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 (32
). 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.
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 (33
). Haemin strikingly increased the number of azoxymethane-induced aberrant crypts (), and the size of foci, by shifting ACF to MACF (). 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 (23
). 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 (34
). 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 (25
), 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 up-regulation of adhesion molecules, the increase in vascular permeability and the granulocytes infiltration (35
). Thus, the chronic inflammation caused by the continuous haem intake might have promoted the ACF growth (36
). In addition, haemin may have weakened the tumour immunosurveillance. Indeed, haem induces haem-oxygenase-1 (HO-1), and the over-expression of HO-1 results in the inhibition of T-cell and NK-cell mediated lysis (37
). 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) (38
). 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.
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% (40
). 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 oxidation-resistant oleic acid, and phenolic compounds, which scavenge LOO. radicals in the oil itself (41
) and in the faecal matrix (42
). Both antioxidant strategies halved TBARs in faecal water (, panel B). This inhibition of lipid peroxidation in faecal water was associated with inhibition of haemin-induced carcinogenesis (, 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 (25
), but contested by Sesink et al
). It is indeed surprising that both antioxidant strategies increased haem concentration in faeces (), 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 (, panel B). The mechanism by which olive oil and antioxidants suppressed the haemin-induced promotion thus appears complex and warrants further studies.
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 (43
). 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 (30
). Newmark et al
) 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 (45
), 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 (24
). Faecal haem was high in rats given a high-calcium diet (), but it was not soluble in the faecal water. This water thus contained no lipid peroxides and showed no cytolytic activity (). 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.
increased the number of aberrant crypts and of ACF in rats (), an effect already observed by Bruce (46
). 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 (). 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 (), but haem concentration in faecal water was higher in haemin-fed rats (). However, this difference cannot explain the differential effect on MACF: rats in groups HH and TG had similar levels of haem in faecal water () but very different numbers of MACF (). 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 (33
). 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 (23
). 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 (33
). Indeed, dietary haemoglobin is three times more efficient than haemin in providing iron for red blood cell synthesis in rats (47
). 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 (). It also suggests that the MACFs were specifically produced by the haemin-induced cytotoxic factor (23
). 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 (48
). 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 (49
). 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 (24
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.