A new method for the determination of the cytotoxicity of bile acids and aqueous phase of stool: the effect of calcium

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Abstract
Soluble secondary bile acids in the colon are supposed to be cytotoxic for normal colonic cells, resulting in an increased compensatory proliferation of colonic crypt cells, which is associated with an increased risk for colonic cancer. We developed a sensitive method to determine cytotoxicity of bile acids in the HT-29 colon cancer cell line, using a tetrazolium-based colorimetric assay. Only in vital cells, tetrazolium-salts are converted into formazan, which can be measured easily. Chenodeoxycholic acid and deoxycholic acid (DCA) were cytotoxic in concentrations above 100 microM, which is in the physiological range for soluble DCA in faeces. Conjugation of bile acids diminished cytotoxicity 7-10 fold. In this concentration range, no effect of calcium or calcium phosphate was demonstrated, suggesting that the effect of calcium on colonic proliferation is not mediated by a precipitation of soluble bile acids in the large bowel. Finally, we could demonstrate a significant correlation between the cytotoxicity of the aqueous phase of faeces and the soluble DCA concentration.