ROLE OF OXIDANT STRESS AND IRON DELOCALIZATION IN ACIDOSIS-INDUCED INTESTINAL EPITHELIAL HYPERPERMEABILITY

Using Caco-2(BBe) monolayers as a model of the intestinal epithelium, we tested the hypothesis that reactive oxygen metabolites contribute t o lactic acid-induced hyperpermeability. Compared to monolayers incuba ted at normal pH (i.e., 7.4) monolayers incubated in medium titrated t o extracellular pH (pH(o)) 5.0 with 10 mM lactic acid demonstrated inc reased permeability to both fluorescein sulfonic acid (FS) and fluores cein isothiocyanate-dextran (average molecular mass = 4000 Da; FD4). L actic acid-induced hyperpermeability to both FS and FD4 was reduced by adding either 30 mu M EUK-8, a superoxide dismutase/catalase mimetic, or catalase (10(4) U/ml). Incubation of monolayers with lactic acid i ncreased cellular malondialdehyde content, a measure of lipid peroxida tion. EUK-8 (30 mu M) completely abrogated this effect. Incubation wit h ferrous sulfate (100 mu M) exacerbated both lactic acid-induced hype rpermeability to FS and lactic acid-induced lipid peroxidation. Iron c helation with 1 mM diethylene triamine pentaacetic acid (DTPA)-trisodi um calcium salt attenuated lactic acid-induced hyperpermeability, wher eas iron-loaded DTPA (1 mM) was not protective. Treatment with DTPA-tr isodium calcium salt also ameliorated lactic acid-induced lipid peroxi dation. Incubation with lactic acid (pH(o) 5.0) for 16 h increased the cellular content of low molecular weight iron species. Incubation wit h lactic acid (pH(o) 5.0) for 24 h significantly increased the-percent age of oxidized protein-bound thiols in Caco-2(BBe) cells. We conclude that lactic acidosis induces hyperpermeability in Caco-2(BBe) monolay ers, in part, via an iron-dependent increase in reactive oxygen metabo lite-mediated damage.