Content uploaded by Denis Corpet
Author content
All content in this area was uploaded by Denis Corpet
Content may be subject to copyright.
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
1
Calcium Carbonate Suppresses Haem Toxicity Markers without
Calcium Phosphate Side Effect on Colon Carcinogenesis
Ossama Allam, Diane Bahuaud, Sylviane Taché, Nathalie Naud,
Denis E. Corpet and Fabrice H.F. Pierre
Université de Toulouse, UMR1089 Xénobiotiques, INRA, ENVT, F-31076 Toulouse, France
Abstract
Red meat intake is associated with increased risk of colorectal cancer. We have previously
shown that haemin, haemoglobin and red meat promote carcinogen-induced preneoplastic
lesions, aberrant crypt foci, in the colon of rats. We have also shown that dietary calcium
phosphate inhibits haemin-induced promotion, and normalizes faecal lipoperoxides and
cytotoxicity. Unexpectedly, high-calcium phosphate control diet-fed rats had more
preneoplastic lesions in the colon than low-calcium control diet-fed rats. The present study
was designed to find a calcium supplementation with no adverse effect, by testing several
doses and types of calcium salts. One in vitro study and two short-term studies in rats
identified calcium carbonate as the most effective calcium salt to bind haem in vitro and to
decrease faecal biomarkers previously associated with increased carcinogenesis: faecal water
cytotoxicity, thiobarbituric acid reactive substances. A long term carcinogenesis study in
dimethylhydrazine-injected rats demonstrated that a diet containing 100 µmol/g calcium
carbonate did not promote aberrant crypt foci, in contrast with previously tested calcium
phosphate diet. The results suggest that calcium carbonate, and not calcium phosphate, should
be used to reduce haem-associated colorectal cancer risk in meat-eaters. They support the
concept that the nature of the associated anion to a protective metal ion is important for
chemoprevention.
Key words: Red meat, Haem, prevention, calcium, colon cancer, Calcium, Colorectal cancer, Lipid peroxidation
Abbreviations: ACF: aberrant crypt foci, MDF: mucin-depleted foci, TBARS: thiobarbituric acid reactive
substances
Introduction
Red and processed meat consumption increases the risk of colorectal cancer, according
to meta-analyses of epidemiological studies [1,2]. In its 2007 report, the World Cancer
Research Fund panel judges that "the evidence that red meat and processed meat are a cause
of colorectal cancer is convincing" [3]. The panel thus recommends: “Limit intake of red meat
and avoid processed meat”. Several meat components have been speculated to explain that
meat-eaters are at a higher risk for colorectal cancer. Meat cooked at a high temperature
contains mutagenic heterocyclic amines that induce colon, mammary and prostate tumours in
rodents and monkeys [4]. However, heterocyclic amines might not play an important role in
colorectal cancer incidence, since (i) chicken intake is a major contributor of heterocyclic
amines intake, but it is not associated with the risk [5], and (ii) doses of heterocyclic amines
that induce cancer in animals are 1000 to 100,000 times higher than doses ingested by humans
[6]. Red meat enhances the formation of putative carcinogenic N-nitroso compounds in
human and rodent faeces [7-9]. N-nitroso compounds level is associated with the promotion
of colon carcinogenesis by cured meat [10]. But N-nitroso compounds brought into rat faeces
by a bacon-based diet do not initiate or promote preneoplastic lesions in rat colon [11]. Red
meat also contains haem, the iron-bearing prosthetic group of myoglobin. Intake of haem iron
is associated with an increased risk of colon cancer among women of the Iowa Women's
Health Study [12]. Intake of black pudding, a blood sausage over-loaded with high haem iron,
hal-00553174, version 1 - 6 Jan 2011
Author manuscript, published in "Br J Nutr (2010) 1-8"
DOI : 10.1017/S0007114510003624
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
2
is associated with increased risk of colorectal cancer among women of the Swedish
Mammography Cohort [13].
Experimental animal studies support the hypothesis that haem iron promotes colorectal
carcinogenesis [14]. We have shown that dietary haem, in the form of haemin (haem
stabilized by a chlorine atom, also called Ferriprotoporphyrin IX chloride), haemoglobin or
red meat, promotes putative precancerous lesions, aberrant crypt foci (ACF) and mucin-
depleted foci (MDF), in the colon of rats given a low-calcium diet [15-17]. This haem-
induced promotion is associated with increased lipoperoxidation and cytotoxicity of faecal
water, and linked to urinary 1,4-dihydroxynonane mercapturic acid (DHN-MA) excretion, a
fat peroxidation biomarker [18]. The addition of calcium phosphate to the diet inhibits
haemin-induced lipoperoxidation, cytotoxicity and promotion of carcinogenesis in rats [17],
and we can explain the lack of effect of meat diets on colon cancer carcinogenesis in previous
study [19] by a high concentration of calcium in the diet. The addition of calcium phosphate
to beef meat diet fully prevents red meat promotion of colon carcinogenesis, but,
unexpectedly, the rats given the no-meat high-calcium phosphate high fat diet had more ACF
and more MDF than the rats given the no-meat low-calcium phosphate diet [16]. In the
absence of meat, dietary calcium phosphate appears to promote carcinogenesis in this rodent
model, which precludes its use in human volunteers.
