dietary meat on endogenous colonic N-nitrosation[END_REF], and possibly in mice [START_REF] Mirvish | N-Nitroso compounds in the gastrointestinal tract of rats and in the feces mice with induced colitis or fed hot dogs or beef[END_REF]. However, a bacon-based diet that increases NOC excretion 10 to 20-times in rats consistently reduces the growth of preneoplastic lesions [START_REF] Parnaud | Effect of meat (beef, chicken, and bacon) on rat colon carcinogenesis[END_REF][START_REF] Parnaud | Endogenous N-nitroso compounds, and their precursors, present in bacon, do not initiate or promote aberrant crypt foci in the colon of rats[END_REF]. Heterocyclic amines, produced during meat cooking, are potent mutagens and carcinogens that are eaten daily by meat eaters [START_REF] Sugimura | Overview of carcinogenic heterocyclic amines[END_REF]. However, some facts suggest that heterocyclic amines may not be major determinants of colon cancer in humans: (i) chicken is an important contributor of heterocyclic amines intake but its consumption is not related to cancer risk [START_REF] Sinha | High concentrations of the carcinogen 2-amino-1-methyl-6phenylimidazo-[4,5-b]pyridine (PhIP) occur in chicken but are dependent on the cooking method[END_REF], (ii) rodent carcinogenic doses are 100-1000 times higher than human exposure [START_REF] Stavric | Biological significance of trace levels of mutagenic heterocyclic aromatic amines in human diet: a critical review[END_REF], and (iii) the colon cancer risk in humans is associated with cooking methods, but not directly with the heterocyclic amines intake [START_REF] Sinha | An epidemiologic approach to studying heterocyclic amines[END_REF].

Recently, a new hypothesis implying haem was proposed to explain the association of colon cancer risk with red meat intake, and the lack of association with white meat intake [START_REF] Sesink | Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme[END_REF]. The haem content of red meat is 10-fold higher than that of white meat. Haem is the iron-bearing prosthetic group of haemoproteins. They include myoglobin in red meat which is made of a single globin chain holding one haem, and haemoglobin in red blood cells which is made of the association of four chains, each holding a haem. These proteins are digested in the upper gastrointestinal tract, delivering haem with ferrous iron oxide to the colon. Haemin has often been used in place of haem delivered from haemoprotein digestion. Haemin is the ferric porphyrin component in haemoglobin, with a freely exchangeable axial chloride group. Sesink et al. studied the effect of haeminsupplemented diet in non-initiated rats. They showed that dietary haemin increases epithelial proliferation [START_REF] Sesink | Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme[END_REF]. This short-term increase in epithelial proliferation is inhibited by calcium, and is associated with cytolytic activity of faecal water [START_REF] Sesink | Red meat and colon cancer: dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by calcium[END_REF]. Furthermore, Sawa et al. showed in vitro that haem induces a non-enzymatic peroxidation of polyunsaturated fatty acids. In contrast, virgin olive oil resists oxidation because it contains monounsaturated oleic acid and antioxidant polyphenols. In vivo, a diet containing both haemoglobin and polyunsaturated fatty acids increases the number of carcinogen-induced adenomas in the colon of rats [START_REF] Sawa | Lipid peroxyl radicals from oxidized oils and heme-iron: implication of a high-fat diet in colon carcinogenesis[END_REF]. We thus think that haem could explain the epidemiological correlation between red meat and colon cancer risk.

These studies may also explain why red meat did not promote carcinogenesis in previous animal studies: the high level of calcium in experimental diets and/or the resistance of dietary fat to peroxidation, may have hindered the promoting effect of haem. Indeed, the AIN-76 standard rat diet contains 5µg/g of calcium, a 200 µmol/g concentration that matches the 180 µmol/g that inhibits the haemin-induced proliferation [START_REF] Sesink | Red meat and colon cancer: dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by calcium[END_REF].

The present study was designed to investigate the effects of diets containing haem, as haemin or haemoglobin, on colon carcinogenesis in rats. Four hypotheses were tested: (i) haemin promotes carcinogenesis, dose-dependently, when added to a low-calcium diet with 5% safflower oil; (ii) free haem (haemin) has not the same effect as protein-bound haem (haemoglobin); (iii) the inhibition of lipid peroxidation inhibits the promotion by haemin; (iv) a high calcium diet reduces the promotion by haemin. The results show that dietary haem dose-dependently increased colorectal carcinogenesis at the aberrant crypt stage in rats, and that haemin was more toxic than haemoglobin. Furthermore, calcium, olive oil or antioxidants suppressed the promotion by haemin. These results suggest preventive strategies against the supposed promotion of colon cancer by haem present in red meat.

Materials and methods

Animals and experimental procedure

Animal care followed the guidelines of the European Council on animals used in experimental studies. Female F344 rats were obtained from Iffa-Credo (Lyon, France) at four weeks of age. One hundred and twenty-four animals were housed two rats per stainless steel, wire-bottomed cage. They were kept in a temperature of 22°C and light-dark cycle (12h on and 12h off), and were allowed free access to the standard AIN-76 diet and tap water. After one week of acclimatisation, the rats received a single i.p. injection of azoxymethane (Sigma Chemical, St Quentin, France; 20mg/kg ip) in NaCl (9 g/l). Seven days later, they were randomly allocated to eleven groups and fed the experimental diets. Body weights were monitored weekly throughout the experimentation, and food and water intakes were measured once a month. Faeces were collected between days 50-51 and 63-64 of the study, and were frozen at -20°C. The animals were sacrificed 97-99 days after the start of experimental diets. The colons were removed and fixed in 10% buffered formalin (Sigma Chemical) before aberrant crypt foci (ACF) scoring.

Diets

Eleven groups of rats were fed for 14 weeks dry powdered AIN76-based purified diets. The control group (N=22 rats) was given a low-calcium diet (20 µmol/g) containing 5% safflower oil. The experimental diets were based on this control diet. Their composition, balanced for fat, protein and iron, is given in Table 1. The diet base was made by the UPAE (INRA, Jouy, France). Haem, bovine haemoglobin, ferric citrate, calcium phosphate, and antioxidants (Sigma Chemical, St Quentin, France), safflower oil (Farberdistelol, Cereol, Mannheim, Germany), and olive oil (Vierge extra, Lesieur, Neuilly, France), were added into the powdered diet in our laboratory. The dose-effect of haemin was studied in three groups of rats (N= 10, 10 and 20 rats) given haemin-supplemented diets (0.25, 0.5, and 1.5 µmol/g respectively). The effect of haemoglobin was studied in two groups of 10 rats, given a diet supplemented with haemoglobin (0.36 and 0.72 µmol/g). Haemoglobin, 0.36 µmol, and haemin, 1.5 µmol, have the same haem content, which enabled the effects of free haemin and of haemoprotein to be compared. To investigate the effect of calcium and of antioxidants, two groups of eight rats were fed a highhaemin diet (1.5 µmol/g) supplemented either with calcium (230 µmol/g) or with butylated hydroxyanisole and rutin (0.05% each). Lastly, the effect of oil was investigated by replacing safflower oil with olive oil (5%) in a high-haemin diet given to eight rats. Because calcium and olive oil might be protective, even in a normal diet without haemin, two other groups of eight rats received haemin-free control diets, supplemented with calcium (230µmol/g), or olive oil (5%). The number of rats per group was calculated to enhance the statistical power.

Accordingly, the basic group size (N= 10) was multiplied by the square root of the number of planned pairwise comparisons. For instance, the group of rats fed the high-haemin diet was compared with 4 groups (control, and high-haemin diet supplemented with calcium, antioxidant and olive oil). Its size was thus increased to N=2x10.

In contrast, hypotheses which were not central to the present study, e.g., the effect of calcium in a control diet, were tested with fewer rats per group (N=8).

TBARs assay of diet

Initially, batches of diet were prepared for three weeks, stored at -20°C and given every other day. However, after two days at room temperature, the high-haemin diets smelt rancid and two rats on high-haemin highcalcium diet died within two weeks. We thus decided to prepare diets weekly. Diets were given daily, and were stored at -20°C for no longer than seven days to reduce lipid peroxidation. Freshly prepared samples were left at room temperature for 24 hours and tested for lipid peroxidation at T0 and T24 h as described by Fenaille et al. [START_REF] Fenaille | Comparison of analytical techniques to quantify malondialdehyde in milk powders[END_REF]. The diet sample (70 mg) was mixed with 700 µl of distilled water. This mixture was homogenised with 560 µl of butylhydroxytoluene (1% in ethanol) and 560 µl of thiobarbituric acid (1% in acetic acid 5%). After centrifugation (2700Xg for 5min), the supernatant (1 ml) was heated for 60 min at 70°C. After cooling, the amount of TBARs was determined as malondialdehyde (MDA) equivalents by measure of the absorbance of the mix at 532 nm against standards (1,1,3,3-tetramethoxypropane at 0, 100, 200, 400, 600, 800 and 1000 µM).

