Rat enterocyte injury by oxygen-dependent processes

Abstract

Neutrophils migrate to areas of inflammation and, when stimulated, produce O2−, H2O2, and other reactive O2 metabolites. To assess the effects of stimulated neutrophils on enterocytes, rat enterocytes were incubated with peripheral neutrophils. To assess cell viability, trypan blue exclusion and lactate dehydrogenase and protein release were measured. When 106 enterocytes/mL were incubated with 2.5 × 105 neutrophils/mL stimulated with phorbol myristate acetate, trypan blue exclusion decreased and lactate dehydrogenase and protein release increased. With the addition of 0.10 mg/mL of Superoxide dismutase, trypan blue exclusion further decreased and lactate dehydrogenase and protein release increased. This suggests that H2O2- or H2O2/ O2 -derived metabolites are more damaging to isolated enterocytes than O2−1. To test this hypothesis, enterocytes were incubated with xanthine and increasing concentrations of xanthine oxidase in the presence and absence of Superoxide dismutase. With increasing concentrations of xanthine oxidase, the cell number decreased and protein release increased. With the addition of Superoxide dismutase, fewer cells were present, suggesting that cell lysis occurred. Protein release was further increased by the addition of Superoxide dismutase. Enterocytes were then incubated with leucine and increasing concentrations of amino acid oxidase. With increasing concentrations of amino acid oxidase, trypan blue exclusion decreased and protein and lactate dehydrogenase release increased. These effects were ameliorated by the addition of 500 IU catalase/mL. These data suggest that O2− and H2O2, whether created by stimulated neutrophils or an enzymegenerating system, are damaging to isolated enterocytes. Superoxide dismutase did not offer enterocytes protection.