Shvedova A A, Kommineni C, Jeffries B A, Castranova V, Tyurina Y Y, Tyurin V A, Serbinova E A, Fabisiak J P, Kagan V E
Health Effects Laboratory Division, Pathology and Physiology Research Branch, NIOSH, Morgantown, West Virginia, USA.
J Invest Dermatol. 2000 Feb;114(2):354-64. doi: 10.1046/j.1523-1747.2000.00865.x.
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.
多种酚类化合物被用于酚醛树脂、油漆、清漆、化妆品和药品的工业生产。已知皮肤接触工业酚类会导致皮疹、皮肤炎症、接触性皮炎、白斑和促进癌症。酚类化合物细胞毒性的生化机制尚不清楚。我们推测酚类化合物的酶促单电子氧化导致苯氧自由基的产生可能是细胞毒性作用的一个重要因素。苯氧自由基很容易被硫醇、抗坏血酸和其他细胞内还原剂（如NADH、NADPH）还原，从而再生母体酚类化合物。因此，酚类化合物可能会经历酶促驱动的氧化还原循环，从而导致氧化应激。为了验证这一假设，我们分析了暴露于苯酚的正常人角质形成细胞中的内源性硫醇、脂质过氧化和总抗氧化储备。使用新开发的基于顺式-紫黄质酸的方法来测定膜磷脂中位点特异性氧化应激，我们发现亚毒性浓度（50微摩尔）的苯酚会导致角质形成细胞中磷脂酰胆碱和磷脂酰乙醇胺（但不是磷脂酰丝氨酸）氧化。苯酚不会诱导由顺式-紫黄质酸标记的角质形成细胞脂质制备的脂质体中磷脂的过氧化。用ThioGlo-1测量表明，苯酚会消耗谷胱甘肽，但不会产生硫自由基，这一点通过我们对GS.-5, 5-二甲基-1-吡咯啉N-氧化物硝酮的高效液相色谱测量得到证明。此外，苯酚会导致蛋白质SH基团显著减少。鲁米诺增强化学发光分析表明，暴露于苯酚的角质形成细胞的总抗氧化储备显著降低。用苯酚孵育预先加载抗坏血酸的角质形成细胞会产生抗坏血酸自由基的电子顺磁共振可检测信号，这表明苯酚的单电子氧化产物、其苯氧自由基的氧化还原循环参与了氧化作用。由于在暴露于50微摩尔或500微摩尔苯酚的角质形成细胞中未观察到细胞毒性，我们得出结论，亚毒性浓度的苯酚会导致显著的氧化应激。