Lipid peroxidation of diets at 24 hours (mg MDA/kg) was as follows: 2.18 for LH diet, 4.17 for MH diet, 9.91 for HH diet, 9.51 for HHCA diet, 9.20 for HHOO diet, and not detectable for the three haemin-free control diets, haemoglobin diets and high-haemin diet added with antioxidants (HHAO).

Assay of ACF

At the end of the study, colons were excised, flushed with Krebs solution (Sigma Chemical), opened longitudinally and fixed flat between coded filter papers in 10% buffered formalin (Sigma Chemical). ACF were scored by Bird's procedure [START_REF] Bird | Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings[END_REF]. The colons were stained with methylene blue (0.1%) for six minutes, and the mucosal side was observed at x32 magnification. A single observer scored all colons blindly. The number of ACF per colon, and the number of aberrant crypts per focus, were recorded. On some colons, no typical ACF were seen, but instead zones where all crypts looked aberrant with slit-like opening, increased blue staining, enlarged size and pericryptal zone. The major differences between these zones and the typical ACF were their larger size and the lack of a defined outline. We decided to name these lesions Major Aberrant Crypt Foci (MACF) (Fig 1 ).

Analysis of haem in total faeces

The amount of haem in faeces was determined according to Van den Berg et al. [START_REF] Van Den Berg | Automating the quantification of heme in feces[END_REF]. An acidified methanolchloroform extract (final concentration of HCl 1 M) was carried out on 20 mg of faeces. After centrifugation, the chloroform phase was recovered and dried under nitrogen. Samples were dissolved in 0.45 ml 250mM KOH, sonicated for 5 min (Cleanet, Hans Grieshaber, Switzerland) and mixed with 0.45ml of distilled water, 3.75 ml of 2-propanol and 0.75 ml of HCL (1.15 M). This mix was homogenised, then centrifuged for 10 min at 1500Xg and the supernatants were assayed for their haem content. Supernatants (50 µl) were mixed with 1 ml of glacial acetic acid. Subsequently, 50 µl of FeSO 4 . 7H 2 O (0.12 M freshly prepared) and HCl (4.5 M) were added.

Samples were immediately mixed and incubated at 60°C for 30 min. Two millilitres of 2-propanol/water (1/1) were added before fluorescence measurement using excitation and emission wavelengths of 400 and 594 nm (JY3D, Jobin-Yvon, France). Blanks were obtained using the same protocol, but without the incubation at 60°C.

Preparation of faecal water for biochemical assays

Faecal water was prepared by reconstituting freeze-dried faeces by adding 1 ml of distilled water to 0.3g of freeze-dried faeces. Samples were incubated at 37°C for one hour and were thoroughly mixed during the incubation time. They were then centrifuged for 15 min at 20000Xg, and supernatants were collected and stored at -20°C until use. Each sample was collected from one cage. One sample thus corresponded to the pooled faeces from two rats.

TBARs assay of faecal water

TBARs were measured in faecal water according to Ohkawa et al. [START_REF] Ohkawa | Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction[END_REF]. Ten µl of faecal water were diluted in 90 µl of distilled water. This sample was mixed with 100 µl of sodium dodecyl sulfate (8.1%) and 1 ml of 2-thiobarbituric acid (TBA, 0.5% in acetic acid 10%). Samples were homogenised and heated at 95°C for one hour. For blanks, thiobarbituric acid was omitted and replaced by 1 ml of acetic acid (10%). After heating, TBARs were extracted with 1 ml of butanol, then centrifuged for10 min at 4000Xg. The amount of TBARs was determined as malondialdehyde equivalents by measure of the absorbance of the butanol extract at 532 nm against a standard (1,1,3,3-tetramethoxypropane at 0, 100, 200, 400, 600, 800 and 1000 µM).

Haem assay

Haem was assayed in the faecal water according to Sesink et al. [START_REF] Sesink | Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme[END_REF]. Briefly, 50 µl of faecal water were diluted in 250 µl of a 5:1 mix of 2-propanol and HCl (1 M). Samples were homogenised and centrifuged 2 min at 10000Xg. Supernatants were assayed for haem as described above.

Cytotolytic activity of faecal water

The cytotolytic activity of faecal water was quantified by potassium-release from erythrocytes as described by Govers et al. [START_REF] Govers | Calcium in milk products precipitates intestinal fatty acids and secondary bile acids and thus inhibits colonic cytotoxicity in humans[END_REF]. Briefly, faecal water was obtained by reconstituting freeze-dried faeces with double distilled water (approximately 30% dry weight). Final osmolarity of each sample was 300 mosmol/L. Then, 10 or 20 µl of faecal water were mixed with saline buffer to 80µl. After a 5 min incubation at 37°C, 20 µl of washed human erythrocytes suspension (final haematocrit 5%) were added. After incubating for 15 min at 37°C, the cytolytic activity was measured.

Statistics

Results, given as mean ± SD, were analysed using Systat 5 software for Windows. Differences between groups were analysed by one-way analysis of variance (ANOVA). When ANOVA showed a statistically significant effect (p<0.05), comparison of each experimental group with the control group was made using Dunnett's test which corrects for multiple comparisons. Most groups were compared with the control group fed a diet without haem. However, according to the tested hypotheses, groups given a high-haemin diet supplemented with a potential inhibitor of haem-promotion were compared with the group fed the high-haemin diet (1.5 µmol/g).

Differences were considered significant when two-sided P was <0.05.

Results

General observations

Rats given a high-haemin diet gained less weight than control rats (-10%). In contrast, for the same or a higher haem intake, haemoglobin diets did not depress the growth of rats (Table 2, panel A). The daily food intake was similar in all groups (11 ± 0.8 g/d, full data not shown), and groups given haemin and haemoglobin diets had haem intakes matching the study design (Table 2, panel A, column 6). However, the haem concentration was higher in the faeces of haemoglobin-fed rats than in haemin-fed rats (+45%, see Table 2 last column). The two strategies to limit the peroxidation in a high-haemin context (antioxidants and olive oil) increased significantly the concentration of haem in faeces (+44% and +86%, Table 2, panel B, last column). These differences in haem faecal concentrations were likely due to differences in faecal daily weight: high-haemin diets increased the faecal excretion (+33%), a laxative effect previously noted by Sesink [START_REF] Sesink | Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme[END_REF], and haem was thus diluted in the faeces. In contrast, haemoglobin was not laxative, and the antioxidants and olive oil normalised the laxative effect of haemin (Table 2).

Effect of diets on carcinogenesis

The number and size of aberrant crypt foci in the colon of rats after 14 weeks on the experimental diets are reported in Table 3 and figure 2. Haemin strikingly increased the size of foci, dose-dependently, from 2.6 in control rats to 6.2, 10.2 and 11.4 crypts/focus in rats fed the haemin diets (all p<0.001). Haemin also increased strongly and dose-dependently the number of aberrant crypts (Fig. 2). The high-haemin diet, but not the low-and medium-haemin diets, increased the number of total foci per colon from 125 to 166 (p<0.001). The haemoglobin diets similarly increased the number of foci from 125 to 162 and 167 (p=0.001, Table 3), but not their size, although the high-haemoglobin diet contained twice the amount of haem as the high-haemin diet. Haemoglobin also increased the total number of aberrant crypts in the colon of rats, but the increase was marginally significant (Fig. 2, p<0.0005 by Student t test for both groups fed haemoglobin, but p>0.05 by Dunnet's test).

Surprisingly, the rats given the low-haemin diet had less foci than control rats given no haem at all (125 foci in controls, 92 in low-haemin group, p<0.01). However, since these foci contained more aberrant crypts than those of control rats, even the low-haemin diet increased the total number of aberrant crypts per colon (324 aberrant crypts in controls, 565 in low-haemin group, p<0.01). Indeed, the specific analysis of ACF and MACF data shows that haemin promoted or induced the large MACF instead of classical ACF, while haemoglobin promoted the classical ACF and not the MACF (Table 3). Both tested levels of haemoglobin produced the same effect on carcinogenesis. It is possible that the lowest level was high enough to reach a plateau of promotion.

