Faecal water genotoxicity
non-cytotoxic, human colon adenocarcinoma
GENOTOXICITY
The human colon adenocarcinoma cell line Caco-2 was used to test faecal water genotoxicity using a CometChip assay (Trevigen), a high-throughput version of the standard alkaline comet assay [Caco-2 cells were purchased from the ECACC and used between passage 10 and 22. Then they were routinely cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 0.1 mM of non-essential amino acids, 100 U/mL of penicillin, 0.1 g/mL of streptomycin, and 20% foetal bovine serum (FBS) (all Lonza UK). In the final passage prior to experimental procedures, the FBS was reduced to 10% of culture medium and experiments were carried out with this as carrier control.The comet assay was performed using the CometChip protocol (Trevigen, USA). To ensure adequate blinding, an independent analyst prepared 10% (v/v) faecal extract in carrier media, prepared by passing the previously prepared faecal water through a 0.22 µm filter immediately prior to treating cells. 10% v/v faecal extract preparations were shown to be non-cytotoxic in a 24 h MTT assay (viability was maintained at over 90%) and thus this concentration was deemed appropriate for genotoxicity.Caco-2 cells were split at 80% confluence, adjusted to 1 × 10Comet tails were assessed using the automated CometChip software (Trevigen, USA) which counted a minimum of 150 cell nuclei per well. Data represents a mean of four wells per treatment. 50 μM H