The present study was designed to determine a calcium supplementation with no
adverse effect by testing different types of calcium salts, and finding the minimum effective
calcium dose.
Materials and Methods
Animals and Diets
Eighty five Fischer 344 female rats were purchased at four weeks of age from Iffa Credo
(St.Germain l‟Arbresle, France). Animal care was in accordance with the guidelines of the
European Council on animals used in experimental studies. The animal colony and staff got
official agreement #31-121 for animal studies by French government.
Short-term studies. Two short-term studies were designed to find the most effective
calcium salt and the minimum effective calcium dose to suppress early biomarkers of haem
toxicity. Rats were housed individually in metabolic cages. The room was kept at a
temperature of 22°C on a 12-h light-dark cycle. After 5 days of acclimatization to the AIN76
powdered diet, rats were given the experimental diets. Body weights and food intake were
monitored at the beginning, and at the end of each study. In the first study, forty rats were
randomly allocated to 8 dietary groups (5 rats per group) for one week. Faeces were collected
at day 7 and were frozen at -20°C. Animals were killed 7 d after the start of the experimental
diets. In the second study, twenty five rats were randomly allocated to 5 dietary groups (5
rats per group) for two weeks. Faeces were collected at day 14 and were frozen at -20°C.
Animals were killed 14 d after the start of the experimental diets.
Long-term study: A three-month study was designed to test the hypothesis that the
minimum effective dose of the most effective calcium salt, selected from short-term studies
results, does not promote carcinogenesis in rats. Twenty rats were housed by pairs in stainless
steel wire bottomed cages. The room was kept at a temperature of 22°C on a 12-h light-dark
cycle. Rats were allowed 7 d of acclimatization to the room and to the control diet, before
being injected i.p. with the carcinogen 1,2 dimethylhydrazine (Sigma chemical, St.Quentin,
France; 190 mg/kg body wt) in NaCl (9 g/L). In most published studies, several carcinogen
injections are given to rats. We reasoned here that the first shot initiates carcinogenesis, but
that the following shots would promote carcinogenesis, blurring diet-induced promotion. We
thus chose a single-shot protocol, following Glauert [20]. Seven days later, rats were
hal-00553174, version 1 - 6 Jan 2011
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
3
randomly allocated to two groups of ten, and allowed free access to calcium carbonate-based
diets for 94 d. We chose to initiate all rats with the carcinogen, since the study was designed
to show dietary promotion of carcinogenesis.
Diets: Experimental diets shown in Table 1 were based on a modified standard AIN-
76 diet [21], prepared and formulated in a powdered form by UPAE (INRA, Jouy-en-Josas,
France). For the first short-term study, high-beef meat diets were formulated to contain 60%
meat (w/w, freeze-dried, 0.6 mmol haem /g of meat), and dibasic calcium phosphate was
included in all diets at a low concentration of 20 µmol/g. To determine, the minimum
effective dose of calcium phosphate, the effect of seven concentrations of calcium was tested
in seven groups of rats given this low-calcium beef meat diet supplemented with calcium
phosphate (doses range: 33 to 250 µmol/g, diet names: Phos33 … Phos250). To compare the
efficacy of calcium phosphate with other salts, the effect of two calcium salts was tested in
two groups of rats given calcium carbonate (Carb250) or calcium gluconate (Gluc250) at the
highest tested dose (250 µmol/g). For the second short-term study, the effect of calcium
carbonate was tested in five groups of rats given a haemoglobin diet, supplying 0.6 mmol/g
haem to mimic the 60% beef meat diet, supplemented with calcium carbonate (doses range:
33 to 155 µmol/g, diet names: Carb33 … Carb155). For the long-term carcinogenesis study,
two experimental diets were formulated to contain two levels of calcium carbonate (33 and
100 µmol/g, diet names: Control Diet and Carb100) added into the control low calcium
phosphate diet.
Preparation of Faecal Water
Faecal pellets were collected for 24 h under each cage (one rat/cage, short-term studies ; two
rats/cage, long-term study), thus leading to five samples per diet. Freeze-dried faeces were
used to calculate dry faecal mass and to prepare faecal water by adding 1 ml of sterilized
water to 0.3 g of dry faeces. Samples were then incubated at 37˚C for one hour, stirring
thoroughly every 20 min, followed by centrifugation at 20 000 g for 10 min. The aqueous
phase was re-centrifuged at the same speed and duration and the subsequent supernatant
(faecal water) collected and stored at –20˚C until use.