Calcium, antioxidants, and olive oil completely inhibited the promoting effect of haemin on the size of foci (Table 3, panel B), and reduced the number of aberrant crypts (p<0.01) in the colon of rats. These three dietary interventions specifically suppressed the haemin-induced MACF, thus normalising the type of ACF. No difference was seen between rats given high-calcium and olive oil control diets and rats given the no-haemin control diet (Table 3, panel C). In addition, macroscopic tumours were detected in the colon of five haem-diet fed rats: two rats given the high-haemin diet, one rat given the medium-haemin diet, and one rat given the highhaemoglobin diet (5/60 haem-fed rats vs. 0/38 control rats, p=0.15 by Fisher's exact test with the approximation of Woolf). Histology showed all tumours were typical adenomas.

Faecal water characteristics

We measured the characteristics of faecal water because, according to studies on bile acids, the soluble fraction of colonic contents would interact more strongly with the mucosa than the insoluble fraction [START_REF] Lapre | Diet-induced increase of colonic bile acids stimulates lytic activity of fecal water and proliferation of colonic cells[END_REF]. As expected, the haem concentration in faecal water depended directly on the level of haemin in the diet (Table 4). Dietary haemoglobin also increased haem concentration in faecal water, but less than haemin: A similar haem concentration was found in rats eating 32 µmol/d of dietary haemoglobin or 15 µmol/d of haemin (Tables 1 and4). Haem can induce the formation of peroxyl radicals in fats, which may be cytotoxic and cleave DNA in vivo [START_REF] Sawa | Lipid peroxyl radicals from oxidized oils and heme-iron: implication of a high-fat diet in colon carcinogenesis[END_REF]. Lipid peroxidation was thus measured in faecal water by the TBARs assay. As shown in Table 4, lipid peroxidation correlated with haem concentration in faecal water (r=0.96 for the 11 groups, r=0.87 for the 116 rats, both p<0.0001). The high-haemin diet thus increased TBARs in the faecal water by ten-fold. Haemoglobindiets also increased TBARs, but to a lesser extent. Furthermore, a cytotoxic factor is found in the faecal water of haemin-fed rats [START_REF] Sesink | Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme[END_REF]. Accordingly, the cytolytic activity of faecal water increased 50-fold in rats fed a highhaemin diet, but, surprisingly absolutely no increase was seen in haemoglobin-fed rats (Table 4). Calcium, antioxidants and olive oil completely suppressed the haemin-induced cytolytic activity of faecal water (Table 4, panel B). The dietary calcium completely inhibited the effects of haemin. Rats fed the high-haemin high-calcium diet had no haem in faecal water, and no increased peroxidation. Sesink et al. suggest that this inhibition comes from the binding and precipitation of haem by calcium phosphate [START_REF] Sesink | Red meat and colon cancer: dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by calcium[END_REF]. Antioxidants and olive oil halved the haemin-induced lipid peroxidation, and reduced the haem concentration, in faecal water of rats given a highhaemin diet. Lastly, calcium, antioxidants or olive oil did not change faecal water values in rats given a diet without haemin (Table 4, panel C).

Discussion

Dietary haem significantly increased the colon carcinogenesis in rats at the aberrant crypt stage. This result supports the hypothesis that haem can explain the association between red meat consumption and colon cancer risk. The low-haemin diet in this study (0.25µmol/g of diet) mimics the haem content of a meat-based Western diet [START_REF] Sesink | Red meat and colon cancer: a possible role for heme[END_REF]. Indeed, this study showed that (i) haem as haemin or haemoglobin increased the number of azoxymethane-induced ACF in the colon of rats; (ii) haemin effect, the strongest promotion ever reported on ACF, was associated with cytotoxicity and lipid peroxidation in faecal water; (iii) haemin effect was inhibited by dietary calcium, by dietary antioxidants, and by olive oil; and (iv) haemoglobin, which raised lipid peroxidation in faecal water but not cytotoxicity, was less potent than haemin to promote colon carcinogenesis. The effect of haemin, antioxidants, calcium and haemoglobin is discussed below.

Haemin was used to explore the effect of haem from red meat because meat haemoproteins are digested in the stomach and the small bowel, and provide free haem to the colon [START_REF] Vaghefi | Influence of the extent of hemoglobin hydrolysis on the digestive absorption of heme iron. An in vitro study[END_REF]. Haemin strikingly increased the number of azoxymethane-induced aberrant crypts (Fig. 2), and the size of foci, by shifting ACF to MACF (Table 3). Macroscopic tumours were seen in rats given haemin for 100 days. To our knowledge, the magnitude of this promotion was the highest ever seen: haemin increased the number of aberrant crypts six-fold, when potent promoters like cooked casein or dextran sulfate increase this number less than two-fold. In faecal water, dietary haemin raised dose-dependently the cytolytic activity, the lipid peroxidation, and the haem concentration: could these changes explain the observed promotion? A faecal cytolytic factor, not yet identified but different from haemin, would increase the epithelial proliferation [START_REF] Sesink | Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme[END_REF]. This factor might be the ACF promoter. Also, the peroxidation of polyunsaturated fatty acids produces aldehydes such as malondialdehyde and 4-hydroxynonenal, which form mutagenic DNA-adducts [START_REF] Bartsch | Exocyclic DNA adducts as oxidative stress markers in colon carcinogenesis: potential role of lipid peroxidation, dietary fat and antioxidants[END_REF]. Sawa et al. showed that haem generates alkylperoxyl radicals (LOO . ) from oxidised oil, particularly from safflower oil. Such radicals can cleave DNA, which would contribute to the colon cancer risk [START_REF] Sawa | Lipid peroxyl radicals from oxidized oils and heme-iron: implication of a high-fat diet in colon carcinogenesis[END_REF], and may also explain the observed ACF promotion. Last, faecal water contained soluble haem, which might directly promote carcinogenesis. Indeed, haem can induce gut inflammation through the upregulation of adhesion molecules, the increase in vascular permeability and the granulocytes infiltration [START_REF] Wagener | Heme is a potent inducer of inflammation in mice and is counteracted by heme oxygenase[END_REF].

Thus, the chronic inflammation caused by the continuous haem intake might have promoted the ACF growth [START_REF] Rhodes | Inflammation and colorectal cancer: IBD-associated and sporadic cancer compared[END_REF]. In addition, haemin may have weakened the tumour immunosurveillance. Indeed, haem induces haemoxygenase-1 (HO-1), and the over-expression of HO-1 results in the inhibition of T-cell and NK-cell mediated lysis [START_REF] Woo | Stress proteininduced immunosuppression: inhibition of cellular immune effector functions following overexpression of haem oxygenase (HSP 32)[END_REF]. A strange finding was that fewer ACF were scored in the gut of rats fed a low-haemin diet, than in rats fed the control diet. However, other agents which promote the growth of ACF, and increase the incidence of chemically-induced cancer, decrease the number of ACF (e.g., cholic acid) [START_REF] Magnuson | Reduction of aberrant crypt foci induced in rat colon with azoxymethane or methylnitrosourea by feeding cholic acid[END_REF]. To prevent the deleterious effect of haemin, several strategies of dietary changes have been tested in this study: the addition of antioxidant agents, the substitution of safflower oil by oxidation-resistant olive oil, and the addition of haemin-blocking calcium phosphate to the diet.

The antioxidants we added to safflower oil, or naturally present in olive oil, completely inhibited the haemin-induced promotion of colon carcinogenesis. Safflower oil is highly susceptible to oxidation, and the high-haemin diet with 5% safflower oil quickly turned rancid (see TBARs assay of diets, in the Materials and Methods section). The addition of two antioxidants into the diet, one soluble in fat, the other soluble in water, completely inhibited the peroxidation of diet. These antioxidants also show direct chemopreventive properties when given during the carcinogen treatment (39), or at concentrations higher than 0.05% [START_REF] Corpet | Most effective colon cancer chemopreventive agents in rats: a systematic review of aberrant crypt foci and tumor data, ranked by potency[END_REF]. However, we think that, in the present conditions (very low levels given post-initiation), their effect was due to the inhibition of haemin-induced peroxidation. Olive oil was added to another diet because it contains the relatively oxidationresistant oleic acid, and phenolic compounds, which scavenge LOO . radicals in the oil itself [START_REF] Kanazawa | Generation of lipid peroxyl radicals from edible oils and their biological activities: a need for consideration for anti-radical components and purification processing[END_REF] and in the faecal matrix [START_REF] Owen | Oliveoil consumption and health: the possible role of antioxidants[END_REF]. Both antioxidant strategies halved TBARs in faecal water (Table 4, panel B). This inhibition of lipid peroxidation in faecal water was associated with inhibition of haemin-induced carcinogenesis (Table 3, panel B). It is thus possible that lipid peroxidation itself is a link between dietary haem and cancer enhancement, as suggested by Sawa and by Owen [START_REF] Sawa | Lipid peroxyl radicals from oxidized oils and heme-iron: implication of a high-fat diet in colon carcinogenesis[END_REF][START_REF] Kanazawa | Generation of lipid peroxyl radicals from edible oils and their biological activities: a need for consideration for anti-radical components and purification processing[END_REF], but contested by Sesink et al. [START_REF] Sesink | Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme[END_REF]. It is indeed surprising that both antioxidant strategies increased haem concentration in faeces (Table 2), but decreased factors apparently not related to oxidation: haem concentration was reduced by 30-40%, and cytolytic activity was fully suppressed, in the faecal water of rats given the diets with olive oil or with antioxidants (Table 4, panel B). The mechanism by which olive oil and antioxidants suppressed the haemin-induced promotion thus appears complex and warrants further studies.

