Red meat enhances the colonic formation of the DNA adduct O6-carboxymethyl guanine: implications for colorectal cancer risk.

Red meat is associated with increased risk of colorectal cancer and increases the endogenous formation of N-nitrosocompounds (NOC). To investigate the genotoxic effects of NOC arising from red meat consumption, human volunteers were fed high (420 g) red meat, vegetarian, and high red meat, high-fiber diets for 15 days in a randomized crossover design while living in a volunteer suite, where food was carefully controlled and all specimens were collected. In 21 volunteers, there was a consistent and significant (P < 0.0001) increase in endogenous formation of NOC with the red meat diet compared with the vegetarian diet as measured by apparent total NOC (ATNC) in feces. In colonic exfoliated cells, the percentage staining positive for the NOC-specific DNA adduct, O(6)-carboxymethyl guanine (O(6)CMG) was significantly (P < 0.001) higher on the high red meat diet. In 13 volunteers, levels were intermediate on the high-fiber, high red meat diet. Fecal ATNC were positively correlated with the percentage of cells staining positive for O(6)CMG (r(2) = 0.56, P = 0.011). The presence of O(6)CMG was also shown in intact small intestine from rats treated with the N-nitrosopeptide N-acetyl-N'-prolyl-N'-nitrosoglycine and in HT-29 cells treated with diazoacetate. This study has shown that fecal NOC arising from red meat include direct acting diazopeptides or N-nitrosopeptides able to form alkylating DNA adducts in the colon. As these O(6)CMG adducts are not repaired, and if other related adducts are formed and not repaired, this may explain the association of red meat with colorectal cancer.

Variability in fecal water genotoxicity, determined using the Comet assay, is independent of endogenous N-nitroso compound formation attributed to red meat consumption.

Red meat consumption causes a dose-dependent increase in fecal apparent total N-nitroso compounds (ATNC). The genotoxic effects of these ATNCs were investigated using two different Comet assay protocols to determine the genotoxicity of fecal water samples. Fecal water samples were obtained from two studies of a total of 21 individuals fed diets containing different amounts of red meat, protein, heme, and iron. The first protocol incubated the samples with HT-29 cells for 5 min at 4 degrees C, whereas the second protocol used a longer exposure time of 30 min and a higher incubation temperature of 37 degrees C. DNA strand breaks were quantified by the tail moment (DNA in the comet tail multiplied by the comet tail length). The results of the two Comet assay protocols were significantly correlated (r = 0.35, P = 0.003), however, only the second protocol resulted in detectable levels of DNA damage. Inter-individual effects were variable and there was no effect on fecal water genotoxicity by diet (P > 0.20), mean transit time (P = 0.588), or weight (P = 0.705). However, there was a highly significant effect of age (P = 0.019). There was no significant correlation between concentrations of ATNCs in fecal homogenates and fecal water genotoxicity (r = 0.04, P = 0.74). ATNC levels were lower in fecal water samples (272 microg/kg) compared to that of fecal homogenate samples (895 microg/kg) (P < 0.0001). Failure to find dietary effects on fecal water genotoxicity may therefore be attributed to individual variability and low levels of ATNCs in fecal water samples.

Haem, not protein or inorganic iron, is responsible for endogenous intestinal N-nitrosation arising from red meat.

Many N-nitroso compounds (NOC) are carcinogens. In this controlled study of 21 healthy male volunteers, levels of NOC on a high (420 grams) red meat diet were significantly greater (P = 0.001) than on a low (60 grams) meat diet but not significantly greater when an equivalent amount of vegetable protein was fed. An 8-mg supplement of haem iron also increased fecal NOC (P = 0.006) compared with the low meat diet, but 35-mg ferrous iron had no effect. Endogenous N-nitrosation, arising from ingestion of haem but not inorganic iron or protein, may account for the increased risk associated with red meat consumption in colorectal cancer.

Effect of vegetables, tea, and soy on endogenous N-nitrosation, fecal ammonia, and fecal water genotoxicity during a high red meat diet in humans.

Red meat increases colonic N-nitrosation, and this may explain the positive epidemiological relationship between red meat intake and colorectal cancer risk. Vegetables, tea, and soy have been shown to block N-nitroso compound (NOC) formation and are associated with protection against colorectal cancer. To determine whether these supplements affect fecal NOC excretion during consumption of a high red meat (420 g/day) diet, 11 male volunteers were studied over a randomized series of 15-day dietary periods. Seven of these subjects completed a further dietary period to test the effects of soy (100 g/day). Soy significantly suppressed fecal apparent total NOC (ATNC) concentration (P = 0.02), but supplements of vegetables (400 g/day as 134 g broccoli, 134 g brussels sprouts, and 134 g petits pois) and tea extract (3 g/day) did not affect mean levels of fecal ATNC, nitrogen and ammonia excretion, and fecal water genotoxicity. However, fecal weight was increased (P < 0.001) and associated with reduced transit time (r = 0.594, P < 0.0001), so that contact between ATNC, nitrite, and ammonia and the large bowel mucosa would have been reduced. Longer transit times were associated with elevated fecal ATNC concentrations (r = 0.42, P = 0.002). Fecal nitrite was significantly suppressed during the tea supplement compared with the meat-only (P = 0.0028) and meat + vegetables diets (P = 0.005 for microgram NO2/g).