The effect of hypoxia/reoxygenation on the cellular function of intestinalepithelial cells

Background: Previously, using in vivo models, we have demonstrated that isc hemia/reperfusion can increase intestinal mucosal permeability, promote bac terial translocation, and induce gut cytokine production. Because of the ce llular heterogeneity of the gut, however, studies investigating the direct effects of hypoxia/reoxygenation on intestinal epithelial cells are confoun ded in in,vivo model systems, Consequently, this study examines oxidant-med iated enterocyte injury using an in vitro intestinal enterocyte hypoxia/reo xygenation model system. Methods: Two intestinal epithelial cell lines, IEC-6 and Caco-2, were seede d onto 3-mu m filters in a Transwell bicameral system and grown until tight junction integrity was established, Cells were subjected to hypoxia in a s ealed chamber with 95% nitrogen and 5% carbon dioxide and incubated at 37 d egrees C for 60 or 90 minutes. Reoxygenation was initiated by replacing the media and putting the cells in an environment of room air plus 5% carbon d ioxide. Permeability and bacterial translocation were assayed by measuring the phenol red concentration and culturing the bacteria that crossed the ce ll monolayer and reached the basal chamber of the bicameral system. Monolay er tight junction integrity was monitored by serial measurements of transep ithelial electrical resistance (TEER), and cell viability was assessed by t rypan blue dye. Results: IEC-6 cell monolayers subjected to 60 or 90 minutes of hypoxia she lved significantly higher permeability to phenol red, with 54 +/- 5% and 57 +/- 5% of the dye crossing the monolayers, respectively, compared with nor moxic control (38 +/- 2%; p < 0.01), Caco- 2 cell monolayers also had incre ased permeability to phenol red, with 24 +/- 6% and 20 +/- 4% of the phenol red crossing the monolayer after 60 or 90 minutes of hypoxia, respectively , compared with 8 +/- 3% in the normoxic controls (p < 0.01), At 3 hours af ter challenge with Escherichia coil, the monolayers subjected to 60 or 90 m inutes of hypoxia had significantly increased bacterial translocation (IEC- 6 cells, p < 0.05; Caco-2 cells, p < 0.01) compared with controls, The incr eased permeability of the hypoxic Caco-2 and IEC-6 monolayers was associate d with a decrease in TEER beginning as early as 1 hour after reoxygenation (p < 0.01). Cell viability, however, was not decreased. Conclusion: These results indicate that hypoxia/reoxygenation can directly impair cellular function as manifested by increased monolayer permeability to phenol red, increased E. coli bacterial translocation, and a decrease in TEER values.