1. Health Benefits PHTYOME FP7-SME-2012-2- PHYTOME (315683) Brussels, November 16, 2015 Prof. dr. Theo M. de Kok Coordinator PHYTOME project Maastricht University, the Netherlands

2. 2 Overview Design human dietary intervention study Outcome measurements Results Interpretation of the key findings

3. 3 Objective Establish the potential health benefits of the consumption of the new type of meat products based on a combination of relevant endpoints. Critical elements in the planning of the investigation: Inclusion criteria What meat types to compare Reference diet Caloric intake Duration of the study What measurements to perform Which samples do we need

4. 4 Study design -Each intervention period = 15 days with controlled diet -Series 1 = normal nitrite; series 2 = low nitrite -X= Sample collection = -Faecal water (24h) -Urine (24h) -Blood -Saliva -Colonic biopsies A: red processed meatB: ‘PHYTOME’ meatO: wash-out C: drinking water 1 2

5. 5 Study design Controlled diet = individual food package -300 grams meat per day spread over breakfast, lunch & dinner -Equal caloric intake for each intervention period -Antioxidants in fruits and vegetables at low, but healthy level -Restricted alcohol & probiotic consumption -Fish: excluded due to presence of high amounts of NOC precursors

6. 6 Analyses Apparent faecal N-nitroso compounds (ATNC) Faecal water genotoxicity (Comet-assay) Analysis of the enzyme activity (nitrate reductase) DNA damage (O 6 -carboxymethylguanine and O 6 -methylguanine) Gene expression analysis (Transcriptomics) Epigenetics (DNA methylation) Compliance markers

7. 7 Data analyses Endpoints, objectives and analyses

8. 8 Inclusion Progress intervention study -Recruitment of volunteers  sampling in 2 hospitals after medical- ethics approval; -80 persons were included over a period of 1 year (2014-2015); -16 drop-outs; -All samples distributed to the various laboratories; -Majority of data have been generated -Data analysis is still ongoing

9. 9 Results Exposure to N-Nitroso compounds (ATNC) ATNC increases after consumption of processed meat ATNC decreases with white meat Meat induced ATNC formation can be (fully) reduced by addition of phytochemicals Drinking water nitrate stimulates ATNC (in mixed diet group)

10. 10 Results Exposure to N-Nitroso compounds (ATNC)

11. 11 Results Exposure to N-Nitroso compounds (ATNC) ATNC increases after consumption of processed meat ATNC decreases with white meat Meat induced ATNC formation can be (fully) reduced by addition of phytochemicals Drinking water nitrate stimulates ATNC (in mixed diet group) The addition of phytochemicals alone can also reduce ATNC (4B1) The combined addition of phytochemicals and reduced nitrite have a stronger reduction of ATNC (4B2) Drinking water nitrate stimulates ATNC formation in combination with both red processed meat and white meat Drinking water nitrate does not stimulate ATNC in the PHYTOME meat group with reduced nitrite (5B2).

12. 12 Results Genotoxicity of faecal water: the capacity to induce DNA breaks in cultured human colon cells.

13. 13 Results Genotoxicity of faecal water: Relatively high genotoxicity at inclusion; Genotoxicity is the lowest during de control period on white meat; No significant differences between normal meat products and PHYTOME meat products; Drinking water nitrate stimulates the capacity to induce DNA damage.

14. 14 Results Nitrate and nitrite in saliva Ratio against control period

15. 15 Results Nitrate and nitrite in saliva No significant impact of meat consumption Levels reduced by half during control period on white meat Marginal increase with drinking water nitrate (mixed group)

16. 16 Genetic and genomics markers Genetic damage: DNA modifications GeneGene expression Biological function Regulation of gene expression (DNA methylation) Gene expression changes

17. 17 Gene expression changes: Health impact assessment Environmental or dietary factor Human disease Exposure results in specific response profiles

18. 18 Gene expression changes: Health impact assessment Specific gene expression patterns after consumption of different types of processed meat

19. 19 Gene expression changes: Health impact assessment Research questions: - Difference normal red meat vs phytome meat: T2 vs T4 - Difference normal red meat vs phytome meat high/low nitrite levels: T2 vs T4B1 or T4B2 Per group - Difference red meat vs white meat: T2 vs T3, T4 vs T3 - Influence meat intake:T2 vs T1 - Variation at the start:All T vs T1 - Relation to NOC levels, genotoxicity, etc

20. 20 Health impact assessment Comparing gene expression changes between groups:

21. 21 Health impact assessment Gene lists and biological processes show that relevant processes in relation to cancer development are differently influenced in de red meat group as compared to the PHYTOME group: Cell cycle control Regulation of APC/C activators between G1/S Separation of sister chromatids APC/C-mediated degradation of cell cycle proteins Gastric Cancer Network 1 Oxidative Stress IL12 signaling mediated by STAT4 Activation of APC/C and APC/C:Cdc20 mediated degradation of mitotic proteins cholesterol biosynthesis I -

22. 22 Conclusions Exposure of the large intestine to ATNC can be reduced by adding natural extracts to processed meat products; Reduction of nitrite in meat products further reduces the formation of ATNC; Complete elimination of nitrite is not necessary to reduce ATNC to background levels; Difference in faecal genotoxicity between white meat and red processed meat is relatively small (both with/without phytochemicals); The impact of drinking water nitrate on ATNC and genotoxicity is significant, particularly when no phytochemicals are added.

23. 23 Conclusions Further exploration of the complex data sets will reveal the molecular mechanisms modified by different meat types and demonstrate the biological plausibility of cancer risk reduction by the PHYTOME concept.

24. 24 Implications Implementation of the PHYTOME concept may contribute to improved European public health; The PHYTOME project demonstrates the importance of a well balanced diet containing a wide variety of phytochemicals and a well controlled intake of dietary nitrate. A final statement on the carcinogenicity of processed meat: ……

25. 25 http://www.phytome.eu