Calcium phosphate was added to a high-haemin diet, as an alternative strategy to reduce the effect of haem. Many studies suggest that calcium can protect against colon carcinogenesis. For instance dietary calcium reduces the recurrence of adenomatous polyps in patients [START_REF] Baron | Calcium supplements for the prevention of colorectal adenomas. Calcium Polyp Prevention Study Group[END_REF]. In volunteers, calcium precipitates the intestinal fatty acids and the secondary bile acids, and thus inhibits colonic cytotoxicity, and, possibly, the promotion by high-fat diets [START_REF] Govers | Calcium in milk products precipitates intestinal fatty acids and secondary bile acids and thus inhibits colonic cytotoxicity in humans[END_REF]. Newmark et al. [START_REF] Newmark | Colonic hyperplasia and hyperproliferation induced by a nutritional stress diet with four components of Western-style diet[END_REF] suggest that 5 mg/g calcium in the AIN-76A rodent diet is equivalent to 2700 mg calcium per day in humans, that is three times the Recommended Dietary Allowance. Using the same proportionality here, the low-calcium (0.79 mg/g) and the high-calcium (9.9 mg/g) diets were equivalent to 427 and 5346 mg calcium per day in humans, respectively. Because the diet contains 500-600 mg/d calcium on average in the USA [START_REF] Newmark | Calcium, vitamin D, and colon cancer[END_REF], the low-calcium diet in this study mimics the intake of a considerable fraction of the American population. In rats given the high-haemin diet, dietary calcium suppressed colon ACF in rats. Calcium reduces the haemin-induced colonic hyper-proliferation, because calcium phosphate precipitates haemin in the gut [START_REF] Sesink | Red meat and colon cancer: dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by calcium[END_REF]. Faecal haem was high in rats given a high-calcium diet (Table 2), but it was not soluble in the faecal water. This water thus contained no lipid peroxides and showed no cytolytic activity (Table 4). Clearly the precipitation of haem by calcium is enough to explain the protection against haem-induced carcinogenesis. In conclusion, both strategies were effective against haemin promotion. Red meat contains no haemin, but myoglobin. We thus also studied the effect of dietary haemoglobin, a haemoprotein similar to myoglobin, but easier to obtain.

Haemoglobin increased the number of aberrant crypts and of ACF in rats (Table 3), an effect already observed by Bruce [START_REF] Bruce | OCI-S-122 Study of raw and cooked haemoglobin effects on azoxymethane induced aberrant crypt foci in F344 rats[END_REF]. The magnitude of the effect (1.6-fold increase) is second only to haemin, dextran sulphate and cooked casein. This result provides a possible explanation for the association observed between red meat consumption, and the colon cancer risk in humans. In contrast with haemin, haemoglobin did not increase significantly the number of aberrant crypts per focus, and was thus less potent than haemin to promote the ACF growth (Table 3). The difference between haemin and haemoglobin was mainly due to the MACFs: frequent in the colon of haemin-fed rats, occasional in the colon of haemoglobin-fed rats. How could this difference be explained? Haemin and haemoglobin led to similar faecal haem excretion: 33% of the haem intake was excreted in both cases (Table 2), but haem concentration in faecal water was higher in haemin-fed rats (Table 4).

However, this difference cannot explain the differential effect on MACF: rats in groups HH and TG had similar levels of haem in faecal water (Table 4) but very different numbers of MACF (Table 3). Haemin and haemoglobin do not have the same fate in the gastro-intestinal tract. Haemin (free haem) is poorly soluble in water, and forms insoluble polymers at low pH in the stomach [START_REF] Vaghefi | Influence of the extent of hemoglobin hydrolysis on the digestive absorption of heme iron. An in vitro study[END_REF]. Haemin thus escapes small intestinal absorption and reaches the colon. In the colon, haemin induces lipid peroxidation and yields the cytotoxic compound described by Sesink et al.: this fat soluble compound is found in the faeces of haemin-fed rats, it shows a specific absorption a 400 nm, but it differs from haemin, porphyrin or bilirubin [START_REF] Sesink | Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme[END_REF]. On the other hand, haemoglobin is water soluble. The hydrolysis of its globin moiety in the upper digestive tract yields haem in a form which may be more available than haemin [START_REF] Vaghefi | Influence of the extent of hemoglobin hydrolysis on the digestive absorption of heme iron. An in vitro study[END_REF]. Indeed, dietary haemoglobin is three times more efficient than haemin in providing iron for red blood cell synthesis in rats [START_REF] Fly | Iron Bioavailability from Hemoglobin and Hemin in Chick, Rat, Cat, and Dog: A Comparative Study[END_REF]. In contrast with haemin-fed rats, animals given a haemoglobin-diet had no cytolytic activity at all in faecal water, which shows that the fates of haemin and of haemoglobin are not the same (Table 4). It also suggests that the MACFs were specifically produced by the haemin-induced cytotoxic factor [START_REF] Sesink | Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme[END_REF]. Haemin has been used in experimental studies to investigate the effect of red meat consumption. The underling hypothesis was that, because globin is digested in the upper tract, haemoprotein and haemin would deliver similar haem-compounds to the colon. The present results clearly show that this is not true. Myoglobin is the main pigment in beef and pork muscles, whereas some haemoglobin is found in white meat. Both haemoproteins hold the haem similarly, and both can initiate the peroxidation of fats [START_REF] Baron | Myoglobin-induced lipid oxidation. A review[END_REF]. We thus suggest that haemoglobin may be a suitable substitute for myoglobin, and thus for red meat studies in rats. In cured meat, however, nitrite reacts with myoglobin to yield nitrosomyoglobin. When ham and hot-dog are cooked, free nitroso-haem is released from the denatured protein [START_REF] Pearson | Advances in Meat Research. Restructured Meat and Poultry Products[END_REF]. Haemin might thus be a suitable model to study the effect of processed meat on colon cancer. Calcium, olive oil and antioxidants protective effects were associated with the inhibition of haemin-induced lipid peroxidation. Lipid peroxidation was associated with the promotion of aberrant crypts by both haemin and by haemoglobin. We thus suppose that calcium, olive oil and antioxidants could counteract the haemoglobin-promotion. The advice to increase consumption of olive oil and of calcium-rich dairy products might be easier to follow than the advice to abstain from consumption of red meat. Indeed, the association between red meat intake and colon cancer was more consistently shown in North-American studies than in European studies. Now, in Europe, the calcium and the olive oil intakes are higher than in the USA [START_REF] Sesink | Red meat and colon cancer: dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by calcium[END_REF].

In conclusion, dietary haemin and haemoglobin increased colorectal carcinogenesis in rats. No known ACF promoter is more potent than haemin. Haemoglobin is close to myoglobin, and it was much less toxic than haemin. This study supports the hypothesis that red meat could promote colon cancer because of its myoglobin content. A diet high in calcium, or with oxidation-resistant fats, may prevent the possible cancer-promoting effect of red meat.

Figure 1 :

 1 Figure 1 : Mucosal surface of colon stained with methylene blue visualised under a light microscope (x40). (A) Aberrant Crypt Focus with four crypts (control diet), (B) Major Aberrant Crypt Focus with twenty crypts (high haemin diet).

