Glutathione oxidation and PTPase inhibition by hydrogen peroxide in Caco-2 cell monolayer

The role of H2O2 and protein thiol oxidation in oxidative stress-induced epithelial paracellular permeability was investigated in Caco-2 cell monolayers. Treatment with a H2O2 generating system (xanthine oxidase + xanthine) or H2O2 (20 mu M) increased the paracellular permeability. Xanthine oxidase-induced permeability was potentiated by superoxide dismutase and prevented by catalase. H2O2-induced permeability was prevented by ferrous sulfate and potentiated by deferoxamine and 1,10-phenanthroline. GSH, N-acetyl-L-cysteine, dithiothreitol, mercaptosuccinate, and diethylmaleate inhibited H2O2-induced permeability, but it was potentiated by 1,3-bis(2-chloroethyl)-1-nitrosourea. H2O2 reduced cellular GSH and protein thiols and increased GSSG. H2O2 mediated reduction of GSH-to-GSSG ratio was prevented by ferrous sulfate, GSH, N-acetyl-L-cysteine, diethylmaleate, and mercaptosuccinate and potentiated by 1,10-phenanthroline and 1,3-bis(2-chloroethyl)-1-nitrosourea. Incubation of soluble fraction of cells with GSSG reduced protein tyrosine phosphatase (PTPase) activity, which was prevented by coincubation with GSH. PTPase activity was also lower in H2O2-treated cells. This study indicates that H2O2, but not O-2(-). or . OH, increases paracellular permeability of Caco-2 cell monolayer by a mechanism that involves oxidation of GSH and inhibition of PTPases.