TBARS and Haem Assay
Thiobarbituric acid reactive substances (TBARS) were measured in faecal water according to
Ohkawa et al. [22], exactly as previously described, and results are given as malondialdehyde
(MDA) equivalent. [15]. Haem contents of freeze-dried faeces and of faecal water were
measured by fluorescence according to Van den Berg et al. [23] and Sesink et al. [14],
respectively, as already described [15]. Both TBARS and heme in faeces were measured on
faecal samples from both short-term studies.
In vitro experiment
Ability of five different calcium salts (1- calcium phosphate, 2-calcium chloride, 3-calcium
gluconate, 4-calcium carbonate, 5-calcium lactate) to bind and to precipitate haem from
bovine haemoglobin solution was studied in vitro. Haemoglobin (Sigma chemical, St.Quentin,
France) 0.36 mmol was acidified at pH 1.4 with HCl 2 mol and then heated one hour at 105°C
to dissociate haem from haemoglobin. This solution was centrifuged at 20 000 g for 10 min.
Supernatant with free haem was collected.
Six concentrations of each of five calcium salts were prepared (25, 50, 75, 100, 125, 150
mmol). According to Sesink [24], one ml haemoglobin added to one ml calcium salt was
incubated for 90 min at 37.5°C in the dark and mixed every 10 min. The mixture was
centrifuged for 2 min at 10 000 g. Supernatant (100 µl) was assayed for remaining haem after
hal-00553174, version 1 - 6 Jan 2011
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
4
20-folds dilution in distilled water, by absorbance measure at 400 nm with a Lambda 2
spectrophotometer (PYE Unicam-PU8600 UV/VIS-spectrophotometer Philips). Different
haemoglobin concentrations were prepared as standard solutions: 1/1 (100%), 1/5, 1/10, 1/20,
and 1/50 in water. The assay was repeated three times and mean values recorded.
ACF Assay
Rats were killed by CO2 asphyxiation in a random order at day 94 of the long-term study.
Colons were excised from rats immediately post mortem, flushed with cold Krebs solution
(Sigma chemical, St.Quentin, France), opened longitudinally and fixed flat between two
sheets of filter paper in 10% formalin (Sigma chemical, St.Quentin, France), marked with a
two-digit blinding code. Colons picked up in random order were stained for 6 min in a 0.05%
filtered solution of methylene blue [25]. Numbers of ACF per colon, and number of crypts in
each ACF, were counted under light microscope at x40 magnification in duplicate by two
readers, blinded for the origin of the colon.
Statistical Analysis
Results were analyzed using Systat 10 software for Windows, and reported as mean ± SD.
Long-term study data were analyzed by Student‟s t test, except ACF data that were analyzed
by two-way analysis of variance (ANOVA: dietary group and reader). Short-term study data
were considered firstly using one-way ANOVA. If a significant difference was found between
groups (P<0.05) then pairwise comparisons were made with fisher LSD test multiple
comparison test.
The meta-analysis of the relation between calcium carbonate and colon cancer in carcinogen-
injected rats was done as a sub-analyses of a study that we published in 2005 [26]. From this
precedent meta-analysis, we selected studies where calcium carbonate had been fed to
experimental groups of rats. The studies were homogeneous (Q Cochran P>0.05), we thus
used the „„Fixed Effects‟‟ model, using EasyMA DOS software (2001 version) to compute
data. Results
Weight and food intake during in vivo studies
Final body weight of rats was 107 2 g after 7 d on experimental diets in the first short-term
study, without significant differences between groups. Food intake was not significantly
different (data not shown), but Gluc250-fed rats tended to eat less than the other groups
(Gluc250: 7.2 2.0 g/d, all other groups: 9.1 1.0 g/d, P=0.09). No difference between
groups body weight and diet intake was observed in the second short-term study or in the
long-term study. Mean body weights were respectively 166 2 g after 14 d, and 201 8 g
after 94 d on the experimental diets.
First short-term study: TBARS of Faecal Water
Haem can induce the formation of peroxyl radicals in fats, which may be cytotoxic and cleave
DNA in vivo. This might explain the promoting effect of haem on colon carcinogenesis. Lipid
peroxidation was thus measured in faecal water by TBARS assay. Calcium can prevent haem-
induced promotion of colon carcinogenesis by precipitating haem in the colonic content, thus
haem concentration in faecal water was measured.
Haem concentration in faecal water from beef meat fed rats was significantly reduced
in rats given 150 and 250 µmol/g calcium phosphate (P<0.01; Figure 1.A), but not in rats
given lower levels of calcium phosphate. Dietary calcium carbonate and gluconate 250µmol/g
produced the same effect on haem than calcium phosphate (Figure 1.A). Surprisingly, haem-
induced lipid peroxidation measured by TBARS level was not significantly reduced by the
hal-00553174, version 1 - 6 Jan 2011
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
5
calcium phosphate diets, but only the high-calcium carbonate diet decrease TBARS level in
faecal water (P<0.01; Figure 1.B).