Figure 2 :

 2 Figure 2: Number of aberrant crypts in the colon of rats after 100 days on experimental diets. A: Effect of haemin (LH, MH, HH) and haemoglobin (HG, TG); B: Effect of calcium (CA), anti-oxidants (AO) and olive oil (OO) in a high-haemin context. *: significantly different from control diet CD (panel A) or from HH diet (panel B) (p<0.01, by Dunnett's test). Haem concentration in diets (µmol/g)

Table 1 :

 1 Composition of experimental diets, given in g per kg of diet.

	Tested factor Control	Haemin		Haemoglobin	Calcium		Anti-	Olive oil
										oxidants
	Group*	CD	LH	MH	HH	HG	TG CDCA HHCA	HHAO CDOO HHOO
	Haemin	0 0.16 a 0.32 0.94	0	0	0	0.94	0.94	0	0.94
	Haemoglobin	0	0	0	0	25	50	0	0	0	0	0
	Ferric Citrate	0.36	0.29 0.24	0	0	0	0.36	0	0	0.36	0
	Rutin+BHA d	0	0	0	0	0	0	0	0 0.5 + 0.5	0	0
	CaHPO 4 .2H 2 O	2.7	2.7	2.7	2.7	2.7	2.7	33.75 33.75	2.7	2.7	2.7
	Methionine	3	3	3	3	3	3	3	3	3	3	3
	Mineral mix b	35	35	35	35	35	35	35	35	35	35	35
	Vitamin mix	10	10	10	10	10	10	10	10	10	10	10
	Choline	2	2	2	2	2	2	2	2	2	2	2
	bitartrate
	b: Mineral mix without CaHPO 4 .2H 2 O
	c: olive oil replaced safflower oil in CDOO and HHOO diets
	d: BHA, butylated hydroxyanisole

* Groups: CD, Control Diet; LH, Low Haemin diet; MH, Medium Haemin diet; HH, High Haemin diet; HG, High haemoGlobin diet; TG, Top haemoGlobin diet; HHAO, High Haemin and AntiOxidants diet; HHCA, High Haemin and Calcium diet; HHOO, High Haemin and Olive Oil diet; CDCA, Control and Calcium diet; CDOO, Control and Olive Oil diet. a: Values are g/kg diet, main differences between diets are printed in bold.

Table 2 :

 2 Effect of diet on body weight, haem intake, concentration in faeces and daily output A: Effect of haemin and haemoglobin; B: Effect of inhibitors in a high-haemin context, and, C: in a low-haemin context.

	A	Haem	No of	Final body weight Faecal output (dry	Haem intake	Haem
		in diet	Rats			weight)		in faeces
	Diets*	µmol/g			g/rat	g/day	µmol/day	µmol/g
	CD	0	22	197 ± 9	0.6 ± 0.1	0	0.04 ± 0.09
	LH	0.25	10	192 ± 8	0.5 ± 0.1	2.9 ± 0.1 a	0.95 ± 0.24
	MH	0.5	10	187 ± 8 a'	0.5 ± 0.1	5.5 ± 0.3 a	2.1 ± 0.7 a
	HH	1.5	20	178 ± 9 a	0.8 ± 0.1 a	15.2 ± 2.2 a	6.6 ± 1.3 a
	HG	1.5	10	193 ± 9	0.6 ± 0.1	16.8 ± 2.0 a	9.6 ± 0.9 a
	TG	3.0	10	190 ± 10	0.6 ± 0.2	31.8 ± 3.2 a	14.7 ± 2.3 a
	B
	HH	1.5	20	178 ± 9	0.8 ± 0.1	15.2 ± 2.2	6.6 ± 1.3
	HHCA	1.5	6		183 ± 5	0.8 ± 0.2	17.8 ± 1.4 b	7.9 ± 0.6
	HHAO	1.5	10	186 ± 9	0.6 ± 0.1 b	14.8 ± 1.3	9.5 ± 1.5 b
	HHOO	1.5	8		180 ± 7	0.6 ± 0. 1 b	16.5 ± 0.8	12.3 ± 1.6 b
	C
	CD	0	22	197 ± 9	0.6 ± 0.1	0	0.04 ± 0.09
	CDCA	0	8		193 ± 5	1.0 ± 0.1 a	0	0.01 ± 0.02
	CDOO	0	8		188 ± 9	0.6 ± 0.1	0	0.04 ± 0.07
	* Dietary groups, see note to table 1.
	a, a': significantly different from control diet CD (a: p<0.01, a': p<0.05 by Dunnett's test)
	b: significantly different from high-haemin diet HH (p<0.01, by Dunnett's test)
	c: haem metabolized was extrapolated by subtracting haem output from haem intake

Table 3 :

 3 Effect of experimental diets on the number and size of aberrant crypt foci and major aberrant crypt foci in the colon of rats after 100 days of nutritional experimentation. A: Effect of haemin and haemoglobin; B: Effect of inhibitors in a high-haemin context, and, C: in a low-haemin context. : significantly different from control diet :CD (p<0.01, by Dunnett's test) b: significantly different from HH diet (p<0.01, by Dunnett's test) c: the median ACF contained 2.6 ± 0.2 aberrant crypts d: the median MACF contained 11.5 ± 0.7 aberrant crypts

	A	Haem in	No of		Total Foci (ACF + MACF)	ACF c	MACF d
		diet	Rats
	Diets*	µmol/g		Foci / colon Size = aberrant	Aberrant crypts /	Foci / colon Foci / colon
					crypts / focus	colon
	CD	0	22	125 ± 25	2.6 ± 0.2	324 ± 70	125 ± 25	0.3 ± 0.8
	LH	0.25	10	92 ± 16 a	6.2 ± 2.0 a	565 ± 193 a	47 ± 23 a	44 ± 20 a
	MH	0.5	10	120 ± 16	10.2 ± 0.9 a	1233 ± 210 a	12 ± 13 a	108 ± 19 a
	HH	1.5	20 166 ± 26 a	11.4 ± 1.1 a	1901 ± 371 a	7 ± 14 a	158 ± 29 a
	HG	1.5	10 162 ± 27 a	3.3 ± 0.4 b	518 ± 119 b	149 ± 23 b	12 ± 8 b
	TG	3.0	10 167 ± 34 a	2.7 ± 0.4	457 ± 119 b	160 ± 32 a.b	7 ± 6 b
	B
	HH	1.5	20	165 ± 26	11.4 ± 1.1	1901 ± 371	7 ± 14	158 ± 29
	HHCA	1.5	6	135 ± 22	2.7 ± 0.4 b	359 ± 38 b	131± 23 b	4 ± 2 b
	HHAO	1.5	10	142 ± 25	2.9 ± 0.3 b	406 ± 85 b	135 ± 25 b	7 ± 7 b
	HHOO	1.5	8	138 ± 31	2.8 ± 0.3 b	389 ± 115 b	132 ± 25 b	7 ± 8 b
	C
	CD	0	22	125 ± 25	2.6 ± 0.2	324 ± 70	125 ± 25	0.3 ± 0.8
	CDCA	0	8	125 ± 11	2.8 ± 0.4	354 ± 28	124 ± 12	1.2 ± 2.3
	CDOO	0	8	123 ± 18	2.4 ± 0.3	302 ± 63	123 ± 18	0.2 ± 0.7

* Dietary groups, see note to table

1

. a

Table 4 :

 4 Effect of experimental diets on concentration of haem, lipid peroxidation and cytotoxicity in faecal water. A: Effect of haemin and haemoglobin diets; B: Effect of antioxidant; high calcium and olive oil diet on a haemin context, C: Effect of calcium and olive oil on control diet.

	A	Haem in	Haem in faecal water	Lipid peroxidation:	Cytotoxicity
		diet		TBARs in faecal water	of faecal water
	Diets*	µmol/g	µM	MDA equivalents (µM)	% K release
	CD	0	2.6 ± 4.5	38 ± 14	2 ± 0
	LH	0.25	36 ± 17 a.b	67 ± 32 b	34 ± 7 a
	MH	0.5	135 ± 44 a.b	168 ± 13 a.b	97 ± 1 a
	HH	1.5	258 ± 91 a	346 ± 26 a	100 ± 1 a
	HG	1.5	158 ± 48 a.b	187 ± 21 a. b	2 ± 0
	TG	3.0	262 ± 68 a	260 ± 28 a.b	2 ± 1
	B
	HH	1.5	258 ± 91	346 ± 26	100 ± 1
	HHCA	1.5	4.7 ± 4.6 b	49 ± 6 b	2 ± 0 b
	HHAO	1.5	160 ± 46 b	138 ± 17 b	2 ± 0 b
	HHOO	1.5	186 ± 61 b	166 ± 27 b	5 ± 4 b
	C
	CD	0	2.6 ± 4.5	38 ± 14	2 ± 0
	CDCA	0	3.8 ± 2.7	28 ± 9	5 ± 3
	CDOO	0	1.6 ± 1.8	45 ± 4	3 ± 3

* Dietary groups, see note to table

1

.

Acknowledgements:

We thank Xavier Blanc (UPAE, INRA) for the preparation of experimental diets, Denise S.M.L. Termont (NIZO) for the determination of cytolytic activity of faecal water, Alexandre Peuch for TBARs assays, Raymond Gazel and Florence Blas Y Estrada for the care of animals, Alain Paris for help in statistical analysis, Amanda Freeman for careful reading of the manuscript, and W.R. Bruce (U.of T.) for helpful advices with the study design. The studies were supported in part by the INRA, the DGER, and by a grant of the région Midi-Pyrénées.


