Natural Compounds to Reduce Nitrite in Meat Products (PHYTOME)

The PHYTOME project (Phytochemicals to reduce nitrite in meat products) is a major European Union (EU) co-funded research project that aims to develop innovative meat products in which the food additive nitrite has been replaced by natural compounds originating from fruits and vegetables. These biologically active compounds, also referred to as phytochemicals, are known to contribute to improved gut health and are added to the meat as natural extracts. In a number of meat products, carefully selected combinations of natural antioxidants and other biologically active compounds occurring in vegetables, fruits and natural extracts such as coffee and tea, will be added during meat processing. Some of these compounds possess an antimicrobial activity allowing them to replace nitrite, whereas others possess a natural red colour that may contribute to the desired appearance of the products. Also, some of these compounds are known to protect colonic cells against damaging effects of cancer causing agents that may be formed in the large intestine after meat consumption. The PHYTOME project will develop new technologies to introduce the natural extracts during processing to different types of meat products. These techniques will guarantee good sensory quality of the product as well as microbiological safety. Once these techniques have been developed and optimized at laboratory scale, the new type of products will be produced on an industrial scale. The health promoting effects of these products will be evaluated in a human dietary intervention study with healthy volunteers. After consumption of a fully controlled diet with either relatively high amounts of the traditional meat products or products produced following the new concept, faeces and colonic material will be collected and investigated for markers of colorectal cancer risk. These investigations will be performed in close collaboration with Research Institutes in the United Kingdom, Belgium, Italy and Greece, and will make use of the newest genomics techniques that are available.

Rationale: The aim of this project is to develop new meat processing technologies, resulting in innovative meat products that have low or no nitrite and that have been shown to contribute to improved human health. This will be achieved by introducing carefully selected mixtures of biologically compounds originating from natural plant extracts. The new meat products will be evaluated in a human dietary intervention study to establish their positive effect on cancer risk markers in colonic tissues using the newest genomics techniques available. Objective: This project aims to evaluate the health impact of newly developed low nitrite containing meat products using genomics markers in a human dietary intervention study. Study design: This study has a cross-over design with only healthy volunteers. Each participant will be asked to donate a urinal, faecal and blood sample and undergo endoscopy after each intervention period. All analyses will be done for each study group separately to examine the overall effects of nitrite levels in meat. Study population: All subjects will be recruited by the University of Maastricht (UM) in the province of Limburg, the Netherlands, using advertisements in local newspapers as well as other media. Healthy subjects of both sexes will be selected based on predefined inclusion criteria (BMI: 18-25; > 18 years) and randomly assigned to one of the different experimental groups. Intervention (if applicable): Subjects will receive a completely controlled diet with 3 different types of meat products according to the study design, with either normal levels, low or no added nitrite. After each of the three intervention periods of 15 days (300 grams meat per day) blood, urine, saliva, mouth wash and faeces will be sampled and stored appropriately at UM for later analysis. Additionally, colonic biopsies will be taken by the department of internal medicine (UM) or at the hospital of Sittard during an endoscopic examination. To evaluate the impact of nitrate in drinking water on the endogenous nitrosation processes in combination with processed red meat intake, there will be and extra intervention period of 7 days were volunteers will be asked to consume drinking water with high nitrate levels according to the Acceptable Daily Intake level (ADI: 3.7mg/kg bodyweight) in combination with 300 grams processed red meat per day. Also after this intervention period volunteers will be asked to collect a blood and saliva sample and 24h urine and faeces samples. Also, colonic biopsies will be taken by the department of internal medicine (UM) or at the hospital of Sittard during an endoscopic examination. Main study parameters/endpoints: Formation of N-nitrosocompounds in human faecal and urine samples Reveal differences in transcriptomic and epigenomic markers after consumption of meat products enriched with natural compounds. These markers can be interpreted as an indicator of reduced cancer risk. Correlating gene expression changes to changes in genotoxic endpoints (DNA damage, reduction in N-Nitroso compounds (NOC)) to reveal the molecular processes involved in cancer risk reduction. The identification of molecular pathways that are crucial in the carcinogenic process will demonstrate a causal association between dietary changes and markers of carcinogenic risk.

