Variability in fecal water genotoxicity, determined using the Comet assay, is independent of endogenous N‐Nitroso compound formation attributed to red meat consumption

Abstract

Red meat consumption causes a dose-dependent increase in fecal apparent total N-nitroso compounds (ATNC). The genotoxic effects of these ATNCs were investigated using two different Comet assay protocols to determine the genotoxicity of fecal water samples. Fecal water samples were obtained from two studies of a total of 21 individuals fed diets containing different amounts of red meat, protein, heme, and iron. The first protocol incubated the samples with HT-29 cells for 5 min at 4 degrees C, whereas the second protocol used a longer exposure time of 30 min and a higher incubation temperature of 37 degrees C. DNA strand breaks were quantified by the tail moment (DNA in the comet tail multiplied by the comet tail length). The results of the two Comet assay protocols were significantly correlated (r = 0.35, P = 0.003), however, only the second protocol resulted in detectable levels of DNA damage. Inter-individual effects were variable and there was no effect on fecal water genotoxicity by diet (P > 0.20), mean transit time (P = 0.588), or weight (P = 0.705). However, there was a highly significant effect of age (P = 0.019). There was no significant correlation between concentrations of ATNCs in fecal homogenates and fecal water genotoxicity (r = 0.04, P = 0.74). ATNC levels were lower in fecal water samples (272 microg/kg) compared to that of fecal homogenate samples (895 microg/kg) (P < 0.0001). Failure to find dietary effects on fecal water genotoxicity may therefore be attributed to individual variability and low levels of ATNCs in fecal water samples.

Adduction of Human p53 Gene by Fecal Water: An In vitro Biomarker of Mutagenesis in the Human Large Bowel

“…Furthermore, there was a 25% loss of ATNC in the necessary fecal water filtering step. Previous assays using fecal water have investigated effects on intact whole cells such as in the Salmonella typhimurium genotoxicity assay and HT29 cells in the comet assay, where systems for repair of DNA damage remain intact (3,5,18). Although the present system may not represent effects in vivo, it nevertheless shows the potential for mutagenesis and thus initiation.…”

“…Fecal water fraction has been reported to be more efficient than fecal solids at altering the growth characteristics of colonocytes rather than homogenate (16,17) and has been used for genotoxicity assays such as the comet assay (6,18,19). However, fecal homogenates have been shown to contain 3 times more ATNC and 100 times more nitrosated heme than fecal water (5,20). Furthermore, there was a 25% loss of ATNC in the necessary fecal water filtering step.…”

“…Seventeen samples of fecal water were extracted from available fecal samples from 10 volunteers taking part in controlled dietary studies as previously described (5,13). Aliquots from defrosted fecal samples that had been snap frozen on dry ice were homogenized for 2 min and then centrifuged at 50,000 Â g for 2 h. Aliquots of the supernatant fecal water were stored at À80jC before analysis.…”

“…Bruce et al (3) first brought to light the mutagenic capacity of human feces, which is known to contain a variety of potentially genotoxic substances including fecapentaenes, bile acids, heterocyclic amines, polycyclic aromatic hydrocarbons, and N-nitroso compounds measured as apparent total nitroso compounds (ATNC; refs. 4,5). However, there have been few studies characterizing the nature of these diverse genotoxins.…”

Cytokinesis-Block Micronucleus Cytome Assays for the Determination of Genotoxicity and Cytotoxicity of Cecal Water in Rats and Fecal Water in Humans

“…These methods include the Comet assay or measurement of DNA adducts (12)(13)(14). The cytokinesis-block micronucleus (CBMN) assay is an alternative well-established approach to measure the cecal or fecal water genotoxicity (15,16).…”