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Increased haem intake through red meat consumption causes a significant increase in fecal N-nitroso compounds (NOCs), measured as apparent total N-nitroso compound levels (ATNC). Many NOCs are known to be carcinogens. To investigate possible genotoxic effects of these NOCs, a polymerase stop assay was used to examine genotoxicity of human fecal water samples. Samples of fecal water were obtained from 16 individuals fed controlled diets containing different amounts of red meat, protein, haem and iron (Cross et al., 2003). Sites of adduction in p53 caused by incubation with fecal waters were characterized using a polymerase stop assay. An amplicon of a region of p53 gene was attached to streptavidin coated magnetic beads (Dynabeads® Dynal, UK) via a biotinylated primer and incubated with fecal water samples. To remove any substances capable of degrading DNA during incubation, fecal waters were filtered through a 5000 MW limit filter (Ultrafree®-MC, Millipore, USA) prior to incubation. DNA salvaged from incubation was analysed for adducts in the sense strand of codons 200-300 that had been induced by the test samples (i.e. NOCs). A fluorescent sequencing primer was annealed to the adducted template and extended by Sequenase® V2.0 polymerase (USB Corp., USA) and the extension products analysed on an ABI 377 Sequencer using Genescan software (Applied Biosystems). Localisation of each polymerase arrest site to a particular base was accomplished by comparison with a sequence of untreated template. Fluorescent intensity (peak height) gave a measure of the level of damage at particular sites. The major site of polymerase termination occurred at pyrimidine sites flanked by guanine on the 5′ side. The remaining adducts were mostly found at thymidine sites. Using incubation times of 2hr, and 24hr, significant formation of adducts were observed. Out of nine subjects studied, significant levels of adducts were detected from just two of these individuals, suggesting a degree of individual variation, however no clear effects of diet were noted. Additional work to analyse adduction in both strands of the entire DNA-binding domain (codons 100-300) is being undertaken. Cross, A.J., Pollock, J.R.A. and Bingham, S.A. (2003) Haem, not protein or inorganic iron, is responsible for endogenous intestinal N-nitrosation arising from red meat. Cancer Research, 63: 2358-2360.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]