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 microM Fe++ +200 microM Fe was without effect on any enzyme activity. 700 microM 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 microM 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 microM 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 microM. Inhibition of catalase with aminotriazole slightly enhanced the toxicity of 700 microns H2O2. In summary, organic hydroperoxides at relatively low concentrations inactive key enzymes of the glutathione pathway, but hydrogen peroxide does not.
Liver Study Unit, Veterans Affairs Medical Center, San Francisco, California 94121.
D A
DA
Vessey
K H
KH
Lee
D000963:Antimetabolites / Q000494:pharmacology; D001555:Benzene Derivatives / Q000494:pharmacology; D019328:Buthionine Sulfoximine; D002118:Calcium / Q000032:analysis; D002374:Catalase / Q000378:metabolism; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D005290:Ferric Compounds / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D005978:Glutathione / Q000502:physiology; D005980:Glutathione Reductase / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015603:Keratinocytes / Q000201:enzymology*; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D010545:Peroxides / Q000494:pharmacology*; D013997:Time Factors; D020122:tert-Butylhydroperoxide
D000963:Antimetabolites; D001555:Benzene Derivatives; D003470:Culture Media; D005290:Ferric Compounds; D005296:Ferrous Compounds; D016877:Oxidants; D010545:Peroxides; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D005954:Glucosephosphate Dehydrogenase; D002374:Catalase; D005980:Glutathione Reductase; D005978:Glutathione; C007164:cumene hydroperoxide; D002118:Calcium