Corpet Denis

Pierre Fabrice

Denis E Corpet
email: <d.corpet@envt.fr>

Point: From animal models to prevention of colon cancer. Systematic review of chemoprevention in min mice and choice of the model system

Keywords: Animals, Anticarcinogenic Agents, therapeutic use, Azoxymethane, diagnostic use, Chemoprevention, Colonic Neoplasms, chemically induced, prevention & control, Diet, Disease Models, Animal, Humans, Mice, Mice, Mutant Strains, Precancerous Conditions, chemically induced, prevention & control, Randomized Controlled Trials as Topic, Rats

The Min ( ( / )) mouse model and the azoxymethane (AOM)-rat model are the main animal models used to study the effect of Apc +dietary agents on colorectal cancer. We recently reviewed the potency of chemopreventive agents in the AOM-rat model (Corpet and Tach , 2002). Here we add the results of a systematic review of the effect of diet and agents on the tumor yield in Min mice, based on é the results of 179 studies, from 71 articles, and displayed also at the website . The efficacy of agents in the Min mouse model and the AOM-rat http://www.inra.fr/reseau-nacre/sci-memb/corpet/indexan.html model correlated (r 0.66, p<0.001), although some agents that afford strong inhibition in the AOM-rat and the Min mouse increase = the tumor yield in the large bowel of mutant mice for

reasons not yet understood. Thus, piroxicam, sulindac, celecoxib, difluoromethylornithine, and polyethylene glycol could promote carcinogenesis in the colon of mice. We also compare the results of rodent studies with those from clinical intervention studies of polyp recurrence. We found that the effect of most of the agents tested is consistent across the animal models, except the above-mentioned puzzling mouse colon. Thus our point is that the rodent models can provide guidance in the selection of prevention approaches to colon cancer, in particular suggesting the likely importance of polyethylene glycol, hesperidin, protease inhibitor, sphingomyelin, physical exercise, epidermal growth factor receptor kinase inhibitor, ( )-catechin, resveratrol, fish oil, curcumin, caffeate and thiosulfonate as preventive agents.

Introduction

Puzzling results were presented at recent meetings of the American Association for Cancer Research. The feeding of the nonsteroidal anti-inflammatory drugs (NSAIDs) piroxicam or sulindac to mutant mice with spontaneous tumors, strikingly increases the tumor yield in their colon ( ). However, NSAIDs are widely accepted chemopreventive agents against colon cancer in humans ( ). These results raise 1-3 4 questions about either the animal model, or the NSAIDs protection. We have thus reviewed the results of dietary chemoprevention studies in animal models of colorectal cancer and compared them with the results of clinical intervention studies, looking for consistency among the models.

Two animal models for preclinical testing of chemopreventive agents

Since 1970 investigators have searched for diets or agents that suppress colorectal tumors in rodents. Rodents have almost no spontaneous colon cancer, and a carcinogen is needed to induce the tumors. Most chemopreventive agents were thus tested in rats, usually male Fisher 344, given AOM injections. AOM is a specific colon carcinogen, like its precursor, dimethylhydrazine. The tumors induced are often mutated on and beta-catenin genes ( ), but seldom [START_REF] Corpet | Most effective colon cancer chemopreventive agents in rats: A review of aberrant crypt foci and tumor data, ranked by potency[END_REF] on the adenomatous polyposis coli ( ) gene, and never on the K-ras 5 % Apc gene ( ). Other rodents (mice), and other colon carcinogens were used less frequently, e.g., specific nitrosamines, and heterocyclic p53 6

amines like 2-amino-1-methyl-6-phenylimidazo 4,5-b pyridine (PhIP). PhIP induces the mutation frequently (40 60 ) ( ) and

[ ]

Apc

-% 7 microsatellite instability ( ), but no or mutations ( ). AOM is not present in our daily diet, but PhIP is. However, PhIP is 8 K-ras p53 9-10 almost never used because AOM is less expensive, more potent, and more convenient to use than PhIP. Chemopreventive treatment can be begun before exposure to the carcinogen and during the initiation phase, during the promotion-progression phase, or through both phases.

The incidence of colon tumors is the major endpoint in most rats studies. Carcinogen-induced tumors produced in the rat colon share many characteristics with human colorectal cancer, except that they have a lower tendency to metastize.

In 1990, the mutant Min mouse was found with multiple intestinal neoplasia ( ). It was shown to have a mutated gene, similar to 11 Apc that in patients with familial adenomatous polyposis, and in many sporadic cancers. This promising animal model mimics the rapid development of adenomatous polyps that affect humans with germline inactivation of one gene. But the mutations observed in Apc K-ras many human tumors were not detected in Min mice polyps ( ), and inactivation, frequent in human cancers, does not raise tumor 12 p53

number in Min mice ( ). Following the Min mouse discovery with truncated in position 850, other mice have been genetically [START_REF] Fazeli | Effects of p53 mutations on apoptosis in mouse intestinal and human colonic adenomas[END_REF] Apc modified so that one or more oncogenes hold a germline mutation (e.g., truncated in positions 716, 1309, or 1638, and mutated Apc Msh2 or ). A mutation on or genes leads to mismatch repair defect, which makes these mice a model for human hereditary Mlh1 Msh2 Mlh1

nonpolyposis colorectal cancers ( ). These mutant mouse models have increased our understanding of carcinogenesis. They have also [START_REF] Dewind | Riele HT Mouse models for hereditary nonpolyposis colorectal cancer[END_REF] provided a model to evaluate the effect of diets and chemopreventive agents. Compared with the AOM-rat model, the use of mutated mice avoids the hazard of carcinogen handling, and leads to shorter studies. Dietary treatments are initiated in mice by the age of 4 5 weeks, when tumors may be already present (except for a few in utero studies). This timing mimics human clinical trials, where dietary treatments are given to adults, likely to bear minute polyps, the visible ones having been removed before randomization. The number of tumors in the small intestine is the primary endpoint in most mouse studies. The major drawback of these mutant models is that, in contrast with the human situation, the mouse tumors occur predominantly in the small intestine and not in the colon.

There are now sufficient results reported from the AOM-rat and Min mouse colon cancer prevention models to make it possible to compare the results of different approaches and to begin to assess which provides the best prediction of response in clinical intervention studies. We consider first the previous review of results of interventions with the rat model, then review results obtained with the Min model.

Review on dietary chemoprevention in the AOM-rat and Min mouse models

Data from our previous systematic review on chemoprevention in the rat model ( ) were gathered from 146 articles with the tumor 15 endpoint, and 137 articles with the aberrant crypt focus endpoint, a putative preneoplastic lesion. Tables were built with potency of each agent or diet to reduce the tumor incidence or the number of aberrant crypt foci in the colon of rats. Both tables are available on a website with sorting abilities, . Agents of outstanding potency, that fully suppress colon http://www.inra.fr/reseau-nacre/sci-memb/corpet adenocarcinoma and/or consistently inhibits adenoma and aberrant crypt foci in several independent AOM-rats studies, were (ranked list): polyethylene glycol (PEG), celecoxib, hesperidin, difluoromethylornithine (DFMO) and piroxicam (combined or not), sulindac (sulfone or sulfide), and ursodeoxycholic acid. In addition, treadmill exercise, and S-methyl-methane-thiosulfonate suppressed carcinoma (supported by a single study each). Last, Bowman-Birk protease inhibitor and sphingomyelin were consistently efficient in AOM-initiated mice ( ). 15

All publications relating to the effect of dietary agents tested in Min mice, and other mice with mutations resulting in intestinal tumors, were identified from three databases, ISI Current Contents, Medline, and the American Association for Cancer Research website, for the period from 1990 to May 2002. Data were gathered from 63 articles and eight meeting abstracts, yielding 179 comparisons between a control and a treated group of mice. A primary table (not shown) was built including the following data: mouse strain, mutation, treatment dose duration and vehicle, the number of intestinal adenomas in treated and control groups, the significance of the treatment effect, and, when reported, the size of intestinal adenomas, and the specific number of colonic adenomas. Some papers did not report small and large bowel data separately. In those cases, we reported the total number of adenomas instead of small intestinal adenomas. This primary table was abstracted to give the efficacy of each treatment to reduce the number of adenomas in the small intestine, and in the colon, of mutated mice (

). The results are reported as a percentage of control values. The table is available on a website with sorting abilities, Table 1 allowing to rank agents by potency, . http://www.inra.fr/reseau-nacre/sci-memb/corpet/indexan.html

The table clearly shows that NSAIDs are, by far, the most potent agents to suppress tumor formation in the small intestine of Min mice. Notably, piroxicam and sulindac decreased the tumor yield by 90 or more in several independent studies. As shown on % Table 1 (ranked by potency, on the website), piroxicam or sulindac were used in all of the top-25 studies but one, which involved the epidermal growth factor receptor kinase inhibitor, EKB-569. Specific anti-cyclooxygenase (COX)-2, like celecoxib or MF-tricyclic, were not more potent than non selective NSAIDs. Other agents were less potent than NSAIDs and the best ones decreased the tumor yield by 60 70 : (

-% +
)-catechin, resveratrol, fish oil (two studies), curcumin, folic acid and caffeic acid phenetyl ester. Following agents were clearly less potent: cellulose, copper, DFMO, PEG, wheat bran, sphingomyelin, uroguanylin and selenium compound p-XSC.

