Inter- and intra-individual variation of faecal water - genotoxicity in human colon cells

Exogenous nutritional factors modulate the faecal contents leading to an en hanced or reduced burden with toxic and cancerogenic factors. These factors are thought to contribute to colon cancer by inducing mutations or enhanci ng proliferation in colon cells. Faecal water more or less causes these eff ects in model systems and thus could be the basis for valuable biomarker ap proaches. Our investigations are aimed at determining geno- and cytotoxicit y of faecal water in human colon cell lines in vitro. We are developing tec hniques for their applicability as biomarker tests during dietary intervent ion studies. Faecal water is isolated by centrifugation of the faeces at 25 000 x g and added to cultured human colon cells (HT29). Membrane damage as assessed by trypan blue exclusion is determined as a measure for cytotoxic ity. Semiquantitative analysis of inducible DNA damage (breaks and alkali l abile sites) are analysed with the single cell microgelelectrophoresis assa y (comet-assay) and oxidised DNA bases by the additional use of repair spec ific enzymes. We have now determined baseline toxic activities and calculat ed inter- and intra-individual and -experimental coefficients of variation for faecal water from different subjects consuming similar or different die ts. Most faecal water induced DNA damage and oxidised DNA bases in HT29 clo ne 19a cells (0.9-9.14 fold and 1.7-4.9 fold, respectively in comparison to the NaCl controls). Intra- and inter-experimental coefficients (CV) of var iation, were in a similar order of magnitude and ranged from 6.9 to 31.4. I n contrast both intra- and inter-individual variability were considerably h igher (CV-ranges of 29.7-76.6 and 21.3-64.0, respectively). Interestingly, these inter-individual values were not lowered when subjects consumed ident ical diets (CV-ranges of 28.4-126.0. However. following intervention with c ertain protective dietary regimens (e.g. lignan containing bread) significa nt reductions of faecal water-induced genotoxicity can be observed. Therefo re, in spite of the expected and observed degrees of variation in this meth odology, effective experimental protocols may still lead to detectable modu lations of the level of toxic and genotoxic effects. (C) 2000 Elsevier Scie nce B.V. All rights reserved.