The role of Calcium, Chlorophyll and other compounds in the prevention of "Colorectal Cancer Risk" associated with red meat consumption
Colon cancer is one of the leading causes of cancer death in Western societies. For the USA in 2003 nearly 110.000 new cases and almost 48.000 deaths were estimated. Incidence rates of colon cancer vary 20-fold between high and low risk countries. Migrant and other epidemiological studies indicate that this variation is due to environmental factors, with diet as a major determinant.
The high intake of red meat is associated with increased colon cancer risk of about 30% for a consumption of 100 g/die. This may be due mainly to the high haem content of red meat that causes the increased endogenous production of nitric oxides compounds (NOCs), that are alkylating agents that can react with DNA. Moreover the catalysis of heme iron is a leading cause of fat peroxidation, forming malondialdehyde (MDA) and 4-hydroxy-nonenal (4-HNE), that cause respectively the formation of DNA adduct (MDA) and cell-death. Cell death causes an increased intestinal mucosa cells proliferation that, combined with the genotoxic and mutagens effect of MDA, promote the cancer progression. In vivo experiments on mouse confirmed that the cured-meat (beacon, hot dog..) cause a 15-20 fold increase in content of nitroso-compounds in faeces of rodents, while a diet high in fresh read meat causes a 3 fold increase of nitroso compound.
The role of calcium
In 2001 an article named Red meat and colorectal cancer: dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by calcium. (Sesink et al) describes how calcium could be a preventive agent for the risk of colorectal cancer development. The researchers have performed in vitro and in vivo experiments using mouse models.
In vitro,calcium phosphate precipitate heme and inhibited heme-induced cytotoxicity.
Fig. 1. Effect of CaPi on the solubility of haem in vitro. Haem (0–400 μM) was incubated in buffer (pH 7.0) in the absence (•) or presence (○) of 10 mM freshly formed CaPi. Results are given as mean of three separate experiments (the SEMs are smaller than the size of the symbols).
In vivo, mouse were divided into four groups fed with diets differing in the content of heme and calcium. The first two groups represent the “control” and received a diet with low heme or low heme supplemented with calcium phosphate (CaPi), the other two groups represent the “treated”, and they were fed with high heme or high heme supplemented with calcium phosphate (CaPi). In rats fed with low calcium and high heme the colonic epithelial proliferation was increased compared with control and also the cytotoxic effects of faecal water. In fact to find differences of the faecal liquid cytotoxicity between the two groups, the researchers collect the faecal water of mice and administered it to a suspension of washed human erythrocyte. Faecal water collected from mice with no calcium supplement is more cytotoxic than water obtained by control mice.
Fig. 2. Effect of freshly formed CaPi on cytotoxicity of faecal water in vitro. Faecal water of control rats and of haem-fed rats was incubated in the absence (filled bars) or presence (hatched bars) of 10 mM CaPi. Bars show the mean ± SEM of three separate experiments.
On the other hand there is also a difference in the solubility of haem in faecal water, evaluating the absorbance rate of the faecal water. The faecal water of mice with low calcium and high haem has a high absorbance at 400 nm suggesting that haem was solubilized in the acqueous phase of faeces. While in the control group (with or without haem), and in the high heme group supplemented with high CaPi the the absorbance was lower, suggesting that haem is less solubilized.
Fig. 3. UV–visible absorbance spectrum of a lipid extract of pooled faecal waters of the different dietary groups. (— • • —) low-calcium control; (— • —) low-calcium haem; (- - - -) high-calcium control; (——) high-calcium haem.
The other interesting thing is that the total content of haem in faeces of rats with “high heme and high calcium” diet is greater of the total content of the “high heme and low calcium” groups, about 83% against 53% of the total haem intake. The reason is probably that if there’s more haem solubilized in the gut, it is metabolised by intestinal epithelial cell into toxic compounds, causing damage to cell surface and DNA. This also suggest that the effect of dietary calcium on faecal haem recovery may limit the diagnostic value of Hemo-Quant method for the diagnostic of intestinal bleeding, and that the large variance in faecal recovery may partly be due to the difference in calcium intake, people with high calcium intake have more haem in faeces compared to people with low calcium intake.
In an article of 2003, Meat and cancer: haemoglobin and haemin in a low-calcium diet promote colorectal carcinogenesis at the aberrant crypt stage in rats. (Pierre et al), the researchers focused their attention on the formation of “aberrant crypt foci”, in the intestinal mucosa of rodents.
Experiments on rats show that the high dietary intake of heme promote the formation of aberrant crypt foci (ACF). In this case the rats were feed not directly with red meat but with different kind of diets supplemented with different concentration of these substances: haem, bovine haemoglobin, ferric citrate, calcium phosphate, antioxidants, safflower oil, and olive oil. The high haemin diet increased the number and the size of ACF. The promotion was also associated with the increased faecal water cytotoxicity. Calcium, olive oil and antioxidant each inhibited the haemin induced ACF promotion. The experiment demonstrate that again the calcium is associated with prevention of colorectal cancer development associated with high red meat intake. This experiment is also important to demonstrate that not the red meat but most it’s high haem content is a promoting agent of carcinogenesis, in fact also using diet without red meat but only with supplement of haemoglobin or haemin there is an increase in number and dimension of ACF.
