Do gastrointestinal bacteria affect the (geno)toxic activity of different meats?

Introduction and Aim: Epidemiological and clinical studies have consistently demonstrated that the consumption of meat, and in particular red and processed meat is correlated with the risk of colorectal tumors. Two independent pathways have been hypothesised as potential link between meat consumption and colon cancer: the N-nitroso compound (NOC) pathway with the potency of alkylating DNA and the fat peroxidation pathway known for its potent aldehydes resulting in mutagenic adducts. The goal of our study was to investigate the formation yield of the NOC-specific DNA adduct O6-carboxymethylguanine (O6-CMG) and of the fat peroxidation product malondialdehyde (MDA) during an in vitro gastro-intestinal simulation and as such asses the geno(toxic) effects of the different meat types. Additionally, this experimental design will indicate the specific digestive process involved in the (geno)toxicity of different meat types, which is up till now unknown. Material and Methods: Consecutive in vitro gastrointestinal digestions (stomach, small intestine and colon) of different meat types (beef, pork, chicken) were performed. To this purpose, six different fecal inocula derived from 6 healthy volunteers, 3 males and 3 females were used to obtain microbiota. For the measurement of O6-CMG, Caco-2 (a human colon carcinoma cell line) DNA was incubated with the different digestive samples. Afterwards the amount of O6-CMG in these samples was quantified by performing an UHPLC-MS/MS analysis, developed and validated especially for this purpose. The MDA concentration was measured by a modified method in accordance with Grotto et al. (2007). Thiobarbituric acid reactive substances (TBARS) were formed from the reaction of MDA with 2-thiobarbituric acid in an acid environment. The absorbance of the coloured complex was measured spectrophotometrically at 532 nm. Results and Discussion: The analysis of the digestive samples derived from stomach, small and large intestinal in vitro digestion of different meat types (beef, pork and chicken) were analysed on the presence of an important NOC-specific DNA adduct O6-CMG. The UHPLC-MS/MS results showed that the formation only occurred in the large intestine and reached the highest yield upon 72 hours of incubation. The amount of O6-CMG formed varied per person (5-1595 μg/kg), however it was clearly observed that red meat (beef higher than pork) consumption led to a higher yield of DNA adduct formation. As for the spectrophotometrical measurement of the main lipid peroxidation end product MDA, the highest yield was observed in the small intestine. Beef digestion showed slightly higher but insignificant differences in MDA concentrations (25-35 mg/kg) compared to the other meats (20-35 mg/kg). Only limited differences (CV < 20%) were observed between the different fecal inocula. Therefore, the highest toxicity response corresponding with red meat digestions in our experimental setup seemed to be correlated with the NOC-specific DNA adduct O6-CMG.Introduction and Aim: Epidemiological and clinical studies have consistently demonstrated that the consumption of meat, and in particular red and processed meat is correlated with the risk of colorectal tumors. Two independent pathways have been hypothesised as potential link between meat consumption and colon cancer: the N-nitroso compound (NOC) pathway with the potency of alkylating DNA and the fat peroxidation pathway known for its potent aldehydes resulting in mutagenic adducts. The goal of our study was to investigate the formation yield of the NOC-specific DNA adduct O6-carboxymethylguanine (O6-CMG) and of the fat peroxidation product malondialdehyde (MDA) during an in vitro gastro-intestinal simulation and as such asses the geno(toxic) effects of the different meat types. Additionally, this experimental design will indicate the specific digestive process involved in the (geno)toxicity of different meat types, which is up till now unknown. Material and Methods: Consecutive in vitro gastrointestinal digestions (stomach, small intestine and colon) of different meat types (beef, pork, chicken) were performed. To this purpose, six different fecal inocula derived from 6 healthy volunteers, 3 males and 3 females were used to obtain microbiota. For the measurement of O6-CMG, Caco-2 (a human colon carcinoma cell line) DNA was incubated with the different digestive samples. Afterwards the amount of O6-CMG in these samples was quantified by performing an UHPLC-MS/MS analysis, developed and validated especially for this purpose. The MDA concentration was measured by a modified method in accordance with Grotto et al. (2007). Thiobarbituric acid reactive substances (TBARS) were formed from the reaction of MDA with 2-thiobarbituric acid in an acid environment. The absorbance of the coloured complex was measured spectrophotometrically at 532 nm. Results and Discussion: The analysis of the digestive samples derived from stomach, small and large intestinal in vitro digestion of different meat types (beef, pork and chicken) were analysed on the presence of an important NOC-specific DNA adduct O6-CMG. The UHPLC-MS/MS results showed that the formation only occurred in the large intestine and reached the highest yield upon 72 hours of incubation. The amount of O6-CMG formed varied per person (5-1595 μg/kg), however it was clearly observed that red meat (beef higher than pork) consumption led to a higher yield of DNA adduct formation. As for the spectrophotometrical measurement of the main lipid peroxidation end product MDA, the highest yield was observed in the small intestine. Beef digestion showed slightly higher but insignificant differences in MDA concentrations (25-35 mg/kg) compared to the other meats (20-35 mg/kg). Only limited differences (CV < 20%) were observed between the different fecal inocula. Therefore, the highest toxicity response corresponding with red meat digestions in our experimental setup seemed to be correlated with the NOC-specific DNA adduct O6-CMG.C