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

Abstract

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 oxidation via radical intermediates. This work analyzed the peroxidase-dependent formation of phenoxyl radicals in HL-60 cells and its contribution to cytotoxicity and genotoxicity. The results showed that myeloperoxidase-catalyzed redox cycling of phenol in HL-60 cells led to intracellular formation of glutathionyl radicals detected as GS-DMPO nitrone. Formation of thiyl radicals was accompanied by rapid oxidation of glutathione and protein-thiols. Analysis of protein sulfhydryls by SDS-PAGE revealed a significant oxidation of protein SH-groups in HL-60 cells incubated in the presence of phenol/H 2O 2 that was inhibited by cyanide and azide. Additionally, cyanide- and azide-sensitive generation of EPR-detectable ascorbate radicals was observed during incubation of HL-60 cell homogenates in the presence of ascorbate and H 2O 2. Oxidation of thiols required addition of H 2O 2 and was inhibited by pretreatment of cells with the inhibitor of heme synthesis, succinylacetone. Radical-driven oxidation of thiols was accompanied by a trend toward increased content of 8-oxo-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 labeling of phospholipids with oxidation-sensitive cis-parinaric acid. Phenol enhanced H 2O 2-dependent oxidation of all classes of phospholipids including cardiolipin, but did not oxidize parinaric acid–labeled lipids without addition of H 2O 2. Induction of a significant hypodiploid cell population, an indication of apoptosis, was detected after exposure to H 2O 2 and was slightly but consistently and significantly higher after exposure to H 2O 2/phenol. The clonogenicity of HL-60 cells decreased to the same extent after exposure to H 2O 2 or H 2O 2/phenol. Treatment of HL-60 cells with either H 2O 2 or H 2O 2/phenol at concentrations adequate for lipid peroxidation did not cause a detectable 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 cell line.