Redox cycling of phenol induces oxidative stress in human epidermal keratinocytes

A variety of phenolic compounds are utilized for industrial production of p henol-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 bi ochemical mechanisms of cytotoxicity of phenolic compounds are not well und erstood. We hypothesized that enzymatic one-electron oxidation of phenolic compounds resulting in the generation of phenoxyl radicals may be an import ant contributor to the cytotoxic effects, Phenoxyl radicals are readily red uced by thiols, ascorbate, and other intracellular reductants (e.g., NADH, NADPH) regenerating the parent phenolic compound. Hence, phenolic compounds may undergo enzymatically driven redoxcycling thus causing oxidative stres s. To test the hypothesis, we analyzed endogenous thiols, lipid peroxidatio n, 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 ph enol at subtoxic concentrations (50 mu M) caused oxidation of phosphatidylc holine and phosphatidylethanolamine (but not of phosphatidylserine) in kera tinocytes. Phenol did not induce peroxidation of phospholipids in liposomes prepared from keratinocyte lipids labeled by cis-parinaric acid. Measureme nts with ThioGlo-1 showed that phenol depleted glutathione but did not prod uce thiyl radicals as evidenced by our high-performance liquid chromatograp hy measurements of GS.-5,5-dimethyl1pyrroline N-oxide nitrone. Additionally , phenol caused a significant decrease of protein SH groups. Luminol-enhanc ed chemiluminescence assay demonstrated a significant decrease in total ant ioxidant reserves of keratinocytes exposed to phenol, Incubation of ascorba te-preloaded keratinocytes with phenol produced an electron paramagnetic re sonance-detectable signal of ascorbate radicals, suggesting that redox-cycl ing of one-electron oxidation products of phenol, its phenoxyl radicals, is involved in the oxidative effects. As no cytotoxicity was observed in kera tinocytes exposed to 50 mu M or 500 mu M phenol, we conclude that phenol at subtoxic concentrations causes significant oxidative stress.