analysis of the area of the lamellae indicated that the edema observed in each group was statistically significant with respect to control; however, no PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19764249 statistically significant difference was seen between the 24 hours and 56 hours exposure groups. BEAS-2B cells exposed to CE for 2 hours showed a dose-dependent decrease in cell diameter measured with Scepter 2.0 cell counter with a 40 m sensor. The ECIS method is a well-established functional assay that utilizes electrical resistance as a measure of permeability and/or integrity of cell monolayers. We assessed the response of bronchial epithelial monolayers to CE by means of ECIS. Real time measurements of electrical resistance were obtained for a range of low CE concentrations over a 46 hours period. The data was normalized with respect to the resistance value of each well 1 hour prior to inoculation with CE. The results obtained indicate that CE caused a reduction in the resistance of the bronchial epithelial monolayer following 46 hours of exposure. These results demonstrate that CE is able to instigate a loss of the barrier function and integrity of the respiratory epithelium of the airway. CE induces caspase-3 activation and subsequent apoptosis The extent of the apoptotic response to CE exposure was evaluated using FITC-Annexin V and PI staining followed by flow cytometry. Flow cytometry analysis demonstrated that CE treatment increased cellular apoptosis. Compared to control, the percentage of apoptotic cells increased from 0.4% to 2.1% after 1 hour and from 4.1% to 8.3% after 4 hours. Cell exposure to CE also showed a significant increase in dead cells. These findings indicate that CE exposure induces epithelial cell apoptosis, which is exposure time and dose dependent. To determine the mechanism of apoptosis elicited by CE, we evaluated the cleavage of caspase-3. The cleavage of caspase-3 marks the point of no return in programmed cell death and is a marker for apoptosis activation. A dose-dependent cleavage of caspase-3 to its active form was observed using western blotting analysis. Caspase-3 activity was also measured via DEVD-pNA colorimetric assay, which showed a seven-fold increase in the induction of cleaved caspase-3 functional activity when compared to the control. We further sought to determine the effects of CE exposure on the respiratory epithelium of explanted mouse tracheal tissue and gill tissues from blue crabs. We observed cleaved caspase-3 positive cells in the epithelium of tracheal explants and crab gill tissues treated with 150 ppm of CE. Overall, these findings suggest that CE-induced epithelial cell apoptosis is caspase-3 dependent. 7 / 23 HO-1 Protects against Corexit-Induced Apoptosis 8 / 23 HO-1 
Protects against Corexit-Induced Apoptosis Fig 1. Morphological changes, reduction in cell diameter, permeability increase and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19762901 disruption of ML-128 intercellular junctions induced by CE. Gills of zebrafish were exposed to either CE 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 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. Cell diameter measurements. BEAS-2B cells were grown to confluence in 65 mm dishes and exposed to 0 to 150 ppm of