Background and Hypothesis: Epidemiological and animal studies indicate that colon cancer is associated with a western style diet (high fat, low vegetable and fruit). It was shown that colon cancer patients have increased concentrations of hydrophobic bile acids in their fecal water. Hydrophobic bile acids, such as deoxycholic acid (DOC), induce apoptosis, DNA damage and oxidative stress. However, chronic exposure to hydrophobic bile acids may lead to the selection of apoptosis resistant cells and thus contribute to cancer development. Since bacteria can deconjugate primary bile acids into more toxic hydrophobic secondary bile acids, we speculate that bacteria may be involved in the development of apoptosis resistance. In this study we tested whether colonic mucosa from germ-free and conventional animals is sensitive to apoptosis induced by DOC. Methods: Wistar rats were raised in germ-free or conventional conditions. Colon cancer was initiated by injecting 9 mg/kg of azoxymethane s.c. once a week for 5 weeks. Furthermore, 2 ml of human bile was injected directly into the cecum 2 times a week for a total of 9 doses, through a surgically created cecal hernia. Rats were sacrificed six months after initiation of treatment and colonic tissue was evaluated for histological changes and apoptosis. Apoptosis capability was evaluated in normal appearing mucosa morphologically and by immunohistochemistry using antibody against cleaved caspase 3, in colonic tissues prior and after stressing tissues ex-vivo in 0.5 mM DOC for two hours. Results: As expected, adenomatous changes and small colonic tumors were found in conventional rats, but none were found in germ-free animals. Morphological and immunohistochemical examination of normal appearing colonic mucosa indicated that the frequency of colonic crypts containing apoptotic cells after ex-vivo incubation in DOC is markedly higher in colonic tissue of germ-free animals compared to the tissue of conventional animals. Conclusion: Normal appearing mucosa from conventional rats was more resistant to apoptosis induced by DOC than mucosa from germ-free rats. Colonic bacteria may contribute to the elevated levels of secondary bile acids in the colon and thus to the selection of cells with an apoptosis-resistant phenotype. This may explain the higher incidence of colon tumors in the conventional compared to germ-free animals. .
[Proc Amer Assoc Cancer Res, Volume 46, 2005]