INACTIVATION OF ENZYMES OF THE GLUTATHIONE ANTIOXIDANT SYSTEM BY TREATMENT OF CULTURED HUMAN KERATINOCYTES WITH PEROXIDES

Either metal ions, H2O2, t-butyl hydroperoxide (tBHP), or cumene hydroperoxide (CHP) was added to the medium of cultured human keratinocytes, and the activities of key peroxide-metabolizing enzymes were examined in a sonicated cell supernatant from the treated cells. 200 muM Fe++ + 200 muM Fe+++ was without effect on any enzyme activity. 700 muM CHP or tBHP decreased glutathione (GSH) peroxidase activity by 90% after 5 h and by 100% at 20 h, even if the CHP or tBHP was removed from the media after 90 min. H2O2 at 700 muM caused a brief 17% decrease in activity, which was followed by complete recovery. GSH peroxidase was found to be rapidly inactivated in vitro by CHP, but the enzyme was also inactivated at 37-degrees-C even in the absence of CHP. GSH prevented both types of inactivation. Consistent with this in vitro data, in vivo depletion of the GSH pool with buthionine sulfoximine led to lower levels of GSH peroxidase and increased sensitivity to peroxide-induced inactivation. Neither GSH reductase nor GSH S-transferase were inactivated by any treatment although CHP did cause a small increase in the activity of the latter, which was not due to induction. The activity of glucose-6-phosphate dehydrogenase was decreased 50% following treatment for 5 h with 700 muM CHP or tBHP, whereas H2O2 treatment caused a brief 15% decline, followed by recovery. The effects of peroxides were not altered by changing the concentration of Ca++ in the media. Catalase was unaffected by concentrations of peroxide up to 700 muM. Inhibition of catalase with aminotriazole slightly enhanced the toxicity of 700 mum H2O2. In summary, organic hydroperoxides at relatively low concentrations inactivate key enzymes of the glutathione pathway, but hydrogen peroxide does not.