Comparison of results obtained with the two prevention models

shows that many agents that suppress tumors in the Min mouse intestine ( ) also decrease the incidence of colorectal Fig. 1 Table 1 cancer in AOM-initiated rats ( ). A significant correlation was found between the efficacy of agents tested in both models (r 0.66, N 36, 15 = = p<0.001). It is clear that the most potent chemopreventive agents in the Min mouse small intestine, are also potent in the colon of AOM-initiated rats, and the animal models thus seem consistent.

Min mice have many tumors in the small intestine (median number 34), but few tumors in the colon (median, 1.0) ( ). In Table 1 contrast, human tumors are rarely found the small intestine, but frequently in the colon. This discrepancy between the mouse model and the human situation led us to examine the effect of diets on the tumor yield in the of mutated mice. We thus calculated the ratio colon between treatment effect in the small intestine and in the colon (two last columns in ). The table was sorted by this ratio, showing Table 1 that the median ratio was 0.95: on average, the agents have similar efficacy on large and small intestinal tumors. The top and bottom of this ranked table are shown on , displaying agents with a ratio below 0.4 or above 2.5. Several studies with NSAIDs and Table 2 peroxisome proliferator-activated receptor (PPAR) agonists show much weaker protection (or specific promotion) to the colon than to the small intestine. In addition high fiber diets, PEG and can increase specifically the tumor yield in the colon. In Citrobacter rodentium contrast resveratrol, folic acid, uroguanylin, selenium in broccoli, and fructo-oligosaccharides afforded a specific protection to the colon ( ). Table 2 Some discrepancies between the small and large bowel are easy to explain, particularly when the effect is modulated by the gut flora.

For instance, fructo-oligosaccharides are not digested in the small intestine, but fermented by the microflora in the colon, where they yield butyrate, a possible apoptosis inducer. It may explain why fructo-oligosaccharides can decrease the tumor yield in the colon of Min mice, but not in their small intestine ( ). In contrast, the promotion of tumors by is limited to the colon, where the 25

Citrobacter rodentium bacterial density is much higher than in the small intestine ( ). The colorectal position of cancers in people is believed to follow, at least 58 in part, the presence of an abundant colonic microflora. From this point of view, the small intestine of Min mice may not be a proper model of the human colon. Also, the tumor promotion by PPAR gamma agonists is much stronger in the colon than in the small intestine.

This pattern reflects PPAR gamma expression, high in the colon, low in the rest of the gut, in both mice and humans ( ). 83

In contrast, it is surprising that the most potent chemopreventive agents in the Min mouse small intestine, also potent in the colon of AOM-initiated rats, could sometimes increase the tumor yield in the colon or in the ileum of mutated mice. The NSAIDs piroxicam, sulindac, and celecoxib strongly decrease the number of tumors in the small intestine of mutant mice (

), and strongly decrease the Table 1 tumor incidence in AOM-initiated rats ( , , ). In contrast, piroxicam increased the number of tumors in the colon of Min mice in four 84 [START_REF] Rao | Chemoprevention of colon carcinogenesis by sulindac, a nonsteroidal anti-inflammatory agent[END_REF] 86 studies out of six (

). In addition, piroxicam caused a ten-fold dose-dependant increase in tumor multiplicity in the distal intestine of Table 1 / mice ( ). In two studies out of three, the sulindac protection in the colon of Min mice is much weaker than in the small intestine ( Msh2--1

). In three studies out of four, no protection is seen in the colon of mice with or 716 mutations ( ). In addition, Table 2 Msh2 Apc Table 1 sulindac treatment significantly increased colonic tumors in four mutated mouse models: Min, 1638, 1638/ , and Apc Apc Apc Mlh1 Mlh1 mice ( , ). For instance, in / mice, sulindac treatment increased the colon tumor incidence from 20 to 91 ( ). Also, a late 2 3

Mlh1+

- % % 3
treatment with a high dose of celecoxib increased the tumor yield in the colon of Min mice ( ). Thus, very potent chemopreventive 45 NSAIDs could promote tumors in the colon of mutated mice.

Other agents than NSAIDs also yield discrepant results in the colon of rats and mice, for instance DFMO, PEG and inulin. DFMO blocks ornithine decarboxylase, it strongly decreases colon tumor incidence in rats ( ), and suppresses polyps in the small intestine of 87 Min mice, but it increased the number of large polyps in the colon of Min mice ( ). PEG, a mild laxative, is a strikingly potent 38 chemopreventive agent in rats ( , ), but in one study out of two ( , ), it strikingly increased the tumor yield in the colon of Min 88 89 65 66 mice (

). Last, inulin, a natural non digestible oligosaccharide, decreases carcinogenesis in rats, but increases the tumor yield in the Table 1 colons of Min mice ( , ).

22 27

The reason for these puzzling discrepancies is unclear. Some differences seem spurious: due to the very low number of tumors in the colon of Min mice, an increase may be seen when there is no true effect. However, the promotion by sulindac or piroxicam is seen in several independent studies. Indeed, in Min mice, differences in key enzymes make small and large bowel mucosas react differently to COX inhibitors. Phospholipases A-2 (PLA-2) are key enzymes at the start of the arachidonic acid cascade, that lead to the promoting prostaglandin E2. Arachidonic acid is released from phospholipids by either the secretory sPLA-2 or the cytosolic cPLA-2 ( ). cPLA-2 is 90 upregulated in tumors from the colon of humans and rats, and from the mouse small intestine ( , ). This difference is not seen in the 91 92 colon of Min mice, where cPLA-2 mRNA is high in both normal and tumor tissues ( ). Moreover, mice with a mutated gene 93 cPLA-2 have smaller tumors in the small intestine than wild controls, but the effect is not seen in the colon. In contrast, sPLA-2 does not seem essential for carcinogenesis in humans ( , ), in PhIP-initiated rats ( ), or in Min mice. Indeed, C57Bl6/Min mice with a mutated 94 95 96 gene have more tumors than AKR/Min mice with the intact gene ( ). COX-2 converts arachidonic acid to prostaglandin. sPLA-2 sPLA-2 97

It is over-expressed in tumors from the colon of humans and rats, and from the mouse small intestine ( ). In -mutated mice, the 92 Apc knocking out of COX-2 dramatically reduces the number and size of small intestinal polyps ( ). Conversely, COX-2 upregulation is 54 associated with the development of polyps in the small intestine. In contrast, COX-2 protein is not over-expressed in colonic polyps. COX-2 expression is higher in the large than in the small bowel mucosa ( ). Thus, prostaglandin producing enzymes are more expressed, 98 but mice have fewer tumors, in the colon than in the small intestine. This may explain that, in several mice studies, NSAIDs do not prevent, but promote, colon tumors. Curiously, the contrasts observed between tumors and normal mucosa in the colon of humans and rats, are better reproduced in the small than in the large bowel of Min mice.

Polyamines levels are lower in the colon than in the small intestine of Min mice. In spite of a high ornithine decarboxylase expression, a colonic antizyme decreases the polyamine pool ( ). This low level of polyamines in the colon may explain why Min mice have few 38 polyps in the colon. Moreover, DFMO treatment reduces polyamine levels in human colon, but not in the colon of Min mice. This may explain why DFMO does not suppress colonic polyps in Min mice. Again, this would suggest that the colon of rats and the small intestine of Min mice are better models than the Min mouse colon.

Comparison of humans data with animal models data

Finally, we would like to know how the results with the two models compare with those obtained to date in clinical trials directed at preventing the recurrence of colonic polyps. How well do the animal models predict what happens in humans? To answer this question we built a table showing the effect of dietary interventions on tumors in rats and mice, and on the recurrence of colonic polyps in humans (

). The mean effect in rats was extracted from a published data base of positive studies ( ), to which were added null and negative Table 3 15

studies. The mean effect in Min mice was calculated from . is obviously a first approach to such a comparison, since no Table 1 Table 3 account was taken of the dose used, and the data presented are not homogeneous across different models.

, none-the-less, shows that the effect of most of the diets or agents is consistent across the various models though discrepancies Table 3 are seen between the effect of agents in humans and in animals as follows.

