title: 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/H2O2 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 H2O2. Oxidation of thiols required addition of H2O2 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 H2O2-dependent oxidation of all classes of phospholipids including cardiolipin, but did not oxidize parinaric acid-labeled lipids without addition of H2O2. Induction of a significant hypodiploid cell population, an indication of apoptosis, was detected after exposure to H2O2 and was slightly but consistently and significantly higher after exposure to H2O2/phenol. The clonogenicity 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/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.
authors.affiliation: Department of Environmental & Occupational Health, University of Pittsburgh, PA 15238, USA.
authors.forename: R
authors.initials: R
authors.lastname: Goldman
authors.forename: G H
authors.initials: GH
authors.lastname: Claycamp
authors.forename: M A
authors.initials: MA
authors.lastname: Sweetland
authors.forename: A V
authors.initials: AV
authors.lastname: Sedlov
authors.forename: V A
authors.initials: VA
authors.lastname: Tyurin
authors.forename: E R
authors.initials: ER
authors.lastname: Kisin
authors.forename: Y Y
authors.initials: YY
authors.lastname: Tyurina
authors.forename: V B
authors.initials: VB
authors.lastname: Ritov
authors.forename: S L
authors.initials: SL
authors.lastname: Wenger
authors.forename: S G
authors.initials: SG
authors.lastname: Grant
authors.forename: V E
authors.initials: VE
authors.lastname: Kagan
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D017209:Apoptosis / Q000187:drug effects; D001205:Ascorbic Acid / Q000378:metabolism; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000633:toxicity; D015227:Lipid Peroxidation / Q000187:drug effects*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D009195:Peroxidase / Q000378:metabolism*; D019800:Phenol / Q000378:metabolism* / Q000633:toxicity*; D010743:Phospholipids / Q000378:metabolism; D011190:Potassium Cyanide / Q000494:pharmacology; D019810:Sodium Azide / Q000494:pharmacology; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D010743:Phospholipids; D013438:Sulfhydryl Compounds; D019800:Phenol; D000080242:8-Hydroxy-2'-Deoxyguanosine; D019810:Sodium Azide; D006861:Hydrogen Peroxide; D009195:Peroxidase; D003849:Deoxyguanosine; D011190:Potassium Cyanide; D001205:Ascorbic Acid