CHARACTERIZATION OF THE OXIDATIVE STRESS INITIATED IN CULTURED HUMAN KERATINOCYTES BY TREATMENT WITH PEROXIDES

Cultured human keratinocytes were treated with H2O2, Fe++/Fe+++, H2O2 + Fe++/Fe+++, t-butylhydroperoxide (tBHP), or cumene hydroperoxide (CHP). Fe++ +/- Fe+++ was without effect on cell viability. Neither CHP, tBHP, nor H2O2 at 200 muM led to alteration of trypan blue exclusion, but with 700 muM CHP or tBHP there was uptake of trypan blue after 20 min and lysis of cells beginning at 4 h of treatment. Lysis occurred even if the organic hydroperoxide was removed from the media after 1 h. Treatment with 700 muM H2O2 resulted in half of the cells becoming permeable to trypan blue by 60 min, but > 80% of the cells remained intact and functional, and eventually recovered their impermeability to trypan blue. No concentration of H2O2, tBHP, or CHP produced significant thiobarbituric acid (TBA)-reactive material, and Fe++/Fe+++, H2O2 + Fe+++, and CHP + Fe++/Fe+++ led to the formation of only small amounts of TBA-reactive material. This was attributed to a lack of polyunsaturated lipid in cells cultured in synthetic media. The activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is sensitive to oxidative damage and thus was used as an indicator of oxidative stress along with the ratio of reduced/oxidized glutathione (GSH/GSSG). Using these two criteria, we found that CHP or tBHP treatment led to an oxidative stress that was more protracted as compared with the effect of H2O2. The organic peroxides also led to depletion of total glutathione, an effect not found with H2O2. It was also found that H2O2 was more rapidly metabolized than the organic peroxides. In summary, cultured human keratinocytes treated with peroxides underwent a number of changes, which included inactivation of GAPDH, a decrease in the ratio GSH/GSSG, and a loss of trypan blue exclusion. However, as long as the duration of this oxidative stress was short, these changes were reversible and the cells survived. Prolonged oxidative stress led to irreversible damage and cell death. H2O2 was rapidly metabolized and relatively well tolerated by keratinocytes. On the other hand, organic hydroperoxides were metabolized more slowly and were lethal at sub-millimolar concentrations. The relative toxicity of organic hydroperoxides is hypothesized to be related to their non-polar nature.