Effect of Colonic Bacterial Metabolites on Caco-2 Cell Paracellular Permeability In Vitro

Abstract

One common effect of tumor promoters is increased tight junction (TJ) permeability. TJs are responsible for paracellular permeability and integrity of the barrier function. Occludin is one of the main proteins responsible for TJ structure. This study tested the effects of physiological levels of phenol, ammonia, primary bile acids (cholic acid, CA, and chenodeoxycholic acid, CDCA), and secondary bile acids (lithocholic acid, LCA, and deoxycholic acid, DCA) on paracellular permeability using a Caco-2 cell model. Paracellular permeability of Caco-2 monolayers was assessed by transepithelial electrical resistance (TER) and the apical to basolateral flux of [ ¹⁴ C]-mannitol. Secondary, but not primary, bile acids increased permeability as reflected by significantly decreased TER and increased mannitol flux. Both phenol and ammonia also increased permeability. The primary bile acid CA significantly increased occludin expression (P < 0.05), whereas CDCA had no significant effect on occludin expression as compared to the negative control. The secondary bile acids DCA and LCA significantly increased occludin expression (P < 0.05), whereas phenol had no significant effect on the protein expression as compared to the negative control. This suggests that the increased permeability observed with LCA, DCA, phenol, and ammonia was not related to an effect on occludin expression. In conclusion, phenol, ammonia, and secondary bile acids were shown to increase paracellular permeability and reduce epithelial barrier function at doses typical of levels found in fecal samples. The results contribute to the evidence these gut microflora-generated products have tumor-promoting activity.

ACKNOWLEDGMENTS

This study was carried out with financial support from the Commission of the European Communities specific RTD program “Quality of Life and Management of Living Resources” (project No. QLK1–CT–1999–00346). It does not necessarily reflect its views and in no way anticipates the Commission's future policy in this area.

Notes

^{a a} Abbreviations are as follows: GAPDH, glyderaldehyde-3-phosphate dehydrogenase; CA, cholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; CDCA, chenodeoxycholic acid. Proteins in lysates of Caco-2 cells exposed to the treatments shown were separated by SDS-polyacrylamide gel electrophoresis and electro-blotted into polyvinylidene difluoride membranes. Membranes were probed for occludin using goat polyclonal antioccludin antibody and for GAPDH (used to check protein loading) using rabbit polyclonal anti-GAPDH antibody. Densitometric analysis of immunoblots was performed using a high-resolution scanner.

^b The value in column 2 represents the normalized value, i.e., the densitometric value for occludin divided by the densitometric value for GAPDH.

^{a c} Occludin expression was significantly increased compared to the negative control (P < 0.05).