title: Redox cycling of phenol induces oxidative stress in human epidermal keratinocytes.
abstract: A variety of phenolic compounds are utilized for industrial production of phenol-formaldehyde resins, paints, lacquers, cosmetics, and pharmaceuticals. Skin exposure to industrial phenolics is known to cause skin rash, dermal inflammation, contact dermatitis, leucoderma, and cancer promotion. The biochemical mechanisms of cytotoxicity of phenolic compounds are not well understood. We hypothesized that enzymatic one-electron oxidation of phenolic compounds resulting in the generation of phenoxyl radicals may be an important contributor to the cytotoxic effects. Phenoxyl radicals are readily reduced by thiols, ascorbate, and other intracellular reductants (e.g., NADH, NADPH) regenerating the parent phenolic compound. Hence, phenolic compounds may undergo enzymatically driven redox-cycling thus causing oxidative stress. To test the hypothesis, we analyzed endogenous thiols, lipid peroxidation, and total antioxidant reserves in normal human keratinocytes exposed to phenol. Using a newly developed cis-parinaric acid-based procedure to assay site-specific oxidative stress in membrane phospholipids, we found that phenol at subtoxic concentrations (50 microM) caused oxidation of phosphatidylcholine and phosphatidylethanolamine (but not of phosphatidylserine) in keratinocytes. Phenol did not induce peroxidation of phospholipids in liposomes prepared from keratinocyte lipids labeled by cis-parinaric acid. Measurements with ThioGlo-1 showed that phenol depleted glutathione but did not produce thiyl radicals as evidenced by our high-performance liquid chromatography measurements of GS.-5, 5-dimethyl1pyrroline N-oxide nitrone. Additionally, phenol caused a significant decrease of protein SH groups. Luminol-enhanced chemiluminescence assay demonstrated a significant decrease in total antioxidant reserves of keratinocytes exposed to phenol. Incubation of ascorbate-preloaded keratinocytes with phenol produced an electron paramagnetic resonance-detectable signal of ascorbate radicals, suggesting that redox-cycling of one-electron oxidation products of phenol, its phenoxyl radicals, is involved in the oxidative effects. As no cytotoxicity was observed in keratinocytes exposed to 50 microM or 500 microM phenol, we conclude that phenol at subtoxic concentrations causes significant oxidative stress.
authors.affiliation: Health Effects Laboratory Division, Pathology and Physiology Research Branch, NIOSH, Morgantown, West Virginia, USA.
authors.forename: A A
authors.initials: AA
authors.lastname: Shvedova
authors.forename: C
authors.initials: C
authors.lastname: Kommineni
authors.forename: B A
authors.initials: BA
authors.lastname: Jeffries
authors.forename: V
authors.initials: V
authors.lastname: Castranova
authors.forename: Y Y
authors.initials: YY
authors.lastname: Tyurina
authors.forename: V A
authors.initials: VA
authors.lastname: Tyurin
authors.forename: E A
authors.initials: EA
authors.lastname: Serbinova
authors.forename: J P
authors.initials: JP
authors.lastname: Fabisiak
authors.forename: V E
authors.initials: VE
authors.lastname: Kagan
mesh_terms: D000975:Antioxidants / Q000032:analysis; D017209:Apoptosis / Q000187:drug effects; D001205:Ascorbic Acid / Q000494:pharmacology*; D001391:Azo Compounds / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003497:Cyclic N-Oxides / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D005231:Fatty Acids, Unsaturated / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D005609:Free Radicals; D005978:Glutathione / Q000031:analogs & derivatives / Q000032:analysis; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008854:Microscopy, Electron; D009570:Nitriles / Q000494:pharmacology; D015388:Organelles / Q000648:ultrastructure; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D019800:Phenol / Q000494:pharmacology; D010636:Phenols / Q000378:metabolism / Q000494:pharmacology; D010743:Phospholipids / Q000032:analysis / Q000302:isolation & purification; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000032:analysis
chemical_list: D000975:Antioxidants; D001391:Azo Compounds; D003497:Cyclic N-Oxides; D005231:Fatty Acids, Unsaturated; D005456:Fluorescent Dyes; D005609:Free Radicals; D009570:Nitriles; D010636:Phenols; D010743:Phospholipids; D013113:Spin Labels; D013438:Sulfhydryl Compounds; C099621:glutathionyl-5,5-dimethyl-1-pyrroline N-oxide; C055792:2,2'-azobis(2,4-dimethylvaleronitrile); D019800:Phenol; D005978:Glutathione; C013641:parinaric acid; D001205:Ascorbic Acid