-NSAIDs strongly decrease the tumor yield in the colon of AOM-injected rats, and in the small intestine of mutant mice. This is consistent with epidemiological studies suggesting that, taken collectively, NSAIDs might decrease the colorectal cancer incidence by 45% in humans ( , ). It is also consistent with the effect of celecoxib and sulindac which decrease the polyp number in familial 4 116 adenomatous polyposis patients trials. However, as detailed above, several independent studies (but not all) show that some NSAIDs can increase the tumor yield in the colon of mutant mice.

-Wheat bran consistently reduces carcinogenesis in animals, but has apparently no significant effect in humans, a discrepancy for insoluble fibers, already pointed out by Giovannucci (

). A soluble fiber, psyllium, decreases carcinogenesis in rats, but increases the 117 tumor recurrence in human volunteers. However, both results are only supported by a single study each. In addition, other soluble fibers similar to psyllium often show promoting properties in AOM-induced rats, an effect that fits the human trial result.

-Rats and mice fed a high fat diet have usually more tumors than controls fed a low fat diet. In rodents, the relationship between the colon cancer incidence and the intake of fat remains true when controlled for calorie consumption. Fatty diets with high linoleic acid content, and n-6-polyunsaturated fatty acids, seem particularly consistent promoters in rodents (

). In contrast, neither human trials nor 105 observation studies support fat, or linoleic acid, as tumor promoters in humans (

), a discrepancy already pointed out by Giovannucci ( 118 ). 117 -Caloric reduction is a strategy that seems very efficient in animals (

). Overnutrition could be seen as the most potent Table 3 " carcinogen in rodents (

). According to Willett, a positive energy balance (caloric intake versus physical activity) is the most powerful " 119 and consistent dietary influence on carcinogenesis ( ). No published human trial specifically tested the effect of caloric reduction. 120 However, a side effect of interventions with low-fat diet, and with fruits and vegetables, was a modest reduction in caloric intake (106-108

). The lack of reduction in polyp recurrence seen in these trials ( ) suggests the caloric reduction was too small to reduce insuline Table 3 resistance, a supposed link between overnutrition and carcinogenesis ( , ). 117 121 -That fruits and/or vegetables consumption protects against colorectal cancer is a dogma supported by many epidemiological studies (

), an association that may have been overstated ( ). This dogma is challenged by all the experimental studies in rats, mice and 122-123 124 humans ( ). Indeed, a mixture of fruits and vegetables reproducing the people typical intake marginally increased the tumor yield in Table 3 most animal studies ( , ), but large amounts of black raspberries or of orange juice can inhibit carcinogenesis in rats ( ). 71 109-111 125

Conclusions

There is a close agreement between the many results obtained in the colon of AOM-initiated rats and in the small intestine of Min mice ( ). There is a reasonable association between these animal studies and the more limited clinical studies ( ). However, Fig. 1 Table 3 some results obtained in the colon of Min mice are discrepant from those of the Min mouse small intestine and the AOM-rats, which suggests they should be disregarded until they can be explained. Many promising agents strongly and consistently suppress tumor formation or growth in the small intestine of Min mice, or in the colon of AOM-injected rats. Some of them have already been tested in completed clinical trials: selenium, celecoxib, aspirin, sulindac, calcium, wheat bran, low fat diet, fruits and vegetables diet, beta-carotene,

vitamin C and E (

). Others are presently under study in humans: ursodeoxycholate, piroxicam, DFMO, and folic acid. Most Table 3 published trials show no reduction in polyp recurrence, and the significant protection afforded by celecoxib, aspirin or calcium was modest (

). We thus need new agents or strategies to reduce cancer load. Table 3 A conservative approach would be to include an agent or a diet in a human clinical trial only when it shows preventive properties in all available models. We think that this approach might be too conservative, and would have disqualified the testing of celecoxib, piroxicam, sulindac, DFMO, calcium and folic acid in humans, since they have shown promoting properties in some preclinical studies. Our point is that it is appropriate to proceed now with the agents that are particularly potent against carcinogenesis in either rats or mice. The data we have summarized and compared would suggest that these include: PEG, hesperidin, Bowman-Birk protease inhibitor, sphingomyelin, physical exercise, and S-methyl-methane-thiosulfonate (from the AOM-rat model), and EKB-569, ( )-catechin, resveratrol, fish oil, + curcumin, and caffeic acid phenetyl ester (from the Min mouse model). These agents showed no toxicity in rodents, and some of them are already used daily by humans on a large scale (PEG, exercise, catechin, fish oil, curcumin). The safety of others, notably EKB-569, still need to be evaluated ( ). 126

Since human studies are extremely long and costly, they require stringent preliminary studies to evaluate side-effects and optimal dosage. In addition, the long term administration of an agent to many people poses ethical problems, as beta-carotene trials in smokers sadly showed. The use of surrogate endpoint biomarkers in step-wise clinical trials might help to decrease both cost and risk. For instance, a short trial on the suppression of aberrant crypt foci in the colon of volunteers ( ) could be done before a standard trial on adenoma 127 recurrence. This approach would be particularly appropriate for agents like PEG that clear the aberrant crypt foci quickly from the mucosa ( ). This strategy could eventually provide evidence for safe dietary interventions for the prevention of colorectal cancer. 89

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43. Sansom OJ , Stark LA , Dunlop MG , Clarke AR Suppression of intestinal and mammary neoplasia by lifetime administration of aspirin in Apc(min/ ) and Apc(min/ ),

+ + Msh2( / ) mice --
. Cancer Res. 61: 7060-7064 2001;

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Fiber

High fiber rodent chow fiber:fat 18:6 vs 0:20% 60

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+.J

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Figure 1

 1 Figure 1Correlation between the effect of various agents on the number of adenomas in Min mice small intestine and on the incidence of colon tumors

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	26.
	NSAID: nonsteroidal anti-inflammatory drug
	AOM: azoxymethane
	Apc : adenomatous polyposis coli
	PhIP: 2-amino-1-methyl-6-phenylimidazo 4,5-b pyridine [ ]
	PEG: polyethylene glycol
	DFMO: difluoro-methylornithine
	COX: cyclooxygenase
	PPAR: peroxisome proliferator-activated receptor
	PLA-2: phospholipase A-2

-A-

16-2002;

28.

  18: 1863-1865 1997; 30. Oshima M , Oshima H , Tsutsumi M , Nishimura S , Sugimura T , Nagao M , Taketo MM Effects of 2-amino-1-methyl-6-phenylimidazo 4,5b pyridine on intestinal

		[	]
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	+ -	--	. Cancer Res. 60: 3191
	-3199 2000;

+ . Nature Med. 4: 1053-1057 1998; 79.

[START_REF] Corpet | Most effective colon cancer chemopreventive agents in rats: A review of aberrant crypt foci and tumor data, ranked by potency[END_REF]

[START_REF] Corpet | Most effective colon cancer chemopreventive agents in rats: A review of aberrant crypt foci and tumor data, ranked by potency[END_REF]

[START_REF] Corpet | Most effective colon cancer chemopreventive agents in rats: A review of aberrant crypt foci and tumor data, ranked by potency[END_REF]

[START_REF] Corpet | Most effective colon cancer chemopreventive agents in rats: A review of aberrant crypt foci and tumor data, ranked by potency[END_REF]

[START_REF] Corpet | Most effective colon cancer chemopreventive agents in rats: A review of aberrant crypt foci and tumor data, ranked by potency[END_REF]

[START_REF] Corpet | Most effective colon cancer chemopreventive agents in rats: A review of aberrant crypt foci and tumor data, ranked by potency[END_REF]

[START_REF] Corpet | Most effective colon cancer chemopreventive agents in rats: A review of aberrant crypt foci and tumor data, ranked by potency[END_REF]

Author Keywords animal-model ; diet ; chemoprevention ; colon-carcinogenesis ; Min-mice ; chemically-induced 10 15 Table 1 Effect of dietary agents on the tumor number in the small intestine and in the colon of Min mice, and other mutant mice. e Number of adenomas in the small intestine of control mice (some studies report total number of intestinal adenomas), and in their colon. f Number of adenomas in treated mice, reported as percent of number in control mice ( smaller than 100 denotes protection). Boldface: significant effect (either decrease or increase).

Category

%

Table 2

Agents with a very different effect on the tumor number in the small intestine and in the colon of Min mice (ratio below 0.4, or above 2.5, data from

). 3 because it is also a low-vit.D and high-fat diet.

" " + % Apc i Significant effect in F344 rats, but no effect in SD rats (not shown). In volunteers, the interventions were in part, or led to, a dietary fat intake reduction. j The above cited low-fat interventions also led to a reduction of caloric intake, estimated as 18 , 10 and 5 respectively.

-% -% -%