Drinking water containing nitrate up to acceptable daily intake level (ADI = 3.7 mg/kg bw) in combination with 300 grams of processed meat, white meat or processed meat enriched with natural compounds

Change in N-nitroso compound (NOC) levels in faeces and urine measured as apparent total nitroso compounds (ATNC) between baseline levels at the start of the intervention and each intervention period

The faecal and urinal level of NOC is measured as Total Apparent Nitroso Compounds (ATNC) and used indicator of colonic endogenous nitrosation. It is known to significantly increase following strictly controlled diets high in red or processed meat.

Change in whole genome gene expression analyses in colonic biopsies and blood between baseline levels and each intervention period (transcriptomics)

Colon biopsies and blood will be analysed for transcriptomic responses to the dietary changes. This will provide information on gene expression changes in the colon epithelium that may be linked with other parameters such as faecal NOC levels. This will also identify molecular pathways that can link dietary composition to processes involved in cancer development.

Change in urinary nitrate and creatine levels between baseline levels at the start of the intervention and each intervention period.

Urine samples (0.25 mL) will be analysed for nitrate and urinary creatinine. Nitrate concentrations (ng/ml) will be divided by creatinine concentrations (mg/100ml) to adjust for the variable hydration of participants.

Blood will be collected which will be used for genotyping of single nucleotide polymorphisms associated with oxidative stress, biotransformation and DNA repair. This will provide information on the relation between genetic variations linked with other tested parameters such as faecal NOC levels.

Change in DNA methylation analyses of DNA isolated from colonic biopsies and blood (epigenetics) between baseline levels at the start of the intervention and each intervention period

Colon biopsies and blood will be analysed for epigenomic responses to the dietary changes. This will provide information on the genes of which the DNA methylation status has changed which might explain their change in gene expression. Epigenetic changes and gene expression changes will be integrated during the analyses.

Change in enzyme activity of nitrate reductase in saliva between baseline levels at the start of the intervention and each intervention period

Microorganisms in the mouth are able to convert nitrate to nitrite and it has been estimated that about 70% of ingested nitrite is formed in this way. Dietary interventions containing nitrate have shown to infuence the activity of the enzyme nitrate reductase.

Change in the microbiome in feacal samples and in saliva between baseline levels at the start of the intervention and each intervention period

Both saliva and fecal specimens will be used for DNA extraction and subsequent analyses. The microbiome is sensitive for dietary changes, and its composition can alter due to different exposures.

Change in faecal water genotoxicity between baseline levels at the start of the intervention and each intervention period

Faecal water can be prepared from faecal matter after which a short-term exposure of an in vitro colon cell line will be performed. DNA damage will subsequently be assessed by the alkaline single cell gel electrophoresis assay or comet assay.

Change in O6-CMG in colonic biopsies between baseline levels at the start of the intervention and each intervention period

O6-CMG is a mutagenic DNA adduct that results from exposure to nitrosated glycine derivatives and has particular potential as a biological marker of DNA carboxymethylation because it does not seem to be repaired by O6-alkylguaninealkyltransferase. O6-CMG has previously been found to increase following a diet high in red meat.

Change in O6-MeG in colonic biopsies between baseline levels at the start of the intervention and each intervention period

Exposure to certain NOCs causes O6-MeG adduct formation. O6-MeG DNA adducts are also mutagenic and have frequently been shown to occur in human CRC tissue. Samples will be analyzed at National Hellenic Research Foundation (NHRF; Greece) using an ELISA method described by Georgiadis et al.

Inclusion Criteria: Healthy with a Body Mass Index (BMI) between 18-25, male or female Between 18-70 years old Exclusion Criteria: Alcohol abuse up to 6 months before participation in this research Current aberrations or insufficiency of kidney, liver, gut, heart or lungs Current presence of persistent inflammation in the gut or liver Current endocrine or metabolic aberrations Current anaemia or infection HIV infection or hepatitis Use of antibiotics and other medication over the last 3 months Current smokers Vegetarians Pregnant women Participants of other intervention studies during this intervention period.

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