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This research project aims to investigate the relevance of elevated apparent total nitrosocompounds in the human colon, resulting from increased red meat consumption.
Study Duration : October 2002 to March 2006
Contractor : Medical Research Council
In the presence of haem from red meat, nitrogenous residue entering the large intestine from high levels of protein form non-volatile N-nitroso compounds (NOC), which are measured as apparent total nitrosocompounds (ATNC) in faecal extracts. Increased intakes of red meat have been shown to increase faecal ATNC levels in a dose responsive manner, the equivalent amount of protein from eggs, milk cheese and vegetable protein have been shown to have no effect. Although many NOC are known carcinogens, the biological relevance of ATNC is unknown.
Faecal samples from volunteers consuming different diets containing red meat, haem, fibre or processed meat will be assessed chemically for levels of ATNC by Thermal Energy Analysis and for genotoxic effects by the Comet Assay, HPRT mutations and the Mouse Lymphoma Assay. N-Proline in faecal samples and O6-carboxymethyl guanine DNA adducts in exfoliated cells will also be measured
This project is a collaboration with Project T01031, The relevance of ATNC to colon carcinogenesis measurement of nitroso compounds by MS or LC-MS or LC-MS-MS.
Human volunteers were given diets with various levels of red meat, fibre, processed meat and iron either from haem or inorganic sources. They were maintained on this diet for between 15 and 21 days. Faecal water and faecal homogenate samples from days 10, 13 and 15 were used for analysis.
To find out whether any N-nitrosocompounds (NOC) formed in the gut were capable of causing damage to DNA, the faecal water samples collected were analysed in the Comet assay (in a colorectal adenocarcinoma cell line), the Mouse Lymphoma assay and the p53 polymerase stop assay.
These three tests showed no correlation between diet and ability to damage DNA. Faecal waters and faecal homogenates were subsequently analysed for their NOC content and this showed that NOC levels were significantly lower in faecal water samples. NOC levels in faecal waters did not vary significantly with diet, though this may be due to loss of NOCs during the preparation process, suggesting that faecal water samples are not appropriate for these analyses. Therefore, the lack of effect in these genotoxicity assays could be due to individual variability and low levels of NOC in the faecal water samples.
Additional studies were conducted on exfoliated colon cells isolated from the surface of stool samples immediately on collection. These cells were analysed for O6-carboxymethyl guanine (O6CMG) adducts using immunohistochemical staining. There were significantly higher proportions of cells staining positive for these adducts from individuals who consumed a red or processed meat diet than in those who consumed a vegetarian diet.
N-nitrosation products of glycine and peptides interact with DNA leading to the formation of O6CMG adducts, which are not repaired, and there is evidence from
in vitro
tests that O6CMG is mutagenic. If this is also shown to be the case
in vivo
and if other adducts are also formed and not repaired, this may explain the association between a diet high in red or processed meat and an increased risk of developing colorectal cancer.
The final report is available from the Agency’s Information Centre.
To obtain a copy, please contact the Enquiry Desk, Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email:
infocentre@foodstandards.gsi.gov.uk
)
Lewin, M.L. Bailey N., Bandaletova, T., Bowman, R., Cross, A.J., Pollock, J. Shuker, D.E.G., Bingham, S.A (2006) Red meat enhances the colonic formation of the DNA adduct O6-carboxymethyl guanine: implications for colorectal cancer risk. Cancer Research. 66 1859- 1865
Cross, A.J., Greetham, H.L., Pollock, J.R.A, Rowland, I.R, Bingham, S.A (2006) Variability in fecal water genotoxicity, determined using the Comet assay, is independent of endogenous N-nitroso compound formation attributed to red meat consumption. Environmental and Molecular Mutagenesis 47 , 179-184
Kuhnle GG, Story GW, Reda T, Mani AR, Moore KP, Lunn JC, Bingham SA. (2007) Diet-induced endogenous formation of nitroso compounds in the GI tract, Free Radic Biol Med. 43 (7):1040-7
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