In vitro study
Any tested concentration of calcium carbonate higher than 25 mMol was much more effective
to precipitate haem than any other tested calcium salt. Proportion of trapped haem was more
than three times higher with calcium carbonate than with calcium phosphate (100 mMol)
(P<0.001; Figure 2).
Second short-term study: TBARS of Faecal Water
Beef meat was replaced by haemoglobin in the second study diets, since we have
demonstrated previously that they produce similar effects on colon carcinogenesis and on
faecal water peroxidation [15]. The high-calcium phosphate diet (250µmol/g) did not prevent
haem toxicity in the 7-d study, in contrast with our previous 50-d study [17]. We suggest this
duration was too short to reduce TBARs level, thus we chose the second short-term study to
last for 14-d. As calcium carbonate was the only effective salt against haem-induced lipid
peroxidation in vivo and the only effective salt to precipitate haem in vitro, this salt was used
to determine the smallest effective calcium dose.
Haem concentration was lower in faecal water from rats given a diet containing at
least 80 µmol/g of calcium carbonate (P<0.001; Figure 3.A). Accordingly, diets containing at
least 80 µmol/g of calcium carbonate reduced haem-induced lipid peroxidation of faecal water
(P<0.001; Figure 3.B).
ACF Assay
We chose to test if a diet containing 100 µmol/g calcium carbonate would promote colon
carcinogenesis, because the second short-term study showed that a 80 µmol/g calcium
carbonate diet reduces faecal TBARS concentration, and that 110 µmol/g seemed twice more
potent than 80 µmol/g to bind faecal haem. In contrast with a calcium phosphate diet [16], the
consumption of a diet containing 100 µmol/g calcium carbonate for 94 days did not increase
significantly the number of ACF per colon, 101 days after a dimethylhydrazine injection
(Figure 4).
Discussion
These data confirm that dietary calcium reduces faecal water lipoperoxides level, a
biomarker linked with haem-induced carcinogenesis promotion. They also establish that the
type of calcium salt is paramount in this protection, and that calcium carbonate is effective
without the promoting side effect of calcium phosphate.
Red meat promotes ACF and MDF in the colon of carcinogen-induced rats, and
promotion by meat is associated with faecal water haem, cytotoxicity and TBARS [15,16].
The mechanism of promotion by haem is not known, and may be linked to lipid peroxidation.
We have explored the effect of faecal water from beef-fed rats on normal (Apc +/+) and
premalignant colonic cells (Apc Min/+). Results show that Apc mutated cells survive haem-
induced faecal lipoperoxides, notably 4-hydroxynonenal (HNE) that is toxic to normal cells.
Selection of mutated cells by cytotoxic lipoperoxides may thus explain haem-promotion of
colon carcinogenesis [27]. Calcium added on top of beef-meat or haemin diets normalizes the
number of ACF and MDF at the same low level as in control group [17]. Calcium phosphate
precipitates haem in the gut, and little haem remains available to induce lipid peroxidation
[17,24]. The trapping of haem by calcium thus abolishes carcinogenesis promotion. In this
study, we established that different calcium salts are able to precipitate haem in vitro and in
hal-00553174, version 1 - 6 Jan 2011
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
6
faecal water of haem-fed rats and by consequence are able to reduce faecal water peroxidation
(Figures 1 and 2). We observed that only high concentrations of calcium phosphate were able
to precipitate haem (Figure 1A. Furthermore the ability of the three tested salts (phosphate,
carbonate and gluconate) to trap haem was very similar in high-calcium diets (Figure 1A).
However, only calcium carbonate decreased significantly the level of lipid peroxidation in
faecal water (Figure 1B). So, to screen more precisely the efficiency of different calcium salts,
we measured the in vitro trapping of haem: Only calcium carbonate precipitated haem
significantly. In conclusion, this study shows for the first time a difference of efficiency
between calcium salts in vitro (figure 2) and in vivo, in the trapping of haem and the
limitation of peroxidation (figure 1B). We have no explanation for the relatively high TBARs
level in controls and in rats given high doses of calcium phosphate or gluconate: we speculate
the beef meat in first study may have contained already oxidized fat or oxidizing agents
different from haem iron (Figure 1B).