Fig. 4. Mucosal surface of colon stained with methylene blue visualized under a light microscope (×40). (A) Aberrant crypt focus with four crypts (control diet). (B) Major aberrant crypt focus with 20 crypts (high-haemin diet).
Fig. 5. Number of aberrant crypts in the colon of rats after 100 days on experimental diets. (A) Effect of haemin (LowH, MediumH, HighH) 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 (A) or from HH diet (B) (P < 0.01). Haem concentration in diets (µmol/g).
The role of chlorophyll
Green vegetables contain chlorophyll, a magnesium porphyrin structurally analogous to haem. In 2005 it was published an article called Green vegetables, red meat and colon cancer: chlorophyll prevents the cytotoxic and hyperproliferative effects of haem in rat colon. (de Vogel et al.), in which researchers demonstrate that chlorophyll has a preventive role against the formation of colon adenoma.
They performed the experiment on rats. The animals were fed with a purified control diet or purified diets supplemented with 0.5 mmol haem/kg, spinach (chlorophyll concentration 1.2 mmol/kg) or haem plus spinach for 14 days. In a second experiment they also studied a group that received haem plus purified chlorophyll (1.2 mmol/kg). Cytotoxicity of faecal water was determined with a bioassay and colonic epithelial cell proliferation was quantified in vivo by [methyl-3H]thymidine incorporation into newly synthesized DNA. Also in this case the total contents of haem in faeces is major when rats were fed with high haem in combination with spinach compared to control and to haem alone. The researchers speculated that chlorophyll and heme, that both are planar hydrophobic porphyrins, can stack together in the hydrophobic phase of the luminal contents and are expelled togheter, increasing the amount of haem in stools.
Fig. 6. Effect of dietary haem, spinach and haem plus spinach on: A) colonic epithelial cell proliferation, determined by in vivo [methyl-3H]thymidine incorporation into colonic mucosa; B) cytotoxicity of faecal water, determined with an erythrocyte bioassay; C) haem excretion in the faeces, determined by HemoQuant.
Exfoliation of colonocytes was measured as the amount of rat DNA in faeces. In both studies haem increased cytotoxicity of the colonic contents ~8-fold and proliferation of the colonocytes almost 2-fold. Spinach or an equimolar amount of chlorophyll supplement in the haem diet inhibited these haem effects completely. Finally, size exclusion chromatography showed that chlorophyll prevented formation of the cytotoxic haem metabolite. In conclusion green vegetables may decrease colon cancer risk because chlorophyll prevents the detrimental, cytotoxic and hyperproliferative colonic effects of dietary haem. The fact that also purified chlorophyll could inhibit this effect is a tangible clue that the preventive action is not only related to fibers contained in vegetables
A general view
In 2011 a review resume the topic of all the precedent studies: Red meat and colon cancer: Should we become vegetarians, or can we make meat safer?. (Denis E. Corpet). In this article also other preventive factors related to the consumption of red meat are considered: polyphenols, Vit C and E. In fact calcium use has a drawback: it binds heme iron and thus reduces its absorption, with iron deficiency as side effects (particularly in menstruating and pregnant women). So it’s important to look for other way to avoid meat promotion without blocking heme iron, by suppressing the fat peroxidation pathway or the N-nitroso pathway. Peroxidation and nitrosation may be reduced by adding antioxidant or antinitrosant additives to meat. In addition, peroxidation is prevented by removing oxygen, and nitrosation is prevented by removing nitrite from meat or from the gastrointestinal tract. Adding antioxidant butylated hydroxyanisole with rutin, or oxidation-resistant olive oil, to a hemin-loaded diet fully prevents the promoting effect of hemin (Pierre et al., 2003). After the catalysis of heme Vit C and Vit E could be active in the prevention of nitrosation while polyphenols could prevent fat peroxidation, but the evidence is not strong as for calcium and chlorophyll and more experiments have to be perform to confirm their biochemical roles.
Fig. 7. Catalytic effect of heme iron on fat peroxidation and N-nitrosation, and their inhibition by dietary means. Consequences for the development of colorectal cancer. Heme iron catalyzes nitrosation and fat peroxidation. End products are N-nitroso compounds (NOCs), malondialdehyde (MDA) and 4-hydroxy-nonenal (4-HNE). These pathways explain, at least in part, the promoting effect of red and cured meat on colorectal cancer. The catalytic effects of heme iron can be inhibited by trapping heme with calcium carbonate or chlorophyll. The endogenous formation of NOCs is inhibited by vitamin C and E. Ongoing studies suggest that specific polyphenols can inhibit fat peroxidation and/or nitrosation.
The aim of this topic is not suggesting to avoid red meat at all but make people aware of risks. Red meat is important for it’s content of iron and protein and can be included in the diet. If the diet is balanced with other compounds, first of all calcium and chlorophyll, followed by vitamins and polyphenols most of the toxic effect of red meat on intestinal mucosa could be reduced. In general is better to stay under a consumption of 50 g/die of red meat, and if possible prefer white meat (because it’s content in heme is lower) and avoid processed meat (like hot dog and bacon, because it is rich in nitrate). Lastly is important a cultural change of meat producers, aiming at find better processing methods and choosing new additives for meat.