Fig. 1

Morphological changes, reduction in cell diameter, permeability increase and disruption of intercellular junctions induced by CE.

(A) Gills of zebrafish were exposed to either CE (150 ppm) or held as controls for 24 hours or 56 hours and stained with H&E. Digital micrographs were obtained at 20 x magnifications. Arrows point to edema and blebbing of gill epithelium. The ratios of gill area/gill length were calculated using Image Software (NIH, Bethesda, MD, USA) and presented as 1-dimensional area measurements. Data is quantified and shown as mean ± SD of three independent experiments. ** p < 0.01 vs control by a one-way ANOVA with HSD test. (B) Cell diameter measurements. BEAS-2B cells were grown to confluence in 65 mm dishes and exposed to 0 to 150 ppm of CE for 2 hours (n = 3). Data are shown as a mean ± SD. * p < 0.05 and ** p < 0.01 vs control by a one-way ANOVA with HSD test. (C) Permeability measurement of the bronchial epithelium of the airway. The sub-acute response to CE exposure was modeled by ECIS. BEAS-2B cells were seeded into the ECIS array. Cells were allowed to cover the gold electrodes in each well of the array prior to exposure to CE (0 ppm to 70 ppm). Real-time measurements of the electric resistance of the bronchial epithelial monolayers were obtained at 64 kHz. Resistance measurements were normalized with respect to the values in each well 1 hour prior to the initiation of the exposure. This time period corresponded to 16 hours after the seeding of the cells and was designated as t = 0 hour in the graph. The data are representative of three independent experiments. (D) BREA-2B cells were culture with or without 100 ppm CE for 1 hour. Protein expression of ZO-1 and actin filaments was detected using immunofluorescence microscopy (original magnification, ×40) with rabbit anti-ZO-1 (green) and phalloidin (red). Representative images captured from BEAS-2B cells are shown.