Our previous study showed that rats eating a high-calcium control diet had more ACF
and more MDF than rats eating a low-calcium control diet [16]. This promotion was observed
with calcium phosphate in a high-fat diet context, but was not seen in a low-fat diet context
without a clear explanation for the high-fat/low-fat discrepancy [17]. Bull et al. also showed
that the promotion of tumours by calcium phosphate was more important in 30%-fat diet-fed
rats than in 3%-fat diet-fed rats [28]. Phosphate, not calcium, might be the promoting nutrient,
an issue already discussed by Bruce [29]. Since our in vitro study indicated that calcium
carbonate was more effective than calcium phosphate to trap haem (Figure 2) and since the
first in vivo study indicated that a high concentration of calcium carbonate was able to
precipitate haem and to limit peroxidation in vivo, we determined the lower effective dose of
calcium carbonate in a second short-term study (Figure 3). We showed that 80 µmol of
calcium carbonate /g of diet is enough to trap haem and to reduce peroxidation. The first aim
of this study was to identify the lower dose and type of calcium sufficient to limit the effect of
haem in the colon without side effects. We thus tested the effect of an addition of 100 µmol of
calcium carbonate /g of diet on the promotion of colon carcinogenesis at the level of ACF
(Figure 4). In contrast with the promoting effect of calcium phosphate at 250 µmol/g [16], we
did not observe a promoting effect of calcium carbonate on ACF. This study shows for the
first time that a calcium salt can inhibit the toxic effect of haem in the colon without side
effect on colon carcinogenesis promotion.
To confirm that calcium carbonate does not promote carcinogenesis in rats, we made a
meta-analysis of published data on calcium carbonate and colon carcinogenesis (Figure 5).
Our previous meta-analysis study shows that calcium (all salts) modestly decreases tumour
incidence in rats and that some calcium salts are more protective than others [26]: calcium
lactate, but not calcium phosphate, reduces chemically-induced carcinogenesis. Figure 5
shows data from a meta-analysis of 11 studies of high calcium carbonate diets on colon
carcinogenesis in chemically-initiated rats; the combined relative risk from all of these studies
was 0.983 (95% confidence interval: 0.902; 1.071, p = 0.69), compared with low calcium
controls. This meta-analysis confirms the lack of promoting side-effect by calcium carbonate
on colon carcinogenesis. These results on rodents are coherent with two randomized, double-
blinded, placebo-controlled trials. Calcium carbonate supplementation was associated with a
significant - though moderate - reduction in the risk of recurrent colorectal adenomas in the
Calcium Polyp Prevention Study [30]. In contrast, daily supplementation of calcium
carbonate (with vitamin D) for seven years to 36,282 postmenopausal women from 40
Women's Health Initiative centres had no effect on the incidence of colorectal cancer among
postmenopausal women [31]. Altogether, these results suggest that calcium carbonate
supplementation to neutralize haem does not bear any risk. However, chelation of dietary
hal-00553174, version 1 - 6 Jan 2011
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
7
heme iron bears the theoretical risk of iron deficiency leading to anaemia: recommendations
should take this in account.
The study also supports the concept that nutrients like calcium may inhibit the toxicity
associated with the excess of another nutrient like haem. This concept differs from the
classical chemoprevention concept, since calcium carbonate does not reduce carcinogenesis in
rats that are not given dietary haem. This specific calcium preventive effect, by interaction,
should be looked for in a cohort study by crossing haem and calcium intake with adenoma or
cancer risk.
Acknowledgments: This study was supported in part by the INRA, the DGER and the French region Midi-
Pyrénées. We thank Xavier Blanc (UPAE) for the preparation of experimental diets, Raymond Gazel and
Florence Blas Y Estrada for the care of the animals.
References
1. Larsson, S.C. and Wolk, A. (2006) Meat consumption and risk of colorectal cancer: a meta-analysis of
prospective studies. Int J Cancer, 119, 2657-2664.
2. Norat, T., Lukanova, A., Ferrari, P. and Riboli, E. (2002) Meat consumption and colorectal cancer risk:
dose-response meta-analysis of epidemiological studies. International Journal of Cancer, 98, 241-256.
3. WCRF, W.C.R.F. (2007) Food, nutrition, physical activity, and the prevention of cancer: a global
perspective. WCRF and American Institute for Cancer Research, Washington DC, 1-537.
4. Sugimura, T., Wakabayashi, K., Nakagama, H. and Nagao, M. (2004) Heterocyclic amines:
Mutagens/carcinogens produced during cooking of meat and fish. Cancer Sci, 95, 290-9.
5. Sinha, R., Rothman, N., Brown, E.D., Mark, S.D., Hoover, R.N., Caporaso, N.E., Levander, O.A.,
Knize, M.G., Lang, N.P. and Kadlubar, F.F. (1994) Pan-fried meat containing high levels of
heterocyclic aromatic amines but low levels of polycyclic aromatic hydrocarbons induces cytochrome
p4501a2 activity in humans. Cancer Research, 54, 6154-6159.
6. Stavric, B. (1994) Biological significance of trace levels of mutagenic heterocyclic aromatic amines in
human diet: a critical review. Food and Chemical Toxicology, 32, 977-994.
7. Bingham, S.A., Pignatelli, B., Pollock, J.R.A., Ellul, A., Malaveille, C., et al. (1996) Does increased
endogenous formation of N-nitroso compounds in the human colon explain the association between red
meat and colon cancer? Carcinogenesis, 17, 515-523.
