Ferric iron increases ROS formation, modulates cell growth and enhances genotoxic damage by 4-hydroxynonenal in human colon tumor cells.

Iron is a relevant risk factor for colorectal cancer due to its genotoxic properties. Here we hypothesised that iron-overload causes other toxic effects, which contribute to carcinogenesis. For this, we investigated formation of reactive oxygen species (ROS), DNA repair, cell growth and glutathione (GSH) in human colon tumor cells (HT29 clone 19A) treated with ferric nitrilotriacetate (Fe-NTA, 0-2000 microM). Intracellular formation of ROS was analysed with the peroxide-labile fluorescent dye carboxy-dichlorodihydrofluorescine-diacetate. DNA repair, reflected as the persistency of DNA damage induced by selected genotoxins, was determined with the Comet assay. Cell growth and GSH were measured by fluorimetrical analysis. Key findings were that ROS formation increased with time (1000 microM Fe-NTA, p < 0.001). DNA damage was largely repaired after 120 min, but was not affected by 10 microM Fe-NTA. In contrast, 10 microM Fe-NTA significantly increased DNA damage induced by 4-hydroxynonenal. Doses of 25 microM Fe-NTA increased cell growth (p < 0.05), whereas high concentrations (2000 microM) resulted in growth arrest (p < 0.05), that was accompanied by increased GSH levels (p < 0.01). In conclusion, high concentrations of Fe-NTA caused cellular effects, which reflect a stress response, and resulted in formation of ROS. Carcinogenic risks from ferric iron could be derived also from lower concentrations, which enhance tumor cell growth and cause progenotoxic effects.

Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the Comet assay: a biomarker model.

Methods are needed to assess exposure to genotoxins in humans and to improve understanding of dietary cancer prevention. The Comet assay was used to detect smoking-related exposures and dietary modulations in target tissues. Buccal scrapings, blood and faeces were collected from 38 healthy male volunteers (smokers and non-smokers) during a dietary intervention study with bread supplemented with prebiotics+/-antioxidants. GSTM1-genotype was determined with PCR. Buccal and peripheral lymphocytes were analysed for DNA damage using the Comet assay. Genotoxicity of faecal water (FW) was assayed in human colon HT29 clone 19A cells. 'Tail intensity' (TI) was used as a quantitative indicator of DNA damage in the Comet assay. Intervention with bread reduced DNA damage in lymphocytes of smokers (8.3+/-1.7% TI versus 10.2+/-4.1% TI, n=19), but not of non-smokers (8.6+/-2.8% TI versus 8.3+/-2.7% TI, n=15). Faecal water genotoxicity was reduced only in non-smokers (9.4+/-2.9% TI versus 18.9+/-13.1% TI, n=15) but not in smokers (15.5+/-10.7% TI versus 20.4+/-14.1% TI, n=13). The Comet assay was efficient in the detection of both smoking-related exposure (buccal cells) and efficacy of dietary intervention (faecal samples). Smokers and non-smokers profited differently from the intervention with prebiotic bread+/-antioxidants. Stratification of data by genotype enhanced specificity/sensitivity of the intervention effects and contributed important information on the role of susceptibility.

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

Exogenous nutritional factors modulate the faecal contents leading to an enhanced or reduced burden with toxic and cancerogenic factors. These factors are thought to contribute to colon cancer by inducing mutations or enhancing proliferation in colon cells. Faecal water more or less causes these effects in model systems and thus could be the basis for valuable biomarker approaches. Our investigations are aimed at determining geno- and cytotoxicity of faecal water in human colon cell lines in vitro. We are developing techniques for their applicability as biomarker tests during dietary intervention studies. Faecal water is isolated by centrifugation of the faeces at 25000xg and added to cultured human colon cells (HT29). Membrane damage as assessed by trypan blue exclusion is determined as a measure for cytotoxicity. Semiquantitative analysis of inducible DNA damage (breaks and alkali labile sites) are analysed with the single cell microgelelectrophoresis assay (comet-assay) and oxidised DNA bases by the additional use of repair specific enzymes. We have now determined baseline toxic activities and calculated inter- and intra-individual and -experimental coefficients of variation for faecal water from different subjects consuming similar or different diets. Most faecal water induced DNA damage and oxidised DNA bases in HT29 clone 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 variation, were in a similar order of magnitude and ranged from 6.9 to 31.4. In contrast both intra- and inter-individual variability were considerably higher (CV-ranges of 29.7-76.6 and 21.3-64.0, respectively). Interestingly, these inter-individual values were not lowered when subjects consumed identical diets (CV-ranges of 28.4-126.0). However, following intervention with certain protective dietary regimens (e.g. lignan containing bread) significant reductions of faecal water-induced genotoxicity can be observed. Therefore, in spite of the expected and observed degrees of variation in this methodology, effective experimental protocols may still lead to detectable modulations of the level of toxic and genotoxic effects.