Myeloperoxidase-catalyzed redox-cycling of phenol promotes lipid peroxidation and thiol oxidation in HL-60 cells

Various types of cancer occur in peroxidase-rich target tissues of animals exposed to aryl alcohols and amines. Unlike biotransformation by cytochrome P450 enzymes, peroxidases activate most substrates by one-electron oxidati on via radical intermediates. This work analyzed the peroxidase-dependent f ormation of phenoxyl radicals in HL-60 cells and its contribution to cytoto xicity and genotoxicity. The results showed that myeloperoxidase-catalyzed redox cycling of phenol in HL-60 cells led to intracellular formation of gl utathionyl radicals detected as GS-DMPO nitrone. Formation of thiyl radical s was accompanied by rapid oxidation of glutathione and protein-thiols. Ana lysis of protein sulfhydryls by SDS-PAGE revealed a significant oxidation o f protein SH-groups in HL-60 cells incubated in the presence of phenol/H2O2 that was inhibited by cyanide and azide. Additionally, cyanide- and azide- sensitive generation of EPR-detectable ascorbate radicals was observed duri ng incubation of HL-60 cell homogenates in the presence of ascorbate and H2 O2. Oxidation of thiols required addition of H2O2 and was inhibited by pret reatment of cells with the inhibitor of heme synthesis, succinylacetone. Ra dical-driven oxidation of thiols was accompanied by a trend toward increase d content of 8-oxa-7,8-dihydro-2'-deoxyguanosine in the DNA of HL-60 cells. Membrane phospholipids were also sensitive to radical-driven oxidation as evidenced by a sensitive fluorescence HPLC-assay based on metabolic labelin g of phospholipids with oxidation-sensitive cis-parinaric acid. Phenol enha nced H2O2-dependent oxidation of all classes of phospholipids including car diolipin, but did not oxidize parinaric acid-labeled lipids without additio n of H2O2 Induction of a significant hypodiploid cell population, an indica tion of apoptosis, was detected after exposure to H2O2 and was slightly but consistently and significantly higher after exposure to H2O2/phenol. The c lonogenicity of HL-60 cells decreased to the same extent after exposure to H2O2 Or H2O2/phenol. Treatment of HL-60 cells with either H2O2 or H2O2/phen ol at concentrations adequate for lipid peroxidation did not cause a detect able increase in chromosomal breaks. Detection of thiyl radicals as well as rapid oxidation of thiols and phospholipids in viable HL-60 cells provide strong evidence for redox cycling of phenol in this bone marrow-derived cel l line. (C) 1999 Elsevier Science Inc.