8. Cross, A.J., Pollock, J.R.A. et al. (2003) Haem, not protein or inorganic iron, is responsible for
endogenous intestinal n-nitrosation arising from red meat. Cancer Research, 63, 2358-2360.
9. Lunn, J.C., Kuhnle, G., Mai, V., Frankenfeld, C., Shuker, D.E., Glen, R.C., Goodman, J.M., Pollock,
J.R. and Bingham, S.A. (2007) The effect of haem in red and processed meat on the endogenous
formation of N-nitroso compounds in the upper gastrointestinal tract. Carcinogenesis, 28, 685-690.
10. Santarelli, R.L., Vendeuvre, J.-L., Naud, N., Taché, S., Guéraud, F., Viau, M., Genot, C., Corpet, D.E.
and Pierre, F.H.F. (2009) Meat processing and colon carcinogenesis: Cooked, nitrite-treated and
oxidized high-heme cured meat promotes mucin depleted foci in rats. Cancer Prevention research, 3,
852-64.
11. Parnaud, G., Pignatelli, B., Peiffer, G., Tache, S. and Corpet, D.E. (2000) Endogenous N-nitroso
compounds, and their precursors, present in bacon, do not initiate or promote aberrant crypt foci in the
colon of rats. Nutrition and Cancer, 38, 74-80.
12. Lee, D.H., Anderson, K.E., Harnack, L.J., Folsom, A.R. and Jacobs, D.R., Jr. (2004) Heme iron, zinc,
alcohol consumption, and colon cancer: Iowa Women's Health Study. J Natl Cancer Inst, 96, 403-7.
13. Larsson, S.C., Rafter, J., Holmberg, L., Bergkvist, L. and Wolk, A. (2005) Red meat consumption and
risk of cancers of the proximal colon, distal colon and rectum: the Swedish Mammography Cohort. Int J
Cancer, 113, 829-34.
14. Sesink, A.L.A., Termont, D.S.M.L., Kleibeuker, J.H. and Vandermeer, R. (1999) Red meat and colon
cancer: the cytotoxic and hyperproliferative effects of dietary heme. Cancer Research, 59, 5704-5709.
15. Pierre, F., Freeman, A., Tache, S., Van der Meer, R. and Corpet, D.E. (2004) Beef meat and blood
sausage promote the formation of azoxymethane-induced mucin-depleted foci and aberrant crypt foci in
rat colons. Journal of Nutrition, 134, 2711-2716.
16. Pierre, F., Santarelli, R., Tache, S., Gueraud, F. and Corpet, D.E. (2008) Beef meat promotion of
dimethylhydrazine-induced colorectal carcinogenesis biomarkers is suppressed by dietary calcium. Br J
Nutr, 99, 1000-6.
hal-00553174, version 1 - 6 Jan 2011
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
8
17. Pierre, F., Tache, S., Petit, C.R., Van der Meer, R. and Corpet, D.E. (2003) Meat and cancer:
haemoglobin and haemin in a low-calcium diet promote colorectal carcinogenesis at the aberrant crypt
stage in rats. Carcinogenesis, 24, 1683-90.
18. Pierre, F., Peiro, G., Tache, S., Cross, A.J., Bingham, S.A., Gasc, N., Gottardi, G., Corpet, D.E. and
Gueraud, F. (2006) New marker of colon cancer risk associated with heme intake: 1,4-dihydroxynonane
mercapturic Acid. Cancer Epidemiol Biomarkers Prev, 15, 2274-9.
19. Parnaud, G., Peiffer, G., Tache, S. and Corpet, D.E. (1998) Effect of meat (beef, chicken, and bacon) on
rat colon carcinogenesis. Nutrition and Cancer, 32, 165-173.
20. Karkare, M.R., Clark, T.D. and Glauert, H.P. (1991) Effect of Dietary Calcium on Colon
Carcinogenesis Induced by a Single Injection of 1,2-Dimethylhydrazine in Rats. Journal of Nutrition,
121, 568-577.
21. American Institute of Nutrition (1977) Report of the American Institute of Nutrition Ad Hoc Committee
on standards for nutritional studies. J Nutr, 107, 1340-1348.
22. Ohkawa, H., Ohishi, N. and Yagi, K. (1979) Assay for lipid peroxides in animal tissues by
thiobarbituric acid reaction., Analytical Biochemistry, vol. 95, pp. 351-358.
23. Van den Berg, J.W., Koole-Lesuis, R., Edixhoven-Bosdijk, A. and Brouwers, N. (1988) Automating the
quantification of heme in feces. Clinical Chemistry, vol. 34, pp. 2125-2126.
24. Sesink, A.L.A., Termont, D.S.M.L., Kleibeuker, J.H. and VanDerMeer, R. (2001) Red meat and colon
cancer: dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by
calcium. Carcinogenesis, 22, 1653-1659.
