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Biological relevance of apparent total nitrosocompounds in the human colon
Project Code: T01030
MRC Dunn Nutrition Unit
Red meat but not white meat is associated with increased colorectal cancer risk and induces endogenous formation of N-Nitrosocompounds (NOC) in the human gastrointestinal tract. The aim of this project was to investigate the biological relevance of Apparent Total N-Nitrosocompounds (ATNC) formed endogenously from red meat in the human gut.
(i) Faecal waters extracted from samples from humans consuming different diets were investigated for genotoxicity in three in vitro assays: the Comet assay using two different protocols; the L5178 in mouse lymphoma cells; and the p53 stop assay
(ii) in an vivo technique, human exfoliated cells were isolated from faeces from individuals consuming different diets and the presence of the NOC specific O6-carboxymethylguanine (O6CMG) adduct examined
iii) faecal water and faecal homogenates were investigated for the presence of group specific NOC. Samples were also sent to Dr J Dennis who was contracted to analyse NOC by mass spectrometry under separate contract (T01031).
(i) In 21 volunteers there was a consistent and significant (p < 0.0001) increase in endogenous formation of NOC with the red meat diet compared with a vegetarian diet as measured as apparent total NOC (ATNC) in faeces. In colonic exfoliated cells, the percentage staining positive for the NOC-specific DNA adduct, O6-carboxymethyl guanine (O6CMG) was significantly (p < 0.001) higher on a high red meat diet. In 13 volunteers, levels were intermediate on the high fibre high red meat diet. Faecal ATNC were positively correlated with the percentage of cells staining positive for O6CMG (r2 = 0.56, p = 0.011). The presence of O6CMG was also shown in HT-29 cells treated with diazoacetate. This study has demonstrated that faecal NOC arising from red meat include direct acting diazopeptides or N-nitrosopeptides, able to form alkylating DNA adducts in the colon. As these O6CMG adducts are not repaired, and if other related adducts are formed and not repaired, this may explain the association of red meat with colorectal cancer.
(ii) In repeat studies of five volunteers fed processed meat, levels of cells staining positive for adduct were similar to red meat (mean processed meat 21.4%, mean red meat 25.2%) and higher than levels on a vegetarian diet (10.5%, p 0.020, 0.049 respectively).
(iii) The in vitro tests using faecal waters showed no effects of diet on genotoxicity. For DNA strand breaks in the Comet assay, the results from two different comet assay protocols were significantly correlated (r = 0.35, P = 0.003) and when conducted at 37ºC with the extended exposure time, resulted in detectable levels of DNA damage. Inter-individual effects were variable and there was no effect of diet (p>0.20), transit time (p = 0.588) or weight (p = 0.705). However there was a significant effect of age (p= 0.019). There was no significant correlation between concentrations of ATNC in the faecal homogenate and the genotoxic potential of faecal water samples using either the first method (r = 0.21, P = 0.08), or the second method (r = 0.04, P = 0.74).
(iv) For the assay for mutation at the thymidine kinase (tk) locus of mouse lymphoma L5178Y cells (MLA) neither two ileostomy nor faecal water samples containing low levels of NOC nor two of those containing high levels were mutagenic.
(v) For the polymerase stop assay, designed to determine sites of adduction in the p53 gene caused by incubation with faecal waters, in provisional results there was no clear effect of diet. However, there was close agreement between the adducted sites and the mutation sites in the IARC database of colorectal cancer. Out of the 45 of the sites which are mutated in colon/colorectal tumours, 30 were adducted by faecal water. Of the 45 Gs that are mutated in colon/colorectal tumours, 26 were adducted by faecal water. Hence 26/30 adduction sites coincided with mutation sites in human colon/colorectal tumours.
(v) ATNC levels were found to be significantly lower in faecal water samples (272 μg/kg) compared to faecal homogenate samples (895 μg/kg) (P <0.0001) and did not vary with diet. Failure to find dietary effects with the in vitro assays could therefore be attributed to the individual variability and comparatively low levels of NOC in faecal water samples.
(vi) In an attempt to characterise the compounds involved in endogenous nitrosation, ileostomy and faecal water samples containing high and low levels of ATNC (from low and high red meat diets) were sent to Dr J Dennis at CSL for mass spectrometry in a linked project (number TO1031). However no conclusive results were obtained (report No TO1031).
(vii) The method applied to detect N-Nitroso compounds as ATNC in the faecal samples relies on the chemical release of NO from nitroso compounds and its detection using chemiluminescence. The chemical denitrosation can also release NO from other types of nitroso compounds, for example from S-nitrosothiols and hence the term ATNC due to this lack of specificity. To investigate the chemistry of NOC in faecal homogenates further, chemiluminescence detection was used to determine types of NOC present by a chemical exclusion technique. In provisional results, this has shown that only a small proportion of the NOC formed in faecal samples are non genotoxic S-nitrosothiols and that the majority probably is nitrosated haem.
(viii) The likelihood that nitrosated haem in faecal material facilitates the formation of direct acting diazopeptides or N-nitrosopeptides, able to form alkylating DNA adducts in the colon, as shown in (i) above is under investigation using funds from elsewhere.
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