McCall Ingrid C, Betanzos Abigail, Weber Dominique A, Nava Porfirio, Miller Gary W, Parkos Charles A
Department of Environmental and Occupational Health, Rollins School of Public Health, Emory University, Atlanta, GA 30322, USA.
Toxicol Appl Pharmacol. 2009 Nov 15;241(1):61-70. doi: 10.1016/j.taap.2009.08.002. Epub 2009 Aug 11.
Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist. We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining. Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains.
土壤和水体中的苯酚污染引起了居住在苯酚生产工厂附近以及含有该化学物质的危险废物场地附近居民的担忧。苯酚，尤其是高浓度时，是一种刺激性和腐蚀性物质，会使人体黏膜成为毒性作用的靶点。然而，关于口服苯酚后的影响的数据很少。我们使用了一种体外模型，该模型采用在可渗透支持物上培养的人肠上皮细胞（SK-CO15）来研究苯酚对上皮屏障功能的影响。我们假设苯酚通过改变紧密连接（TJ）蛋白表达来破坏上皮屏障。使用跨上皮电阻（TER）和异硫氰酸荧光素标记的葡聚糖通透性测量来确定苯酚对上皮屏障功能的剂量反应效应。我们通过免疫荧光和蛋白质印迹分析研究了苯酚诱导的细胞形态变化以及几种紧密连接蛋白的表达。通过MTT、台盼蓝、碘化丙啶和TUNEL染色评估对细胞活力的影响。暴露于苯酚导致TER降低，异硫氰酸荧光素标记的葡聚糖的细胞旁通量以剂量依赖的方式增加。紧密连接蛋白1（claudin-1）和紧密连接蛋白相关蛋白1（ZO-1）从紧密连接向细胞质的移位与苯酚处理后观察到的通透性增加相关。此外，TER的降低与膜筏标记物分布的变化相关，表明苯酚对膜流动性有介导作用。这些观察结果与苯酚对细胞活力的影响无关，因为在不导致细胞死亡的苯酚剂量下就出现了通透性增强。总体而言，这些发现表明苯酚可能会短暂影响细胞膜的脂质双层，从而使含有紧密连接的微结构域不稳定。