25. Bird, R.P. (1987) Observation and quantification of aberrant crypts in murine colon treated with a colon
carcinogen: preliminary findings. Cancer Lett, 37, 147-151.
26. Corpet, D.E. and Pierre, F. (2005) How good are rodent models of carcinogenesis in predicting efficacy
in humans? A systematic review and meta-analysis of colon chemoprevention in rats, mice and men.
Eur J Cancer, 41, 1911-22.
27. Pierre, F., Tache, S., Gueraud, F., Rerole, A.L., Jourdan, M.L. and Petit, C. (2007) Apc mutation
induces resistance of colonic cells to lipoperoxide-triggered apoptosis induced by faecal water from
haem-fed rats. Carcinogenesis, 28, 321-7.
28. Bull, A., Bird, R.P., Bruce, W.R., Nigro, N. and Medline, A. (1987) Effect of calcium on azoxymethane
induced intestinal tumors in rats. Gastroenterology, 92, #1332.
29. Bruce, W.R., Giacca, A. and Medline, A. (2001) Possible mechanisms relating diet to colorectal cancer
risk. Europ. Conf. Nutr. Cancer. IARC, June 21-24, 2001, Lyon France, 1-7.
30. Baron, J.A., Beach, M., Mandel, J.S., Vanstolk, R.U., Haile, R.W., et al. (1999) Calcium supplements
for the prevention of colorectal adenomas. New England Journal of Medicine, 340, 101-107.
31. Wactawski-Wende, J., Kotchen, J.M., Anderson, G.L., Assaf, et al. (2006) Calcium plus vitamin D
supplementation and the risk of colorectal cancer. N Engl J Med, 354, 684-96.
32. Nelson R.L., Tanure J.C., Andrianopoulos G. (1987) The effect of dietary milk and calcium on
experimental colorectal carcinogenesis. Dis Colon Rect, 30, 947–949.
33. Behling A.R., Kaup S.M., Choquette L.L., et al. (1990) Lipid absorption and intestinal tumour
incidence in rats fed on varying levels of calcium and butterfat. Br J Nutr, 64, 505–513.
34. Pence BC, Dunn DM, Zhao C, et al. (1995) Chemopreventive effects of calcium but not aspirin
supplementation in cholic acid-promoted colon carcinogenesis: correlation with intermediate endpoints.
Carcinogenesis, 16, 757–765.
35. Pence BC, Dunn DM, Zhao C, et al. (1996) Protective effects of calcium from non fat dried milk against
colon carcinogenesis in rats. Nutr Cancer, 25, 35–45.
36. Belbraouet S, et al. (1996) Dietary calcium salts as protective agents and laminin P1 as a biochemical
marker in chemically induced colon carcinogenesis in rats. Cancer Detect Prev, 20, 294–299.
37. Adell-Carceller R, Segarra-Soria M, et al. (1997) Inhibitory effect of calcium on carcinogenesis at the
site of colonic anastomosis: an experimental study. Dis Colon Rect, 40, 1376–1381.
38. Quilliot D, Belbraouet S, Pelletier X, et al. (1999) Influence of a high-calcium carbonate diet on the
incidence of experimental colon cancer in rats. Nutr Cancer, 34, 213–219.
39. Molck AM, Poulsen M, Meyer O. (2002) The combination of 1alpha,25(OH2)-vitamin D3, calcium and
acetylsalicylic acid affects azoxymethane-induced aberrant crypt foci and colorectal tumours in rats.
Cancer Lett, 186, 19–28.
hal-00553174, version 1 - 6 Jan 2011
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
9
Table 1: Formulation of diets (g/100g)c
First short-term study: Beef meat-based diets
Second short-term study: Casein-based diets
Long-term study
Phos20
Phos33
Phos55
Phos90
Phos150
Phos250
Carb250
Gluc250
Carb33
Carb55
Carb80
Carb110
Carb 155
Control
Diet
(Carb33)
Carb100
Beef
60.0
60.0
60.0
60.0
60.0
60.0
60.0
60.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Haemoglobin
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.63
0.63
0.63
0.63
0.63
0.0
0.0
Safflower Oil
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
Lard
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
Casein a
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
59.0
59.0
59.0
59.0
59.0
59.0
59.0
Corn Starch
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
Sucrose
12.8
12.6
12.3
11.9
11.1
9.7
10.6
2.5
16.0
15.8
15.5
15.2
14.8
16
15
Cellulose
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5
5
Methionine
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
Mineral mix b
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.5
Vitamin mix b
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Choline bitartrate
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
Ca. phosphate
0.2
0.4
0.7
1.1
1.9
3.3
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
Ca. carbonate
0.0
0.0
0.0
0.0
0.0
0.0
2.5
0.0
0.3
0.5
0.8
1.1
1.5
0.3
1.0
Ca. gluconate
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10.6
0.0
0.0
0.0
0.0
0.0
0.0
0.0
a. Low-calcium casein
b. AIN76 mix, but 500 g/kg of dibasic calcium phosphate replaced by sucrose in mineral mix
c. Phos20 to Phos250 are low-calcium beef diet supplemented with calcium phosphate from 20 µmol/g to 250 µmol/g. Carb250 and Gluc 250 are low-calcium beef diet
supplemented respectively with calcium carbonate and calcium gluconate at 250 µmol/g. Carb33 to Carb155 are low-calcium haemoglobin diet supplemented with calcium
carbonate from 33 µmol/g to 155 µmol/g. Control diet and Carb100 low-calcium haemoglobin diet supplemented with calcium phosphate from 33 µmol/g to 100 µmo
hal-00553174, version 1 - 6 Jan 2011
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
10
Figure 1: Effect of diets on haem and TBARS in faecal water after the first short term study
(A) Haem in faecal water. (B) TBARS (MDA equivalents) in faecal waters as marker for lipid
luminal peroxidation. * Significantly different from Phos20 (P<0.01, by fisher LSD test).
