Inhibition of Intercellular Communication in BALB/3T3 Fibroblasts by Cigarette Smoke Condensates

Abstract

In vitro tests for blockage on gap junctional intercellular communication (GJIC) provide a valuable information in predicting tumor promoter activity of various chemicals. Both crude cigarette smoke condensates (CSC) and 12-O-tetradecanoylphorbol-l3-acetate (TPA) inhibited GJIC of BALB/3T3 fibroblasts in a dose-dependent manner, in which the concentrations of CSC of main stream and TPA needed to inhibit 50% of the initial rate of fluorescence recovery (IC50) were estimated to be 40 and 0.085μg/ml, respectively, as determined by fluorescent return after photo-bleaching (FRAP) on the interactive laser cytometer. Among the fractionated samples of CSC, the neutral aromatic fraction, which contained about 12% of CSC substances, showed the highest GJIC inhibitory activity (IC50=15μg/ml) . The aqueous fraction, which comprised about 51% of CSC, showed the next higher GJIC inhibitory activity (IC5057μg/ml) . Immunoblot analysis indicated that TPA induced hyperphosphorylation of Ser/Thr residues of connexin 43 in the fibroblasts, whereas no significant changes in the phosphorylation of connexin 43 was detected by treatment of the fibroblasts with any of the CSC samples. These results indicate that TPA and CSC disfunctioned connexin 43 through the different action mechanisms.