Aim: The aim of the project is to understand the role of the intestinal microbiome as determinant of the effect of diet on colorectal cancer risk and to identify specific microbiome/metabolomic profiles associated with cancer risk. We are comparing in humans (WP1) and experimental animals (WP2-WP3-WP4), a high-risk meat-based diet (MBD) with a pesco-vegetarian diet (PVD) associated with a lower CRC risk and a MBD diet supplemented with tocopherol (MBD-T medium risk). 

Results: For the WP1 in human subjects, total cholesterol, triglyceride, and ferritin levels significantly changed between diets.  Results indicate that the MBD was associated with an increase in markers of colon cancer risk as faecal waters (FW) genotoxicity, whereas no effect of the PVD was observed. Instead, a complete protection from the effect of the MBD was found upon supplementation of the meat-based diet with alpha-tocopherol. No differences were found in mutagenicity tests on FW pre and post the dietary intervention.  For the WP2 urinary and fecal lipid peroxidation biomarkers indicate an increase in luminal lipid peroxidation in the colon of the rats fed meat based diets when compared to the control or the PVD, with no significant effect of alpha-tocopherol diet supplementation. In addition, intestinal permeability was decreased by the PVD, with no effect of MBD and MBD-T. The analysis on microbiota composition indicated that the dietary intervention significantly modified bacterial communities generating clearly distinguishable community profiles, especially for the PVD. Notably, the lowest difference was observed between the MBD and MBD-T. All groups showed lower bacterial richness compared to T0, except for the MBD-T diet. Notably, all samples yield a DNA which was successfully amplified and analyzed for the bacterial community, but not for the fungal community except for the PVD group where it was possible to assess the presence of a fungal community. Through Metabolomics analysis, we found that a set of seven metabolites were able to classify different diet groups. Subsequently expanding those to another set of 50 highly correlated metabolites which are highly correlated. For the WP3, results showed a significantly lower number of colon and total tumours in the rats treated with the PVD, while MBD and MBD-T presented similar numbers. Apoptosis in the normal mucosa was significantly higher in the PVD group compared with the two groups fed with meat. Targeted metagenomics  indicated a significant difference of the bacterial community between the 3 diets, in particular of the PVD with respect to all others, while no difference between the MBD and MBD-T diet were highlighted.

Impact: The results will provide fundamental insight into the role of microbiota in determining the effect of diet, in particular red/processed meat intake, on CRC risk. Preliminary results are already suggesting differences between the PVD and meat based diet on a variety of CRC risk biomarkers.

The MeatIC project is the first controlled dietary intervention study to understand the contribution of microbiome in red/processed meat-mediated CRC risk, that we are conducting in parallel in humans and in three relevant models of colon carcinogenesis. The project is focused on the role of microbiome profile and colon metabolites on CRC risk (in humans and animals) and will determine whether reduced intake of red and processed meat will reduce the level of toxic metabolites. We are comparing in human’s volunteers and experimental animals (Chemically-induced carcinogenesis with Azoxymethane (AOM) in rats and colon carcinogenesis in Pirc rats, mutated in Apc gene), a high-risk meat-based diet (MBD) with a pesco-vegetarian diet (PVD) associated with a lower CRC risk. An additional arm is a MBD diet supplemented with tocopherol (MBD-T), possibly reducing risk. Moreover, to better understand the contribution of diet-induced gut microbiome changes on the CRC and to demonstrate causality between dysbiosis and pathology, we performed a transplant of faeces from rats fed the three diets into germ–free rats. The intestinal microbiome and metabolomics profiles associated with these diets will be correlated with carcinogenesis measuring surrogate biomarkers in the humans and tumorigenesis in rats fed the same diets.

Regarding the human study, total cholesterol, triglyceride, and ferritin levels significantly changed between diets.  Results indicate that the MBD was associated with an increase in markers of colon cancer risk as faecal waters (FW) genotoxicity, whereas no effect of the PVD was observed. Instead, a complete protection from the effect of the MBD was found upon supplementation of the meat-based diet with alpha-tocopherol. No differences were found in mutagenicity tests on FW pre and post the dietary intervention.

The chemically-induced colon carcinogenesis in rats’ experiment, the analyses of urinary and faecal lipid peroxidation biomarkers indicate an increase in luminal lipid peroxidation in the colon of the rats fed meat-based diets when compared to the control or the PVD, with no significant effect of alpha-tocopherol diet supplementation. In addition, intestinal permeability was decreased by the PVD, with no effect of MBD and MBD-T. The analysis on microbiota composition indicated that the dietary intervention significantly modified bacterial communities generating clearly distinguishable community profiles, especially for the PVD. Notably, the lowest difference was observed between the MBD and MBD-T. All groups showed lower bacterial richness compared to T0, except for the MBD-T diet. Notably, all samples yield a DNA which was successfully amplified and analysed for the bacterial community, but not for the fungal community except for the PVD group where it was possible to assess the presence of a fungal community. Through Metabolomics analysis, we found that a set of seven metabolites were able to classify different diet groups. Subsequently expanding those to another set of 50 highly correlated metabolites which are highly correlated. 

The colon carcinogenesis experiment in Pirc rats mutated in Apc, showed a significantly lower number of colon and total tumours in the rats treated with the PVD, while MBD and MBD-T presented similar numbers. Apoptosis in the normal mucosa was significantly higher in the PVD group compared with the two groups fed with meat. Targeted metagenomics indicated a significant difference of the bacterial community between the three diets, in particular of the PVD with respect to all others, while no difference between the MBD and MBD-T diet were highlighted. 

The results will provide fundamental insight into the role of microbiota in determining the effect of diet, in particular red/processed meat intake, on CRC risk. Our results are already suggesting differences between the PVD and meat based diet on a variety of CRC risk biomarkers.