Data are means, bars are standard deviations (n = 5 in each group).
0
50
100
150
200
250
Phos20 Phos33 Phos55 Phos90 Phos150 Phos250 Carb250 Gluc250
Diets
Haem (µmol/L)
0
20
40
60
80
100
120
140
160
Phos20 Phos33 Phos55 Phos90 Phos150 Phos250 Carb250 Gluc250
Diets
MDA Equivalents (µM)
Note: Phos20 to Phos250 are low-calcium beef diet supplemented with calcium phosphate from 20 µmol/g to
250 µmol/g. Carb250 and Gluc 250 are low-calcium beef diet supplemented respectively with calcium carbonate
and calcium gluconate at 250 µmol/g.
*
*
*
*
*
A
B
hal-00553174, version 1 - 6 Jan 2011
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
11
Figure 2: Effect of calcium salts on haem solubility in vitro. Haem (0.36 mM) was incubated
with six different concentrations of calcium salts (150, 125, 100, 75, 50, 25 mMol). Data are
means, bars are standard deviations (n = 3). * Significantly different from other salts.
0
10
20
30
40
50
60
70
80
90
025 50 75 100 125 150 175
Concentration of calcium (mmol/l)
% of heam trapped
Ca Carbonate
Ca Phosphat
Ca Chloride
Ca Gluconat
Ca lactat
*
*
*
*
*
hal-00553174, version 1 - 6 Jan 2011
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
12
Figure 3: Effect of calcium carbonate-based diets (30 to 155 µmol/g) on faecal water values
after the second short term study A. Haem concentration in faecal water (µmol/L) B. TBARS
concentration in faecal water, expressed as µmol/L MDA equivalents. * Significantly
different from Carb30 by fisher LSD test (P< 0.05). Data are mean, bars are standard
deviation (n=5 in each group).
0
10
20
30
40
50
60
70
Carb33 Carb55 Carb80 Carb110 Carb155
Diets
Haem (µmol/l)
0
10
20
30
40
50
60
70
Carb33 Carb55 Carb80 Carb110 Carb155
Diets
MDA Equivalents (µM)
Note: Carb33 to Carb155 are low-calcium haemoglobin diet supplemented with calcium carbonate from 33
µmol/g to 155 µmol/g.
*
*
*
*
*
*
A
B
hal-00553174, version 1 - 6 Jan 2011
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
13
Figure 4: Effect of calcium salts in diet on putative precancerous lesions (ACF) per rat colon
101 d after the injection of dimethylhydrazine. Hatched bars are results of this study with 100
µmol/g of calcium carbonate. Solid bars are previous results with 250 µmol/g of calcium
phosphate (Pierre et al 2008 [16]). Results are expressed after normalization of the Control
Diet groups to 100. * Significantly different control group (CD).
0
20
40
60
80
100
120
140
160
180
Control Diet Carb 100 Control Diet CaPhos250
Diets
ACF / colon
Note: The Control Diet was a low-calcium haemoglobin diet supplemented with calcium carbonate at 33 µmol/g.
Carb100 and CaPhos250 are low-calcium haemoglobin diet supplemented respectively with calcium carbonate
(100µmol/g) and calcium gluconate ( 230 µmol/g).
*
hal-00553174, version 1 - 6 Jan 2011
British Journal of Nutrition, 2010, Allam et al., Calcium carbonate blocks haem without side effect. Authors’ Version
14
Figure 5: Meta-analysis of the relation between calcium carbonate and colon cancer in
carcinogen-injected rats. The common relative risk with 95% confidence was calculated from
11 studies.*
* Solid black circles correspond to the study specific relative risk. The open white circle represents the common
relative risk. Horizontal lines are 95% confidence intervals. References are indicated between brackets
[32]
[33]
[33]
[34]
[34]
[35]
[35]
[36]
[37]
[38]
[39]
hal-00553174, version 1 - 6 Jan 2011
