CACO-2 CELL-METABOLISM OF OXYGEN-DERIVED RADICALS

Reactive oxygen metabolites have been associated with gastrointestinal injury and may play a role as mediators of inflammation. The effect of oxygen metabolites on Caco-2 cell viability (trypan blue exclusion and Cr-51 release), hexose monophosphate shunt activity, and glutathione was assessed. Caco-2 cells were incubated with amino acid oxidase, xanthine oxidase, menadione, and t-butylhydroperoxide in the presence and absence of superoxide dismutase, catalase, mannitol, and butylated hydroxytoluene. With amino acid oxidase, trypan blue exclusion decreased (P < 0.01) and Cr-51 release, oxidized glutathione, and shunt activity increased (P < 0.05). The addition of catalase attenuated these changes. Trypan blue exclusion decreased (P < 0.005) and Cr release, oxidized glutathione, and shunt activity increased (P < 0.01) with xanthine oxidase. The addition of superoxide dismutase caused a further increase in Cr-51 release, oxidized glutathione, and shunt activity (P < 0.01), which was prevented by the addition of catalase or mannitol. t-Butylhydroperoxide did not effect Cr-51 release or trypan blue exclusion, but oxidized glutathione and shunt activity increased (P < 0.01). The increase in shunt activity was prevented by preincubation with butylated hydroxytoluene (P < 0.01). Menadione did not alter trypan blue exclusion or Cr-51 release, but caused an increase in oxidized glutathione and shunt activity (P < 0.001). The increase in shunt activity was attenuated by preincubation with butylated hydroxytoluene (P < 0.001). Menadione also caused a depletion of total glutathione. Thus, Caco-2 cells are sensitive to oxidant injury and in all four systems increase in shunt activity and oxidized glutathione occurred at concentrations lower than those that caused cell injury, suggesting the shunt via the glutathione cycle is important in Caco-2 cell metabolism of oxidant species.