Philip Morris Products S.A., R&D, Rue des Usines 90, 2000 Neuchatel, Switzerland.
Toxicol Lett. 2013 Jun 7;219(3):248-53. doi: 10.1016/j.toxlet.2013.03.028. Epub 2013 Apr 1.
Inhibition of gap-junctional intercellular communication (GJIC) via exposure to various toxic substances has been implicated in tumor promotion. In the present study, cigarette smoke total particulate matter (TPM), a known inhibitor of GJIC, were used to characterize a new GJIC screening assay in three independent experiments. The main features of this assay were automated fluorescence microscopy combined with non-invasive parachute technique. Rat liver epithelial cells (WB-F344) were stained with the fluorescent dye Calcein AM (acetoxymethyl) and exposed to TPM from the Kentucky Reference Cigarette 2R4F (a blend of Bright and Burley tobaccos) and from two single-tobacco cigarettes (Bright and Burley) for 3h. Phorbol-12-myristate-13-acetate (TPA) was used as positive control and 0.5% dimethyl sulfoxide (DMSO) as solvent control. The transfer of dye to adjacent cells (percentage of stained cells) was used as a measure of cellular communication. A clear and reproducible dose-response of GJIC inhibition following TPM exposure was seen. Reproducibility and repeatability measurements for the 2R4F cigarette were 3.7% and 6.9%, respectively. The half-maximal effective concentration values were 0.34ng/ml for TPA, 0.050mg/ml for the 2R4F, 0.044mg/ml for the Bright cigarette, and 0.060mg/ml for the Burley cigarette. The assay was able to discriminate between the two single-tobacco cigarettes (P<0.0001), and between the single-tobacco cigarettes and the 2R4F (P=0.0008, 2R4F vs. Burley and P<0.0001, 2R4F vs. Bright). Thus, this assay can be used to determine the activity of complex mixtures such as cigarette smoke with high throughput and high precision.
抑制缝隙连接细胞间通讯（GJIC）通过暴露于各种有毒物质已被牵连在肿瘤促进。在本研究中，香烟烟雾总颗粒物（TPM），已知的GJIC 的抑制剂，被用来描述在三个独立的实验中的一个新的 GJIC 筛选试验。这个试验的主要特点是自动荧光显微镜结合非侵入性降落伞技术。大鼠肝上皮细胞（WB-F344）用荧光染料 Calcein AM（乙酰氧甲基）染色，并暴露于来自肯塔基州参考香烟 2R4F（一个混合的光明和白肋烟）和两个单一烟草香烟（光明和白肋烟）的 TPM 3 小时。佛波醇-12-肉豆蔻酸-13-醋酸盐（TPA）被用作阳性对照，0.5%二甲基亚砜（DMSO）作为溶剂对照。染料转移到相邻细胞（染色细胞的百分比）被用作细胞通讯的衡量标准。在 TPM 暴露后，GJIC 抑制的清晰和可重复的剂量反应可见。2R4F 香烟的重复性和可重复性测量值分别为 3.7%和 6.9%。TPA 的半最大有效浓度值为 0.34ng/ml，2R4F 为 0.050mg/ml，光明香烟为 0.044mg/ml，白肋烟为 0.060mg/ml。该试验能够区分两种单一烟草香烟（P<0.0001），以及单一烟草香烟和 2R4F（P=0.0008，2R4F 与白肋烟和 P<0.0001，2R4F 与光明）。因此，该试验可用于确定具有高通量和高精度的复杂混合物（如香烟烟雾）的活性。
